Islam News Room - Months Named for Pagan Gods?
How do names of Ancient False Gods get easily accepted into modern day society?
Origin of the Names of the Months begins with the Roman Sun Worshippers Calendar, having names of the months indicating the beginning of the "birth of the sun" (March or mars) and ending with the 12th or "shortest month of the sun". The most sun was in the 5th and 6th months: quintilis (5 or July) and sextilis (6 or August).
The Gregorian, calendar was instituted by Pope Gregory XIII, in 1592.
Pope Gregory XIII, of the Catholic Church - did change the official Roman, Latin calendar which started the first month with (March) Mars, to (Januay) Janus being the first month of the year.
But he is not the only one who has played with the "Sun Calendar", not the least at all.
"Janus" the Roman god of doors, beginnings, sunset and sunrise, had one face looking forward and one backward. Formerly, the numeric month of eleven for the Roman Latin calendar.
"februare" - Latin: meaning to purify. Romans celebrated the festival of forgiveness of sins in the middle of this month. This was originally the 12th or last month, and shortest month of the Roman calendar, having only 29 days, except every fourth year it would have 30 days. This was changed by August Caesar (see below).
"aprilis" (Roman name in Latin), perhaps derived from aperire, (Latin to open, as in opening buds and blossoms) or maybe even from Aphrodite, original Greek name of Venus.
"maia" Roman goddess, mother of the god Mercury, whose father was the god Jupiter, and she was the daughter of the god they called Atlas.
"Juno" chief Roman goddess. Female godesses were popular amongst elite of the Roman empire. Some women were used as prophets or seers.
"septem" - Latin for the seventh month in the Julian or Roman calendar. This was also established in the reign of Julius Caesar.
"octo" - Roman Latin for eight in the Julian calendar. Note the word "octagon" or eight sided coming from the same root. Catholic countries adopted the Gregorian calendar in 1582, skipping 10 days that October, correcting for too many leap years.
"novem" - number nine on Roman latin calendar. This name has more or less remained the same over 2,000 years.
"decem" - number ten, Julian or Roman calendar year's tenth month (decem, Latin for ten). Still essentially the same meaning.
We see now the names of the months on the sun calendar are more than just some words. These had meanings and were considered by the people of the times to be more or less sacred or holy to them.
For these reasons it would be wise to consider the calendar of the Jews and Muslim (based on the stages and sightings of the moon) as more valid, at least in respect to the naming convention.
- It was in 44 B.C. when Julius Caesar ordered the longest month of the year with the most sunshine, to be renamed for him (Julios), as this month had the most days and hours for sun worship and he claimed to be born of the sun god in that month.
- Not to be outdone by his predecessor (Julius Caesar), August wanted the same number of days in "his month".
- One more day was needed to bring August (formerly sextilis) to the level of July (formerly "quintilis").
So they took one day away from the last month of the year (February was the 12 month for the Romans).
This is how we end up with only 28 days in February, except 29 days every four years.
- Incidentally, the word "Catholic" is still used exactly today as it was 2300 years ago (Yes, even before Christ): ROMAN CATHOLIC CHURCH (Universal Church of Rome): Catholic - "universal".
The original Roman year had 10 named months Martius "March", Aprilis "April", Maius "May", Junius "June", Quintilis "July", Sextilis "August", September "September", October "October", November"November", December "December".
There are likely two unnamed months in the dead of winter when not much happened in agriculture.
The year began with Martius "March".
Numa Pompilius, the second king of Rome circa 700 BC, added the two months Januarius "January" and Februarius "February".
After Februarius there was occasionally an additional month of Intercalaris "intercalendar".
This is the origin of the leap-year day being in February. In 46 BC, Julius Caesar reformed the Roman calendar (hence the Julian calendar) changing the number of days in many months and removing Intercalaris.
Julius Caesar reformed the Roman calendar (hence the Julian calendar) in 46 BC.
In the process, he renamed this month after himself.
Augustus Caesar clarified and completed the calendar reform of Julius Caesar.
In the process like Julius, he also renamed this month after himself.
September had 30 days, until Numa, then it had 29 days, then after Julius it became 30 days long.
These sources are somewhat inconsistent. I have chosen interpretations that are predominate among sources or that seem most reasonable.


Let's be perfectly clear: The Hamilton Beach Breakfast Sandwich Maker ($29.99) is not a gourmet, high-tech, fancy cooking tool for super-serious cooks.
What it is, though, is fun. And it's well-built and sturdy, considering what it's designed to do. And it works just like it's supposed to.
The whole idea is to make a breakfast sandwich like the ones from fast-food restaurants. You could certainly do that with normal cooking equipment - a toaster, frying pans, egg rings - but that's really not the point. This gadget makes the process a little more user-friendly. Or maybe I should say kid-friendly.
Or cooking-challenged friendly. Because this isn't really like cooking. It's a simple assembly process that just about anyone could manage.
The idea is that you start with the bottom of a muffin, add meat, cheese or vegetables on top, then close that section and put an egg on the next layer and put the top piece of bread on top of the egg. Close it up, and in about five minutes, the egg is cooked, the bread is toasted, and everything is very hot. Slide the bottom out from under the egg, and there's a complete sandwich.
Letting kids make their own fast-food-style breakfast sandwiches might help picky kids get involved with cooking - which is supposed to help them become more adventurous - and mom could limit the selections to healthier choices.
Younger kids would still need supervision, because this machine does get hot while it's cooking, but they could choose their ingredients and make their own special breakfast sandwiches with a little help. Older kids could work unsupervised, making breakfast or an after-school snack.
Kids with allergy issues who can't have fast-food style sandwiches might also get a kick out of making their own sandwich that looks like the real thing. And since this is small, it's something that could be brought along on trips to make a safe breakfast in a hotel room or in someone's home. Or an adult with allergy issues that prevents them from using the communal toaster and microwave might keep something like this in their office.
Since it takes about five minutes for an egg to cook in the sandwich, this isn't the best bet for someone who needs to make a half-dozen breakfast sandwiches. In that case, you might as well break out the frying pan and egg rings. But for someone single, a breakfast sandwich could be done in the time it takes to brew coffee.
Besides breakfast sandwiches, I've used this to make English muffin pizzas and hot ham-and-cheese sandwiches on biscuits, so it's not all about breakfast.
Is this for everyone? Absolutely not. Is it for some people? Definitely yes.


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Commission reassures member states about meat market crisis risks
By Gerardo Fortuna | EURACTIV.com
19-03-2019 (updated: 01-04-2019 )
Ireland's beef sector is the most exposed to the UK's no deal tariff plans. [Photo: SHUTTERSTOCK]
Languages: Français
Some EU ministers raised concerns about external market disturbances in the meat sector coming from Ukraine and Brexit during a meeting on Monday (18 March) but Commissioner for Agriculture Phil Hogan pledged to fight fire with fire.
An item regarding the safeguarding of the EU meat market was included on the agenda of yesterday's EU Agrifish Council thanks to pressure from Poland, which was concerned about imbalances in the sector caused by Ukrainian poultry imports.
A number of member states led by Ireland also jumped at the chance to raise concerns on the same subject but with a Brexit angle, following the announcement of the UK's no deal tariff schemes last week.
May's government proposed plans to slash tariffs to zero on 87% of goods imports for 12 months as a temporary response if the UK ends up leaving the EU without a deal.
In a press conference, EU agriculture boss Phil Hogan cited the availability of suitable market measures in the event of any significant market disruption.
"We have a lot of experience in dealing with these market disturbances related to market developments," the Commissioner said, mentioning actions implemented by the EU in the past to cope with the Bovine spongiform encephalopathy (BSE) outbreak and the Russian embargo.
Hogan reaffirmed his belief that the UK temporary tariff plan is illegal under the WTO rules, evidenced by the fact that Westminster itself only considers it a temporary measure.
"There is a long way to go in terms of the schedule of tariffs that will be acceptable to be negotiated under the WTO rules," he added.
UK may have to hold EU elections, May warns after MPs rule out 'no deal' Brexit
Theresa May warned MPs on Wednesday (13 March) that she could be forced to request a lengthy extension to Brexit talks, which would mean UK involvement in the European elections in May, after suffering another parliamentary defeat to her Brexit plans.
A diplomatic source confirmed that, during the debate over the meat sector in the Council, Brexit was considered as a possible market disturbance capable of affecting Ireland and some other member states directly, as well as the rest of the EU indirectly.
France, Germany, Belgium, the Netherlands, Ireland and also Denmark are the most exposed countries by this proposed schedule of tariffs, according to the Commissioner.
Irish beef and dairy sectors could have been badly affected, as Ireland exports over 50% of its meat to the UK. According to the Irish farmer's association, if meat exports will be subject to tariffs there will be a direct hit of almost €800 million on the sector.
"It is worth noting that Ireland exports into the UK a significant amount of beef, but that it also represents 75% of total exports of beef to the UK," Hogan recalled.
Ireland will seek EU aid to support livestock in case of hard Brexit
Ireland will seek hundreds of millions of euros in aid from Brussels if Britain crashes out of the EU without a deal, the agriculture minister was quoted Thursday (3 January) as saying.
The Commissioner added that Brexit is likely to happen and, since its impact is likely to be quite dynamic, the EU must be prepared for all scenarios.
However, an EU source said that the Commissioner did not go into details on contingency planning.
The UK representative also took the floor during the Council, explaining the precautionary ratio behind the no deal tariff scheme but reiterating that the UK government wants a deal.
According to a source, the UK stressed that going to zero tariffs is actually not feasible because other trade partners could ask for the same treatment, which is something that the UK cannot extend to other WTO members.
As regards market disturbances coming from Ukraine, it emerged from the debate among EU ministers that the EU is not facing a real crisis yet, but more a problem of overproduction from the Eastern neighbour, also with a seasonal trait.
Ukraine became a major supplier of poultry meat to the EU, having exported 123,000 tons in 2018. About 15% of the estimated chicken meat imports in 2019 is also expected to come from Ukraine.
Hogan announced that the EU is preparing an amendment to the Deep and Comprehensive Free Trade Agreement (DCFTA) with Ukraine in order to limit the import of certain poultry products.
The amendment covers fraudulent imports of chicken breasts into the EU, as some Ukrainian breeders and manufacturers export a huge amount of chicken through the broader duty-free category "other bone-in chicken parts" contained within the DCFTA.
Agribusiness drives development technologies in Ukraine, deputy minister says
Once regarded as the breadbasket of Europe, Ukraine is now ready to be the 'supermarket of the world' as processed and high-end product manufacturing is booming, Ukraine's deputy agriculture minister, Olga Trofimtseva, told EURACTIV.com.
The EU and Ukraine have provisionally applied the DCFTA since 1 January 2016, as part of a broader association agreement dating back to November 2014.
After the entry into force of the DCFTA, Ukrainian agricultural and food exports to the EU increased by 32%. Bilateral trade in agrifood produce has grown 7% compared to the same period in 2017, while Europe had an overall trade surplus of €3.5 billion with Ukraine in 2017.
Some member states like Denmark argued that, although there might be a problem in the meat sector, the EU needs to stick with its commitment to free market and free trade agreements.
Other member states, particularly Spain and Portugal, also advocated vigilance, particularly when it comes to pig meat and the risk of swine fever.
[Edited by Sam Morgan]
DCFTA
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redpajama_set_name: RedPajamaCommonCrawl


**Due to coming changes in our factory's layout, the Greenhouse will no longer be available for Pins & Needles. This will be the last on-site craft night we will be hosting for 2018. We hope you can join us for one last hurrah!** Please join us in the Greenhouse for a knitting, crocheting, and sewing social! Bring your project of choice and enjoy crafting and conversation with fellow makers monthly throughout the year! All crafts are welcome, and this is a floating event, so come when you can and stay as long as you like. Friends are also welcome to attend--we love to meet new folks! Want to sew with us? All skill levels are welcome! We'll have a sewing expert on hand at each Pins & Needles to get you started with a simple project, help you through a tricky part in your pattern, or to simply cheer you on in whatever it is you're sewing at the moment. Please let us know when you RSVP if you're planning to bring your own sewing machine or need to reserve one of ours. The Greenhouse is equipped with ironing & cutting stations and basic tools & sewing notions that you are welcome to use while you're here. We can't wait to see what you're working on this month!
Please join us in the Greenhouse for a knitting, crocheting, and sewing social! Bring your project of choice and enjoy crafting and conversation with fellow makers monthly throughout the year! All crafts are welcome, and this is a floating event, so come when you can and stay as long as you like. Friends are also welcome to attend--we love to meet new folks! Want to sew with us? All skill levels are welcome! We'll have a sewing expert on hand at each Pins & Needles to get you started with a simple project, help you through a tricky part in your pattern, or to simply cheer you on in whatever it is you're sewing at the moment. Please let us know when you RSVP if you're planning to bring your own sewing machine or need to reserve one of ours. The Greenhouse is equipped with ironing & cutting stations and basic tools & sewing notions that you are welcome to use while you're here. We can't wait to see what you're working on this month!
Please join us in the Greenhouse for our 3rd Annual Rare Bear (http://www.rarescience.org/rare-bear-program/) Sew-In for RARE Science (http://www.rarescience.org/), a non-profit organization that helps find cures for kids with rare disease. The RARE Bear Project is a community driven initiative and is supported by the ever-growing RARE Bear Army of volunteers that help make one-of-a-kind bears for special one-of-a-kind kids. We'll provide fabric + notions for folks to create fun bears to donate to RARE Science. Any fabric or embroidery thread donations you have for this cause are welcome, too! All ages and all skill levels are welcome to attend, so please do bring your children and your friends! There is plenty ironing, pinning and stuffing to be done in addition to simple sewing, so help in any way you like. This event is floating, so come when you can and stay as long as you like. Though the Greenhouse has 6 sewing machines to share, we strongly encourage those who do have a portable sewing machine to bring it with them! Please let us know if you'll need to borrow a machine. If you'd like to bust your stash and make some RARE bears to donate ahead of time, you can do so by joining the RARE Bear Army today to receive sewing instructions and necessary custom RARE Bear supplies (http://www.rarescience.org/rare-bear-army-contact/)! We'll provide these instructions and supplies at our sew-in, too! You're welcome to drop your RARE Bear at Spoonflower anytime before or during our event! We look forward to seeing you in the Greenhouse to sew for a great cause!
Please join us at Spoonflower to sew head scarves to benefit the Duke Cancer Patient Support Program, which offers counseling, support groups and a wig and turban program to help patients and families cope with the impact of cancer on their lives. We'll provide the knit fabric for you to create brightly colored headscarves to donate to DCPSP. Any fabric donations you have for this cause are welcome, too! All ages and all skill levels are welcome to attend, so please do bring your children and your friends! There is plenty of cutting and pinning to be done in addition to simple sewing, so help in any way you like. This event is floating, so come when you can and stay as long as you like. The Greenhouse will be equipped with several sewing machines to share if you do not have your own. Please let us know if you'll need to borrow a machine. If you have your own sewing machine, we encourage you to bring your own! If you'd like to bust your stash and make some turbans or headscarves to donate ahead of time, you can do so by following these simple instructions (http://www.sewing.org/html/turban.html)! We'll provide these instructions and supplies at our sew-in, too! You're welcome to drop your turbans at Spoonflower anytime before or during our event! We look forward to seeing you at Spoonflower in The Greenhouse to sew for a great cause!
Please join us in the Greenhouse for a knitting, crocheting, and sewing social! Bring your project of choice and enjoy crafting and conversation with fellow makers monthly throughout the year! All crafts are welcome, and this is a floating event, so come when you can and stay as long as you like. Friends are also welcome to attend--we love to meet new folks! Want to sew with us? All skill levels are welcome! We'll have a sewing expert on hand at each Pins & Needles to get you started with a simple project, help you through a tricky part in your pattern, or to simply cheer you on in whatever it is you're sewing at the moment. Please let us know when you RSVP if you're planning to bring your own sewing machine or need to reserve one of ours. The Greenhouse is equipped with ironing & cutting stations and basic tools & sewing notions that you are welcome to use while you're here. We can't wait to see what you're working on this month! As we enter into a season of giving and gifting, November's Pins & Needles will have a twist -- a craft swap! Bring any fabric, yarn, notions or other craft supplies that you would like to de-stash and donate to the swap. One man's old notions is another man's holiday miracle! Don't have swag to swap? No problem! This is completely voluntary.
Please join us in the Greenhouse for a knitting, crocheting, and sewing social! Bring your project of choice and enjoy drinks, snacks and conversation with fellow crafters quarterly throughout the year! All crafts are welcome, and this is a floating event, so come when you can and stay as long as you like. Friends are also welcome to attend--we love to meet new folks! Want to sew with us? All skill levels are welcome! We'll have a sewing expert on hand at each Pins & Needles to get you started with a simple project, help you through a tricky part in your pattern, or to simply cheer you on in whatever it is you're sewing at the moment. Please let us know when you RSVP if you're planning to bring your own sewing machine. The Greenhouse is equipped with ironing & cutting stations and basic tools & sewing notions that you are welcome to use while you're here. We also have several sewing machines that folks new to sewing are welcome to use, just be sure to let us know that you need one. We can't wait to see what you're working on this month!


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We welcome walk ins anytime and they are treated on a first come, first serve basis. To guarantee a spot/availability, book an appointment.
Although consultations are important, a formal consultation session is not required. You are more than welcome to come by at anytime to discuss and plan your tattoo. All of the artists and management are friendly, well versed, and knowledgeable in the field.
Once an artist has been decided for your tattoo, you choose an appointment date and time, and leave $100 deposit. You will be asked to fill out a consent form when you arrive for your appointment so please provide valid identification. Piercings do not require appointments or deposits, but we do recommend calling in advance.
We require 48 HOURS notice to change the appointment date. To cancel an appointment would be to forfeit your deposit. Deposits are non-refundable.
Pieces under 3 hours will be charged a flat rate based on the size and detail of the artwork.
We recommend all clients resting well before their appointments. Make sure you are well fed, hydrated and not under any influence of alcohol or abusive substances. Bring a comfortable/loose change of clothes if the placement of your tattoo is not easily accessible (clothes may get ink stains). Feel free to help your artist by carefully shaving the area where your tattoo will go the day of your appointment.


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Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury
Yuanyuan Cheng1,#, Liangkai Cheng1,#, Xiang Gao2, Sixuan Chen1, Peng Wu1, Caiyan Wang1, , Zhongqiu Liu1,
1. Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine, Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, School of Pharmaceutical Sciences, Guangzhou Univ Chinese Med, Guangzhou, Guangdong, 510006, China
2. School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, 361102, China
# Yuanyuan Cheng and Liangkai Cheng contribute to the work equally.
✉ Corresponding authors: Caiyan Wang, Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine, Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China. Phone: 86-020-39358875; Fax: 86-020-39358071; Email: wangcaiyanedu.cn. Zhongqiu Liu, Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine, Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China. Phone: 86-020-39358061; Fax: 86-020-39358071; Email: liuzqedu.cn. More
Cheng Y, Cheng L, Gao X, Chen S, Wu P, Wang C, Liu Z. Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury. Theranostics 2021; 11(2):861-877. doi:10.7150/thno.48436. Available from https://www.thno.org/v11p0861.htm
Background and Purpose: Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from Ilex pubescens, has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA.
Methods: The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA.
Results: PBA protected cardiomyocytes against OGD/R in vitro and LAD-induced MI/RI in vivo. PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1.
Conclusions: Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.
Keywords: pubescenoside A, myocardial ischemia-reperfusion injury, Kelch ECH-associating protein 1, nuclear factor erythroid 2-related factor 2, covalent modification
Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases [1]. Keap1 has five domains: the NTR, BTB, IVR, Kelch (DGR) and CTR domains [2]. Cysteines play important roles in the on-off switch for the functions of Keap1; for example, Cys151, Cys273, and Cys288 are well recognized as the key cysteines for Keap1 [3, 4]. Keap1 has several partners, such as P53-induced protein with a death domain (PIDD), nuclear factor erythroid 2-related factor 2 (Nrf2) and p62 [5, 6]. The BTB domain and Kelch domain can regulate Nrf2 ubiquitination and degradation [7, 8]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor and sensitive to redox potential [9]. It induces several antioxidative proteins and antioxidants to suppress reactive oxygen species (ROS) [10, 11]. Recent studies have revealed that several small molecules could modify these cysteines and affect the interaction between Keap1 and Nrf2 [12, 13]. However, few of them are being evaluated in clinical trials, because the activation and selection of these cysteines by the molecules are unclear.
Myocardial infarction is the leading cause of cardiovascular disease-related death worldwide [14]. Reperfusion is an accepted therapy for myocardial infarction [15]. However, reperfusion can increase the infarct size by 50% at some time, because myocardial ischemia-reperfusion (MI/R) initiates oxidative stress and inflammation, which aggravates the injury [16, 17]. The NLRP3 inflammasome controls interleukin-1β (IL-1β) and IL-18 induction. Interestingly, inhibition of the NLPR3 inflammasome decreases cardiac infarct size, ameliorates adverse cardiac remodeling, and improves cardiac function in an animal model of MI/R [18, 19]. Excessive ROS triggers the activation of the NLRP3 inflammasome [20, 21]. ROS scavengers or knockdown of NADPH oxidase can inhibit the NLRP3 inflammasome [20, 22]. Therefore, modification of cysteines on Keap1 to affect the Nrf2-related antioxidative signaling pathway might ameliorate NLRP3 inflammasome-related myocardial ischemia reperfusion.
Ilex pubescens has been widely used in treating cardiovascular diseases in traditional Chinese medicine therapeutics [23-25]. Our previous studies have demonstrated that pubescenoside A (PBA) is an active compound from I. pubescens (Fig. 1A), which has antithrombotic and anti-inflammatory effects [23]. However, the underlying mechanism and the direct target for MI/R injury are still unknown.
During the early stages of AMI/R, the NLRP3 inflammasome specks can be detected in the endothelial cells, cardiomyocytes, and fibroblasts [16, 23]. When the inflammatory cells infiltrate the heart, the majority of NLRP3 inflammasome specks can be found in macrophages [23]. Moreover, cardiomyocytes injured are the major feature of AMI/R, and NLRP3 inflammasome activation in cardiomyocytes under oxygen-glucose deprivation and reperfusion (OGD/R) insult has been established [15]. Thus, we chose cardiomyocytes and macrophages as representative cell types to study. In the present study, we first found that PBA could protect cardiomyocytes against oxygen-glucose deprivation and reperfusion injury in vitro and significantly ameliorate MI/R injury in vivo. Furthermore, PBA could block NLRP3 inflammasome activation. Importantly, our results demonstrated that PBA could covalently bind to the conserved Cys77 and Cys434 residues of Keap1, disturbing the interactions of the Cul3-Keap1 ubiquitin ligase complex and the Keap1-Nrf2 complex, subsequently activating the Nrf2 signaling pathway. Our findings revealed that PBA might be used as a lead compound for the discovery of new therapeutic drugs for the treatment of MI/R injury.
PBA protects against myocardial ischemia-reperfusion injury
We initially evaluated the cytotoxicity of PBA on the growth of RAW264.7 macrophages, H9c2 cells and primary neonatal mouse cardiomyocytes using an MTT assay. As shown in Figure S1, PBA did not exhibit any cell toxicity under 240 μM in RAW264.7, H9c2 cells and primary cardiomyocytes. Thus, 30 μM or less of PBA was chosen in the following assays. The damage to cardiomyocytes in ischemic conditions is induced by reduction of glucose and oxygen. Oxygen and glucose deprivation/reperfusion (OGD/R) insult is recognized as a classic method to stimulate the pathological process of ischemia-reperfusion in vitro. Subsequently, we performed the OGD/R model in H9c2 cells and primary cardiomyocytes to investigate the cardioprotective effect of PBA.
After overnight culture, H9c2 cells and primary cardiomyocytes were exposed to 6 h or 3 h of OGD insult in the following experiments. H9c2 cells were treated with various doses of PBA and diazoxide (positive control drug) before being subjected to OGD. Then, the cells were reperfused and maintained for 18 h. Cell viability was detected by MTT assays. As shown in Figure 1B-C, PBA treatment (10 and 30 μM) protected H9c2 or primary cardiomyocytes against OGD/R induced injury. The protective effect of PBA seemed equal to that of 100 μM diazoxide.
Next, we used the LAD-induced myocardial ischemia-reperfusion model in mice to examine the cardioprotection of PBA. PBA administration at the dose of 30 mg/kg could significantly reduce myocardial ischemia-reperfusion induced cardiac infarct size and the level of CK-MB (Figure 1D-F). LVESD and LVSV enlarged after AMI/R, while LVEF, LVSF, +dp/dtmax and -dp/dtmax were decreased after AMI/R. However, PBA administration could improve cardiac function by increasing LVEF, LVFS, +dp/dtmax and -dp/dtmax and decreasing LVESD and LVSV (Figure 1G-M). H&E staining showed that AMI/R disrupted myofibrillar structure and caused neutrophil/macrophage clusters accumulation. PBA at the dose of 30 mg/kg not only preserved myofibrillar structure but also reduced the recruitment of inflammatory cells (Figure 1N).
PBA protected cardiomyocytes against ischemia-reperfusion. (A) Chemical structure of PBA. (B and C) The effect of PBA on the OGD/R-induced H9c2 cells and primary mouse cardiomyocytes. H9c2 cells and primary mouse cardiomyocytes were cultured in 96-well plates. After removal of complete DMEM, DMEM without glucose and serum was added, and the cells were treated with different concentrations of PBA. The cells were cultured under OGD conditions as described in the Method section for 6 h and under normal conditions for another 18 h. The results are normalized to expressed as the mean ± SD (n = 5). ###p < 0.001, the control vs OGD/R; **p < 0.01, ***p < 0.001, OGD/R+PBA or OGD/R+diazoxide vs OGD/R. (D and E) The effect of PBA on myocardial ischemia-reperfusion injury in a mouse model. Detection and quantification of MI/R injury. After 30 min of ischemia and 24 h of reperfusion, the heart tissue sections were stained with 1% TTC and 1% Evans blue. The infract size was determined. The results are expressed as the mean ± SEM (n = 6) and data were analyzed by t-tests. ***p < 0.01, the AMI/R + PBA group vs the AMI/R group. (F) Level of cardiac CK-MB. After 30 min of ischemia and 24 h of reperfusion, the blood samples were collected and measured for CK-MB ELISA kit. Values represent the mean ± SEM (n = 6) and data were analysed by one-way ANOVA. ###p < 0.001, the sham group vs the AMI/R group; ***p < 0.001, the AMI/R +PBA group vs the AMI/R group. (G) Representative echocardiographic image under M-mode. (H-M) Analysis of left ventricular end systolic diameter (LVESD), left ventricular end systolic volume (LVSV), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), the maximum rate of rise of left ventricular pressure increase (+dp/dtmax) and the maximum rate of rise of left ventricular pressure decrease (-dp/dtmax) at 24 hours after AMI/R. # p < 0.05, ##p < 0.01, the sham group vs the AMI/R group; *p < 0.05, ** p < 0.01, *** p < 0.001, the AMI/R + PBA group vs the AMI/R group. (N) Histopathological examination of mouse hearts. The cardiac tissues were stained with H&E stain. Representative images were selected from three groups. The black arrows represent inflammatory cell infiltration, and the red arrows represent disrupted myofibrillar structure. Scale bar: 50 μm.
PBA suppresses activation of the NLRP3 inflammasome in macrophages, OGD/R-induced H9c2 cells and primary cardiomyocytes
To evaluate the effect of PBA on the NLRP3 inflammasome, we first used an established method (LPS plus ATP) to induce the NLRP3 inflammasome in RAW264.7 cells. As shown in Figure 2A, PBA attenuated LPS/ATP-mediated NLPR3 induction as well as cleaved-caspase1 and IL-1β. And immunofluorescent staining in Figure S2 showed that PBA significantly reduced LPS/ATP-induced ACS and Caspase1 expression. To confirm the role of the PBA-mediated NLRP3 inflammasome in myocardial ischemia-reperfusion, we ascertained the efficacy of PBA in OGD/R-induced cardiomyocytes. Exposure of H9c2 cells and primary cardiomyocytes to OGD conditions (6 h or 3 h) followed by reperfusion (18 h) significantly increased the NLRP3, cleaved-caspase1 and mature IL-1β levels. However, OGD/R-induced inflammasome activation in cardiomyocytes was substantially inhibited by PBA in a dose-dependent manner (Figure 2B-C). In addition, as shown in Figure 2D, PBA administration also attenuated NLRP3 inflammasome activation in heart tissue of AMI/R-injured mice.
PBA activates the Nrf2 pathway and induces protective enzymes
ROS play an important role in the activation of the NLRP3 inflammasome. We found that PBA could significantly suppress the LPS plus ATP-induced ROS in macrophages and the OGD/R-induced ROS in H9c2 cells (Figure 3A). To explore the mechanism underlying the PBA-mediated reduction of ROS, we determined the effect of PBA on Nrf2 nuclear translocation. PBA significantly promoted Nrf2 translocation into nucleus (Figure 3B). Moreover, as shown in Figure 3C, PBA strongly induced Nrf2 expression in cardiomyocytes under OGD/R condition. Nrf2, as a transcription factor, induces the expression of a panel of antioxidant enzymes, such as heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase 1 (NQO1). As shown in Figure 3D, PBA increased HO-1 and NQO1 expression at the protein level in a dose-dependent manner in both RAW264.7 and H9c2 cells. In addition, to investigate whether the cardioprotective effect of PBA is associated with Nrf2, we used Nrf2 specific small interfering RNA to knock down the expression of Nrf2. The results in Figure 4A show that H9c2 cells were more sensitive to OGD/R-induced injury after Nrf2 siRNA transfection. Moreover, the cardioprotection of PBA was blocked in the Nrf2 knockdown H9c2 cells. Consistent with in vitro study, the cardioprotective effect of PBA on Nrf2-/- mice under AMI/R injury was disappeared. Specifically, the decrease effect of CK-MB level and the increase effect of -dp/dtmax and -dp/dtmax by PBA were blocked in Nrf2-/- mice under AMI/R injury (Figure 4B-E). H&E staining in Figure 4F shows that PBA administration in Nrf2-/- mice could not suppress inflammatory cells infiltration and improve disturbed myofibrillar structure. These data suggested that Nrf2 was involved in the cardioprotective and NLRP3-suppressive effects of PBA.
PBA directly targets Keap1 at Cys77 and Cys434
Keap1 is a cysteine-rich protein and negatively regulates the activation of Nrf2 based on cysteine-induced conformational changes. The Co-IP results showed that PBA significantly disturbed the complex of Keap1 and Nrf2. To evaluate whether PBA could directly bind to intracellular Keap1 and subsequently affect Nrf2 activity, we used a pull-down assay using PBA conjugated Sepharose 4B beads. The result in Figure 5A shows that Keap1 could bind to PBA-Sepharose 4B beads but not bind to Sepharose 4B beads only. Recombinant human Keap1 was purified from Escherichia coli BL21 cells for further analyses. To identify the residues that contributed to this interaction, we performed an impartial mass spectrometry (MS) analysis to detect cysteine residues modified by PBA in vitro. The MS analysis in Figure 5B-C of chymotrypic peptide covering Cys434 showed an increase of 442.14 in mass, demonstrating that Cys434 was modified by PBA.
In addition to Cys434, covalent modifications of PBA at Cys77, Cys23, Cys38, Cys273 and Cys236 were similarly discovered from the MS study (Figure 5C). Covalent modification of these six cysteines could similarly serve to inactivate Keap1. Thus, we separately mutated these cysteine residues to serine residues to determine which residues were indispensable for binding to PBA. As shown in Figure 5D, mutants of Cys434, Cys77, Cys23 and Cys38 lost the capacity to bind to PBA, while other mutants retained their high binding affinity for PBA. Considering that Cys23 and Cys38 are located in the NTR domain of Keap1 without possible function on Nrf2, we speculated that the two key residues Cys77 and Cys434 are indispensable for its interaction with PBA to regulate the Nrf2 signaling pathway.
PBA suppresses NLRP3 inflammasome activation. (A) PBA inhibited NLRP3 inflammasome activation in RAW264.7 cells. The cells were pretreated with PBA at the indicated concentration for 1 h and then stimulated with 200 ng/mL LPS for 6 h. Then, 2 mM ATP was added to the culture medium for another 20 min. Protein was isolated and subjected to SDS-PAGE. Data are shown as the mean ± SD, n = 3. ###p < 0.001, the control vs LPS+ATP; *p < 0.05, **p < 0.01, LPS+ATP+PBA vs LPS+ATP. (B and C) The effect of PBA on NLRP3 inflammasome activation in OGD/R-induced H9c2 cells and primary cardiomyocytes. H9c2 cells or primary cardiomyocytes were subjected to OGD for 6 h or 3 h, and reperfusion for 18 h. Then, proteins were isolated for SDS-PAGE for analysis of NLRP3, cleaved-caspase1 and mature IL-1β. Data are shown as the mean ± SD, n = 3. #p < 0.05, ##p < 0.01, the control vs OGD/R; **p < 0.01, ***p < 0.001, OGD/R+PBA vs OGD/R. (D) The effect of PBA administration on NLRP3 inflammasome activation in heart tissue of AMI/R-induced mice. Data are shown as the mean ± SEM, n = 5. ## p < 0.01, ###p < 0.01, the sham group vs the AMI/R group; ** p < 0.01, *** p < 0.001, the AMI/R + PBA group vs the AMI/R group.
PBA activates the Nrf2 signaling pathway. (A) PBA decreased ROS production. Cells were treated with the indicated concentrations of PBA, and then stimulated with LPS plus ATP, and the ROS level was determined by the fluorescent probe DCFH-DA. The results are expressed as the mean ± SD (n = 3). (B) PBA promoted Nrf2 translocation into the nucleus. The cells were treated with PBA for 6 h. After isolation of the proteins in the cytosol and nucleus, the protein extract was subjected to SDS-PAGE for western blotting analysis. Other cells were used for Nrf2 immunofluorescence staining. Scale bar: 10 μm. (C) PBA increased Nrf2 expression and HO-1 expression in the OGD/R-induced cardiomyocytes. *p < 0.05, **p < 0.01, ***p < 0.001, OGD/R + PBA vs OGD/R. (D) The effects of PBA on HO-1 and NQO1 expression. Cells were treated with PBA for 24 h. *p < 0.05, **p < 0.01, PBA treatment vs the control.
PBA suppresses Keap1-regulated ubiquitination of Nrf2 by disrupting the interaction between Cul3 and Keap1
Alkylation of the BTB domain of Keap1 by covalent modifiers contributes to the disruption of the Keap1-Cul3 interactions, leading to Nrf2 activation. Next, we investigated the effect of PBA on the modulation of Nrf2 ubiquitination as Nrf2 stability is controlled by ubiquitin-mediated proteasomal degradation. First, 3X Flag-Keap1, Myc-Nrf2 and Ub plasmids were transfected into 293T cells and then, the cells were treated with 30 μM PBA for 20 h and 5 μM MG132 for another 4 h. Cell lysates were pulled down using anti-Myc beads, and immunopurified Nrf2 was examined for the occurrence of Ub by western blotting. As shown in Figure 6A-B, both PBA and tBHQ significantly suppressed Keap1-dependent Nrf2 ubiquitination. When mutant Cys77S and Cys434S-Keap1 vectors were transfected into 293T cells, PBA was unable to protect Nrf2 from ubiquitination, which indicated that PBA prevented Keap1-driven ubiquitination of Nrf2 in a Cys77-dependent manner. As shown in Figure 6E-F, PBA could disrupt the interaction of Keap1 and Cul3 in WT-Keap1 cells, while the disruption was blocked in the Cys77S and Cys434-Keap1 mutant cells.
PBA also disturbs the interaction between Keap1 and Nrf2
To investigate the effect of PBA on the interaction of Keap1 and Nrf2, we transfected 293T cells with Flag-Keap1 and Myc-Nrf2 plasmids, and treated them with 30 μM PBA for 24 h. The protein expression of Nrf2 was measured by western blotting analysis followed by immunoprecipitation with Flag beads. As shown in Figure 6C-D, PBA disrupted the interaction between Keap1 and Nrf2, while tBHQ did not affect it. Furthermore, when Cys77S and Cys434S-Keap1 mutant vectors were transfected into 293T cells, PBA was unable to block the Keap1-Nrf2 complex. Therefore, these results suggest that alkylation by PBA could trigger dissociation of the Keap1-Nrf2 complex. Importantly, shown in Figure 5G, Cys77S and Cys434S-Keap1 mutant vectors blocked the protective effect of PBA against OGD/R injury on primary cardiomyocytes.
PBA attenuates myocardial ischemia-reperfusion via the Nrf2 signaling pathway. (A) PBA protected cardiomyocytes against OGD/R-induced cytotoxicity in a Nrf2-dependent manner. H9c2 cells were transfected with siNC and Nrf2 siRNA for 24 h. Then, the cells were treated with the indicated concentrations of PBA before OGD/R insult. Cell viability was measured by MTT assays. The results are expressed as the mean ± SD (n = 5). ##p < 0.01, OGD/R vs control; *p < 0.05, **p < 0.01, OGD/R vs OGD/R + PBA. (B) The effect of PBA on LAD-induced Nrf2-/- mice. (C) Level of cardiac CK-MB in Nrf2-/- mice. After 30 min of ischemia and 24 h of reperfusion, the blood samples were collected and measured for CK-MB ELISA kit. Values represent the mean ± SEM (n = 4) and data were analysed by one-way ANOVA. #p < 0.05, the sham group vs the AMI/R group; *p < 0.001, the AMI/R + PBA group vs the AMI/R group. (D and E) Analysis of the maximum rate of rise of left ventricular pressure increase (+dp/dtmax) and the maximum rate of rise of left ventricular pressure decrease (-dp/dtmax) at 24 hours after AMI/R in Nrf2-/- mice. ##p < 0.01, ###p < 0.001, the sham group vs the AMI/R group. (N) Histopathological examination of mouse hearts in Nrf2-/- mice. The cardiac tissues were stained with H&E stain. Representative images were selected from three groups. The black arrows represent inflammatory cell infiltration, and the red arrows represent disrupted myofibrillar structure. Scale bar: 50 μm. (D) The suppressive effect of PBA on the NLRP3 inflammasome was blocked in Nrf2-/- mice. After reperfusion, heart tissue protein was collected for WB analysis. Data are shown as the mean ± SEM, n = 5. ###p < 0.01, the sham group vs the AMI/R group.
PBA directly targets Keap1. (A) Keap1 was pulled down by PBA-Sepharose4B. Total cell lysates were harvested. The lysates were incubated with PBA-Sepharose 4B or Sepharose 4B at 4 °C. After removing the unbound protein, the eluted protein was subsequently detected by immunoblotting for the Keap1 protein. (B and C) Cys434 was covalently modified by PBA. Human His-Keap1 was expressed in BL21 cells. The recombinant Keap1 was incubated with PBA (protein-to-compound molar ratio, 1:20) at 37 °C for 2 h before MS analysis. (D) Other modified cysteines detected by LC/MS/MS. (E) Cys434 and Cys77 of Keap1 were indispensable for its interaction with PBA. The recombinant Keap1 mutant proteins were expressed in BL21 cells and incubated with PBA-Sepharose 4B (200 μl). After removal of the unbound protein, the eluted protein was subsequently detected by immunoblotting for the Keap1 protein.
Thermodynamic analysis of Keap1 and mutant interactions with PBA
Cysteine is an essential amino acid in proteins, and the functional group -SH is a key element in the active centre. Cysteine is the on-off switch for the functions of Keap1, and we demonstrated that the compound PBA derived from herbs can interact with six key cysteines in Keap1. However, the detail mechanism of the binding manner, especially the selection of the combining order with different cysteines is still unclear. From the ITC results shown in Figure 7A-H, (Table 1), we found that PBA had a different preference for cysteines based on the binding constant. The Kd of the interaction between the WT Keap1 and PBA was 2.61 μM, as determined by ITC. Similarly, the Kd values of the mutants Cys77S and Cys434S were 19.5 μM and 13.9 μM respectively. The Kd value of Cys23S, Cys38S, Cys226S and Cys273S were 9.78, 8.59, 8.12 and 3.80 μM, respectively. Collectively, the order in which PBA bound to Keap1 on the cysteines was as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273 (Figure 7I), which was consistent with the results from the pull-down assays in Figure 5D. In addition, we constructed the double mutant Cys77S/Cys434S and the Kd was 38.6 μM, which was ~15-fold weaker than that of the WT. These results strongly support the contention that PBA preferentially bound to Cys77 and Cys434.
Thermodynamic parameters for binding of PBA to Keap1 mutants at 25 ℃.
Alanine mutation
Kd (μM)
△H (kcal/mol)
T△S (kcal/mol)
△G (kcal/mol)
WT 2.61e-6±0.769e-6 -3.31±0.199 4.31±0.00 -7.62±0.00
C23S 9.78e-6±1.24e-6 -3.62±0.241 3.22±0.00 -6.84±0.00
C38S 8.59e-6±0.908e-6 -4.59±0.199 2.32±0.00 --6.91±0.00
C77S 19.5e-6±4.81e-6 -5.65±0.866 0.78±0.00 -6.43±0.00
C226S 8.12e-6±0.894e-6 -5.11±0.279 1.84±0.00 -6.95±0.00
C273S 3.80e-6±1.13e-6 -3.87±0.271 3.53±0.00 -7.40±0.00
C434S 13.9e-6±3.63e-6 -4.76±0.367 1.87±0.00 -6.63±0.00
C77S/C434S 38.6e-6±24.0e-6 -4.92±0.637 1.11±0.00 -6.03±0.00
Kd value is kind of the dissociation constant and △H, T△S, △G are the change in binding enthalpy, entropy, and Gibbs energy, respectively. -RT InKd =△G =△H-T△S, where T and R are the absolute temperature and gas constant, respectively. Values are shown as means ± standard deviations from triplicate runs.
PBA suppresses Keap1-mediated ubiquitination of Nrf2 by disrupting the interaction between Cul3 and Keap1. (A and B) PBA suppressed Nrf2 ubiquitination in a Keap1-Cys77 and C434-dependent manner. First, 293T cells were transfected with WT-Keap1 or C77S and C434S-Keap1, Myc-Nrf2, and Ub plasmids for 24 h and treated with 30 μM PBA. The cell lysates were immunoprecipitated using anti-Myc beads, and the ubiquitination level was evaluated using the anti-Ub antibody. (C and D) PBA induced the dissociation of the Nrf2-Keap1 complex. Then, 293T cells were cotransfected with WT-Keap1 and Flag-Nrf2 plasmids for 24 h and treated with 30 μM PBA for 12 h. The cell lysates were immunoprecipitated with the anti-Flag antibody, and the level of Nrf2 was assessed using the anti-Nrf2 antibody. (E and F) PBA disrupted the interaction between Cul3 and Keap1. The 293T cells were transfected with the WT-Keap1 or C77S-C434S-Keap1 plasmids for 24 h and treated with 30 μM PBA for 12 h. The cell lysates were immunoprecipitated with the anti-Flag antibody, and the level of Cul3 was measured using the anti-Cul3 antibody. #p < 0.05, ##p < 0.01, ###p < 0.001, the control vs DMSO; *p < 0.05,**p < 0.01, ***p < 0.001, PBA or tBHQ vs DMSO. (G) C77S and C434-Keap1 mutant blocked the protective effect of PBA against OGD/R-induced primary mouse cardiomyocytes injury. Primary mouse cardiomyocytes were transfected with mouse WT-Keap1 or C77S and C434S-Keap1 mutant vectors. After 24 h, the cells were treated with the indicated doses of PBA and subjected to OGD conditions as described in the Method section for 3 h and under normal conditions for another 18 h. Cell viability was measured by MTT assays. #p < 0.05, ###p < 0.001, the control vs OGD/R; **p < 0.01, ***p < 0.001, OGD/R+PBA vs OGD/R.
Representative ITC titration profiles of the titration of PBA with mutation the Keap1 mutant protein. Keap1-WT (A), Keap1-C23S (B), Keap1-C38S (C), Keap1-C77S (D), Keap1-C226S (E), Keap1-C273S (F), Keap1-C434S (G), Keap1-C77S/C434S (H). The top graphs represent the raw ITC thermograms, and the bottom graphs represent the fitted binding isotherms. The dissociation constant (Kd) is shown below the curves. (I) Schematic representation of the Keap1-PBA interaction order. Keapl is composed of the BTB (white cyan barb), IVR (brown helical), and Keap1-Kelch (Khaki concave) domains. The Keapl homodimer binds with one Neh2 domain of Nrf2 and Cul3. The Cyan crescent represents PBA, the gray-green circle represents cysteine and the number represents the position of cysteine.
The binding mode of PBA with the Keap1-Kelch and Keap1-BTB domains is revealed by covalent docking
To reveal the possible interaction mode of PBA and Keap1, we carried out covalent docking studies [26]. The docking score ranged from -4.412 to -5.565 for Keap1-Kelch and -1.964 to -4.898 for Keap1-BTB, and the lowest docking score was selected for the following study (Table 2). As shown in Figure 8A-D, the results of covalent docking of PBA with Keap1-Kelch domain revealed that PBA occupied a shallow groove formed by the residues Asn414, Arg380, Asp389, Ser431, Hid432, Gly433, Cys434, Ile135, Hid436, Thr481, Gly480, Asp479, Arg459, Ile461 and Ile435. Among them, PBA interacted hydrophobically with Ser431, His436, Asn414 and Thr481, and the polar interactions were found to be with Gly433 and Gly480.
Docked complex of PBA with the Keap1-Kelch and Keap1-BTB domains. (A) Structural view of PBA with the Keap1-Kelch domain. (B) The PBA and Keap1-Kelch domain 2D interaction diagram; purple lines represent hydrogen bonds, black lines represent covalent bonds. (C) The surface picture of the Keap1-Kelch domain with PBA bound in the region of Cys434. (D) Details of the interactions between PBA and the Keap1-Kelch domain. The hydrogen bonds are presented as yellow dashed lines. Cys434 of the Keap1-Kelch domain forms a covalent bond with PBA. (E) Structural view of PBA with the Keap1-BTB domain; yellow dashed lines indicate hydrogen bonds. (F) PBA and the Keap1-BTB domain 2D interaction diagram; purple lines represent hydrogen bonds, black lines represent covalent bonds. (G) The surface picture of the Keapl BTB domain with PBA bound in the region of Cys77. (H) Details of the interactions between PBA and the Keapl-BTB domain. The hydrogen bonds are presented as yellow dashed lines. Cys77 of the Keapl-BTB domain forms a covalent bond with PBA.
The docking scores of Keap1-Kelch, Keap1-BTB with PBA.
Docking score
Keap1-Kelch -4.412 -5.565 -4.656 -4.652 -5.436
Keap1-BTB -1.964 -2.963 -4.898 -2.907 -3.925
The docking score was obtained from the project table after docking completed. Red numbers are the lowest docking score and selected it for study.
Hydrogen bonding interactions are also involved in the binding of the Keap1-Kelch domain and PBA. The residues Arg380, Gly480 and Asp479 formed hydrogen bonds with hydroxyl groups with PBA, respectively. Interestingly, Cys434 formed one covalent bond with PBA, and hydrogen bond formed between the nitrogen of Cys434 and the oxygen of PBA, which could enhance the binding affinity between PBA and Cys434. Therefore, the covalent docking results of the Keap1-Kelch domain provided further evidence that PBA directly targeted Keap1 at Cys434 and played a critical role.
In addition, the results of the docking studies in Figure 8E-H showed that PBA interacted hydrophobically with the lipophilic pocket of Keap1-BTB through the Met94, Val98, Met120, Cys77, and Leu76 residues. The polar interactions were found to be with Gln74, Gln75 and Gln118. Moreover, PBA occupied a surface cavity formed by Arg116, Glu 117, Gln118, Gly119, Met120, Gln74, Gln75, Leu76, Cys77, Asp78, His96, Lys97 and Thr80. Importantly, apart from a covalent bond with Cys77, the two adjacent residues Leu76, Asp78 also formed two hydrogen bonds with PBA, Gly119 formed hydrogen bonds with two hydroxyl groups adjacent to PBA, and Gly119 and Glu117 formed hydrogen bonds with the same hydroxyl group. These interaction modes may significantly increase the binding affinity of PBA for the BTB domain.
The NLRP3 inflammasome plays an important role in the pathophysiology of myocardial ischemia reperfusion [19, 27]. Suppression of the NLRP3 inflammasome can reduce IL-1β release, subsequently attenuating the inflammatory process and protecting cardiomyocyte death against the ischemia/reperfusion injury [22]. Our study found that PBA could significantly inhibit the NLRP3 inflammasome in both macrophages and cardiomyocytes, subsequently reducing the cardiac infarct size. Redox signaling is a critical factor that modulates NLRP3 inflammasomes [28, 29]. Decreasing ROS level can block NLRP3 inflammasome activation [30]. Nrf2 is a transcription factor and is sensitive to redox potential. This molecule induces antioxidative proteins and antioxidants to suppress ROS [31]. Our results showed that PBA could significantly reduce ROS production, promote Nrf2 translocation into nucleus, and upregulate the expression of downstream proteins (HO-1、 NQO1).
Keap1 is a partner of Nrf2. Keap1 could regulate Nrf2 ubiquitination and translocation into nucleus [32]. In our present study, we discovered that PBA activated the Nrf2 signaling pathway by covalent modification of Keap1 at cysteine residues. By pull-down assays, we found that PBA directly bound to Keap1. Notably, we used LC/MS/MS and cysteine mutagenesis to show that PBA could bind to Cys77 and Cys434. Given the possible cysteine modifications of PBA, we used an ITC assay to detectthe binding capacity of PBA with WT Keap1 and mutant Keap1. We found that the Cys77S (Kd = 19.5 μM) and Cys434S (Kd = 13.9 μM) mutants showed a lower binding affinity with PBA than the WT (Kd = 2.61 μM). Finally, we found that the order of selection was Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which was consistent with our LC/MS/MS data. From the structure analysis, Cys77 and Cys434 are located in the functional domain for Nrf2 activation, while Cys23 and Cys38 might affect other functions; and we will clarify this issue in future studies.
To gain insight into the differences between PBA and existing inhibitors [33, 34], we revealed the binding mode of PBA with Keap1 by covalent docking (Figure 8). We downloaded six inhibitor complex structures acting on the Kelch domain from PDB for comparison (Figure S3). Our results showed that the main inhibitory site of the traditional inhibitor of the Keap1-Kelch domain is the active site of the central cavity in the Kelch domain, while PBA interacts with the Keap1-Kelch domain on Cys434, which occupies a shallow groove surface. More importantly, PBA forms a covalent bond with Cys434 and produces hydrogen bonding. For the BTB domain, we also downloaded five sets of inhibitors for comparison, such as the inhibitors affecting Cys151. PBA binds to a completely different surface of Keap1. Moreover, we found that both Leu76 and Asp78 play important roles in the helping formation of covalent bonds between Cys77 and Cys434 with PBA.
A small molecule covalently binding to a specific cysteine of Keap1, such as Cys151, Cys273 and Cys288, could activate Nrf2 [4, 35]. In the present study, we indicated the critical role of Cys77 and Cys434 covalently bonding with PBA in activation of Nrf2. Cys77 is located at the BTB domain for affecting Cul3 and Keap1 interaction, while Cys434 is located at the Kelch domain for regulating Keap1 and Nrf2 interaction. Our results showed that PBA could modify Keap1 by preferentially forming a covalent bond with Cys77 and Cys434. PBA interacted with Cys77 leading to Cul3 dissociation from the Keap1-BTB domain. On the other hand, PBA acting on Cys434 could cause the Keap1 with Nrf2 dissociation easily. Blocking both cysteines could decrease Nrf2 ubiquitination and promote Nrf2 translocation into the cell nucleus. Compared with the traditional Keap1 inhibitor acting on the Keap1-Kelch domain or the Keap1-BTB domain, PBA could not only form stronger covalent bonds, but could also produce two kinds of effects, which is better for preventing drug resistance for Keap1 mutants.
Although there are several Nrf2 activators that directly induce Nrf2 expression or inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a noncovalent mechanism [36, 37], PBA provides a new Nrf2 activator with an accurate Keap1 modification mechanism. In addition, the Keap1-Nrf2 signaling pathway also contributes to detoxification [38]. Thus, PBA might be a potential compound with low off-target toxicity and therapeutic effects. As described above, we confirmed that PBA protected the heart against ischemia-reperfusion via the Nrf2 pathway in vivo and in vitro.
In conclusion, the present study provides primary evidence that PBA improved myocardial ischemia reperfusion injury via suppression of NLRP3 inflammasome activation. We also found that PBA could modify the Cys77 and Cys434 residues of Keap1 to regulate Nrf2 ubiquitination and translocation to the nucleus, subsequently attenuating myocardial ischemia-reperfusion injury. Additionally, the cysteines of Keap1 have different degrees of activation by PBA: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which gives us deeper insight into the cysteine sensitivity of Keap1.
PBA was isolated from I. pubescens and identified by Dr. Peng Wu from Guangzhou University of Chinese Medicine. Antibodies against Keap1 (10503-2-AP) was purchased from Proteintech Group, Inc. (Chicago, IL, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E12-052), HO-1 (E1A5393), NQO1 (E2A6437), cullin-3 (Cul3, E2A7225V), Flag (E1T508-2) and β-actin (E12-051) antibodies were purchased from Enogene Company (Nanjing, Jiangsu, China). Ubiquitin (Ub, sc-8017), caspase-1 (sc-56036), caspase-1 p20 (sc-398715) and interleukin-1 beta (IL-1β, sc-52012) antibodies were bought from Santa Cruz Biotechnology (Santa Cruz CA, USA). Nrf2 (12721) and lamin B1 (13435) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Nrf2 antibody (ab137550) for immunofluorescence staining was purchased from Abcam (Cambridge, UK). NLPR3 (NBP-2-12446) antibody was purchased from Novus Biologicals, LLC (Centennial, CO, USA). MG-132 (HY-13259) and tBHQ (HY-100489) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).
Cell culture and treatment
Rat H9c2 cells, human embryonic kidney 293T cells and RAW264.7 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% foetal bovine serum (FBS, Gibco, USA), 100 U/ml penicillin and 100 μg/mL streptomycin (PS, Gibco, USA). The cells were incubated at 37 °C with 5% CO2.
For OGD/R treatment, H9c2 cells were washed twice with phosphate buffered saline (PBS). The medium was changed to glucose and serum free DMEM, and H9c2 cells were incubated in an incubator with 95% N2 and 5% CO2 at 37 °C. After 6 h, the medium was replaced with DMEM containing 10% FBS and 1% PS. Then H9c2 cells were returned to a normal incubator with 5% CO2 balanced with air at 37 °C for the indicated time.
Based on the previous study [15], RAW264.7 cells were treated with different concentrations of PBA 1 h before 200 ng/mL lipopolysaccharide (LPS) stimulation for 6 h. Then, 2 mM adenosine triphosphate (ATP) was added and incubated for 20 min at 37 °C with 5% CO2.
Primary neonatal mouse cardiomyocytes isolation
The heart was removed from the neonatal C57BL/6 mice and cut into 1 mm3 pieces in ice PBA buffer. The tissues were digested with the mixture of 0.125% trypsin (Gibco, 15090-046) and 0.1% type II collagenase (Gibco, 17101-015) 6 min at 37 °C for three times until the tissues were completely digested. The cells were harvested and seeded in a dish. After 90 min of incubation, the cell culture medium was collected and centrifuged. The centrifuged cardiomyocytes were harvested, seeded on 0.1 % gelatin-coated culture plates with DMEM containing 10% FBS and 0.1M Brdu, and incubated in a 37°C incubator.
Cell viability measurement
Cell viability was determined by MTT assays. PBA was prepared to a stock solution dissolved in dimethyl sulfoxide (DMSO). RAW264.7, H9c2 cells, primary mouse cardiomyocytes were treated with different doses of PBA, and DMSO only as control for 48 h for the toxicity. In addition, H9c2 cells or primary mouse cardiomyocytes were treated with the indicated doses of PBA, diazoxide as positive control, DMSO only as control, then cultured with or without OGD/R treatment. At the end of the experiments, MTT was added into each well at the concentration of 0.5 mg/ml, and incubated at 37 °C for 4 h. After removal of the supernatant, 150 μL of DMSO was added to solubilize the formazan products. And the solution was measured at the absorbance of 490 nm by a spectrophotometer (Thermo Fisher Scientific, MA, USA). The results of the MTT assay in all groups were normalized to the control group with DMSO only treatment.
Intracellular ROS detection
The fluorescent probe DCFH-DA was used to measure intracellular ROS levels. RAW264.7 cells were treated with 100 μg/mL LPS plus ATP as mentioned above. H9c2 cells underwent OGD/R. After the appropriate treatment, the cells were incubated with DCFH-DA at a concentration of 10 μM for 30 min. The cells were washed with PBS three times. A fluorescence microscope and microplate reader were used to measure the level of DCFH-DA fluorescence at the excitation wavelength of 488 nm and the emission wavelength of 525 nm.
For transient transfection, HiPerFect Transfection Reagent (Qiagen, German) was used according to the manufacturer's instructions. Nrf2 siRNA (h) (sc-156128) and control siRNA-A (sc-37007) were bought from Santa Cruz Biotechnology. Briefly, H9c2 cells were seeded in 96-well plate at the density of 2x104 cells per well or 12-well plate at the density of 3x105 cells per well. According to preliminary results, transfection with siRNA for 24 h could get a better knockdown effect. Therefore, after transfection with siRNA for 24 h, H9c2 cells were treated with the indicated doses of PBA (which is consistent to the assay above mentioned) before OGD/R insult.
Western blotting analysis
Total protein was isolated from H9c2 cells, primary mouse cardiomyocytes, RAW264.7 cells and heart tissue using 1X RIPA buffer containing phosphatase and proteinase inhibitors at 4 °C. After measurement of the concentration, the cell lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in Tris-buffered saline-Tween 20 (TBST) buffer with 5% BSA for 1 h and incubated with primary antibodies (1:1000 dilution) overnight at 4 °C. After the membranes was washed by TBST buffer for three times, they were incubated with horseradish peroxidase (HRP)-conjugated secondary mouse or rabbit antibodies (1:3,000 dilution) at room temperature (RT) for 1.5 h and then detected by SuperEnhanced chemiluminescence (ECL) reaction reagents (GBCBIO Technologies Inc., Guangzhou, Guangdong, China). In brief, drop evenly the ECL working liquid onto the membrane at the ratio of 1 mL ECL working liquid and 10 cm2 of membrane. After letting the membranes stand for 1-2 min, the bands of the respective proteins were detected with the exposure time of 10 ~60 s.
Immunofluorescence staining
According to previous experiences, Nrf2 nucleus translocation could be detected at 6 h. Therefore, briefly, the H9c2 cells were seeded in 15 mm confocal dishes at the density of 2x105 overnight, and incubated with 30 μM PBA for 6 h. RAW264.7 cells were also seeded in in 15 mm confocal dishes at the density of 3x105 overnight and treated with 30 μM PBA for 1 h prior to 200 ng/mL LPS stimulation for 6 h and then induced by 2 mM ATP for another 20 min. After treatment, cells were fixed and incubated with primary antibodies against Caspase-1, ASC, and Nrf2 at 4 °C overnight. After several washes with PBS, the bound antibodies were detected by Alexa Fluor 594-conjugated goat anti-rabbit or Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody at RT for 1 h. The cell nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI, Solarbio Life Science, Beijing, China) at room temperature for 10 min. The cells were examined on a laser scanning microscope (Carl-Zeiss, Jena, Germany). Five or six fields of view per sample were randomly selected to be captured.
Pull-down assay
PBA-Sepharose 4B beads were created by dissolving PBA in coupling buffer (100 mM NaHCO3, pH 8.3, 0.5 M NaCl). After washing with 1 mM HCl medium, Sepharose 4B beads were mixed with PBA coupling buffer and slowly rotated overnight at 4 °C. Excess PBA was washed away with coupling buffer and any remaining active groups were blocked with 0.1 M Tris-HCl buffer (pH 8.0) for 2 h. The medium was then washed with 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl followed by a wash with 0.1 M Tris-HCl (pH 8.0) that also contained 0.5 M NaCl. PBA-Sepharose 4B beads were ready for use in the pull-down assay. The RAW264.7 cellular supernatant fraction (600 µg) was incubated with either PBA-Sepharose 4B or Sepharose 4B as a negative control in reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP-40, 2 µg/mL of BSA, 0.02 mM PMSF, 1 × protease inhibitor cocktail). After incubation with gentle rocking overnight at 4 °C, the beads were washed five times with wash buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP-40, 0.02 mM PMSF) and proteins bound to the beads were analyzed by immunoblotting assay with a Keap1 antibody.
Animal and drug administration
The experimental procedures and protocols were approved by the Committee on Ethical USE of Animals of International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine (No. IITCM-20180115). Adult male C57BL/6 mice were purchased from the Laboratory Animal Center, Sun Yat-Sen University (Guangzhou, Guangdong, China), and Nrf2 knockout mice were obtained from Riken. Adult male C57BL/6 or Nrf2 knockout mice were treated with 30 mg/kg PBA or an equal volume of vehicle 15 min prior to reperfusion. Mice were divided into 6 groups: (1) Wild-type sham (n = 10): C57BL/6 mice received vehicle, as the sham group; (2) Wild-type AMI/R group (n = 10): C57BL/6 mice received vehicle (i.p.) 15 min before reperfusion; (3) Wild-type AMI/R plus PBA group (n = 10): C57BL/6 mice received 30 mg/kg PBA (i.p.) 15 min before reperfusion; (4) Nrf2 knockout sham (n = 8) : Nrf2 knockout mice received vehicle (i.p.), as in the sham group; (5) Nrf2 knockout AMI/R group (n = 8): Nrf2 knockout mice received vehicle (i.p.) 15 min before reperfusion; (6) Nrf2 knockout AMI/R plus PBA group (n = 8): Nrf2 knockout mice received 30 mg/kg PBA (i.p.) 15 min before reperfusion. PBA was dissolved in 0.9% NaCl buffer containing 2% EtOH. The vehicle was 0.9% NaCl buffer containing 2% EtOH.
Model of AMI/R
The C57BL/6 mice or Nrf2 knockout mice underwent myocardial ischemia-reperfusion injury as described previously. In brief, mice were anaesthetized by i.p. injection of sodium pentobarbital at a dose of 50 mg/kg and ventilated under a pressure-control VentStar R415 ventilator (RWD Life Science Inc., Shenzhen, China). Following thoracotomy between the 3rd and 4th intercostal spaces, the mouse heart was exposed. The LAD was ligated with an 8-0 silk suture and the PE10 tube for 30 min. PBA and vehicle were i.p. injected 15 min prior to reperfusion. After 30 min of ischemia, the suture with PE10 tube was removed. At the end of 24 h of reperfusion, the LAD was relegated, and 1% Evans blue dye was injected to measure the area at risk (AAR). The hearts of the mice were collected, and frozen at -80 °C for 10 min and cut into 5 slices. The slices were incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Santa Cruz, USA) at 37 °C for 15 min and fixed with 4% paraformaldehyde overnight. The slices were captured and the areas of AAR and IA were quantified by using ImageJ software.
Hemodynamic measurement
Hemodynamic measurements were performed using a PV-loop catheter (Transonic Systems Inc., Ithaca, NY, USA). Hemodynamic measurements were analysed using Ponemah 7700 modules software according to a standardized protocol. The maximum rate of rise of left ventricular pressure increase (+dp/dtmax) and the maximum rate of rise of left ventricular pressure decrease (-dp/dtmax) were calculated for all mice.
Echocardiographic detection
Echocardiography was performed on mice at the end of reperfusion. In brief, mice were re-anesthetized with isoflurane and subjected to the Vevo 2100 System (VisualSonics Inc., Toronto, Canada). M-mode was used to obtain LV dimensions in diastole and systole according to the standard procedures and calculations. Left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic volume (LVSV) and left ventricular end diastolic volume (LVDV) were measured. And the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) values were calculated using Vevostrain software as parameters indicating cardiac function.
LC/MS/MS detection of modified peptides
Keap1 recombinant protein (1 mg/mL) in PBS buffer was mixed with 50 μM PBA, and incubated at 30 °C for 2 h. After removal of the small molecules by centrifugation, the product was dissolved in 0.1 M Tris-HCl, pH 8.0 containing 8 M urea. Then, 50 mM NH4HCO3 was added to adjust the pH value. Trypsin was added at a ratio of 1:100. The mixture was incubated in a 37 °C water bath for 16 h. After digestion, the mixture was subjected to a C18 column to remove salt. Finally, the peptides were eluted by the buffer containing 700 μL of 100% ACN, 299 μL of double distilled H2O and 1 μL of 0.1% FA. Then, the peptides were analysed by LC/MS/MS.
Protein cloning, expression and purification
The Keap1 was amplified by PCR using a human cDNA library, and was subcloned into the vector pET28-TEV, which contained one tobacco etch virus (TEV) protease cleavage site at the upstream multiple cloning site. The protein was expressed in E. coli BL21 (DE3) cells and incubated at 37 °C until the OD600 value reached 0.6, using 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight induction at 18 °C. The cells were isolated by centrifugation, resuspended in ice-cold lysis buffer and lysed by cryogenic overpressure cell breaker at 1,200 psi. The lysates were centrifuged for 60 min at 14,000 rpm to obtain the supernatant. Gravity flow purification of Keap1 protein was obtained at room temperature using 1 mL of Ni-NTA and eluted in buffer containing 250 mM imidazole, followed by 1 mL of HiTrap QHP for the second step purification. The final protein was purified and validated by Superdex 200 10/300GL resin.
The mutant plasmid DNA was obtained by PCR. The PCR products were cleaved by DpnI restriction enzymes. The restriction endonuclease-cleaved mixture was subsequently transformed into E. coli DH5α. Colonies were evaluated by colony PCR and sequenced. The mutant plasmid was transformed into E. coli BL21 (DE3) after successful sequencing. The steps for expression and purification of all recombinants were described above.
ITC titration experiments
Isothermal calorimetry (ITC) titration experiments were performed at 25 °C. WT Keap1, Keap1 mutants and PBA were dissolved in binding buffer (40 mM Tris-HCl pH 8.0, 250 mM NaCl). In a typical experiment, 60 μL of PBA (1 mM) was injected 19 times at 2-min intervals from a stirring syringe into the sample cell containing 300 μL of WT protein or mutants (30 μM). The first injection was 0.4 μL of PBA, and the subsequent 18 injections were 2 μL each time. The site-binding models were determined by using MicroCal Origin software.
The receptors Keap1-Kelch and Keap1-BTB were obtained from PDB (PDB ID: 3WN7, 5DAF) in PDB format for docking purposes. Schrodinger software was used for molecular docking. The protein was prepared by assigning bond orders, adding hydrogens, setting proper ionization states of residues, and capping. The protein was then refined with H-bond assignment (water orientations, at neutral pH). The ligands were prepared in ligprep with the following parameters: OPLS3e, do not change ionization states, generate tautomers and stereoisomers (create all combinations of specified chiralities and determine chiralities from 3D structure) with at most 32 ligands to be made. Finally, the ligands were covalently docked by the covalent docking method and the reaction type was selected as Michael addition. The latest best pose for PBA was selected using the docking score.
Statistics analysis
The results are presented as the mean ± SEM or the mean ± SD from at least three independent experiments. Data analysis was performed using a two-tailed unpaired Student's t test for two-group comparisons. Multiple comparisons were carried out by one-way analysis of variance (ANOVA), followed by Dunnett's test with GraphPad Prism 8 (GraphPad Software, Inc, La Jolla, CA, USA). P < 0.05 were considered statistically significant.
Keap1: Kelch ECH-associating protein 1; Nrf2: nuclear factor erythroid 2-related factor 2; MI/R: myocardial ischemia-reperfusion; PBA: pubescenosides A; LAD: left anterior descending artery; Cys77: Cysteine 77; Cys434: Cysteine 434; PIDD: protein with a death domain; ROS: reactive oxygen species; IL-1β: interleukin-1β; HO-1: heme oxygenase-1; NQO1: NAD(P)H quinone dehydrogenase 1; Cul3: Cullin-3; Ub: ubiquitin; FBS: fetal bovine serum; PS: penicillin and streptomycin; PBS: phosphate buffered saline; LPS: lipopolysaccharide; ATP: adenosine triphosphate; HRP: horseradish peroxidase; RT: room temperature; ECL: enhanced chemiluminescence; DAPI: 4', 6-diamidino-2-phenylindole; TTC: 2,3,5-triphenyltetrazolium chloride; AAR: area at risk; IPTG: isopropyl β-D-1-thiogalactopyranoside; ITC: isothermal calorimetry; ANOVA: analysis of variance.
Supplementary figures and tables.
This work was supported by the National Natural Science Foundation of China (81930114, 81803765, 31700625), Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine (2018B030322011), Key-Area Research and Development Program of Guangdong Province (No. 2020B1111100004) and Scientific Research Team Training Project of GZUCM (No. 2019KYTD201).
Y.C. and S.C conducted cell and animal-based experiments, and wrote the manuscript. P.W. isolated and identified PBA. X.G. and P.P. performed LC/MS/MS experiments for binding site analysis. L.C. produced the expression and mutation constructs and ITC assays. Z.L. and C.W. supervised the work and wrote the manuscript.
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Corresponding authors: Caiyan Wang, Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine, Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China. Phone: 86-020-39358875; Fax: 86-020-39358071; Email: wangcaiyanedu.cn. Zhongqiu Liu, Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine, Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China. Phone: 86-020-39358061; Fax: 86-020-39358071; Email: liuzqedu.cn.


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4 Scientific Reasons Why Saving Endangered Species Is Important
Keeping endangered species alive benefits our food sources, our environment, our health, and our economy.
By Alex Daniel November 27, 2019
By Alex Daniel
The importance of saving endangered species has been all over the news in recent years. But there are still those out there who wonder why, with approximately 7.7 million animal species on the planet, losing a few hundred or even a few thousand is such a huge deal. Well, believe it or not, protecting endangered species has major implications that extend far beyond simply keeping cute animals alive. From their role in agricultural production to their contributions to medicine, these animals do far more than many of us realize. Here are some of the reasons why saving endangered species is of the utmost importance. And for more on how you can help, check out 17 Small Things You Can Do to Help Endangered Species.
They are the sources of medicines, from antibiotics to anti-cancer agents.
Those species whose lives are on the line could potentially save your life down the road. Biological diversity has been vital to the development of all types of medicine. In fact, according to one 2012 paper published in the Journal of Advanced Pharmaceutical Technology & Research, nearly half of all drugs created within the last 30 years "are either directly or indirectly from natural products." And these natural products are disappearing at a rapid rate, leaving the animals who live off them dying as a result.
"A few hundred wild species have stocked our pharmacies with antibiotics, anti-cancer agents, pain killers, and blood thinners," notes the U.S. Fish and Wildlife Service. "The biochemistry of unexamined species is an unfathomed reservoir of new and potentially more effective substances." Basically, there's no telling what potentially life-saving sources could be lost as more plants and animals go extinct.
What we do know is that some of the animals on the verge of extinction harbor definitive medicinal resources. For example, the blood of a particular type of horseshoe crab that's on the decline due to overfishing has been used to detect harmful bacteria in medical devices and drugs. And an experimental drug derived from the saliva of Gila monsters, a venomous lizard in the Southwestern U.S. and parts of Mexico, has been found to help people with type 2 diabetes keep their blood sugar at healthy levels, according to Michelle Magee, MD, director of the MedStar Diabetes Institute.
In short, every species lost is the loss of a wonder drug that may save human lives.
They are the first signal of serious environmental issues.
There is no better way to track the health of our ecosystems and the species that live in them than through living organisms. For instance, freshwater mussels that filter water from the ocean are often the first to signal pollution issues, and the drop in population of peregrine falcons alerted the world to the dangers of DDT. These creatures often serve as "canaries in the coal mine," alerting us to dangerous environmental conditions when we would never realize otherwise. If these species go extinct, we lose these essential and natural monitors of our environment. And FYI, those freshwater mussels species are going extinct. So, if you want to help keep the environment healthy, check out these 30 Easy Ways to Make Your Home More Eco-Friendly.
They help keep crops (and by proxy, humans) healthy.
Farmers frequently use insects to help ensure the health of their crops. Without them, that could mean less healthy food for us. "Farmers are using insects and other animals that prey on certain crop pests," the U.S. Fish and Wildlife Service points out. "These are called 'biological controls,' and in many cases they are a safe, effective, and less expensive alternative to synthetic chemicals." Without animals naturally doing the dirty work of pesticides, we're exposing our crops, and thereby ourselves, to dangerous chemicals.
But, according to a 2019 meta-analysis published in the journal Biological Conservation, more than 40 percent of insect species are threatened with extinction. "Insects constitute the world's most abundant and speciose animal group and provide critical services within ecosystems," the study authors explain. Potential extinction, the researchers conclude, "cannot be ignored and should prompt decisive action to avert a catastrophic collapse of nature's ecosystems."
They boost the economy.
While endangered species provide seriously pivotal services, they're also quite the commodity, too. Ecotourism generates billions of dollars every year throughout the world—bird-watchers alone spend nearly $41 billion annually on travel and equipment. Though it's not often something that people think about, the less we do to protect animal species, the more we risk slashing an important sector of the economy. And for more on animals that need protecting, read up on All the Endangered Species in the World.
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Internet, meet your new BFFs.
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33 Adorable Animals That Are Actually Deadly
Pandas aren't so cute once you learn of their murder potential.
17 Small Things You Can Do to Help Endangered Species
You can make a greater impact than you realize.
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Some sub-zero temperatures won't scare these intrepid animals.


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Home Betting Slot Machines: 5 of the Best
Slot Machines: 5 of the Best
The thrill of playing slot machines is one which millions of people around the globe have enjoyed at some point during their lives.
Our love affair with slots often starts during childhood, with visits to amusement arcades in seaside holiday resorts first introducing us to a new world of wonderment.
The attraction of slots continues into our adult years, with casinos, bingo halls and bars amongst a plethora of land-based venues where games are available.
Modern slots are very different from the earliest incarnations, with advancements in technology allowing developers to create some magnificent creations.
However, at the core of every good slot game is simplicity – a factor that helps to make them hugely appealing to all types of players.
Whether you play purely for fun, for a few dollars or you are a high roller, slots are generally much easier to understand than other casino games like blackjack, poker and roulette.
Perhaps the most tempting aspect of slots are the progressive jackpots on offer, which can grow into truly life-changing amounts of money.
The enduring popularity of slots means that there are now thousands of different games to choose from, so read on as we look at five of the very best in the business.
1. Cleopatra
Source: TotalSporty
Cleopatra slots have become a hugely recognisable part of the online casino scene since the first version was launched and numerous operators currently feature the game on their sites.
Wink Slots are a great example of this in action, with the company rating Cleopatra as one of the best slot machines that has ever been made.
It is very difficult to argue with that viewpoint, as the game's 'It's Your Lucky Day' strapline offers a glimpse into the treasures that await within.
Players are taken back in time to ancient times courtesy of Egyptian themed symbols that can unlock the door to a potential king's ransom.
Cleopatra has spawned many subsequent sequels, while numerous developers have tried to imitate it, but much like many popular movie series the original version remains the best of the bunch.
With a wide range of features and the opportunity to win up to 10,000 coins per active payline, Cleopatra is a slot game that you simply cannot afford to miss.
2. Wheel of Fortune
Source: Casino in Switzerland
Based on the iconic game show of the same name, the Wheel of Fortune slot is firmly established as one of the best slot games in the business.
The television programme is the longest-running syndicated game show in the United States and its features have transferred perfectly into the slot arena.
Developed by International Game Technology (IGT), the game uses the same exciting sounds and graphics from the TV show.
When the wheel symbol appears on the reels, you even hear a crowd chanting 'Wheel of Fortune' just as they do when the show opens.
The unusual 3-4-5-4-3 layout of the reels can initially be tricky to get to grips with, but free play mode allows players to try out the game before spending real money.
There are two exciting bonus rounds on Wheel of Fortune, with one of them offering players the opportunity to win eye-watering amounts of money.
3. Starburst
Source: NetEnt Games
Starburst is another exciting slot game that is now commonly featured online and it is easy to see why it is hugely popular with players across the world.
The 5-reel, 3-row slot boasts 10 fixed paylines that take you on a spectacular voyage into the deepest extremes of outer space.
You can play Starburst in practice play mode to fully familiarise yourself with the game, before taking the plunge and playing for real money.
The Starburst Wilds are the path to riches on the slot, activating a feature that has the potential to generate massive cash payouts.
Players can win a maximum of 50,000 coins on Starburst, regardless of whether they choose to play on desktop or mobile devices.
A Return to Player (RTP) percentage of more than 96.1 percent is extremely competitive, helping Starburst stand head and shoulders above many of its rivals.
4. Deal or No Deal
Source: as an NZ online casino
If you are a fan of the long-running TV show, it is highly likely that the Deal or No Deal slot will be right up your street.
It closely follows the format of the game show via its special features, most notably during the jackpot bonus game.
This requires you pick your box, before revealing the others in the hope that you manage to hang on to the highest value prizes.
Deal or No Deal offers three bonus features in total, in addition to giving players the opportunity to win three different progressive jackpots.
The starting values are £10,000, £50,000 and £100,000, all of which continue to build with every spin until a jackpot is paid out, at which point they are reset.
Free Spins, Win Multipliers and Wild Substitutions add further depth to a slot that provides one of the most immersive gameplay experiences around.
5. Wizard of Oz
Source: dbestcasino.com
Based on the classic 1939 musical fantasy film of the same name, the Wizard of Oz slot machine is a true legend of the iGaming industry.
The game contains five reels, 30 bet lines and a host of features including free spins, a bonus round, wild symbols, scatter symbols and bumper jackpots.
Where Wizard of Oz really shines is in the nostalgia stakes, with players immediately able to identify with the characters in the game.
Dorothy, the Cowardly Lion, Tin Man and Scarecrow are all included, along with the legendary yellow brick world leading towards the Emerald City.
Glenda the Good Witch is also worth keeping an eye on, as she can appear on any of the five reels and turn them into expanding reels.
While other slots may have surpassed Wizard of Oz from a purely graphics perspective, very few of them can match the sheer charm this game exudes.
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