Natural compounds as safe therapeutic options for ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology. Several conventional treatments for UC such as corticosteroids, immunosuppressive agents, tumor necrosis factor antagonist, integrin blockers, and interleukin antagonist, and salicylates are available but are associated with the various limitations and side-effects. None of the above treatments helps to achieve the ultimate goal of the therapy, i.e., maintenance of remission in the long-term. Natural remedies for the treatment of UC show comparatively less side effects as compared to conventional approaches, and affordable. The current review presents details on the role of herbal drugs in the treatment and cure of UC. Google, PubMed, Web of Science, and Scopus portals have been searched for potentially relevant literature to get the latest developments and updated information related to use of natural drugs in the treatment of UC. Natural products have been used over centuries to treat UC. Some of the essential herbal constituents exhibiting antiulcerogenic activity include gymnemic acid (Gymnema sylvestre), shagoal (Zingiber officinale), catechin (Camellia sinensis), curcumin (Curcuma longa), arctigenin (Arctium lappa), and boswellic acid (Boswellia serrata). Although many plant-derived products have been recommended for UC, further research to understand the exact molecular mechanism is still warranted to establish their usefulness clinically.

Introduction
According to the World Health Organization (WHO) report, more than 80% of the world's population relies on the traditional system of medicine for their health problems (World Health Organization 2019). Traditional medicines, mainly herbal products, serve as a lead compounds for identifying other bioactives as these have been used for thousands of years for treating various types of diseases and have the advantages of lower side effects, better availability and cost effectiveness (Choi et al. 2016;Huang et al. 2010;Lin et al. 2014). As the prevalence of chronic diseases, including cardiovascular system disorders, diabetes, cancer, ulcerative colitis (UC), and acquired immunodeficiency syndrome (AIDS) is increasing day by day; herbal medicines have gained popularity in the healthcare system and have been recommended to be used globally for these diseases. Moreover, several clinical and pre-clinical studies have been conducted for evaluation of the effectiveness and safety of such herbal remedies (Choi et al. 2016;Quansah and Karikari 2016). The UC, a type of inflammatory bowel disease (IBD), generally affects the mucosal lining of colon resulting in inflammation and ulcers.

Epidemiology
The IBD is a collective term used for a group of chronic manifestations that affect the small and large intestine and is a common cause of gastrointestinal morbidity (Fruet et al. 2012;Zois et al. 2010). The risk factors for IBD involve the overproduction of free radicals and decreased antioxidant capacity (Aleisa et al. 2014;Parfenov 2012). The two primary forms of IBD are UC and Crohn's disease (CD). According to WHO, the prevalence of UC is estimated to be 200-250 per 100,000. It is more common in western countries and is increasing worldwide (Annaházi and Molnár 2014;Campbell et al. 2001;Porter et al. 2020). UC affects both sexes equally and can start at any age; however, the primary age of onset of the disease is 15-30 years (Annaházi and Molnár 2014).

Etiology
The exact cause of UC is not known to date. It is multifaceted disorder where genetic factors, infective agents, oxidative stress, dysfunction of immune regulation, overproduction of prostaglandin (PG) E2 and the loss of tolerance of the luminal microbiota are key contributors to the development of this disease (Awaad et al. 2013;De Almeida et al. 2013;Fruet et al. 2012;Zhang et al. 2006). Among all, oxidative stress contributes the most, in which interplay between reactive oxygen species (ROS) and reactive nitrogen species (RNS) is responsible for many physiological functions and colorectal pathological processes. Therefore, there has been an increase in interest in the potential uses of exogenous antioxidants to treat and prevent oxidative gastrointestinal disorders (Aleisa et al. 2014). UC is also initiated and promoted by release of inflammatory cytokines by macrophages, B-cells, and T-cells. Various pro-inflammatory cytokines involved in articular cartilage destruction are tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and transforming growth factor-β) (TGF-β) (Patil and Moss 2008;Toshifumi 2003;Clinton 2009).

Symptoms of ulcerative colitis
The UC exhibits many characteristic features like chronic remitting, relapsing course, inflammatory nature, and unknown causes (Bamias et al. 2005;Hirten and Sands 2021;Samanta et al. 2012;Hendrickson et al. 2002). Some other symptoms are fatigue, tiredness, fever, nausea, diarrhea, bloody stool, anorexia, weight loss, malaise, delayed growth, arthritis, and sometimes anemia (Sninsky 2010). Although the transformation of UC to CD is not frequent, the pathological finding performed during clinical studies confirmed the first case of the progress of UC to CD (Satish Chandra Yadav 2021).

Available treatment approaches for ulcerative colitis
The ultimate goals of currently used antiulcerogenic drugs are not only to control disease progression but also to induce a quick remission and to maintain it for a long time along while preventing complications of the disease itself, minimize disability, and hence improving patient life and expectancy (Annaházi and Molnár 2014;Hanauer 2008;Probert et al. 2014). The choice of therapy depends on the severity of the condition, i.e., the extent of colon involvement and its localization. Further treatment depends upon the primary response of induction therapy (Meier and Sturm 2011;Theede et al. 2013;Sharma and Mishra 2014).
Therapy for UC consists of the following two steps: the first-line treatment is to induce remission (with induction agents) and resolve all inflammatory symptoms while the second is to maintain remission (with maintenance agents) (Nanda and Moss 2012;Dalal 2007). Most of these objectives are achieved by the combination of salicylates (like mesalazine and olsalazine); immunomodulators (like azathioprine, 6-mercaptopurine, cyclosporine, and methotrexate); corticosteroids (like methylprednisolone, and prednisolone); tumor necrosis factor signalling inhibitor (like infliximab, adalimumab and golizumab); integrin blocker (like vedolizumab, natalizumab etrolizumab); Janus kinase (JAK) inhibitor (like tofacitinib); and interleukin antagonist (like mirikizumab and ustekinumab) (Sands et al. 2014;Witaicenis et al. 2012). Along with the same, colectomy (surgical treatment) may be an alternative choice in case of life-threatening complications. The potential therapeutic agents for the treatment of UC and their targets are tabulated in Table 1. In addition to conventional therapies, some unconventional treatments, including leukocytapheresis, inorganic nitrite or nitrate, and fecal bacteriotherapy, have been explored to treat UC (Yokoyama et al. 2014;Jädert et al. 2014;Borody et al. 2003).

Drawbacks of conventional treatment approaches
The pharmacological therapies used for UC are associated with one or more side effects, which render them unsuitable for regular use. The conventional therapy is the treatment regimen, which is widely accepted and used by most of the healthcare professionals. The main adverse effects reported after using conventional therapy of UC include fever, nausea, headache, kidney damage, myopathy, myalgia, edema, neoplasia, congestive heart failure, tuberculosis, tremor, and hirsutism (Yokoyama et al. 2014). Side effects observed on using 5-aminosalicylates include bronchitis, arthralgia, headache, dizziness, abdominal cramps, and minor metabolic disorders (Patil and Moss 2008;Miehlke et al. 2014). Corticosteroids, though effective for UC when immediate remission is required, are also not free from side effects. The significant adverse effects of corticosteroids include edema, moon face, acne, mood disturbances, adrenal suppression, congenital fetal abnormalities, cushingoid face, gastric ulceration, and osteoporosis. Moreover, their long-term use may cause ocular side effects because of steroid-induced cataract and hyperglycemia, and chances of severe relapse have also been reported (Hanauer 2008;Kondamudi et al. 2013;Nunes et al. 2013). The safety profile of biologicals has also been studied. Their side effects include leukoencephalopathy, hypersensitivity, myalgia, neoplasia, congestive heart failure, tuberculosis, and malaise (Sands et al. 2014;Kondamudi et al. 2013;Langan et al. 2007). The most common side effects of immunosuppressants are hepatitis, pancreatitis, bone marrow toxicity, and leukopenia (Meier and Sturm 2011;Xu et al. 2004;Carter et al. 2004;Bamba et al. 2011) . Surgical treatment (colectomy) in UC offers a better quality of life in life-threatening complications untreatable with medical therapy. Risk of colorectal cancer, sexual dysfunction, female infertility, reoccurrence of inflammation, and psychological disorders are associated with colectomy (Ingrid Ordás et al. 2012;Coviello and Stein 2014).

Herbal approaches for the treatment of ulcerative colitis
Herbal products are being used worldwide for their therapeutic potential in various ailments. The phytoconstituents such as catechins, flavonoids, terpenes, alkaloids, anthocyanins, quinines, and anthoxanthins having anti-inflammatory and antioxidant effects, can modulate the expression of proinflammatory signals and are considered potential agents for the treatment of UC (Zhang et al. 2006). All these agents act by multiple mechanisms, including suppression of TNF-α, IL-1β, cyclooxygenase (COX), lipoxygenase (LOX), and nuclear factor κB (NF-κB). Various bioactive principles of the plants, including gymnemic acid, shagol, catechin, curcumin, glycyrrhizin, boswellic acid, aloein, arctigenin, and cannabidiol, have been successfully employed to treat UC (Huang et al. 2010;Borrelli et al. 2009;Sałaga et al. 2014;Arun et al. 2014;Hsiang et al. 2013;Brückner et al. 2012). The chemical structures of some active constituents responsible for antiulcer activity have been represented in Fig. 1.
The role of aloe in the treatment of UC is mainly due to PGE2 and IL-8 secretion inhibition, which in turn, is responsible for its anti-inflammatory nature. It is further reported to inhibit ROS by phorbol 12-myristate 13-acetate (PMA) stimulated human neutrophils (Wan et al. 2014).  Bamba et al. (2011), Carter et al. (2004, Kawakami et al. (2015), Meier and Sturm (2011) Surgical treatment --GIT disturbance, post-operative sitespecific infections and psychological disadvantages Meijs et al. (2014), Patel et al. (2013), Soon et al. (2014) Protective and therapeutic effects of AV gel on UC in acetic acid (AA)-induced colitis in rats have been evaluated by Bahrami et al. Reduction in inflammation, ulcer score, and tissue damage in AV-treated (50 and 300 mg/kg AV gel) rats compared with negative control animals (treated with 2 mL water), proved the usefulness in UC (Fig. 2). Pre-treatment with AV gel (50 and 300 mg/kg AV gel) reduced inflammation, lesions to serous layer and fibrosis and the results were found to be similar to positive control animals (treated with sulfasalazine 100 mg/kg) showed therapeutic effects   (Bahrami et al. 2020) in colitis animals (Bahrami et al. 2020). Hassanshahi et al. estimated the healing effect of AV gel in AA induced UC in rats. Histologically, it has been observed that AV gel treatment reduced and healed colon tissue damages in induced colitis. Also, this gel reduced apoptosis in rat's colon, which showed a considerable decrease in Bax messenger ribonucleic acid (mRNA) expression and significantly increased B-cell lymphoma 2 (BCL-2) mRNA expressions. Further, the histopathological data have indicated protective effect of AV gel in colon, which was supported by reduced cell infiltration and appearance of normal tissue ( Fig. 3) (Hassanshahi et al. 2020).

Arctigenin
Arctium lappa (AL) (Compositae), commonly known as Bardana or burdock, is widely used for various pharmacological activities such as diuretic, depurative, digestive, Fig. 3 Representative of the microscopic slide sections of the colon samples in rats: A Normal colon sample magnification (× 40), showed the normal structure of mucosa with an intact epithelial surface, submucosa, and muscular layer. B Normal colon sample, magnification (× 100), showed the normal structure of layers. C Acetic acidinduced colitis with no treatment, magnification (× 40), showed an inflammatory reaction, presence of ulcer, inflammation, edema, and diffuse infiltration of leukocytes in the submucosal layer. D Acetic acid-induced colitis with no treatment, magnification (× 200), showed inflammation in the submucosal layer with ulceration and edema. E Acetic acid-induced colitis + oral administration of sulfasalazine, magnification (× 40), revealed inflammatory reaction in the serosa layer. F Acetic acid-induced colitis + oral administration of sulfasalazine sample, magnification (× 100) revealed inflammatory reaction with the dominance of lymphocyte and fewer neutrophils with tissue necrosis. G, H Acetic acid induced colitis + oral administration of aloe vera gel, magnification (× 40), and (× 100), revealed the improvement of inflammation and the normal tissue (Hassanshahi et al. 2020) 1 3 anti-inflammatory, antiulcer, antioxidant, antimicrobial, antirheumatic, and antiallergic (De Almeida et al. 2013;Zhao et al. 2014;Al-Snafi 2014;Kenny et al. 2014;Wang et al. 2014;Predes et al. 2011;El-Kott and Bin-Meferij 2015;Maghsoumi-Norouzabad et al. 2016;Liu et al. 2014). Its antiulcer activity is attributed to arctigenin and other secondary metabolites like dicaffeoylquinic acid, caffeoylquinic acids, chlorogenic acid, and caffeic acid (Chen et al. 2004;Jiang et al. 2016;Carlotto et al. 2015;Liu et al. 2012;de Almeida et al. 2012). Onopordopicrin, a secondary metabolite of AL, also has a protective effect on gastric mucosa and can be an effective remedy for UC. Huang et al. investigated the protective role of AL in a dextran sodium sulphate (DSS)-induced murine model of UC. The alteration in mean body weight and disease activity index (DAI) of diseased and AL-treated animals was found to be significant. Moreover, the histological findings showed that AL treatment could prevent mucosal edema, submucosal erosions, ulceration, inflammatory cell infiltration, and colon damage (Fig. 4). In case of control animals, the architecture of colon was found to be normal, whereas, pre-treatment with AL showed slight cell infiltration without any abnormality of crypt cells. Therefore, AL can be considered as effective in suppressing DSS-induced colitis and also for prevention of bloody diarrhea (Huang et al. 2010). The possible mechanism involved in protection is down regulation of inflammatory mediators like IL-6, TNF-α, macrophage inflammatory protein-(MIP)-2, monocyte chemo attractant protein (MCP)-1, mucosal vascular addressin cell adhesion molecule (MAdCAM)-1, intercellular adhesion molecule (ICAM)-1, T helper cell (Th) 1, Th17, inducible nitric oxide synthase (iNOS), mitogen-activated protein kinase (MAPK), and vascular cell adhesion protein (VCAM)-1 at both protein and mRNA levels in colonic tissues (Huang et al. 2010;Maghsoumi-Norouzabad et al. 2016). Pomari et al. studied the effect of AL extract in treating UC. It has been found that AL elevates activities of antioxidant enzymes glutathione (GSH), superoxide dismutase (SOD), reduces lipid peroxidation (LPO), and prevents the formation of ROS; hence it can effectively be used for the treatment of UC (Pomari et al. 2014).
Wu et al. evaluated the anti-colitis effect of arctigenin and arctiin in DSS-induced colitis in mice. The comparative studies indicated that reduction in weight loss, DAI, and histological damage in the colon were better observed with arctigenin. Furthermore, arctigenin recovered the loss of intestinal epithelial cells (E-cadherin-positive cells) and decreased the infiltration of neutrophils myeloperoxidase (MPO)-positive cells and macrophages (CD68-positive cells) and also caused down-regulation of TNF-α, IL-6, MIP-2, MCP-1, MAdCAM-1, ICAM-1, and VCAM-1. The above findings clearly indicate that arctigenin, not arctiin, is the active ingredient of AL for attenuating colitis (Fig. 5) ).

Boswellic acid
Boswellic acid (BA), obtained from Boswellia serrata, is a pentacyclic compound along with its various derivatives such as acetyl-11 keto-β-boswellic acid (AKBA), and 11-keto boswellic acid. The biological potential of BA and its various derivatives has been measured in the treatment of diseases like UC, asthma, bronchitis, laryngitis, cancer, inflammation, and pain (Iram et al. 2017;Anthoni et al. 2006;Ebrahimpour et al. 2017). In clinical evaluation, BA has been reported to reduce ulcer index, ulcer area in patients suffering from UC and was found to be well tolerated with minor gastrointestinal tract (GIT) disturbances (Algieri et al. 2015). Its anti-ulcer activity is attributed to the inhibition of pro-inflammatory enzymes such as COX-2, LOX-5, NF-ĸB, and leukotriene B4 (LTB4) (Ebrahimpour et al. 2017). Chande et al. reported that in patients with collagenous colitis, B. serrata extract was found to be effective in ameliorating disease process as compared to placebo (Chande et al. 2008). In another study, Catanzaro et al. evaluated the antiinflammatory activity of B. serrata extract (BSE) and AKBA in colonic epithelial cell monolayers exposed to hydrogen peroxide (H 2 O 2 ) or interferon (INF)-γ, TNF-α, an in vitro model of intestinal inflammation. Pre-treatment with BSE and AKBA significantly reduced functional and morphological alterations and the NF-κB phosphorylation induced by the inflammatory stimuli. Along with the same, BSE and AKBA also counteracted the increase of ROS caused by H 2 O 2 exposure, therefore protecting the intestinal epithelial barrier from inflammatory damage and supported its use as a safe adjuvant for UC patients (Catanzaro et al. 2015). Roy et al. aimed to investigate the anti-inflammatory potential of AKBA against DSS-induced colitis in Swiss albino mice. Reduction in soreness and histopathological studies revealed that the chemo-protective effect of AKBA was attributed to anti-proliferation, apoptosis, and anti-inflammation ( Fig. 6) (Roy et al. 2020).
Efficacy of Persimmon-derived tannin, i.e., condensed catechin, has been evaluated on a murine model of UC using DSS as ulcerogen by Kitabattake et al. The reduction in disease activity and inflammation through alteration of the microbiota composition and immune response established it as a promising candidate for UC therapy (Kitabatake et al. 2021).  Effects of the ethanol extract of ALF on DSS-induced colitis in mice. Mice were treated with 3.5% DSS in drinking water for 7 days followed by drinking water for 3 days. ALE (25, 50, 100 mg/ kg) were administrated orally once a day for 10 consecutive days. Mice were sacrificed at day 10. A DAI as the average score of body weight loss, stool consistency and rectal bleeding was scored from 0 to 4 (n = 7-8). B The colon length of each group at day 10 (n = 7-8).
C The levels of MPO in colon (n = 7-8). D Histological changes of colon, characterized by distinct infiltration of inflammatory cells (arrow) and crypt destruction (solid circle) (magnification × 200). E Histological scores of colon from each group (n = 6). All data are presented as mean ± SEM. ## P < 0.01 vs. normal group, *P < 0.05, **P < 0.01 vs. DSS group. ALE ethanol extract of ALF, MES mesalazine  Curcumin Curcumin (diferuloylmethane) is a primary natural polyphenol found in the rhizome of Curcuma longa L. (Zingiberaceae). It is used to manage oxidative and inflammatory conditions, metabolic syndrome, arthritis, anxiety, and hyperlipidemia (Aggarwal and Harikumar 2009;Jurenka 2009;Anand et al. 2008). Chandan et al. investigated the efficacy of curcumin in ameliorating 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Results demonstrated the improvement in both wasting and histopathological signs in murine experimental colitis (Chandan et al. 2020;Sugimoto et al. 2002). Its postulated mechanism of action is suppression of NF-κB mediated IL-1β/TNF-α, which makes it an effective treatment option for inflammatory disorders (Sugimoto et al. 2002). Toden et al. investigated the anti-inflammatory activity of essential turmeric oils (ETO-curcumin) in an animal model of DSS-induced colitis against standard curcumin. ETO-curcumin improved DAI dose-dependently, while the anti-inflammatory efficacy of standard curcumin remained constant, suggesting that ETO-curcumin may provide superior anti-inflammatory efficacy compared to standard curcumin. The up-regulation in gene expression of anti-inflammatory cytokines in the colon, i.e., IL-10, IL-11, and transcription factor of regulatory T-cells, i.e. Forkhead box P (FOXP)-3, further suggested that combined use of ETO and curcumin can afford better protection in UC ( Fig. 7) (Toden et al. 2017).

Glycyrrhizin
Glycyrrhiza glabra (Fabaceae), known as licorice, has been used to treat various ailments such as gastritis, bronchitis, ulcer, constipation, adrenal insufficiency, and allergy (Kim et al. 2006;Dogan and Ugulu 2013). Along with glycyrrhizin, it also contains other bioactive principles like glycyrrhizic acid, glycyrol, and sterol ( Yellow arrows represent the reduced infiltration of mixed inflammatory cells and well maintained tissue architecture (Roy et al. 2020) control group suggested its role as an anti-inflammatory agent. Further decrease in mediators of oxidative stress and inflammatory cytokines, down regulation of NF-ĸB, and up regulation of Nrf2 clarify its role in treating UC ( Fig. 8) (Liu et al. 2018). Glycyrrhizin acts by inhibiting nitric oxide (NO), NF-ĸB, IL-6, IL-1β, TNF-α, and suppressing PGE 2 level in lipopolysaccharide (LPS) stimulated macrophage (Kim et al. 2006;Dogan and Ugulu 2013).

Gymnemic acid
It is obtained from Gymnema sylvestre (GS) (Asclepiadaceae), also known as Gurmur, which is native to India and also found in tropical forests of Africa, Australia, and Indonesia (Arun et al. 2014;David and Sudarsanam 2013;Gurav et al. 2007). Therapeutically, Gymnemic acid (GA) and its derivatives have been used to treat various diseases like diabetes, infection, inflammation, and oxidative stress  9). The anti-ulcerative activity of GS was attributed to inhibition of TNF-α, SOD, CAT, GSH, IL-1β, IL-6, PGE, and NO (Aleisa et al. 2014).

Lonicerin
Lonicerin obtained from Lonicera japonica Thunb. known as Japanese honeysuckle has been used as antibacterial, antiinflammatory, antiviral, antiendotoxin, blood fat reducing, and antipyretic (Shang et al. 2011 Five specimens were selected randomly for histopathological study. Data are expressed as the means and individual histopathological scores, **P < 0.01 (Liu et al. 2018) natural killer (NK) cells and cytotoxic lymphocyte (CTL) activities were observed in DSS-treated mice. Improvement in the number of intestinal probiotics (Bifidobacterium and Lactobacilli) and decrease in number of the pathogenic bacteria (Escherichia coli and Enterococcus) has been observed with LJP-treated rats in a dose-dependent manner (Zhou et al. 2021). Lee et al. explored the effect of butanol extract of L. japonica in reducing the DSS-induced colitis and crypt injury in mice. The effectiveness of L. japonica in alleviating colitis was observed in dose dependent manner and was also found to be comparable with standard control i.e. 5-amino salicylic acid (5-ASA) as no distortion of crypt and cell infiltration was observed in treatment control group animals ( The protective effect of LJE against histological damage of colonic mucosal layer as relatively intact epithelium was observed as compared to DSS treated animals ( Fig. 11) (Park et al. 2013). Lv et al. verified the potency of lonicerin in UC as it disrupts the NLRP3-ASC-pro-caspase-1 complex assembly dose-dependently and therefore alleviates colitis. Therefore, lonicerin can be considered as a potent antiinflammatory epigenetic agent and a novel approach to treat UC (Lv et al. 2021).

Shagoal
Ginger, Zingiber officinale Roscoe (Zingiberaceae), is a natural dietary rhizome with various biological properties and activities. The health benefits of ginger are attributed to numerous biological components, including gingerols, gingerdiols, shogaols, paradols, and zingerones. Further, shogaol, a primary active ingredient of ginger, exists in various forms such as 4-, 6-, 8-, 10-, and 12-shogaol (Gupta et al. 2021). Guo et al. (2021) investigated the therapeutic action of ginger in DSS-induced UC in male BALB/c mice and reported that ginger alleviated colitis-associated pathological Fig. 9 Histopathological sections of colons from rats stained with H&E (× 400). Colonic microscopic image of A normal rat colon from Cont group with intact mucosal layer and epithelial; B acetic acid (AA) treated rat colon with diffused active colitis, extensive damage including edema in submucosa and chronic inflammatory cells infil-trate with widely ulcerating mucosa, and hemorrhages; C-E dose dependent reparative epithelial changes and ulcer healing with lymphoid follicle in colon of GS treated rats (50, 100 and 200 mg/kg, respectively); F attenuated cell damage with complete ulcer healing in MES treated group (Aleisa et al. 2014) changes and decreased the mRNA expression levels of IL-6 and iNOS. Zhang et al. (2017) explored the potency of orally administered siRNA-CD98/ginger-derived lipid vesicles (GDLVs) targeting specifically the colon tissues, which resulted in reduced expression of CD98 in colitis, thereby suggesting the use of these nanovesicles for UC. Hassan and Hassan explored the effect of shogoal in DSS-induced UC in BALB/c mice and compared their effect to that of an immunosuppressant drug, 6-thioguanine. The reduction in DAI and the histopathological score of shogoal treated rats demonstrated its beneficial role in treating UC. In Figs. 12 and 13, the efficacy of different concentration of shogoal was shown in proximal and distal parts of colon, respectively. The positive control group (DSS-exposed animals without treatment) showed focal epithelial ulceration with transmural infiltration of inflammatory cells whereas intact epithelial surface with normal epithelial cells infiltration was observed in negative control group animals. Shogoal showed protective effect in dose dependent manner with mild infiltration of inflammatory cells. Further, the histological index score of the proximal colon of mice was found to be maximum i.e. 5 for DSS-exposed animals without treatment and 0 for DSSexposed shogoal treated (40 mg/kg BW) animals (Fig. 12).
However, the histological index score of the distal colon of mice was found to be maximum 6 and minimum 1 for DSS-exposed animals without treatment and DSS-exposed shogoal treated (40 mg/kg BW) animals, respectively ( Fig. 13) (Hassan and Hassan 2018). It has been further documented that the antiulcerogenic activity of shagoal is due to the suppression of NF-kβ, TNF-α, and IL-1 β signalling pathway (Hsiang et al. 2013;Banerjee et al. 2011).

Miscellaneous phytoconstituents
Tannins obtained from rhatany root (Krameria triandra), wine grape seed (Vitis vinifera), and Scotch pine bark (Pinus sylvestris) have also been used effectively for UC. Their efficacy is directly related to the presence of proanthocyanidin. Higher the proanthocyanidin content; more is the ability to combat inflammation by inhibiting NF-κB p65 activity, decreased matrix metalloproteinase (MMP) production responsible for damage on GI mucosa (Clinton 2009). Cinnamon oil also has a role in preventing colonic damage in a dose-dependent manner and has a considerable effect on body weight gain recovery (Bujňáková et al. 2013).
Bruckner et al. studied the effect of polyphenol epigallocatechin-3-gallate (EGCG) of tea in DSS-induced colitis Treatment dose was 1 mg/kg, 10 mg/kg and 100 mg/kg, respectively, and 5-ASA was 100 mg/kg (Lee et al. 2011) mice. The reduced level of malondialdehyde (MDA) and MPO as well as enhanced expression of SOD, glutathione peroxidase (GPO), and pro-inflammatory cytokines have depicted its potential to treat UC (Brückner et al. 2012).
Liu et al. described the protective effect of a polysaccharide from Rheum tanguticum as an antiulcerogenic agent in TNBS-induced UC in rats. Significant inhibition of NF-Κβ, Th1/Th2 cytokine production was observed in a dose-dependent manner (Liu et al. 2003(Liu et al. , 2005(Liu et al. , 2008(Liu et al. , 2009. The safety and efficacy of ethanolic extract of Scorzonera alexandrina were evaluated in Wistar albino rats with AA-induced colitis. The findings demonstrated a significant reduction in inflammation and acute colonic damage due to the presence of luteolin and luteolin 7-O-glycoside. The results were associated with the ROS scavenging property of the plant (Akkol et al. 2012;Donia 2016). Witaicenis et al. (2012) explored the anti-inflammatory activity of 4-methylesculetin, a natural coumarin, obtained from Scopolia carniolica, in TNBS-induced colitis rat and a significant decline in the level of reduced IL-1β, TNF-α, and oxidative stress, confirmed its role as antiulcer agent. Typha angustifolia has been tested for its anti-inflammatory activity, and results have shown attenuation in GSH depletion and decrease in MPO, and alkaline phosphate (AP) activity because of which it can be used in UC (Fruet et al. 2012;Chen et al. 2017). The possible mechanism of AP involved in curing colitis may be attributed to its dephosphorylation of pro-inflammatory molecules such as LPS, flagellin and adenosine triphosphate, which are released from cells under stressed conditions during inflammation (Lukas et al. 2010;Bilski et al. 2017).
The role of cannabidiol (CBD), a non-psychotic component of Cannabis sativa, was investigated in a murine model of dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats by Borrelli et al., and the effect of CBD in the change of body weight and colon weight/colon length ratio was determined. Results indicated that treatment with CBD (1-10 mg/kg) significantly reduced the colonic damage associated with DNBS administration. No significant change in COX-2 expression was observed; however, over-expression of iNOS, nitrile production, IL-1β, and IL-10 was found to be declined up to a considerable extent (Borrelli et al. 2009).
Kotakadi et al. estimated the effect of Ginkgo biloba extract (EGB) in amelioration of inflammatory injury in TNBS-induced colitis in rats with different doses. The inflammatory response was assessed by histology and measurement of MPO, GSH, TNF-α, and IL-1β levels in the colon mucosa. It significantly decreased the colonic MPO activity, TNF-α, and IL-1β levels. The increased GSH concentration was observed; hence it can be used to treat UC Fig. 11 Effects of the water extract of Lonicera japonica (LJE) on histological findings and score in dextran sulphate sodium (DSS)induced colitis. The images a-e are the representative histological findings of all groups: a normal, b DSS, c DSS þ LJE 20 mg/kg, d DSS þ LJE 100 mg/kg and e DSS þ LJE 500 mg/kg groups, cryptal grand, surface of the epithelium;, neutrophils. The graph shows the histological scores. The score of the normal group is zero. Values are means (n = 8 for each group), with their standard errors represented by vertical bars. Mean values were significantly different compared with the DSS group: *P < 0·05, **P < 0·01 (Park et al. 2013) due to its scavenging activity (Kotakadi et al. 2008). The antiulcer activity of many herbal products has been summarized in Table 2.
Clinical data has depicted that patients with UC may have deficiency of many micronutrients such as vitamins and minerals due to loss of appetite, reduced absorption by the colon, and colonic diarrhea, and/or maybe due to different types of medication therapies. So, it is essential to overcome these deficiency states for which different kinds of nutraceuticals can be used. The role of probiotics in UC has been explored, and it has been documented that these agents can be used alone or in combination with other anti-ulcer agents.
Different probiotics such as Lactobacillus salivarius, Lactobacillus acidophilus, and Bifidobacterium bifidum along with mesalazine have been administered to UC patients for two years, and the response was evaluated according to the Modified Mayo Disease Activity Index. So, probiotics can help to avoid long-term use of corticosteroids in mild to moderate UC and can be used for induction of remission (Valdovinos et al. 2017;Palumbo et al. 2016;Mallon et al. 2006;Shigemori and Shimosato 2017;Hevia et al. 2015).
The role of Escherichia coli Nissle (E. coli/EcN) in patients suffering from UC has been demonstrated, and it has been reported that E.coli is effective and safe in maintaining remission in patients suffering from UC (Gallo et al. 2016;Fábrega et al. 2017;Scaldaferri et al. 2016).
A non-comparative clinical trial using a combination of probiotics (Bifidobacterium, lactobacillus, and streptococcus), commonly known as VSL#3, has been carried out, and down expression of toll like receptor (TLR) 2 and TLR4 was observed. Further, the intestinal epithelial up-regulation of protective IL-10 and down-regulation of IL-12, IL-17 and IL-23 has been achieved, which indicated its role in treatment of UC (Yao et al. 2017;Zhang et al. 2016).
The impact of polysaccharides from Chrysanthemum morifolium Ramat on the gut microbiota was assessed in ulcerative rats. Physiological investigations recommended that Chrysanthemum polysaccharides had quite defensive consequences for UC. It decreases the level of pro-inflammatory cytokines (such as IL-23, IL-6, TNF-α, and IFN-λ) and increases the level of anti-inflammatory mediators (like IL-4, IL-10, IL-11), thus re-establishing the state of eubiosis and restoring the immune system (Yao et al. 2017).
The role of B. subtilis has been evaluated in DSS-induced UC in mice, and its efficacy has been assessed by performing alcian blue staining, cytokine level by enzyme linked immunosorbent assay (ELISA), and microbiota composition. The effect is achieved by mucosal repairing and microbiota balance. Lactobacillus rhamnosus derived soluble protein acts by increasing mucus production in colonic epithelium. It causes thickening of mucus layer by modulating EGF factor Sun et al. 2016;Chapman et al. 2007).
Although many trials have been carried with phytoconstituents for UC (Table 3) but they were not able to establish much clinical efficacy due to a lack of data comparison with standard drugs. Hence, more studies need to be carried out to assess the role of natural compounds in UC. Along with the same, the safety profile of herbal products should also be done to determine toxic reactions and should be compared with conventional drugs. Moreover, the identification of active moieties in such products should be done to identify the new lead molecule.

Safety concerns of herbal products used in UC
Although, a large number of herbal products have been explored for treatment of UC but safety profile of herbs should be considered for being used in human beings. A double blind, randomized and placebo controlled study of AV gel was performed to evaluate its efficacy and safety in patients suffering from mild to moderate colitis. Forty four out patients were randomly chosen and AV gel or placebo treatment twice daily was given. The protective effect of AV gel was assessed by primary (clinical, sigmoidoscopic and histological remission) and secondary (colitis activity index, Baron score, histology score, C-reactive protein) outcomes. Adverse effects reported by patients were minor and were not directly correlated with consumption of AV gel as the side effects such as bloating, foot pain, sore throat, and acne were also reported by patients on placebo treatment, which advocated AV gel as safe for curing UC (Langmead et al. 2004).  (Hassan and Hassan 2018) In another study, the sub-acute, acute and genotoxicity of A. vera soft gelatin capsules (ASC) were estimated in ICR male and female rats. The acute toxicity study was estimated at a dose of 15,000 mg/kg body weight, whereas for subacute study, the blended dose in range of 832.5 to 3330 mg/ kg was used. No changes in body weight, behavior, biochemical, histopathalogical parameters and mortality were observed, which indicated that lethal dose of ASC is above 15,000 mg/kg. Genotoxicity of ASC was determined using Ames test (10,000 mg/kg) and no evidence of bone marrow micronucleus and testicular chromosome abnormality was found, hence can be considered safe on oral administration (Biancone et al. 2008).
In a recent study, the toxicity of AL fruit extract was determined in female Wistar rats using acute and repeated models. In acute toxicity study, the animals were administered two different doses i.e. 1000 and 5000 mg/kg, whereas for sub-acute toxicity study, 300 mg/kg dose was given for a period of 4 weeks. As, no mortality was observed in animals, hence can be considered as safe therapeutic option (Yaghoubi et al. 2019).
The safety profile of B. serrata extract and AKBA was demonstrated as no alteration in intestinal cell viability, barrier functions and integrity of biomarkers was observed; therefore, these can be used as a safe adjuvant for UC patients (Catanzaro et al. 2015). The double-blind placebo controlled randomized study was performed in 108 outpatients with CD and clinical remission and rate of relapse were determined after oral administration of B. serrata extract, Boswelan (3 × 2 capsule/day; 400 mg) for 52 days. The results indicated that B. serrata can be tolerated safely for treatment of IBD (Holtmeier et al. 2011).
No toxicity and mortality was observed in mice treated with different doses (700, 1400 and 2800 mg/kg) of C. CAT catalase, COX-2 cycloxegenase-2, GSH glutathione, IFN-γ interferon-gamma, IL interleukin, LOX-5 lipoxegenase-5, LTB 4 leukotriene B4, p38 MAPK P-38 mitogen-activated protein kinases, MCP-1 monocyte chemo attractant protein-1, MIP-2 macrophage inflammatory protein-2, MPO myeloperoxidase, NF-kβ nuclear factor-Kappa β, NO nitric oxide, PGE 2 prostaglandin E 2 , ROS reactive oxygen species, Th1 type-1 T helper, Th2 type-2 T helper, SOD superoxide dismutase, TNF-α tumor necrosis factor-alpha   sinensis extract, indicating safety profile of tea (Olayinka et al. 2018). Further, genotoxicity study of catechin was estimated by micronucleus and big blue transgenic rodent mutation assays in ICR mice after single or multiple oral administration of catechin preparation and lack of significant mutagenic and clastogenic concern confirmed its potential and safety in human beings (Ogura et al. 2008). Chandan et al. (2020) has shown promising effect of curcumin in TNBS induced UC and it was observed that curcumin at a dose of 0.75-7.5 g/kg/day did not cause any abnormality in mice. Acute and sub-acute toxicity of C. longa extract was evaluated at dose 30-240 mg/kg in Wistar rats using Organization for Economic Co-Operation and Development (OECD) 425 and 407 guidelines, respectively. No risk of toxicity was observed at any selected dose of curcumin, which suggested the safety of curcumin (Kamsu et al. 2019).
A large number of studies have indicated non-toxic nature of glycyrrhizin as it neither exhibit teratogenic nor mutagenic effects and its daily recommended dose can be up to 0.015-0.229 mg/kg of the body weight (Isbrucker and Burdock 2006).
The acute toxicity of homeopathic preparation of Gurmur (G. sylvestre) was determined in Sprague Dawley rats. No significant difference was observed in haematological, biochemical and histopathological parameters of placebo and Gurmur treated animals. Further, no mortality was observed in animals and can be recommended at a dose of 300 mg/kg body weight safely (Shukla et al. 2020).
Further, safety assessment of fermented Phylloporia ribis (Lonicera japonica Thunb) was performed in Sprague-Dawley rats and no adverse effects were observed in animals in both acute and sub-chronic toxicity study, indicating its potential in treatment of UC (Lu et al. 2014).
Another double blind, randomized, controlled study indicating the efficacy of ginger capsule in UC patients was performed. The down-regulation of inflammatory mediators and high sensitivity of C-reactive protein (hs-CRP) showed protective effect in treatment of UC and can be given up to 3 g/kg (Shayesteh et al. 2020).
Various reported studies have demonstrated that herbal products are an effective and safe option for treatment of UC.

Marketed herbal formulations for ulcerative colitis
A large number of herbal products such as Kutajghan vati, Vatsakadi churna, Arjuna capsule, and pitta balance capsule are mainly available in Indian market for the treatment of UC.
Arjuna capsule/tablet containing active constituent of Arjuna (Terminalia arjuna) is prescribed as two capsules of 500 mg each twice a day. It is manufactured by various Indian pharmaceutical companies like The Himalaya Drug Company (Himalaya 2021), Indian Herbo Pharma (Indian Herbopharma 2021), and Sona Health care (Sona Health Care 2021).

Concluding remarks and future perspectives
Although numerous conventional and non-conventional treatment options are available for UC, all of these suffer from various drawbacks such as safety, efficacy, and high cost. Usually, the therapy of UC requires treatment and maintenance of reemission for the entire life period, so these side effects assume much more significance. Herbal products are alternative medicines used to relieve UC with much milder side effects as compared to those associated with the present medicine system. According to a study conducted by WHO, 80-85% of the world population relies on plantderived products that offer much promise for the treatment of UC but still require further investigation in preclinical and clinical fields to prove their safety, efficacy, and usefulness.
Funding This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data availability Enquiries about data availability should be directed to the authors.

Declarations
Conflict of interest Authors confirm that there are no known conflicts of interest associated with this work.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.


Stemazole Promotes Oligodendrocyte Precursor Cell Survival In Vitro and Remyelination In Vivo

Maintaining the normal function of oligodendrocyte precursor cells (OPCs) and protecting OPCs from damage is the basis of myelin regeneration in multiple sclerosis (MS). In this paper, we investigated the effect of stemazole, a novel small molecule, on the promotion of oligodendrocyte precursor cell survival and remyelination. The results show that stemazole enhanced the survival rate and the number of clone formation in a dose-dependent manner and decreased the percentage of cell apoptosis. In particular, the number of cell clones was increased up to 6-fold (p < 0.001) in the stemazole group compared with the control group. In vivo, we assessed the effect of stemazole on recovering the motor dysfunction and demyelination induced by cuprizone (CPZ). The results show that stemazole promoted the recovery of motor dysfunction and the repair of myelin sheaths. Compared with the CPZ group, the stemazole group showed a 30.46% increase in the myelin area (p < 0.001), a 37.08% increase in MBP expression (p < 0.01), and a 1.66-fold increase in Olig2 expression (p < 0.001). Histologically, stemazole had a better effect than the positive control drugs. In conclusion, stemazole promoted OPC survival in vitro and remyelination in vivo, suggesting that this compound may be used as a therapeutic agent against demyelinating disease.

Introduction
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system that gives rise to focal lesions in the gray and white matter and diffuse neurodegeneration in the entire brain [1,2]. In 2020, 2.8 million individuals were living with MS worldwide [3]. MS is a lifelong, chronic and recurrent disease of the central nervous system that can lead to paralysis, blindness and even death without timely treatment [4].
The pathology of MS involves the reduction in nerve cells, the destruction of myelin sheaths and the disintegration of axons in the white and gray matter [4]. Remyelination is the process in which new myelin sheaths are restored to demyelinated axons, reinstating saltatory conduction and resolving functional deficits [5]. The formation of myelin originates from the proliferation, migration and differentiation of oligodendrocyte precursor cells (OPCs) [6,7]. The failure of remyelination is related to OPCs that do not function as they should [8,9]. Therefore, maintaining the normal function of cells and protecting them from injury is the basis of myelin regeneration [9,10].
As a complex disease, some hypotheses of MS pathogenesis, including primary neurodegenerative disease and primary inflammatory disease, have been proposed. According to the different pathogenesis hypotheses, treatment strategies of neuroprotection and myelin regeneration therapy and immunotherapy have been proposed [11,12]. Currently, based on immunotherapy, many disease-modifying therapy (DMT) drugs have been developed. Most of these drugs only relieve symptoms and reduce the recurrence of the disease by modulating the immune response and reducing inflammation but fail to address Int. J. Mol. Sci. 2022, 23, 10756 2 of 12 the desired long-term outcome of remyelination and the recovery of neurological function. Due to the limitations of immunotherapy, MS therapy targeting neuroprotection and myelin regeneration has attracted increasing attention. The core of neuroprotection and myelin regeneration therapy is to improve nerve injury and restore myelin regeneration [1]. Dimethyl fumarate (DMF,  is an approved drug with neuroprotective effects [13,14]. It can reduce the recurrence rate and prolong the progression of disability [14,15]. Thus, DMF was used as a positive control drug to compare the neuroprotective effect of stemazole on multiple sclerosis. Based on different hypotheses, different animal model studies have focused on experimental autoimmune encephalomyelitis (EAE) and CPZ models. The EAE model is mainly applied to screen drugs related to the immune system [16]. The CPZ model is a toxin-induced model that simulates the process of myelin damage and myelin regeneration [17] which is suitable for screening drugs with neuroprotective and remyelination functions [18]. Based on the PubMed database, we searched for relevant studies of demyelination animal models published in the last 20 years. The results show that the number of studies related to the EAE model tended to level off as of 2013, however, the number of CPZ model studies has shown a sharp increase since 2013. The increasing number of CPZ model studies indicates the increasing importance of neuroprotection and myelin regeneration therapies. In addition, the increasing number suggests that this is a suitable animal model for simulating behavioral and histological myelin injuries.
Stemazole (C 9 H 9 N 5 OS 2 , chemical name: N-(4-(4,5-dihydro-5-thioxo-1,3,4-oxadiazol-2yl)phenyl) hydrazine carbothioamide) is a novel small molecule that has neuroprotective pharmacological activity [19][20][21][22][23] and promotes cell survival [23,24]. Stemazole can promote the survival of stem cells by preventing apoptosis under nutrient deficiency or injury conditions [23]. Additionally, stemazole can significantly improve memory impairment in a rat model of AD [20] and has a therapeutic effect on motor nerve damage in a PD mouse model [21]. Stemazole has revealed new pharmacological activity in preventing and treating neurodegenerative diseases by promoting the repair potential of stem cells. Due to the pharmacological activity of stemazole, we hypothesized that it may promote myelin regeneration, improve nerve injury and restore nerve function in the treatment of MS [21,22]. In this study, we investigated whether: (i) stemazole could promote OPC survival and (ii) stemazole could sufficiently improve mouse motor dysfunction and remyelination in a CPZ-mediated demyelination animal model.

Stemazole Promotes Oligodendrocyte Precursor Cell Viability
To investigate the ability of stemazole to promote cell survival and viability, cells were examined after 4 days by a cell viability assay. To explore whether stemazole could affect OPC survival, we measured the viability of OPCs cultured with different concentrations of stemazole (1-30 µM) (Figure 1). Compared to OPCs cultured without stemazole, the viability of OPCs was significantly enhanced by stemazole: at concentrations of 3, 5, 10, 15, 20, 25, and 30 µM, the cell viability increases were 10.37%, 11.63%, 12.37%, 23.78%, 18%, 18.03%, and 12.56%, respectively. The results indicate that stemazole can increase the viability of OPCs in a dose-dependent manner.

Stemazole Enhances the Survival of Disaggregated Oligodendrocyte Precursor Cells
Colony formation experiments reflect the survival rate of cell inoculation, indicating the number of adherent cells that survive and form clones after inoculation. To further investigate the effect of stemazole on OPCs, we inoculated disaggregated OPCs in a medium with different concentrations of stemazole for 4 days. The Giemsa staining results showed that stemazole increased the number of OPCs in a dose-dependent manner (Figure 2A,B). In contrast, most OPCs did not survive in disaggregated cell cultures without stemazole. Specifically, compared with cells cultured without stemazole, the colony formation rate was considerably increased after treatment with stemazole (1, 3, 5, 10, and 30 μM), achieving approximately 226%, 375%, 503%, 746%, and 966% improvement, respectively. A significant increase in OPCs was observed when the dose of stemazole was 3 μM (p < 0.05). Moreover, the colony formation ability was increased by approximately 6-fold upon treatment with 30 μM stemazole compared with untreated OPCs (p < 0.001). Taken together, when cultured with stemazole, the individual OPCs were more likely to be adherent and clonal. These results indicate that stemazole notably promotes the survival of disaggregated OPCs in a dose-dependent manner.

Stemazole Enhances the Survival of Disaggregated Oligodendrocyte Precursor Cells
Colony formation experiments reflect the survival rate of cell inoculation, indicating the number of adherent cells that survive and form clones after inoculation. To further investigate the effect of stemazole on OPCs, we inoculated disaggregated OPCs in a medium with different concentrations of stemazole for 4 days. The Giemsa staining results showed that stemazole increased the number of OPCs in a dose-dependent manner (Figure 2A,B). In contrast, most OPCs did not survive in disaggregated cell cultures without stemazole. Specifically, compared with cells cultured without stemazole, the colony formation rate was considerably increased after treatment with stemazole (1, 3, 5, 10, and 30 µM), achieving approximately 226%, 375%, 503%, 746%, and 966% improvement, respectively. A significant increase in OPCs was observed when the dose of stemazole was 3 µM (p < 0.05). Moreover, the colony formation ability was increased by approximately 6-fold upon treatment with 30 µM stemazole compared with untreated OPCs (p < 0.001). Taken together, when cultured with stemazole, the individual OPCs were more likely to be adherent and clonal. These results indicate that stemazole notably promotes the survival of disaggregated OPCs in a dose-dependent manner.

Stemazole Enhances the Survival of Disaggregated Oligodendrocyte Precursor Cells
Colony formation experiments reflect the survival rate of cell inoculation, indicating the number of adherent cells that survive and form clones after inoculation. To further investigate the effect of stemazole on OPCs, we inoculated disaggregated OPCs in a medium with different concentrations of stemazole for 4 days. The Giemsa staining results showed that stemazole increased the number of OPCs in a dose-dependent manner (Figure 2A,B). In contrast, most OPCs did not survive in disaggregated cell cultures without stemazole. Specifically, compared with cells cultured without stemazole, the colony formation rate was considerably increased after treatment with stemazole (1, 3, 5, 10, and 30 μM), achieving approximately 226%, 375%, 503%, 746%, and 966% improvement, respectively. A significant increase in OPCs was observed when the dose of stemazole was 3 μM (p < 0.05). Moreover, the colony formation ability was increased by approximately 6-fold upon treatment with 30 μM stemazole compared with untreated OPCs (p < 0.001). Taken together, when cultured with stemazole, the individual OPCs were more likely to be adherent and clonal. These results indicate that stemazole notably promotes the survival of disaggregated OPCs in a dose-dependent manner.

Stemazole Prevents Apoptosis of Oligodendrocyte Precursor Cells
Apoptotic cells were analyzed by flow cytometry after annexin V and propidium iodide staining. As shown in Figure 3, the apoptosis distribution results show that the percentages of early and late apoptosis were 12.89% in the control group, 7.69% in the 3 µM stemazole group, 7.98% in the 10 µM stemazole group and 8.2% in the 30 µM stemazole group. Different concentrations of stemazole (3, 10, and 30 µM) reduced the proportion of early and late apoptotic cells. Therefore, 3 µM stemazole decreased the percentage of cell apoptosis by 5.2% and served as the optimal concentration. The above results suggest that the proportion of apoptotic cells reached 12.89% in the culture condition of 10% serum, which caused slight damage to the cells. The addition of the small molecule stemazole conferred resistance to apoptosis in disaggregated OPCs.

Stemazole Prevents Apoptosis of Oligodendrocyte Precursor Cells
Apoptotic cells were analyzed by flow cytometry after annexin V and propidium iodide staining. As shown in Figure 3, the apoptosis distribution results show that the percentages of early and late apoptosis were 12.89% in the control group, 7.69% in the 3 μM stemazole group, 7.98% in the 10 μM stemazole group and 8.2% in the 30 μM stemazole group. Different concentrations of stemazole (3, 10, and 30 μM) reduced the proportion of early and late apoptotic cells. Therefore, 3 μM stemazole decreased the percentage of cell apoptosis by 5.2% and served as the optimal concentration. The above results suggest that the proportion of apoptotic cells reached 12.89% in the culture condition of 10% serum, which caused slight damage to the cells. The addition of the small molecule stemazole conferred resistance to apoptosis in disaggregated OPCs.

Stemazole Improves Motor Dysfunction and Neurological Impairment In Vivo
Given that MS patients have the pathological characteristics of demyelination and the clinical symptoms of paralysis and cognitive neurological disorders, we chose the CPZ model to simulate the clinical symptoms and demyelination process of the patients. Moreover, to investigate the therapeutic effect of stemazole on the demyelination model, we studied the recovery of motor coordination and memory impairment in behavioral tests and remyelination degree in histological tests on CPZ-mediated demyelination animals.

Stemazole Improves Motor Coordination
The rotarod test was used to determine the movement coordination ability of the experimental animals. Compared with the control group, a significant decrease in the CPZ group was noted over time, achieving an approximately 130 s decline (p < 0.001). Compared with the CPZ group, the mice treated with stemazole at doses of 3, 10 and 30 mg/kg had longer persistence times, achieving approximately 72.63 s, 77.25 s and 80.13 s improvements, respectively. Compared with the CPZ group, the endurance time of the DMF group increased by 74.13 s, however, this result was not statistically significant ( Figure  4A). After the induction of CPZ, the mice in the CPZ model group could hardly persist on the rotarod. Compared with the CPZ group, either 10 mg/kg or 30 mg/kg stemazole significantly promoted motor coordination and balance ability. However, 30 mg/kg DMF did not enhance the movement coordination ability. These results show that stemazole significantly recovered the movement coordination ability of demyelination animals in the CPZ model, which had a better effect than the positive drug.

Stemazole Improves Motor Dysfunction and Neurological Impairment In Vivo
Given that MS patients have the pathological characteristics of demyelination and the clinical symptoms of paralysis and cognitive neurological disorders, we chose the CPZ model to simulate the clinical symptoms and demyelination process of the patients. Moreover, to investigate the therapeutic effect of stemazole on the demyelination model, we studied the recovery of motor coordination and memory impairment in behavioral tests and remyelination degree in histological tests on CPZ-mediated demyelination animals.

Stemazole Improves Motor Coordination
The rotarod test was used to determine the movement coordination ability of the experimental animals. Compared with the control group, a significant decrease in the CPZ group was noted over time, achieving an approximately 130 s decline (p < 0.001). Compared with the CPZ group, the mice treated with stemazole at doses of 3, 10 and 30 mg/kg had longer persistence times, achieving approximately 72.63 s, 77.25 s and 80.13 s improvements, respectively. Compared with the CPZ group, the endurance time of the DMF group increased by 74.13 s, however, this result was not statistically significant ( Figure 4A). After the induction of CPZ, the mice in the CPZ model group could hardly persist on the rotarod. Compared with the CPZ group, either 10 mg/kg or 30 mg/kg stemazole significantly promoted motor coordination and balance ability. However, 30 mg/kg DMF did not enhance the movement coordination ability. These results show that stemazole significantly recovered the movement coordination ability of demyelination animals in the CPZ model, which had a better effect than the positive drug. , and Swimming tracks (D) were recorded via a video tracking camera system. Data represent the means ± SD (n = 10 duplicates). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the CPZ group. ### p < 0.001 vs. the control group.

Stemazole Improves Memory Impairment
The Morris water maze (MWM) was used to test the learning and memory abilities of the experimental animals. To measure the learning and memory ability of mice, the time that the mice spent finding the platform was recorded. According to the results, the mice treated with 10 and 30 mg/kg stemazole exhibited a significantly shorter time to find the platform than the CPZ group, with reductions of 68.25 s and 64.75 s, respectively. Mice in the DMF group had the best recovery effect, reaching a healthy level by extending the time of searching for the platform by 84.63 s. Compared with the CPZ group, the stemazole and DMF treatments both reduced the time required by mice to find the platform ( Figure 4B).
To evaluate the ability of the mice to retain their memory of the platform's spatial location after learning to find it, the platform was removed. Then, the paths the mice swam were observed ( Figure 4D). The CPZ group circled the edge, and the other five groups repeatedly passed by the platform. Compared with the control group, the stay time of the mice in the CPZ group was significantly reduced by 25% ( Figure 4C The aforementioned statistics demonstrate that CPZ intoxication reduced the animal capacity of spatial learning, memory and spatial impairment. After treatment with stemazole and DMF, the above symptoms were significantly ameliorated.

Stemazole Promotes Remyelination
The most typical pathological histological feature of MS is myelin loss. The assessments of myelin, MBP proteins and OL-specific transcription factor (Olig2) [25,26] are key indicators for the identification of remyelination. Therefore, we used histology, immunochemistry and immunofluorescence staining to comprehensively evaluate remyelination.
We investigated the myelin status in the corpus callosum region using Luxol Fast Blue staining. The data indicated a significant loss of myelin in the CPZ group, with a 56.02% decrease compared with the control group. Compared with the CPZ group, the

Stemazole Improves Memory Impairment
The Morris water maze (MWM) was used to test the learning and memory abilities of the experimental animals. To measure the learning and memory ability of mice, the time that the mice spent finding the platform was recorded. According to the results, the mice treated with 10 and 30 mg/kg stemazole exhibited a significantly shorter time to find the platform than the CPZ group, with reductions of 68.25 s and 64.75 s, respectively. Mice in the DMF group had the best recovery effect, reaching a healthy level by extending the time of searching for the platform by 84.63 s. Compared with the CPZ group, the stemazole and DMF treatments both reduced the time required by mice to find the platform ( Figure 4B).
To evaluate the ability of the mice to retain their memory of the platform's spatial location after learning to find it, the platform was removed. Then, the paths the mice swam were observed ( Figure 4D). The CPZ group circled the edge, and the other five groups repeatedly passed by the platform. Compared with the control group, the stay time of the mice in the CPZ group was significantly reduced by 25% ( Figure 4C The aforementioned statistics demonstrate that CPZ intoxication reduced the animal capacity of spatial learning, memory and spatial impairment. After treatment with stemazole and DMF, the above symptoms were significantly ameliorated.

Stemazole Promotes Remyelination
The most typical pathological histological feature of MS is myelin loss. The assessments of myelin, MBP proteins and OL-specific transcription factor (Olig2) [25,26] are key indicators for the identification of remyelination. Therefore, we used histology, immunochemistry and immunofluorescence staining to comprehensively evaluate remyelination.
We investigated the myelin status in the corpus callosum region using Luxol Fast Blue staining. The data indicated a significant loss of myelin in the CPZ group, with a 56.02% decrease compared with the control group. Compared with the CPZ group, the myelin area of the mice treated with 10 and 30 mg/kg stemazole was increased by 19.85% and 30.46%, respectively. Compared with the CPZ group, the myelin area of the mice treated with DMF was increased by 20.22%. Above all, compared with the control group, approximately 50% damage was induced by CPZ. After CPZ removal, stemazole substantially accelerated the remyelination process in a dose-dependent manner ( Figure 5). Stemazole (30 mg/kg) significantly promoted remyelination in the corpus callosum, which had better effects than the same dose of the positive control drug. myelin area of the mice treated with 10 and 30 mg/kg stemazole was increased by 19.85% and 30.46%, respectively. Compared with the CPZ group, the myelin area of the mice treated with DMF was increased by 20.22%. Above all, compared with the control group, approximately 50% damage was induced by CPZ. After CPZ removal, stemazole substantially accelerated the remyelination process in a dose-dependent manner ( Figure 5). Stemazole (30 mg/kg) significantly promoted remyelination in the corpus callosum, which had better effects than the same dose of the positive control drug. We further studied the degree of remyelination in the corpus callosum after 2 weeks of stemazole treatment using the immunohistochemical staining of the myelin marker MBP ( Figure 6). The data indicate a 29.9% increase in MBP expression in the CPZ group compared with the control group; treatment with 3, 10 and 30 mg/kg stemazole promoted remyelination with improvements of 15.45%, 37.08% and 29.35%, respectively, in comparison with the CPZ group. The area of myelin in the DMF group increased the expression of MBP up to 16.28%. The data indicate that CPZ could decrease the expression of MBP up to 70.1% compared with the healthy level. Treatment with 10 mg/kg and 30 mg/kg stemazole clearly promoted MBP expression. Compared with 30 mg/kg stemazole, the same dose of the positive control drug did not show a significant effect on promoting myelin regeneration. These results show that stemazole could significantly promote myelin regeneration and had a better effect than the positive control drug. We further studied the degree of remyelination in the corpus callosum after 2 weeks of stemazole treatment using the immunohistochemical staining of the myelin marker MBP ( Figure 6). The data indicate a 29.9% increase in MBP expression in the CPZ group compared with the control group; treatment with 3, 10 and 30 mg/kg stemazole promoted remyelination with improvements of 15.45%, 37.08% and 29.35%, respectively, in comparison with the CPZ group. The area of myelin in the DMF group increased the expression of MBP up to 16.28%. The data indicate that CPZ could decrease the expression of MBP up to 70.1% compared with the healthy level. Treatment with 10 mg/kg and 30 mg/kg stemazole clearly promoted MBP expression. Compared with 30 mg/kg stemazole, the same dose of the positive control drug did not show a significant effect on promoting myelin regeneration. These results show that stemazole could significantly promote myelin regeneration and had a better effect than the positive control drug.
We also evaluated the Olig2 expression using immunofluorescence staining (Figure 7). The results indicate a significant reduction in Olig2 expression in the CPZ group compared to the control group (42.94 vs. 29.91, p < 0.05). The group treated with stemazole (3, 10 and 30 mg/kg) showed an improvement in Olig2 expression, achieving approximately 1.66-, 1.66-and 1.57-fold increases, respectively. A significant increase in the DMF group (1.33 times, p < 0.05) was also observed. In addition, the group treated with stemazole (3, 10 and 30 mg/kg) showed an improvement in Olig2 cells/mm 2 , achieving approximately 3.09-, 3.19-and 3.64-fold increases. The results show that treatment with stemazole increased the expression of oligodendrocytes (OLs) more effectively than the positive control drug. We also evaluated the Olig2 expression using immunofluorescence staining ( Figure  7). The results indicate a significant reduction in Olig2 expression in the CPZ group compared to the control group (42.94 vs. 29.91, p < 0.05). The group treated with stemazole (3, 10 and 30 mg/kg) showed an improvement in Olig2 expression, achieving approximately 1.66-, 1.66-and 1.57-fold increases, respectively. A significant increase in the DMF group (1.33 times, p < 0.05) was also observed. In addition, the group treated with stemazole (3, 10 and 30 mg/kg) showed an improvement in Olig2 cells/mm 2 , achieving approximately 3.09-, 3.19-and 3.64-fold increases. The results show that treatment with stemazole increased the expression of oligodendrocytes (OLs) more effectively than the positive control drug. Taken together, stemazole recovered the learning ability and movement coordination ability of demyelination animals. In addition, stemazole increased the expression of OLs and promoted remyelination by histology. Moreover, stemazole performed significantly better than positive control drugs in promoting the recovery of myelin.

Discussion
Stemazole is a novel small molecule previously found to have neuroprotective effects The olig2 cells/mm 2 were compared with that of the control and expressed as a percentage of the control value using ImageJ software. Data represent the means ± SD (n = 3 duplicates). * p < 0.05, ** p < 0.01 *** p < 0.001 vs. the CPZ group. # p < 0.05, ### p < 0.001 vs. control group.
Taken together, stemazole recovered the learning ability and movement coordination ability of demyelination animals. In addition, stemazole increased the expression of OLs and promoted remyelination by histology. Moreover, stemazole performed significantly better than positive control drugs in promoting the recovery of myelin.

Discussion
Stemazole is a novel small molecule previously found to have neuroprotective effects against neurodegeneration [20]. In our research, we studied the effect of stemazole on promoting OPC survival in vitro and remyelination in vivo to investigate its therapeutic potential for multiple sclerosis. The results show that stemazole promotes the survival, attachment and apoptosis resistance of OPCs. Stemazole enhanced the cell viability, formation number, and colony size in a dose-dependent manner. In addition, 10 and 30 mg/kg stemazole promoted the recovery of motor dysfunction and remyelination in the CPZinduced demyelination model, which performed better than DMF. This finding indicates that stemazole promotes oligodendrocyte precursor cell survival, protects the cells from damage in vitro and improves motor dysfunction and neurological impairment in vivo.
Neuroprotection and myelin regeneration are therapeutic strategies that are widely studied in the treatment of multiple sclerosis. OPCs undergo cell death upon disaggregation into single cells or upon starvation, precluding differentiation. According to the in vitro results, OPCs caused slight injury and apoptosis in 10% serum culture. Stemazole significantly enhanced cell viability, increased the number of colonies formed in a dosedependent manner and antagonized the proportion of apoptotic cells. In our previous study, the results indicated that stemazole could antagonize cell apoptosis by reducing the caspase 2 protein, which is well known to be involved in apoptosis [27]. Stemazole could also prevent cell apoptosis due to disaggregation or starvation without compromising self-renewal, proliferation and pluripotency [23]. For this reason, we believe that stemazole can promote cell survival and maintain the normal function of cells.
CPZ is a copper chelating agent commonly used as a neurotoxicant to induce demyelination and neurodegeneration [28]. The CPZ model simulates the process of demyelination and remyelination and serves as a suitable model for neuroprotection and myelin regeneration therapy. In the past 20 years, CPZ model studies have been widely conducted to develop neuroprotective therapies. In this study, we selected a CPZ-induced model to study whether stemazole can promote remyelination and restore nerve function. According to the in vitro results, stemazole significantly prolonged the persistence time on the rotarod, reduced the time to find the platform and increased the time in the quadrant of the original platform in behavioral studies, suggesting that stemazole effectively improved animal motor balance and the learning and memory ability of demyelinated mice. Histologically, stemazole promoted myelin area, MBP content and Olig2 expression in demyelinated mice. Compared with the CPZ group, stemazole increased the myelin area by 30.46% (p < 0.001), MBP expression by 37.08% (p < 0.01) and Olig2 expression by 1.6-fold (p < 0.001). However, compared with the stemazole group, the DMF group did not show a better promoting effect on myelin area, Olig2 expression or MBP content. Stemazole both promoted OPC survival in vitro and protected myelin from CPZ-induced toxicity in vivo. Stemazole has revealed pharmacological activity in preventing and treating neurodegenerative diseases by promoting the repair potential of cells. The neuroprotective effects of stemazole may be important for its therapeutic efficacy.
DMF is an approved drug that relieves neuroinflammation and oxidative stress damage to reduce nerve injury [15]. Because DMF has neuroprotective pharmacological activity, the drug was selected as a positive control. According to the behavioral studies, DMF almost restored neurological function to a healthy level. According to histological studies, DMF could partially reverse the damage to the myelin sheath. The recovery effect of DMF is still unable to reach a healthy level and is slightly inferior to the effect of stemazole. We hypothesize that DMF could restore neurological function, which may be related to its ability to alleviate neuroinflammation and antioxidative damage caused by CPZ. However, DMF does not promote the regeneration of myelin; thus, it cannot restore myelin to a healthy level. Although DMT drugs, such as DMF, modulate the immune process to alleviate disease recurrence, the effect of promoting myelin regeneration is not sufficient. Thus, more work on drug screening is urgently needed.
A limitation of this study was that it only studied the pharmacodynamics of stemazole, and its mode of action needs to be explored.

Chemicals and Reagents
Stemazole was synthesized by the Key Laboratory of Radiopharmaceuticals. The purity of stemazole was determined to be >98% by high-performance liquid chromatography (HPLC), and the structure of stemazole was confirmed by high-resolution mass spectrometry and nuclear magnetic resonance. CPZ, Giemsa, and Luxol Fast Blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). The rabbit anti-MBP antibody (Cat No. 10458-1-AP), rabbit anti-Olig2 antibody (Cat No. 13999-1-AP), immunohistochemistry kit and immunofluorescence kit were purchased from Proteintech (Wuhan, China).

OPC Culture
OPCs were purchased from Otwobiotech (Shenzhen, China). The purity of this cell line was determined to be >90%. The cells were maintained in 90% DMEM-HG supplemented with 10% FBS at 37 • C in a 5% CO 2 -humidified atmosphere.

Cell Viability Assay
The cells were seeded at 3000 cells per well in a flat-bottomed 96-well plate and exposed to 1, 3, 5, 10, 15, 20, 25 or 30 µM stemazole dissolved in PBS for 4 days. Cell viability was quantitatively analyzed using the Cell Titer-Glo™ luminescent cell viability assay, which is based on the luciferase/luciferin reaction. After mixing and incubating for 10 min at ambient temperature, the luminescence value was measured at 595 nm with a Multilabel Counter (PE, 1420, VICTOR3™).

Clone Formation Assay
To assess clone formation, 1000 single cells were seeded into a flat-bottomed 6-well plate and exposed to 1, 3, 5, 10 or 30 µM stemazole for 4 days. The day on which OPCs were seeded was defined as Day 0. The number of colonies formed was measured by Giemsa staining.

Annexin V-PE Apoptosis Detection Assay
For annexin V staining and fluorescence-activated cell sorting, dissociated cells were seeded at 3 × 10 5 cells in a 60 mm dish and treated with 3, 10, or 30 µM stemazole for 4 days. The cells were then collected, resuspended, centrifuged for 5 min, the supernatant was discarded, and the cells were gently resuspended by adding 195 µL Annexin V-PE binding solution. Then, 5 µL Annexin V-PE was added. The samples were incubated for 10-20 min at room temperature in the dark. Flow cytometry was performed immediately, and Annexin V-PE showed red fluorescence. The day on which OPCs were seeded was defined as Day 0.

Animals and Treatment Procedures
Female C57BL/6 mice (8 weeks) were fed 0.2% (w/w) cuprizone mixed with ground standard rodent chow. The CPZ diet was maintained for 5 weeks, after which the CPZ-infused feed was removed, and the animals were given standard chow. The mice were administered a daily intraperitoneal injection of stemazole dissolved in normal saline (3, 10 and 30 mg/kg) and a daily intragastric injection of DMF (30 mg/kg) after CPZ withdrawal. The treatment lasted for 2 weeks. Experimental procedures involving animals were approved by the Ethics Committee of Beijing Normal University (BNU/EC/01/2011) (Figure 8).
standard rodent chow. The CPZ diet was maintained for 5 weeks, after which the CPZinfused feed was removed, and the animals were given standard chow. The mice were administered a daily intraperitoneal injection of stemazole dissolved in normal saline (3, 10 and 30 mg/kg) and a daily intragastric injection of DMF (30 mg/kg) after CPZ withdrawal. The treatment lasted for 2 weeks. Experimental procedures involving animals were approved by the Ethics Committee of Beijing Normal University (BNU/EC/01/2011) ( Figure 8).  Motor function was assessed using the rotarod test. Before the formal experiment, the mice (n = 10) were trained to adapt to the rotating cylinder environment operating at 20 rpm for 3 consecutive days by DXP-3 (Chinese Academy of Medical Sciences, Beijing, China). The movement was maintained for 300 s. During the formal test periods, the time spent on the rotating bar until the mice fell from the cylinder was recorded.

Morris Water Maze
Spatial learning and memory were tested using the Morris water maze behavior test. The water maze device (Chinese Academy of Medical Sciences, China) consists of a circular pool divided into four quadrants, and a translucent platform below the water surface was hidden in the center of the quadrant. In the navigation task, the time required for the mice to find the underwater platform within a 120 s period was recorded. In the exploration experiment, the platform was removed, and the time spent by the animal in the target quadrant (the quadrant where the platform was originally placed) and the number of times it entered the quadrant within 60 s were recorded. 4.5. Histology, Immunochemistry and Immunofluorescence Staining 4.5.1. Luxol Fast Blue (LFB) Staining LFB was performed to evaluate the degree of demyelination. The sections (n = 5) were immersed in 0.01% LFB solution and incubated at 60 • C for 2 h. The sections were differentiated in lithium carbonate solution for 20 s followed by 70% ethyl alcohol for 20 s. The sections were dehydrated in graded alcohols. Then, the samples were dehydrated, cleared and mounted.

Immunochemistry Staining
Immunochemistry staining was performed to evaluate MBP expression. The sections (n = 3) were fixed in 4% paraformaldehyde, incubated with rabbit anti-MBP (1:200) antibody at 4 • C overnight and stained with an immunohistochemistry kit following the manufacturers' instructions.

Immunofluorescence Staining
Immunofluorescence staining was performed to evaluate the expression of the transcription factor Olig2. The sections (n = 3) were fixed in 4% paraformaldehyde, incubated with rabbit anti-Olig2 (1:200) antibody at 4 • C overnight and stained with an immunofluorescence kit following the manufacturers' instructions.

Data Analysis
In our experiments, the data were analyzed using GraphPad Prism 5. Data are presented as the means ± SD. Differences between groups were analyzed using one-way ANOVA followed by Dunnett's post hoc test. Student's t test was used to compare two sets of data. A value of p < 0.05 was regarded as significant.

Conclusions
In conclusion, our experimental data show that stemazole promotes OPC survival in vitro, improves behavioral motor dysfunction and reduces the degree of demyelination in vivo. These results indicate that stemazole may be used as a potential therapeutic agent against MS.