Effects And Mechanisms Of Three Nature Compounds Of Traditional Chinese Medicine Inhibiting Janus Kinase On Experimental Colitis In Mice

Background: Inflammatory bowel disease(IBD)is an idiopathic intestinal inflammatory disease involving ileum,rectum and colon.It is characterized by aggressiveness,destructiveness,refractory and recurrent attacks,including two common types of ulcerative colitis(UC)and Crohn’s disease(CD).The incidence rate of ulcerative colitis is about three times that of Crohn’s disease.The clinical manifestations of inflammatory bowel disease are diarrhea,abdominal pain,bloody stool and weight loss.The incidence rate of inflammatory bowel disease has increased year by year,which brings about heavy financial worries and heavy pressure among the patients and their families.In Europe and the United States,0.5% of the population suffers from inflammatory bowel disease.It has also spread in newly industrialized countries such as Asia,South America and the Middle East.Thus it has developed into a global disease.At present,it is dissatisfactory for the treatment methods and effects of inflammatory bowel disease.(1)surgical treatment: affecting the quality of life,only limited lesions are suitable for surgical treatment.(2)Traditional drugs treatment:The main drugs of treating IBD incude 5-aminosalicylate,sulfasalazine,steroid hormones,immunosuppressants,etc.,the overall effective rate is 30% to 40%,with great toxic and side effects.(3)Biological agent treatment: By December 2021,eight biological agents have been approved to cure ulcerative colitis and Crohn’s disease,including anti-TNF monoclonal antibodies(infliximab,adalimumab,golimumab,certolizumab pegol),anti-integrin monoclonal antibodies(natalizumab,vedolizumab)and anti-interleukin 12 and 23 monoclonal antibodies(ustekinumab,ustekinumab).Overall,these biological agents were effective in one third of patients,tolerated in one third and ineffective in one third,with side effects.(4)Other treatment methods are not mature: probiotics,vitamins,fish oil,acupuncture,psychotherapy,sports,flagellates,fecal bacteria transplantation,stem cell therapy,etc.The pathology mechanism of inflammatory bowel disease has been considered to be a combination of genetic and environmental factors,in which the mechanisms of immunity,infection,flora imbalance,intestinal barrier dysfunction and endoplasmic reticulum stress have been discovered.Cytokines,a class of biological activities and produced by immune cells and non immune cells,have a variety of biological activities..Janus kinase(JAK)receptor complex can bind more than 50 different cytokines and play an important role in innate and adaptive immunity.Cytokines bind to cell surface receptors,making the intracellular fragments of receptors activate JAK protein and start the phosphorylation of its downstream STAT dimer.Then the Phosphorylated STAT dimer enters the nucleus,affecting the transcription of target genes.The genetic variation and dysfunction of JAK–STAT signaling pathway are closely related to the pathogenesis of autoimmune diseases(such as inflammatory bowel disease,severe combined immunodeficiency,multiple sclerosis,rheumatoid arthritis,and psoriasis etc.).Therefore,JAK inhibitors have become a promising means for the treatment of autoimmune diseases such as inflammatory bowel disease,and have also shown good therapeutic effects in recent five years.As a small molecule targeted drug,JAK inhibitors have similar efficacy and safety to biological agents.They can be taken orally and are easy to preserve.They are far better patient compliance than biological agents,but they are also very expensive and have certain side effects.Traditional Chinese medicine is the treasure of the Chinese nation.The background information of traditional Chinese medicine is very rich and well documented.For example,Zhang Tingdong and his team found that arsenic,a traditional Chinese medicine,plays a significant role in the treatment of acute promyelocytic leukemia.At present,arsenic combined with all trans retinoic acid has become the global standard drug for the treatment of acute promyelocytic leukemia.Tu Youyou and her team extracted artemisinin from Artemisia annua by low-temperature extraction.The combination therapy based on artemisinin drugs is the most effective and important means to treat malaria at present.JAK inhibitor in nature compounds of Traditional Chinese Medicine is not only cheap,but also easy to apply,which has great research and application values.We found a strong binding between arbutin,puerarin,Ophiopogonin D and JAK protein by database screening and molecular docking.Furthermore,it deserves further study whether they can inhibit JAK/STAT signaling pathway to alleviate inflammatory bowel disease,and what their exact mechanisms are.Experiment 1: Arbutin inhibiting JAK alleviates experimental colitis in DSS miceObjective: To investigate the role of arbutin in experimental colitis in mice and its mechanism based on JAK / STAT signal pathway.Methods:(1)in vivo experiment: Mice were free to drink water containing 2.5%dextran sodium sulfate(DSS)to make an experimental colitis model.At the same time,mice were gavaged with arbutin(50mg/kg,100mg/kg)every day for 7 days.The general indicators such as body weight,food intake and disease index were collected.After 7days,the mice were killed,the weight and length of spleen and colon were measured,and the tissues and serum were collected.Hematoxylin/eosin(H&E)staining,immunohistochemistry and light microscope were used to observe the histopathological damage.Western blot analysis(WB)and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression levels of inflammatory cytokines and key proteins.(2)In vitro experiment: Rat intestinal epithelial cell line IEC6 and mouse monocyte macrophage line raw246.7 were used In vitro culture.The cell inflammatory response model was made with bacterial lipopolysaccharide(LPS).The inhibitory effect of arbutin on inflammation and the protective effect on intestinal epithelial function were detected by Western blotting,immunofluorescence and flow cytometry,and its mechanism was explored based on JAK / STAT signaling pathway.Results: Arbutin alleviated the weight loss,colon length reduction,intestinal pathological damage,and inhibited the overexpression of inflammatory cytokines such as TNF-α,IL-1β,IL-6,i NOS and COX2 in colitis mice.Arbutin decreased the expression level of myosin light chain kinase(MLCK),an index of intestinal mucosal permeability,and reversed the abnormal expression of Bcl-2,an index of intestinal epithelial apoptosis.Arbutin inhibited the phosphorylation of JAK2 in vivo and in vitro.Conclusion: Arbutin has a protective effect on experimental colitis in mice.Arbutin can inhibit JAK-STAT signaling pathway by inhibiting JAK2 phosphorylation.Experiment 2: Puerarin ameliorates 5-fluorouracil-induced intestinal mucositis in mice via JAK / STAT signaling pathwayObjective: To investigate the effect of puerarin on experimental colitis in mice based on JAK / STAT signal pathway.Methods:(1)in vivo experiment: Mice were intraperitoneally injected5-fluorouracil(5-FU)(75mg/kg)to make a experimental colitis model,with puerarin(50mg/kg,100mg/kg)intraperitoneal injection for 7 days.The general indexes such as body weight,food intake and disease index were collected.After 7 days,the animals were killed to measure the weight and length of spleen and colon.The tissues and serum were collected for hematoxylin/eosin(H&E)staining and immunohistochemistry.The histopathological damage was observed by optical microscope.Western blot analysis(WB)and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression levels of inflammatory cytokines and key proteins.(2)In vitro experiment:Rat intestinal epithelial cell line IEC6 and human colon adenocarcinoma Caco-2 cells were cultured in vitro,and the cellular inflammatory response model was made with bacterial lipopolysaccharide(LPS).The inhibitory effect of puerarin on inflammation and the protective effect on intestinal epithelial function were detected by Western blot,immunofluorescence and flow cytometry,whose mechanisms were explored on JAK /STAT signal pathway.Results: Puerarin alleviated the weight loss and intestinal pathological damage,and inhibited overexpression of inflammatory cytokines such as TNF-α,IL-1β,IL-6,i NOS and COX2 in colitis mice caused by 5-FU.It also reversed the increase of intestinal mucosal permeability index myosin light chain kinase(MLCK)and the increase of intestinal epithelial cell apoptosis index Bax / Bcl-2 ratio.Both in vivo and in vitro experiments puerarin inhibited the phosphorylation of JAK2.Conclusion: Puerarin has a protective effect on experimental colitis in mice by inhibiting JAK2 phosphorylation to regulate JAK-STAT signaling pathway.Experiment 3: Ophiopogonin D inhibiting epithelial NF-κB signaling pathway protects against experimental colitis in miceObjective: To investigate the effect of Ophiopogonin D on experimental colitis in mice via NF-κB signaling pathway.Methods:(1)in vivo experiment:Mice were free to drink water containing 2.5%dextran sodium sulfate(DSS)to make an experimental colitis model.At the same time,mice were gavaged with Ophiopogonin D(10mg/kg,40mg/kg)once a day for 7days.The general indexes such as body weight and food intake were collected.After 7days,the mice were killed,and the tissues and serum were collected for hematoxylin /eosin(H&E)staining,immunohistochemistry and optical microscope to observe the histopathological damage.Western blot analysis(WB)and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression levels of cytokines and key proteins.(2)In vitro experiment: Rat intestinal epithelial cell line IEC6 and mouse monocyte macrophage line raw246.7 were used to verify the therapeutic effect of Ophiopogonin D on intestinal inflammation and the protective effect of intestinal epithelium in vitro,based on NF-κB signaling pathway.Results: Ophiopogonin D alleviated the symptoms of colitis in mice,and reversed the abnormal changes of inflammatory cytokines,intestinal epithelial cell apoptosis indexes Bax,Bcl-2,CL-caspase3,intestinal mucosal barrier function indexes MLCK and occludin.In vivo and in vitro it was confirmed that Ophiopogonin D played a role to alleviate cilitis by inhibiting NF-κB signaling pathway.Conclusion: Ophiopogonin D can effectively alleviate experimental colitis in mice,and its effect may be related to the inhibition of NF-κB signaling pathway.