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Gaia's Dreams by Alan AtKisson (Pre-Beta)
Category Archives: World
Basic Concepts, Change & Transformation, Economics & Business, Forecasts & Scenarios, Future: Study of, Future: Visions of, Humans: Future of, Science & Technology
Notes: Future Shock
4 March 2018 Alan AtKisson	Leave a comment
In 1970, Alvin Toffler (and his wife Heidi, they wrote as a couple but used only his name) published a book that took the world by storm, selling millions of copies and introducing a phrase — "Future Shock" — that soon earned a place in the dictionary.
Future Shock was not just a book about what was likely to happen, based on trends and patterns (though it did plenty of predicting). It was a book about how human beings were reacting to a feeling that the future was arriving faster than they could prepare for it. The principal thesis of the book is preserved in its dictionary definition: "the physical and psychological distress suffered by one who is unable to cope with the rapidity of social and technological changes" (Merriam-Webster).
The Tofflers (Alvin died in 2016) were surprisingly skilled at extrapolating from the trends of the 1960s into the world that later emerged, accurately predicting personal computers, the Internet, "Information Overload," the sharing economy, telecommuting, the role of artificial intelligence in the workplace, and the rise of informal and less-hierarchical organizational structures. Their famous "errors" look wise in hindsight: Heidi predicted throwaway paper clothes, which did not happen; but the throwaway society certainly did. Today's "fast fashion" garments may not be made out of paper, but they are not much longer-lasting.
Future Shock was not the first book to claim that "This lifetime is … different from all others because of the scale and scope of change," nor was it the last. But it was the first to bring the speed of change into global public discourse as a topic of debate, and to highlight the counter-intuitive features of exponential growth — a topic that would stand at the center of another mega-bestseller that followed just two years later, The Limits to Growth. The Tofflers noted that accelerating economic expansion — Japan was the China of their day, its GDP growing at an average of 9.8% per year — translated into "a doubling of the total output of goods and services in the advanced societies about every fifteen years." The individuals coming of age in the 1960s might live to see a world 32 times as "big" in economic terms (after five doublings) because of the compounding impact of exponential growth.
As it happens, the world economy has doubled in size more than four times since 1950. Graphs of that growth resemble rocket launches.
The Tofflers were not the world's first futurists — the term had emerged during World War II, in connection with US think tanks — but they were the first to popularize the profession on the world stage. Alvin even taught the first known university course on the subject of "future studies" in 1966.
In introducing the systematic study of the future to a mass audience, they were careful not to let it be reduced to mere trend-spotting and prediction. Instead, they gave the popular understanding of future studies a decidedlhy philosophical and analytical character. Here they are writing on the advancement of technology, in words that seem to presage the significantly more developed and coherent theories of contemporary thinkers such as W. Brian Arthur (The Nature of Technology: What it is and How it Evolves, 2009):
"[T]echnological innovation does not merely combine and recombine machines and techniques. Important new machines do more than suggest or compel changes in other machines — they suggest novel solutions to social, philosophical, even personal problems. They alter [humanity]'s total intellectual environment — the way [we think and look] at the world." (Future Shock, p. 29)
The Tofflers noted that for humans to avoid the "shock" part of "future shock," they would need to become more adaptable, flexible, and ready for the unfamiliar and unpredictable. It was wise advice then, and it also seems to have anticipated the emergence of an entirely new area of scientific inquiry, which is today called "resilience". Becoming more resilient was imperative, wrote the Tofflers, because the advancement of technological civilization "offers no surcease from change. It offers no return to the familiar past. It offers only the highly combustible mixture of transience and novelty."
While the Tofflers are usually remembered as cheerleaders for the future, their agenda was far more radical. In their book's final pages, they called for a massive slow-down in the pace of economic, industrial and social change in order "gently guide our evolutionary destiny." Their diagnosis of where "this wild growth, this cancer in history" was leading reads less like a prediction, and more like a hauntingly accurate premonition of the early decades of the 21st century:
"[B]efore we can build a humane future, [we must] halt the runaway acceleration that is subjecting multitudes to the threat of future shock while, at the very same moment, intensifying all the problems they must deal with — war, ecological incursions, racism, the obscene contrast between rich and poor, the revolt of the young, and the rise of a potentially deadly mass irrationalism." (Future Shock, p. 486)
Today's "ecological incursions" are happening at the scale of planetary ecosystems. The global "contrast between rich and poor" is wider and more obscene than ever. Phrases like "climate change" or "mass migration" or "school shooting" were not even in our lexicon when "future shock" was invented, nor was "social media," which has become the most powerful technology ever known for spreading "mass irrationalism" in the form of conspiracy theories and fake news.
Today the "runaway acceleration" of human history appears to be speeding up rather than slowing down. The ever-faster arrival of "the future" may have become less and less shocking to us since the Tofflers' time. It may even be that the majority of humanity is not just accepting, but even desiring, of the "novelty and transience" they worried about.
But that does not mean that we have solved the problem of collaboratively making the future that we are creating more humane, more livable, more sustainable. In fact, we appear to have just caught up to the Tofflers' vision of how we might start.
In the closing pages of 1970's Future Shock, the Tofflers laid out a suggested program of activity that bears a striking resemblance to something that finally occurred, at the international level, in the years 2012 to 2015. They called for "a movement" that would "broaden and define in social, as well as merely economic terms, the goals of 'progress.'" They imagined gatherings happening "in each city, in each neighborhood" whose purpose was to "assign priorities to specific social goals for the remainder of the century." They even proposed the use of large-scale, participatory simulation games, involving thousands or even millions of people at a time, to help us "formulate goals for the future."
In 2015, the largest-ever gathering of heads of state convened in New York City, at the United Nations headquarters, to formally approve a document that had been developed with the input of hundreds of thousands of people, from all around the world. Called "Transforming Our World," the document included 17 broad "Sustainable Development Goals", and 169 prioritized sub-goals, to guide global development through the year 2030.
It was not exactly what the Tofflers had in mind. But it was astonishingly close.
Additional sources to review:
Future Shock at 40: What the Tofflers Got Right (and Wrong)
https://www.fastcompany.com/1695307/future-shock-40-what-tofflers-got-right-and-wrong
Future Shock on Wikipedia
https://en.wikipedia.org/wiki/Future_Shock
"Future Shock" Documentary Film on YouTube (starring Orson Welles):
Climate, Economics & Business, Goals & Planning, Science & Technology, Uncategorized
Notes: Science-Based Targets
19 February 2018 Alan AtKisson	Leave a comment
"Setting greenhouse gas emission reduction targets in line with climate science is a great way to future-proof growth."
This headline from the first page of the website for the Science-Based Targets Initiative sums up their approach succinctly. It involves (a) using scientific methods and existing scientific consensus (b) to determine an organization's fair share of greenhouse emissions reduction, on a timeline that also aligns with science-based international climate agreements, while (c) maintaining something close to business as usual — "growth" in this case means steadily increasing revenues, profits, and market share — in the process.
Here is the formal definition:
Targets adopted by companies to reduce greenhouse gas (GHG) emissions are considered "science-based" if they are in line with the level of decarbonization required to keep global temperature increase below 2°C compared to pre-industrial temperatures, as described in the Assessment Report of the Intergovernmental Panel on Climate Change (IPCC).
[Applies to the 4th or 5th AR (Assessment Report) of IPCC as well as modeling of the IEA (International Energy Agency).]
The technical note in brackets is part of the formal definition. While this specificity makes the definition seem extremely robust, it is important to keep in mind that that the IPCC's work is based on numerous climate models, which in turn rely on both past scientific data and a great many assumptions about the future. One of those assumptions is how much carbon dioxide will be removed from the atmosphere in the future, by technologies that either have not yet been developed or that will be scaled up to enormous proportions in the coming decades. If those technologies do not materialize, the "level of decarbonization required" is likely to be much higher.
The Initiative, run by a consortium of prominent global organizations, also presents science-based targets — "SBTs" — as the best way to minimize uncertainty (i.e. "future-proof") with regard to business prospects. Predicting future business success is much less certain than predicting future global temperature rise, so this promise of future business success, implied by the SBT Initiative, is not based on similarly scientific methods.
The concept of SBTs does not just apply to the operations of a company, but to its core products as well. The SBT Initiative has even described how oil and gas companies can participate, by setting science-based targets and timelines for steadily reducing their production of fossil fuels and replacing these products with other, renewable forms of energy products and services.
Note that the phrase "science-based targets," used in connection with climate change and greenhouse gas emissions reduction, has been around since at least 1992*; but it only began to gain serious traction after the Paris Agreement of 2015, which established a broad international consensus around the goal of limiting global temperature rise to 2 degress C or less.
* See, for example, "Convention on climate change: economic aspects of negotiations," OECD, 1992, p. 22-23.
Arts & Literature, Change & Transformation, Economics & Business, Future: Visions of, Science & Technology
Notes: The Viridian Design Movement (1999-2008)
In 1999, science-fiction writer Bruce Sterling gave a speech in which he called the growing problem of greenhouse gas emissions "an aesthetic problem." Our reliance on fossil fuels and other pollutants was creating an ugly world, even though it was largely invisible to our senses. He believed the best hack for starting an environmental revolution was not technical or political, but artistic.
"We're in trouble because we live in filth and we can't see it," said Sterling. "We're like eighteenth century people who lived before germ theory. We're ignorant of the squalor that surrounds us, and we have bad taste."
Sterling determined to attack the problem head-on, and he single-handedly launched a new design movement to "Change what people see. Change how they see." Sterling wanted to use art and design to help people visualize a future that was a brighter and more attractive shade of green — and start making it real.
He called the movement "Viridian" because that was the name for a shade of green that was slightly unnatural — cool, techy, and environmentally friendly without being predictably so.
Sterling's initiative was bold and quirky, and he was well-known in sci-fi and futurist circles, so he immediately attracted a following. He wrote a manifesto and a set of design principles, and he declared himself the Viridian Movement's "Pope-Emperor." Then he appointed a "Curia" of friends, advisors and fellow-travelers, to help spread the word. (I was lucky enough to be one of these.) The Viridian Movement, operating through Sterling's email list, quickly spawned a number of design competitions, whose purpose was to call into being the kinds of design innovations Sterling felt to be lacking — things like spore-based ink to help people "embrace decay," or a graphic symbol to highlight the fact that we were already in a state of "greenhouse disaster."
Being on the slightly more practical side, I decided to organize an international Viridian Design Competition around one of Sterling's best-loved ideas (conceived with colleague Stefan Jones): an electricity meter that was actually fun to look at, and that would tell you clearly when you were destroying the climate, and when you were saving it.
With a $10,000 grant from an enlightened philanthropist, I ran the contest though Donella Meadows' Sustainability Institute (where I was the unpaid Director of Arts & Culture) and in partnership with a prominent international sustainability network known as the Balaton Group. That group's global experts in climate science, modelling, renewables, and energy efficiency were the judges. And our winner, Inci Mutlu's "Wattbug", went on to receive media exposure in the New York Times and Wired magazine.
So did the Viridian Movement help create the future — that is, the present in which we are living now? Did it "introduce something new into the probability stream" as Elon Musk puts it? Or just accelerate what was bound to happen anyway?
Today, "smart meters" that provide immediate visual feedback on energy consumption are everyday items in the world's electricity grids — though none are as cute as the Wattbug, and they would have arrived, eventually, anyway. Sustainable design is an increasingly normal part of life, though not in the radical ways described by Sterling at the end of the 20th century. New, more mainstream-friendly design movements for sustainability have emerged such as the one I helped launch with the Norwegian government's Center for Design and Architecture, organized around the UN's Sustainable Development Goals. (It's called the Oslo Manifesto.)
In 2008, Sterling himself seemed to sense that the Viridian moment was over and abdicated from his self-appointed overlord position, four years ahead of the movement's scheduled "Expiration Date." Having an expiration date was itself a Viridian idea; but the zeitgeist was also changing. Al Gore and the IPCC had won the Nobel Peace Prize, which reduced the need for specialized awareness-raising on climate change. The economic meltdown now called the "Financial Crisis" was hitting its peak, which reduced interest in flashy new design ideas for a time. Sterling had just published a thoughtful book on design, called "Shaping Things," that he considered "very Viridian without coughing up that fact in a hairball." (Sterling, who is from Texas, has both a large personality and a penchant for colorful metaphors.)
Meanwhile, both the ideas and many of the people from the Viridian list had been absorbed into a then-new thing, called a "blog", that went by the name of Worldchanging and promoted the alternative phrase "bright green." (I was part of that, too.)
So in his final "Viridian Note" — the epistles that Pope-Emperor Sterling had been regularly sending out to the masses for nearly a decade — he gracefully admitted that "the world has become a very different place," and that the growing calls for change in finance and politics went a bit beyond the design focus of Viridian. But his parting words were still a broadside against traditional green thinking or the notion that we should just consume less, an approach that he disparagingly called "hairshirt-green" (after the very uncomfortable goat-hair garments that Christian penitents and ascetics used to wear).
"Hairshirt-green is the simple-minded inverse of 20th-century consumerism. Like the New Age mystic echo of Judaeo-Christianity, hairshirt-green simply changes the polarity of the dominant culture, without truly challenging it in any effective way. It doesn't do or say anything conceptually novel — nor is it practical, or a working path to a better life."
Sterling challenged his readers, in a more open-ended, less green-authoritarian way, to simply "re-think your relationship to material possessions in terms of things that occupy your time. The things that are physically closest to you. Time and space." You spend a lot of time with your stuff, and it takes up space. So acquire quality. Ditch what you don't need and don't use. This was ultimately far more important, opined the author of the Viridian Manifesto, than whether the object in question was "green."
From this future vantage point, I can see the "streams of probability" that Viridian touched, as it tried to create a certain quirky vision. It lifted the topic of green design out of the environmental muck and into flashy, digitized lights of Silicon Valley. It was a brilliant act of conceptual art, the impacts of which are impossible to measure. I know it affected a certain moment in my life, inspired certain of my actions, and that it did that for hundreds of other people, who then went on to affect (or infect) other people, with the idea of making "green" more attractive, cool, and relevant. It probably "accelerated the inevitable."
But the boldness of Sterling's vision — his willingness to just write a manifesto and launch a movement, armed with nothing but an email account and some name recognition — inspires me still today. I doubt, for example, that we would have thought up the Oslo Manifesto in 2016 if my Viridian memories hadn't been lurking, quite unconsciously, in the background of my mind.
So hats off to Viridian! It may have expired — but like true design classics, it never actually went out of style.
Basic Concepts, Change & Transformation, Future: Study of, Science & Technology
Quote: A branching stream of probabilities (Musk)
Quote 19 February 2018 Alan AtKisson Leave a comment
I look at the future from the standpoint of probabilities. It's like a branching stream of probabilities. And there are actions that we can take that affect those probabilities, that accelerate one thing, or slow down another thing, [or] introduce something new into the probability stream.
— Elon Musk, TED interview, April 2017
Economics & Business, Future: Visions of, Humans: Future of, Science & Technology
Notes: Elon Musk's vision of the future
Screenshot of Elon Musk's 2017 TED interview. Click to view.
It is widely known that Elon Musk — founder of Tesla, SpaceX, the Boring Company (for revolutionizing tunnel technology), and most recently Neuralink (for developing computer-mind interfaces) — wants humans to live on Mars. Why?
Intellectually, he describes the reason this way: if we "stay on Earth forever, there will be some eventual extinction event." [1] Apparently his biggest worry is "World War III".[2] Musk and others believe that spreading humans out into the solar system is the only real insurance against these existential risks to humanity's survival.
But that's just the intellectual rationale. Musk's most powerful motivations for heading to Mars appear to lie a bit deeper in his psyche.
In a revealing 2017 interview [3] on the TED channel, Elon Musk often seemed to be searching for the most efficient and effective words that would convey his thoughts clearly but in an entertaining fashion. As you watched him parse the interviewer's less-than-clear questions, which were laced with hero worship ("Yeah," Musk would say, in a disappointed tone that suggested he was thinking, "I guess you felt you had to ask that question that way, but you could have asked it much better") one could easily imagine why he wants to create brain-computer interfaces. There was often a methodical jerkiness to this delivery, as though having to squeeze his big, fast thoughts through the slow, cumbersome instrument of an ordinary voice, in a body, on a stage, was just so damn slow.
Musk in public is one of the most future-focused people I have ever seen, though his passion for vision is deeply influenced by his sense of the past. He reminded the TED audience that past civilizations built up impressive technologies only to forget them later (the ancient Egyptians, the Romans). He summarized the history of the US space program in a few sentences:
"If you look at the progress in space, in 1969 you were able to send somebody to the moon. 1969! Then we had the space shuttle. The space shuttle could only take people to low Earth orbit. Then the space shuttle retired and the United States could take no one to orbit. So that's the trend." He made a gesture with his hand, slicing downward from left to right. "The trend is down to nothing."
This trend greatly disturbs him. "People are mistaken when they think that technology automatically improves. It does not automatically improve. It only improves if a lot of people work very hard to make it better." Without that continuous focus and effort, says Musk, technology often degrades.
Musk often appears in the media as a single genius, reshaping the future, but of course the greatest part of his accomplishment resides not just in "dreaming big," as the TED interviewer kept insisting, but in building companies around those visions, hiring brilliant talent, and motivating them to "work very hard to make it better."
But what motivates Musk, at least as he reports in this interview, is not a messianic urge to save humanity. It is his inability to live without the possibility of realizing his vision of the future.
"It's important to have a future that's inspiring and appealing," he says. "There have to be reasons that you get up in the morning, and you want to live." And what makes Musk want to live? He does not wake up in the morning torn, as E.B. White famously wrote, "between a desire to improve the world and a desire to enjoy the world." Improving the world is absolutely what matters most to Elon Musk. Because Elon Musk is in love with the future.
"What do you love about the future?" he said to the audience at TED, though he was referring to himself, and to the question he wakes up with in the morning. "If we're not out there — if the future does not include being out there among the stars, and being a multi-planet species — I find that incredibly depressing."
Elon Musk is not trying to save the planet, or save humanity. "I'm just trying to think about the future and not be sad."
[1] Elon Musk, "Making Humans a Multi-Planet Species," New Space, June 2017.
[2] Olivia Solon, "Elon Musk: we must colonise Mars to preserve our species in a third world war," The Guardian, 11 Mar 2018
[3] TED.com, "Elon Musk at TED: The Future We're Building – and Boring", April 2017
Goals & Planning, Science & Technology
Notes: China intends to take the lead on AI
"In July, China unveiled a plan to become the world leader in artificial intelligence and create an industry worth $150 billion to its economy by 2030."
"As China Marches Forward on A.I., the White House Is Silent," New York Times, 16 Feb 2018
Artificial Intelligence may be the technology that is currently most linked, in the public mind, to expectations about the future — even though there are many other technologies that are likely to change our lives just as much, or more. The announcement of an "AI Race," much like the "Space Race" of the 1960s, suggests that the public hype around AI will only increase in the coming decade.
Animals, Brain & Mind, Ecosystems, Humans: Study of, Philosophy & Ethics
Quote: The deadliest predator on the planet (Raillton)
The deadliest predator on the planet is not the strongest or swiftest, but the one with the longest time horizon of anticipation, Homo sapiens.
— Peter Raillton
Writing in the introduction to Homo Prospectus, Seligman et al., 2016, p. 4
Climate, Earth, Ecosystems, Science & Technology
Film: A Beautiful Planet
Screenshot from the website for "A Beautiful Planet" (click to visit)
When these breathtaking views of our planet begin to fill the domed screen of an IMAX theater, filmed at the International Space Station by astronauts living there, it is difficult not to be moved. (Although the young students all around me in the theater, at Stockholm's Natural History Museum, had no trouble not being moved. At lunch, some of them confessed to falling asleep.)
"A Beautiful Planet" begins with a simulated faster-than-light trip into the Milky Way galaxy and its hundreds of billions of stars, which underscores the absolutely non-special status of the star we know as the Sun.
But somehow, all this stellar ordinariness only enhances the planetary uniqueness of Earth — covered with glittering water and air, just warm enough for life, protected from solar radiation by a magnetic field, which reveals itself in the shimmering green curtain of the northern lights.
Familiar and unfamiliar places float by — Paris, Florida, massive lightning storms over the Congo, the enormity of the ocean, Arctic icescapes, great river deltas — and the message digs deeper and deeper into your consciousness: this planet is alive. We are part of that life.
By the end of the film, the astronauts whose pictures and voices have filled one's head and heart have begun to speculate about whether another "Goldilocks Planet" (a planet where everything is "just right") might host life as we know it, or at least something similar. Of course, given the vastness of space, that is very statistically probable.
But without real (as opposed to simulated) faster-than-light travel, we are not likely to find out. And until then, there is only Earth — Gaia — our spaceship, our extraordinarily beautiful home, captured in all its glory in this wonderful film.
Object: The Antikythera mechanism
The Antikythera mechanism, used in ancient Greece to predict the movements of celestial bodies. Photo: Wikipedia
The Antikythera mechanism is considered the oldest known machine for accurately predicting the future based on scientific observations and mathematical calculations — in this case, the positions of the stars, the orbit of the moon, the four-year cycle of the Olympic Games, and astrological features that were important to the Greeks.
Computer-generated reconstruction of the Antikythera mechanism's display face. Image: Wikipedia
Discovered in an ancient shipwreck off the coast of Greece in 1902, the mechanism dates to approximately 100 BCE. A complex set of 37 gears powered the mechanism, like a clockwork, and a display face indicated when future events would occur (see the computer model reconstruction). The knowledge of science and technology that are reflected in the Antikythera mechanism were lost to the Western world for well over a thousand years, only reappearing again in Europe during the 14th Century.
Cities, Future: Visions of, World
Notes: The city as a symbol of the future
King's Dream of New York – Book Cover by Moses King, 1908
The city has been a visual symbol of the future for well over a century, and the "futureness" of it is always reflected in the same way: by increasing the density, verticality, and mobility. From "King's Dream of New York" — the book cover from 1908 pictured here — to science fiction icons such as Luc Besson's The Fifth Element or Ridley Scott's Blade Runner, popular culture places us in a future vision by showing us a city that holds more people that we imagine possible, housed in gigantic buildings that dwarf any current city, flitting about in vehicles that do not yet exist (usually flying ones).
Although urban visions are changing to accommodate the needs of climate change, and green urbanism is a mainstream feature of both past and future city planning, people still think of the future as being "more of the same" in these classically urban terms: dense, vertical, mobile.*
And of course, we must add the word technological, for the city is the opposite of our agrarian, low-tech past. Agriculture and rural life is, in fact, a symbol of the past, just as the city is a symbol of the future — except when agriculture involves hydroponics powered by solar cells in a modern, urban building. This expanding practice is sometimes called "vertical farming".
* With thanks to Jeremiah B.C. Axelrod who points out these qualities in Inventing Autopia: Dreams and Visions of the Modern Metropolis in Jazz Age, University of California Press, 2009
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Frontiers in Pharmacology
Ethnopharmacology
Cheorl-Ho Kim
Sungkyunkwan University, South Korea
William Chi-Shing Tai
Hong Kong Polytechnic University, Hong Kong
Sung-Kwon Moon
Chung-Ang University, South Korea
Front. Pharmacol., 12 December 2018 | https://doi.org/10.3389/fphar.2018.01441
Anti-allergic Inflammatory Effects of the Essential Oil From Fruits of Zanthoxylum coreanum Nakai
Rui Hong Guo1, Jung Up Park1, Se Jin Jo1, Jae Hun Ahn2, Jong Hwan Park2, Ji Yoon Yang3, Sung Suk Lee3, Mi Jin Park3* and Young Ran Kim1*
1College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju, South Korea
2Laboratory Animal Medicine, College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea
3Division of Wood Chemistry, Department of Forest Products, National Institute of Forest Science, Seoul, South Korea
Zanthoxylum coreanum Nakai is a rare shrub which grows in Korea and China. Pericarp of Z. coreanum has been used as a crude medicine, but there are few researches about the pharmacologic activities. The present study was designed to investigate the anti-allergic inflammatory activities of the essential oil from fruits of Zanthoxylum coreanum Nakai (ZCO). Our findings showed that ZCO inhibited both the IgE-antigen complex or PMA/A23187-induced β-hexosaminidase release and IL-4 production dose-dependently in RBL-2H3 mast cells, and confirmed that ZCO at the tested concentrations did not show cytotoxicity to RBL-2H3 cells by MTS assay. Additionally, we found that ZCO showed the significant inhibition on LPS-induced overproduction of TNF-α, IL-6 and NO. Consistently, the protein levels of iNOS and COX-2 were also remarkably decreased by ZCO treatment. Herein, Our mechanistic studies revealed that ZCO significantly suppressed the activation of transcription factor NF-κB in PMA-activated 293T cells, and further inhibited NF-κB p65 translocation into the nucleus in LPS-stimulated RAW264.7 cells. Further investigation identified that ZCO down-regulated LPS-induced phosphorylation of MAPK (JNK, ERK, and p38) signal pathway. For incremental research, we established an DNCB-induced atopic dermatitis model in BALB/c mice, and found that ZCO remarkably inhibited DNCB-induced ear swelling and AD-like symptoms. Based on these findings, ZCO is suggested to have a therapeutic potential for the allergic inflammatory diseases.
Allergic diseases are inflammatory disorders which have become a worldwide clinical health problem. With the allergy patients increasing annually due to various factors, approximately 10 ∼ 20% of the world population is affected by allergies (Ring et al., 2014; Do et al., 2017; Appel et al., 2018). Most allergy patients are genetically predisposed to produce IgE antibody. Mast cells express a high-affinity IgE receptor on membranes, which is important to the pro-inflammatory allergic response. When an IgE-antigen binds with FcεRI (high-affinity receptor for the Fc region of immunoglobulin E), the receptor is activated, and occurs complex biological reactions causing allergic reactions, including inflammatory disorder (Bochner and Schleimer, 2001; Yang et al., 2016; Appel et al., 2018). Also, a combined treatment with PMA/A23187 has been used widely for mast cell activation, because they are known to induce the generation of inflammatory cytokines (Hong et al., 2010; Vo et al., 2011). RBL-2H3 cells, a mast cell line, are originated from rat basophilic leukemia, and have been used to study IgE-FcεRI interactions and degranulation. Furthermore, RBL-2H3 cells are a useful model for in vitro screening of anti-allergic agent candidates (Tang et al., 2015). β-hexosaminidase, a granule-associated exoglycosidase, is stored in secretory granules of mast cells, and has been used to monitor mast cell degranulation just as histamine has been used (Dinneswara Reddy et al., 2012; Zhang et al., 2013). During the pathogenesis of allergic disease, IL-4 is also crucial for the induction of IgE synthesis and mast cell development (Nabeshima et al., 2005).
Inflammation is a complex mechanism involving the activation and deactivation of immune cells, which could result in cellular and tissue damage causing the chromic disease (Alessandri et al., 2013; Ahmad et al., 2017). Macrophages are key immune cells in initiating and maintaining the inflammatory response. Lipopolysaccharide (LPS), a principle component of the outer membrane of Gram-negative bacteria, could induce the macrophage cells' inflammation reaction, and stimulate the production of inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-α and IL-6 (Yi et al., 2013; Chen et al., 2014). This could be used to assess the anti-inflammatory activities of samples. Nuclear factor kappa B (NF-κB) is an important regulator of allergic inflammation, which controls the expression of pro-inflammatory mediators (Kim et al., 2018). When stimulated, NF-κB translocates to the nucleus, and regulates the expression of various transcription factors leading to the expression of pro-inflammatory enzymes like cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), which are responsible for the stimulation of inflammatory signaling molecules (Ahmad et al., 2017). Apart from NF-κB, MAPKs also plays an important role in the regulation of the cell growth, cell differentiation, and cellular pro-inflammatory molecules (Huang et al., 2012; Song et al., 2018).
Essential oils have been used to treat allergy and inflammation as cosmetic materials. In the present study, we obtained essential oils by hydro-distillation from some tree plants or fruits and screened the anti-allergic or inflammatory activities. The essential oil from fruits of Zanthoxylum coreanum Nakai (ZCO) showed the most inhibitory activity on the mast cell degranulation. Zanthoxylum coreanum Nakai, Korean lime tree, is a rare shrub which only grows in Korea and China. In Korea, it is called wang-cho-pi tree. Pericarp of Z. coreanum has been used as a crude medicine for the treatment of ozena, rheumatoid, nasal sinusitis, sore throat, etc. in Korea. Previous study has reported that Z. coreanum showed the antiviral activity against picornaviruses (Choi, 2016). However, there are few researches about the pharmacologic activities of Z. coreanum. Here, we studied the anti-allergic inflammatory effects of ZCO on degranulation and IL-4 production in RBL-2H3 cells, inflammatory cytokines production such as NO, TNF-α, and IL-6, and the protein expression levels of iNOS and COX-2 in LPS-induced RAW264.7 macrophage cells. For incremental research, we investigated the mechanism related to the NF-κB and MAPKs signal pathways. In addition, in vivo experiment was designed to investigate the effect of ZCO on DNCB-induced AD-like symptoms. To our knowledge, this is the first evidence for the effects of ZCO on allergic inflammation.
The fruits of Z. coreanum were used in this study. The fruits were collected in experimental forest of National institute of forest science located on Jin-ju city, republic of Korea, in August 2017. Taxonomical identifications were established by the ecologist Dr. Hwa-Ja Hyeon at warm temperate and subtropical forest research center of national institute of forest science and the voucher specimen code was WTFRC 10030535.
Essential Oil Extraction
The fruits of Z. coreanum were hydro-distillated at atmospheric pressure, using a clevenger-type apparatus. A 10 L round-bottom flask containing 2099.8 g of fruits was placed in heating mantle. In this flask, the fruits were mixed in 5 L distilled water. Then the flask was connected with clevenger type apparatus. The fruits of Z. coreanum were extracted for 14 h. The collected essential oil was dried over anhydrous sodium sulfate and filtered through 0.45 μm membrane disk-filter. The essential oil obtained was transferred to sealed dark vials and stored at 4°C for further analysis.
The essential oil content was determined on the basis of oven dry matter and measurements were carried out in triplicates. The yield (%) of essential oil was calculated using the following formula:
Essential oil yield(%) = mass of essential oil obtained (g)mass of oven dry matter (g)×100
Gas Chromatography–Mass Spectrometry (GC–MS) Analysis
Coupled GC-MS analysis was performed on a Thermo Scientific Model ISQ LT equipped with both a flame ionization detector (FID) and a mass spectrometer (MS). GC/MS analysis solution was prepared by dissolving 4 μL oil to 1.0 mL dichloromethane solution (containing 100 ppm methyl undecanoate), and 1 μL was injected. A VF-5MS GC column (60 m × 0.25 mm × 0.25 μm film thickness; Agilent Technologies) was used. Carrier gas was helium at a constant flow rate of 1.0 mL/min. Injection temperature was 250°C with a split ration 1:20. The oven temperature was maintained at 50°C for 5 min, then increased at 10°C/min to 65°C held for 30 min, then at 5°C/min to 120°C held for 10 min, then at 5°C/min to 210°C held for 10 min, and finally at 20°C/min to 325°C held for 10 min. For FID detection, temperature was set to 300°C, air flow to 350.0 mL/min, hydrogen flow to 35.0 mL/min, and make-up gas (helium) flow to 40.0 mL/min. Mass interface temperature was 250°C and ion source temperature was 250°C. Mass scan data was acquired in EI mode at 0.2 s scan time rate with a scan range from 35 amu to 550 amu.
The identification of peaks was performed by comparing peak average mass spectrum of peak with an electronic library database (NIST/EPA/NIH Mass Spectral Library, version 2.0 g). In addition, the identity of the compounds was assigned by comparison of the koviats retention indices (KI, Table 1) determined in relation to a homologous series of n-alkanes (C7-C30).
Table 1. Chemical compositions of ZCO.
Cell Culture and Reagents
The rat basophilic leukemia cell line RBL-2H3 was supplied from the Korean Cell Line Bank (Seoul, South Korea). RBL-2H3 cells were cultured in Minimum Essential Medium (MEM) (Welgene, South Korea) supplemented with 10% FBS (Gibco, Rockville, MD, United States) and 1% penicillin-streptomycin under an atmosphere of 5% CO2 in a humidified 37°C incubator. RAW264.7 macrophage cells, IgEL b4 cells and 293 T cells were purchased from American Type Culture Collection (Manassas, VA, United States) and were cultured in Dulbecco Modified Eagle's medium (DMEM) with 10% FBS and 1% penicillin-streptomycin. Calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Quercetin, 1-Chloro-2,4-dinitrobenzene (DNCB) and dexamethasone were purchased from Sigma-Aldrich (St. Louis, MO, United States). Albumin from bovine serum, 2,4-dinitrophenylated (DNP-BSA) was purchased from Invitrogen by Thermo Fisher Scientific (Eugene, OR, United States). rIL-4 and mTNF-α were purchased from R&D system Inc (Minneapolis, MN, United States). mIL-6 was purchased from Biolegend (San Diego, CA, United States).
Stock solutions of ZCO were diluted using the mixture solution of DMSO and ethanol (1:1) to a concentration of 20% and filter-sterilized, and then diluted with phosphate buffered saline (PBS) for the working concentration.
Measurement of Cell Viability
RBL-2H3 cells, RAW264.7 cells, and 293T cells were seeded into 96-well plates (SPL life Sciences Co., Pocheon, South Korea) at 1.5 × 105 cells/mL overnight under an atmosphere of 5% CO2 in a humidified 37°C incubator, then the cells were treated with ZCO at the concentration of 0.0025, 0.005, 0.01% for 24 h. Cell proliferation was assayed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega, Madison, WI, United States) according to manufacturer's instructions, and absorbance was read with a microplate reader (BioTek, Winooski, VT, United States) at 490 nm.
β-Hexosaminidase Release by RBL-2H3 Cells
β-hexosaminidase is a marker of mast cell degranulation (Dinneswara Reddy et al., 2012). RBL-2H3 cells were incubated into 48-well plate (2 × 105 cells/mL) in MEM with 10 % FBS and IgE anti-DNP at 37°C overnight. After washing 2 times with siraganian buffer (SB) pH 7.2, 119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2⋅6H2O, 25 mM PIPES [piperazine-N, N′-bis (2-ethanesulfonic acid), 40 mM NaOH], the cells were incubated with SBC buffer (siraganian buffer containing 5.6 mM glucose, 1 mM CaCl2, and 0.1% BSA). Then cells were pretreated with ZCO at the concentration of 0.0025, 0.005, 0.01% or the positive control quercetin (20 μM) respectively, for 1 h prior to stimulation with DNP-BSA (1 μg/mL) (Thermo Fisher Science, OR, United States) for 30 min at 37°C and stopped on ice for 5 min, and the cell supernatants (50 μL) collected by centrifugation were incubated with 50 μL of 1 mM 4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma, St Louis, MO, United States) in 0.1 M sodium citrate (pH 4.5) at 37°C for 1 h. The reaction was terminated with 200 μl/well of carbonate buffer containing 0.1 M Na2CO3 and 0.1 M NaHCO3 (pH 10), and the absorbance was measured at 405 nm with a microplate reader (BioTek, Winooski, VT, United States).
In addition, RBL-2H3 cells incubated into 48-well plate (3 × 105 cells/mL) in MEM with 10% FBS overnight were washed 2 times with SB buffer, and then incubated with SBC buffer. Cells were then pretreated with ZCO at the concentration of 0.0025, 0.005, and 0.01% or quercetin (20 μM) for 1 h prior to stimulation with 30 ng/mL of PMA and 350 ng/mL of A23187 for 1 h at 37°C, and stopped on ice for 5 min, then cell supernatants were collected for β-hexosaminidase activity assay, as described previously (Su et al., 2018).
Rat IL-4 Secretion by RBL-2H3 Cells
To measure the IL-4 concentrations in the culture media, RBL-2H3 cells were incubated into 48-well plate (2 × 105 cells/mL) in MEM with 10% FBS and IgE anti-DNP at 37°C overnight. After washing 2 times with MEM, the cells were pretreated with ZCO at the concentration of 0.0025, 0.005, and 0.01% or quercetin (20 μM) respectively, for 2 h and then treated with DNP-BSA (1 μg/mL) for 16 h at 37°C. The cell-free supernatants were collected and IL-4 concentrations were measured using ELISA kit (R&D, Minneapolis, MN, United States) according to the manufacturer's instruction.
In addition, RBL-2H3 cells were incubated into 48-well plate in MEM with 10% FBS overnight and pretreated with ZCO at the concentration of 0.0025, 0.005, and 0.01% or quercetin (20 μM) respectively, for 2 h. Then cells were stimulated with 30 ng/mL of PMA and 350 ng/mL of A23187 for 16 h. The supernatants were collected for the measurement of IL-4 levels.
NF-κB Reporter Assay
293T cells were seeded into poly-D-lysine hydrobromide (Sigma, MO, United States) coated 96 well plate at 1.5 × 105 cells/mL, and then transient transfection of a reporter plasmid pNF-κB-SEAP (Clontech Laboratories, Palo Alto, CA, United States) was performed to cells with HilyMax for 4 h. After transfection, the cell medium was replaced with fresh DMEM overnight. Then cells were treated with ZCO at the concentration of 0.0025, 0.005, and 0.01% or the positive control Bay11-7082 (20 μM) for 2 h prior to stimulation with 3 ng/mL of PMA (Sigma, St. Louis, MO, United States) for 24 h. The supernatants (10 μL) were incubated with Quanti-Blue (100 μL) (Invitrogen, Carlsbad, MA, United States) for 1 h, and the absorbance was read at 630 nm with an ELISA microplate reader (BioTek, Winooski, VT, United States).
NF-κB Staining
RAW264.7 cells were plated into an 8-well glass chamber plate (Thermo Fisher Scientific, United States) at 1 × 105 cells/mL overnight. The cells were pretreated with ZCO (0.01%) or Bay11-7082 (20 μM) for 2 h prior to stimulation with 1 μg/mL of LPS (Sigma Co., MO, United States) for 2 h, and then fixed with 4% paraformaldehyde (Molecular Probes, Inc., Eugene, OR, United States) and permeabilized with 0.1% triton for 15 min. The NF-κB p65 protein was detected by immunostaining using a polyclonal-anti-NF-κB p65 antibody (Invitrogen, Carlsbad, MA, United States) and Alexa Fluor 488-conjugated anti-rabbit IgG antibody (Molecular Probes Invitrogen, MA, United States). Actin was visualized by staining with Alexa Fluor 594 conjugated phalloidin. The cells were mounted by using Prolong gold anti-fade reagent with DAPI (Molecular Probes). Bay 11-7082 (Sigma, MO, United States) was used as the positive control for the NF-κB inhibitors. Fluorescence images were acquired using Laser Scanning Confocal Microscope System (Leica TCS SP5/AOBS/Tandem, Germany) at Korea Basic Science Institute, Gwangju Center).
Nitric Oxide Analysis
RAW264.7 cells were plated into 48-well plates (SPL Life Sciences Co., Pocheon, South Korea) at 2 × 105 cells/mL and treated with ZCO (0.0025, 0.005, and 0.01%) or the positive control Bay11-7082 (20 μM) for 2 h before stimulation with 500 ng/mL of LPS. After 24 h of incubation, nitric oxide (NO) production in the culture supernatants was measured using Griess Reagent [1% sulfanilamide in 5% H3PO4, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride] and incubated for 30 min at room temperature. The absorbance was read at 570 nm using an ELISA microplate reader (BioTek, Winooski, VT, United States).
Measurement of Pro-inflammatory Cytokines Production
RAW264.7 cells were seeded into 48-well plates (SPL Life Sciences, Pocheon, South Korea) at 2 × 105 cells/mL and treated with ZCO (0.0025, 0.005, and 0.01%) or the positive control Bay11-7082 (20 μM) for 2 h before stimulation with 500 ng/mL of LPS. The supernatants were collected after incubation for 24 h to measure the TNF-α (R&D system, Minneapolis, MN, United States) and IL-6 (Biolegend, CA, United States) with an ELISA kit according to manufacturer's instructions.
Western Blot Analysis
RAW264.7 cells cultured in a 6-well plates were harvested in an ice-cold cell culture lysis reagent (Promega, Madison, WI, United States). The protein of the cell lysates was determined using the Bradford reagent (Bio-Rad laboratories, CA, United States). Equal amounts of protein extracts (20 μg) for iNOS or COX-2 determination or 50 μg protein for MAPK pathway determination in a lysis buffer were subjected to 10% SDS-PAGE analysis and then electro-transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA, United States). Following blocking of the non-specific binding sites with 5% skin milk at room temperature for 2 h, the membrane were incubated with primary antibodies directed against polyclonal antibodies specific to COX-2 (1:1000, Cell Signaling Tech., Danvers, MA, United States), iNOS (1:1000, Santa Cruz, CA, United States), or β-actin (1:1000, Santa Cruz, MA, United States), p-p38, p-38 (1:500, Cell signaling Tech, MA, United States), p-ERK, ERK (1:500, Cell signaling Tech, MA, United States), p-JNK, JNK (1:500, Cell signaling Tech, MA, United States), and NF-κB p65 (1:1000, Santa Cruz, MA, United States), IkB-α (1:1000, Cell signaling Tech, MA, United States), LaminB (1:1000, Santa Cruz, MA, United States) overnight at 4°C, and then were incubated with horseradish-peroxidase- conjugated second antibodies at room temperature for 1 h. Immunoreactive proteins were visualized using an ECL Western blot detection system (Advansta, Menlo Park, CA, United States).
Subcellular Fractionation
RAW264.7 cells cultured in 6-well plates were pretreated with ZCO (0.005 and 0.01%) or Bay11-7082 (10 μM) for 4 h, and then stimulated with LPS (1 μg/mL) for 30 min. RAW264.7 cells were lysed in a hypotonic buffer (EDTA 5 mM, dithiothreitol (DTT) 5 mM, HEPES 10 mM (pH 7.5), MgCl2 5 mM, protease inhibitor cocktail) on ice for 30 min, and then centrifuged at 1300 × g for 10 min at 4°C. The supernatants with cytosolic proteins were transferred into new iced tubes. The pellets were lysed with hypertonic buffer [HEPES 10 mM (pH 7.9), MgCl2 1.5 mM, NaCl 100 mM, DTT 0.5 mM, EDTA 0.1 mM, protease inhibitor cocktail] on ice for 1 h, and then centrifuged at 13000 × g for 10 min. Supernatants with nuclear protein were collected.
Induction of Atopic Dermatitis in the Mouse Ear
All animal experiments were approved by the Institutional Animal Care and Use Committee of Chonnam National University, and the protocol was approved by the Committee. The induction of AD-like lesions by DNCB was performed according to our previous research (Park et al., 2017). Female BALB/c white mice (7 weeks) were divided into 5 groups (n = 4), and control group was sensitized by the application of 150 μL of the mixture of acetone and olive oil (4:1), and other groups were sensitized by the application of 2 % 1-Chloro-2,4-dinitrobenzene (DNCB, Sigma, MO, United States) dissolved in the mixture of acetone and olive oil (4:1) on the shaved abdominal skin. After 4 days' rest, 20 μL of ZCO (1 and 2%) or dexamethasone (1%, Sigma, MO, United States) was pretreated in both sides of the ears for 30 min, and followed by application of 20 μL of 1% DNCB. After 3 h, repeated one time. On day 6, the same application was repeated as the day 5. From day 7, ears were treated with 20 μL of ZCO or dexamethasone only. Ear thickness was daily measured with a digimatic micrometer (Ozaki seisakusho, Tokyo, Japan) (Approval Number: CNU IACUC-YB-2018-72).
Histological Analysis
The ears of the mice were fixed in 10% neutral formalin for 24 h. The tissues were processed with alcohol and xylene series and embedded in paraffin. The paraffin blocks were sectioned at 2 μm, and then stained with hematoxylin and eosin, and examined under microscopy (Kang et al., 2017). Histological changes (acanthosis, thickening of the subepidermal layer, cellular infiltration) were evaluated semiquantitatively on a grade of 1–5 (1 = no change from negative control group; 5 = maximum change).
All statistical calculations were performed using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, United States). All experiments were repeated at least three times, and the data were obtained at least in triplicate. The results are presented as the means ± SEM. Statistical comparisons were evaluated using student's t-test with a P-value < 0.05 considered statistically significant.
Chemical Composition of ZCO
The yield of ZCO was about 2.1%. The chemical compositions of ZCO are shown in Table 1 and 37 constituents were identified. β-Ocimene was predominant component, accounting for 24.48% of the total. It was followed by (-)-α-pinene (16.56%), 4-carvomenthenol (11.61%), sabinene (10.81%), linalool (10.09%), o-cymene (3.56%), β-phellandrene (3.15%), limonene (2.63%), and α-terpineol (1.74%) (Table 1).
Effect of ZCO on the Cell Viability
Effect of ZCO on cell viability was evaluated by MTS, respectively, in RBL-2H3, RAW264.7 and 293T cells to ensure that its anti-allergic inflammatory effects was not due to the cell death in each condition. Treatment with ZCO for 24 h at the concentrations of 0.0025, 0.005, and 0.01% showed no significant cytotoxic effects. In addition, ZCO at the concentrations of 0.0025 and 0.005% showed the increased cell growth of RBL-2H3 and RAW264.7 cells (Figure 1).
Figure 1. Effect of ZCO on the cell viability. (A) RBL-2H3 cells, (B) RAW264.7 cells, and (C) 293T cells were seeded into 96-well plate overnight, and then were treated with ZCO (0.0025, 0.005, and 0.01%) for 24 h. The cell viability was tested by MTS assays. The data are representative of three independent experiments and expressed as mean ± SEM (∗P < 0.05; ∗∗∗P < 0.001 versus with control group).
Effect of ZCO on β-Hexosaminidase Secretion in RBL-2H3 Cells
Mast cells contain a large amount of cytoplasmic granules, which activation caused the process of degranulation (Zeng et al., 2016). The release of β-hexosaminidase can be used to quantify the extent of degranulation (Huang et al., 2015). Pre-treatment with ZCO significantly suppressed the degranulation either in IgE-antigen complex or PMA/A23187-stimulated RBL-2H3 cells. In addition, ZCO treatment reduced the degranulation in RBL-2H3 cells in a dose-dependent manner, and ZCO at the concentration of 0.01% in the PMA/A23187- stimulated RBL-2H3 cells showed the similar inhibition of β-hexosaminidase release to the positive control quercetin, with an IC50 value of 0.0066%, which indicated that ZCO may be a promising new anti-allergic agent (Figure 2).
Figure 2. Effect of ZCO on β-hexosaminidase secretion in RBL-2H3 cells. (A) RBL-2H3 cells incubated into 48-well plate (2 × 105 cells/mL) in MEM with 10% FBS and IgE anti-DNP a 37°C overnight were washed with SB buffer, and then were pretreated with ZCO (0.0025, 0.005, and 0.01%) or quercetin (20 μM) with SBC buffer for 1 h prior to stimulation with DNP-BSA (1 μg/mL) for 30 min. The supernatants were collected for β-hexosaminidase assay. Experiments were conducted in quadruplicate and expressed as mean ± SEM (∗P < 0.05; ∗∗∗P < 0.001 versus with DNP-BSA group). (B) RBL-2H3 cells incubated into 48-well plate overnight were washed with SB buffer, and then pretreated with ZCO or quercetin with SBC buffer for 1 h prior to stimulation with 30 ng/mL of PMA plus 350 ng/mL of A23187 for 1 h. The supernatants were collected for the detection of β-hexosaminidase (∗∗∗P < 0.001 versus with PMA/A23187 group). +++means the significance compared with the mock cells (deal with nothing).
Effect of ZCO on IL-4 Production in RBL-2H3 Cells
Modulation of inflammatory cytokines from mast cells is one of the key indicators of reduced allergic symptoms, and IL-4 is considered the most important therapeutic step for allergic inflammation diseases (Hoesel and Schmid, 2013). So, we examined whether ZCO could regulate a pro-inflammatory cytokine IL-4 in RBL-2H3 cells. The inhibitory effects of ZCO on IL-4 release were measured in sensitized RBL-2H3 cells stimulated by DNP-BSA or PMA/A23187. ZCO inhibited IL-4 secretion from DNP-BSA-stimulated RBL-2H3 cells with an IC50 value of 0.0034%, or PMA/A23187-stimulated RBL-2H3 cells with an IC50 value of 0.0059% (Figure 3), suggesting that ZCO can modulate mast cell-mediated allergic inflammatory responses.
Figure 3. Effect of ZCO on IL-4 production in RBL-2H3 cells. (A) RBL-2H3 cells were sensitized with IgE overnight at 37°C, followed by pretreatment with ZCO (0.0025, 0.005, and 0.01%) or quercetin (20 μM) for 2 h before stimulation with 1 μg/mL DNP-BSA for 16 h. The supernatants were analyzed for IL-4 production. Experiments were conducted in quadruplicate and expressed as mean ± SEM (∗∗∗P < 0.001 versus with DNP-BSA group). (B) RBL-2H3 cells incubated into 48-well plate overnight were pretreated with ZCO or quercetin for 2 h prior to stimulation with 30 ng/mL of PMA plus 350 ng/mL of A23187 for 16 h. The supernatants were collected and IL-4 cytokine was tested by using ELISA kit (∗∗∗P < 0.001 versus with PMA/A23187 group). +++means the significance compared with the mock cells (deal with nothing).
Effect of ZCO on the Production of Inflammatory Mediators in LPS-Activated RAW264.7 Cells
To investigate the anti-inflammatory effects of ZCO, their ability to modulate NO synthesis, TNF-α and IL-6 cytokines were measured in LPS-stimulated RAW264.7 cells. The results demonstrated that ZCO exhibited marked activity against TNF-α release in a dose-dependent manner, with an IC50 value of 0.0068% (Figure 4A). In addition, pre-treatment with ZCO significantly inhibited IL-6 secretion, with an IC50 value of 0.0023%, which were significantly different from the control group. The suppressive effects of 0.01% of ZCO approached the effect of the reference compound Bay11-7082 at 20 μM (Figure 4B). NO production was also significantly decreased by ZCO, with an IC50 value below 0.0025%, which implied anti-inflammatory activity in LPS-induced RAW264.7 macrophage cells (Figure 4C).
Figure 4. Effect of ZCO on the production of inflammatory mediators in LPS-activated RAW264.7 cells. RAW264.7 cells seeded into 48-well plate were pretreated with ZCO (0.0025, 0.005, and 0.01%) or Bay11-7082 (20 μM) for 2 h prior to addition of LPS (500 ng/mL) for 24 h. TNF-α (A) and IL-6 (B) in the supernatants was measured by ELISA and NO production (C) in the supernatants was detected by Griess reagent. Experiments were conducted in quadruplicate and expressed as mean ± SEM (∗∗∗P < 0.001 versus with LPS group). +++means the significance compared with the mock cells (deal with nothing).
Effect of ZCO on the Expression of COX-2 and iNOS in LPS-Activated RAW264.7 Cells
Pro-inflammatory enzymes, such as iNOS and COX-2, cause inflammation. Thus, we also examined whether ZCO could affect iNOS and COX-2 protein expressions by Western blot analysis. RAW264.7 cells were pretreated with ZCO (0.01 and 0.005%) or celecoxib (10 μM) for 2 h prior to addition of LPS (200 ng/mL). Stimulation of the RAW264.7 cells with LPS resulted in accumulation of iNOS and COX-2 proteins, as determined by Western blot analysis. ZCO significantly reduced the levels of iNOS protein. Also, ZCO decreased the COX-2 protein expression in LPS-activated RAW264.7 cells, especially the 0.01% of ZCO showed the remarkable effect on the inhibition of COX-2 and iNOS expression (Figure 5).
Figure 5. Effect of ZCO on the expression of COX-2 and iNOS in LPS-activated RAW264.7 cells. RAW264.7 cells cultured into 6-well plate were pretreated with ZCO (0.01 and 0.005%) or celecoxib (10 μM) for 2 h, and then LPS (200 ng/mL) was added to cells for 16 h. The protein levels of iNOS and COX-2 were detected by Western blot analysis. Celecoxib was used as a control for the COX-2 inhibitor, β-actin was used as the control protein.
Effect of ZCO on PMA-Induced NF-κB Activation in 293T Cells
NF-κB is an important factor in the regulation of immune responses in allergic diseases, and has been considered a prototypical pro-inflammatory signaling pathway (Kim et al., 2017). The effect of ZCO on NF-κB activation was tested in 293T cells. PMA increased NF-κB transcription, which was decreased by ZCO in a dose-dependent manner, with an IC50 value of 0.0065%. ZCO at the concentration of 0.01% showed the most significant reduction on the activation of NF-κB transcription (Figure 6A).
Figure 6. Effect of ZCO on NF-κB transcription and translocation. (A) Effect of ZCO on PMA-induced NF-κB activation in 293T cells. 293T cells seeded into poly-D-lysine hydrobromide coated 96-well plate were transfected with a reporter plasmid, pNF-κB-SEAP for 4 h. Cells were treated with ZCO (0.0025, 0.005, and 0.01%) or Bay11-7082 (20 μM) for 2 h prior to stimulation with 3 ng/mL of PMA for 24 h. The supernatants were collected for NF-κB activity assay. Experiments were conducted in quadruplicate and expressed as mean ± SEM (∗∗∗P < 0.001 versus with PMA group). (B) Effect of ZCO on NF-κB translocation into nucleus. RAW264.7 cells in 8-well glass chamber plate were pre-incubated with ZCO (0.01%) or Bay11-7082 (20 μM) for 2 h prior to addition of LPS (1 μg/mL). The cells were immunostained with a polyclonal anti- NF-κB p65 antibody and Alexa Fluor 488-conjugated 2nd antibody. Bay 11-7082 was used as a control for the NF-κB inhibitor. (C) Cytosolic and nuclear fractions were isolated from RAW264.7 cells. The protein level of NF-κB p65 in the nuclear fractions and IkB-α in the cytosolic fractions were detected by Western blot analysis. +++means the significance compared with the mock cells (deal with nothing).
Effect of ZCO on NF-κB Translocation in LPS-Activated RAW264.7 Cells
RAW264.7 cells were pretreated with ZCO (0.01%) or Bay11-7082 (20 μM) for 2 h prior to the stimulation of LPS (1 μg/mL). The effect of ZCO on LPS-induced NF-κB pathway was tested. LPS caused the translocation of NF-κB from the cytosol to the nucleus, which was inhibited by ZCO (Figure 6B). We also confirmed NF-κB translocation by Western blot analysis. Cytosolic and nuclear fractions were isolated from RAW264.7 cells. Treatment with ZCO decreased the level of NF-κB p65 proteins in the nuclear fractions and elevated the level of IkB-α in the cytosolic fractions (Figure 6C). These results indicated that ZCO exerted an anti-allergic inflammation effect through the inhibition of NF-κB translocation by the induction of LPS.
Effect of ZCO on MAPKs in LPS-Activated RAW264.7 Cells
Apart from NF-κB, MAPKs are also upstream modulators of inflammatory mediators and cytokines responses. In particular, JNK, ERK, and P38 play important roles in the activation of NF- κB (Huang et al., 2012; Yu et al., 2018). To further investigate whether ZCO exerted the inhibitory effects on inflammation through regulating MAPK pathway, we examined the MAPK phosphorylation by Western blot analysis. RAW264.7 cells were pretreated with ZCO for 4 h, and then stimulated with LPS (1 μg/mL) for 30 min. Phosphorylation levels of JNK, ERK, and p38 were increased in cells treated with LPS alone, which were remarkably inhibited by ZCO treatment. Non-phosphorylated JNK, ERK, and p38 have no changes in cells treated with LPS or LPS and ZCO (Figure 7). These results indicated that ZCO exerted an anti-allergic inflammation effect via the inhibition of MAPK pathway in LPS-stimulated RAW264.7 cells.
Figure 7. Effect of ZCO on MAPKs in LPS-activated RAW264.7 cells. RAW264.7 cells cultured into 6-well plate were pretreated with ZCO (0.01 and 0.005%) or Bay11-7082 (10 μM) for 4 h, and then stimulated with LPS (1 μg/mL) for 30 min. Cell lysates (50 μg) were used for Western blotting of the target proteins. Phosphorylated MAPKs were detected by using anti-phospho-JNK, anti-phospho-ERK or anti-phospho-p38 antibody. The membrane was stripped and reassessed with the antibodies targeting non-phosphorylated MAPKs.
Effect of ZCO on Atopic Dermatitis in Mice
We determined the effect of ZCO on AD-like skin lesions in mice. DNCB treatment elicited severe ear swelling and AD-like skin lesions such as large ulcers and hyperkeratosis, which were as an index of skin inflammation, and this swelling peaked at day 7 after sensitization. The positive control dexamethasone remarkably decreased the ear thickness, and showed no AD-like skin lesions. In addition, ZCO treatment inhibited DNCB-induced ear swelling and AD-like skin lesions comparable to dexamethasone (Figures 8B,C).
Figure 8. Effect of ZCO on Atopic Dermatitis in Mice. (A) Experimental design for the induction of AD. 5 days after the first boost with 2% DNCB (150 μL/mouse) to the abdomen, 1% DNCB was applied to both sides of the ears on day 6 and 7. Drug were pre-applied to the ear for 30 min. After 3 h, repeated one time. From day 8, drugs were applied only. (B) Representative photographed example of the effects of ZCO on DNCB-induced AD in the ear. (C) Ear thickness was daily measured with a digimatic micrometer. Data are presented as mean ± SEM (∗∗∗P < 0.001 versus with DNCB group). (D) Histological analysis of AD mice. Representative photomicrographs of ear sections were stained with H&E staining (original magnification: × 100). (E) Histological changes (acanthosis, thickening of the subepidermal layer, cellular infiltration) were evaluated semiquantitatively on a grade of 1–5 (1 = no change from negative control group; 5 = maximum change). ∗∗means that P < 0.01 versus with DNCB group.
A microscopic analysis of the ear by H&E staining showed many alternations. Compared with the DNCB-induced AD-like skin lesions, ZCO treatment significantly decreased the DNCB-induced epidermal and dermal thickness and infiltration of inflammatory cells, which was comparable to the positive control dexamethasone (Figures 8D,E).
In the present study, we had screened 15 essential oils from natural plants on anti-allergic inflammatory activities, and the essential oil from Zanthoxylum coreanum Nakai (ZCO) showed the highest inhibitory activity on the mast cell degranulation. We identified 37 constituents from ZCO (Table 1) and β-Ocimene was predominant component (24.48%) followed by (-)-α-pinene (16.56%), 4-carvomenthenol (11.61%), sabinene (10.81%), linalool (10.09%), o-cymene (3.56%), β-phellandrene (3.15%), limonene (2.63%), and α-terpineol (1.74%). Some studies have demonstrated that glycoproteins from Zanthoxylum piperitum DC fruits or leaves have the anti-allergic or anti-inflammation effect (Lee and Lim, 2009a,b, 2010; Park et al., 2009). However, there are few researches about the pharmacologic activities of Z. coreanum.
IgE-mediated allergic reactions through the FcεRI receptor is known to be the main mechanism of mast cell activation (Krystel-Whittemore et al., 2015). In this study, we investigated whether ZCO inhibits mast cell degranulation using RBL-2H3 cells. First, we found ZCO profoundly affected anti-DNP IgE-induced local allergic reaction and PMA plus A23187-induced systemic allergic reaction. Degradation of granules in mast cells leads to the release of the enzyme β-hexosaminidase along with histamine. Therefore, β-hexosaminidase is often used as a marker to measure the efficacy of new drugs in preventing mast cell activation and degranulation (Huang et al., 2014; Zeng et al., 2016). Using this assay, we confirmed that ZCO significantly inhibited anti-DNP IgE-mediated and PMA/A23187-stimulated β-hexosaminidase release (Figure 2). Mediators such as cytokines (IL-4, TNF-α, etc.) from the cells are involved in the late phase reaction of allergy. Also, IL-4 could modulate the inflammatory response owing to its ability to affect adhesion molecule expression and cytokine production in endothelial cells (Brown and Hural, 1997; Do et al., 2017). Our results exhibited dose-dependent effects of ZCO against both anti-DNP IgE-mediated and PMA/A23187-stimulated IL-4 production (Figure 3). These results indicate that ZCO may be a promising new anti-allergic inflammatory agent.
Macrophage cells are the key immune cells in the initial period of inflammation. After macrophages are activated by LPS, a series of inflammatory mediators will be released (Jeong et al., 2013; Hu et al., 2014). TNF-α and IL-6 cytokines are key pro-inflammatory cytokines. Therefore, LPS induced macrophages have usually been used for assessing the anti-inflammatory effects of various agents. In this paper, the anti-inflammatory activities of the ZCO by reducing inflammatory factors (NO, TNF-α, and IL-6) production in LPS-activated murine macrophages inflammation were studied, and ZCO showed the significant reduction on the TNF-α and IL-6 production, respectively, with an IC50 value of 0.0068 and 0.0023% (Figures 4A,B). NO is an endogenous free radical which is low in the human body. Inhibition of NO production is usually used as an important treatment for inflammation-related diseases (Zhang et al., 2017). LPS-induced RAW264.7 macrophages could release large amounts of NO, and iNOS is the main enzyme to catalyze NO production in acute and chronic inflammation (Chen et al., 2017). COX-2 is an inducible isoform of cyclooxygenase which mainly exerts its important role in the inflammation (Yi et al., 2013). We found that treatment with ZCO dramatically inhibited LPS-induced overproduction of NO in RAW264.7 macrophages (Figure 4C). Consistently, the expression of the iNOS and COX-2 protein were remarkably decreased by ZCO, as determined by Western blot analysis (Figure 5).
NF-κB and MAPK pathways are two major mechanisms which regulate LPS-induced inflammatory cytokines production. Activation of NF-κB by the phosphorylation and degradation of IκB-α causes the translocation of NF-κB p65 to the nucleus. This, in turn, activates the transcription of specific target genes such as TNF-α and IL-6 (Liu and Malik, 2006; Hong et al., 2010; Yi et al., 2013). Also, NF-κB induces both iNOS and COX-2 (Park et al., 2004). Therefore, inhibition of NF-κB activity is an important target for anti-inflammation treatment. In this study, ZCO significantly inhibited PMA-induced NF-κB activity in 293T cells (Figure 6A). In addition, LPS-induced translocation of NF-κB p65 to the nucleus was significantly reduced by ZCO in RAW264.7 cells (Figures 6B,C). This means that NF-κB pathway is involved in the anti-inflammatory effects of ZCO. MAPKs phosphorylation are also crucial events in the allergic inflammation response. The maximal MAPK (JNK, ERK, p38) expression is known to occur 10 ∼ 30 min after LPS stimulation (Reddy and Reddanna, 2009; Song et al., 2018). RAW264.7 cells were pretreated with ZCO for 4 h, and then stimulated with LPS for 30 min. We found that LPS-induced overexpression of phosphorylated JNK, ERK, and p38 levels was reduced by ZCO treatment (Figure 7). These results suggest that ZCO exerts anti-allergic inflammatory effects by inhibition of NF-κB activation and MAPK phosphorylation.
Atopic dermatitis (AD) is a chronic allergic skin inflammation disease associated with a combination of intense pruritus, eczematous skin disorder, scratching, and cutaneous sensitization with allergens (Choi et al., 2013). Our further investigation also researched the effects ZCO of on DNCB-induced AD-like symptoms in mice model, and found that ZCO treatment inhibited DNCB-induced ear swelling and AD-like skin lesions (Figure 8).
The present study reveals that the ZCO inhibits the degranulation and IL-4 production both in IgE/Ag and PMA/A23187-activated mast cell. In addition, ZCO is an effective inhibitor of LPS-induced pro-inflammatory mediators in RAW264.7 macrophages through suppressing the NF-κB and MAPK signal pathways. In vivo, ZCO treatment inhibited DNCB-induced ear swelling and AD-like skin lesions. These findings suggest that ZCO may serve as a potential therapeutic candidate for the treatment of anti-allergic inflammatory disease by the inhibition of NF-κB activity and MAPKs phosphorylation. Further studies are needed to characterize some pharmacologic activities of the 37 Chemical compositions validated from ZCO.
RG carried out major experiments and drafted the manuscript. JP and SJ supported with the study design and reviewed the protocol. JHA and JHP did the histological analysis. JY and SL carried out the essential oil extraction. YK and MP conceived the project, provided reagents and conceptual design, and wrote the manuscript.
This study was carried out with the support of Forest Science Technology Program (Project No. FP0900-2016-01) provided by National Institute of Forest Science.
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Keywords: Zanthoxylum coreanum Nakai, degranulation, inflammatory mediators, iNOS, COX-2, NF-κB, MAPK, atopic dermatitis
Citation: Guo RH, Park JU, Jo SJ, Ahn JH, Park JH, Yang JY, Lee SS, Park MJ and Kim YR (2018) Anti-allergic Inflammatory Effects of the Essential Oil From Fruits of Zanthoxylum coreanum Nakai. Front. Pharmacol. 9:1441. doi: 10.3389/fphar.2018.01441
Received: 02 July 2018; Accepted: 22 November 2018;
Cheorl-Ho Kim, Sungkyunkwan University, South Korea
Sung-Kwon Moon, Chung-Ang University, South Korea
William Chi-Shing Tai, Hong Kong Polytechnic University, Hong Kong
Copyright © 2018 Guo, Park, Jo, Ahn, Park, Yang, Lee, Park and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Mi Jin Park, lionpmj@korea.kr Young Ran Kim, kimyr@jnu.ac.kr; kimyr@chonnam.ac.kr

redpajama_set_name: RedPajamaCommonCrawl


My 7-day challenge was "to not use any products on my hair for styling purposes, simply just shower and use shampoo. The reason I decided on this challenge was that I wanted to see how I would look with my not done and what kind of reactions I would get.
Typically I get up at 6:15 am and take a shower then brush my teeth and get dressed and then spend at least 10 mins doing my hair. I leave conditioner in after the shower, then blow dry my hair, add the curler enhancer, and leave it like that.
Going into the challenge, my biggest concern was that I felt like my hair was going to look terrible, and I thought people at school would make fun of the way my hair looked. However, all of these concerns weren't necessary as many people gave me compliments as some people said they liked my messy look.
Not everyone was so kind though as some people said I looked like a "train wreck". I felt kinda bad at first like it would seem like I was just a sloppy person, but I started not to care and just forget about it. Eventually, I got use to not doing my hair in the morning and it actually felt good.
The other effect that the challenge had on me was few days my sleeping schedule. I got use to not having my hair done and it actually affected me as I started waking up later than usual and I would barely get to school on time.


9/01/2011 · Best Answer: The manual for your motherboard, my friend, states: "The front panel audio header supports HD audio by default. If your chassis provides an AC'97 front panel audio module, refer to the instructions on how to activate AC'97 functionality via the audio software in Chapter 5, "Configuring 2/4/5.1/7.1-Channel Audio.""... Hello guys I have a slight issue with my front panel connector. I.e my 2 USB ports, E-Sata slot, audio and microphone jacks on the front of the case all fail to work as soon as I connect them to the motherboard.
8/06/2011 · unplug the front audio panel connectors to see if the problem goes away. maybe you dont have the connections right. maybe the connectors themselves are bad.


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