Study On The Pharmacodynamic Mechanism Of Wuji Wan In The Treatment Of Inflammatory Bowel Disease Based On Ion Channel/PPARγ

Background and objectiveInflammatory bowel disease(IBD)is a chronic,non-specific inflammatory disease of the bowel,including ulcerative colitis(UC)and Crohn’s disease(CD),Clinical patients often appear abdominal pain,diarrhea,blood in the stool and other symptoms,if not effectively controlled,may appear digestive tract bleeding,intestinal obstruction,perforation and even cancer.The disease has a long course,is prone to relapse,and the pathogenesis is not clear.Although the commonly used drugs such as 5-aminosalicylic acid and glucocorticoid have good curative effect,long-term administration has more adverse reactions.In fact,PPAR y receptor is highly expressed in gastrointestinal colon epithelium,macrophages,and lymphocytes.These results suggest that PPAR γ may be a potential therapeutic target for IBD.At the same time,the imbalance of T cell subsets will produce the corresponding immune response,promoting the development of IBD disease course.In addition,different cell types in the intestinal epithelium,such as intestinal cells,cluster cells and goblet cells,sense internal or external stimuli through potassium ion channels and calcium ion channels and maintain the integrity of the intestinal epithelium.Ion channels also play a key role in synchronous signaling between different cellular components that coordinate immune responses in the gastrointestinal tract.Only when ion channels are functioning properly can ions and organic materials function properly in the gut.However,patients with IBD suffer from abnormal gastrointestinal motility,obstruction of ion channel activity,abdominal distension,diarrhea and other symptoms due to nervous and inflammatory sensitivity and gastrointestinal mucosal barrier dysfunction,suggesting that the study on ion channel activity is also a potential treatment strategy for IBD.Wuji Wan is used to treat epigastric burning pain,vomiting and acid swallowing,abdominal pain and diarrhea caused by liver fire invading the stomach and disharmony between the liver and stomach.Studies have shown that Coptis has good effects on hyperlipidemia,hypertension,diarrhea,inflammation and other aspects.Paeoniae alba can play a role by antibacterial,vasodilator,analgesic,anti-inflammatory,immune regulation,etc.Fructus officinalis and its main components have many pharmacological effects,such as anti-gastric ulcer,anti-inflammation,analgesic and anti-diarrhea.Our research group has been working on the compatibility mechanism of Wuji Wan.Previous studies have shown that Wuji Wan has a good therapeutic effect on postinfectious irritable syndrome(PI-IBS)and diarrheal irritable bowel syndrome.It can improve viscera hypersensitivity and gastrointestinal motility.The clinical symptoms of PI-IBS(diarrhea with abdominal distension and increased fecal mucus)are highly similar to those of IBD.IBS-like symptoms exist in some patients with IBD(especially CD).Currently,these clinical signs may fully meet the diagnostic criteria for IBS,leading to delayed diagnosis of IBD.The above-mentioned action characteristics of Wuji Wan has potential advantages in relieving clinical symptoms of IBD,controlling inflammatory processes and enhancing patients’ immunity.However,there is no relevant research on whether Wuji Wan has a pharmacodynamic effect on IBD.After the complex compatibility of TCM components is absorbed into the body,many "prototype components/metabolites" mixed pharmacodynamic substance groups will be generated,and the body may also produce endogenous active/interfering substances.Therefore,the overall pharmacodynamic of TCM compounds must be able to fully or well reflect the overall effects of these mixed pharmacodynamic/endogenous substance groups,so as to objectively evaluate the overall pharmacodynamic of the compound.Therefore,the study on the overall pharmacodynamic substance group in Wuji Wan can better reflect the objective pharmacodynamic effect of Wuji Wan.At present,there are few researches on pharmacodynamic substances in Wuji Wan.Therefore,the purpose of this study was to explore the pharmacodynamic effect of Wuji Wan on IBD induced by 2,4,6-Trinitrobenzenesulfonic acid(TNBS).On the one hand,starting from the perspective of PPARγ,the mechanism of action of Wuji Wan on IBD was preliminarily explored at the cellular and molecular levels.On the other hand,the multi-angle therapeutic effect of Wuji Wan on IBD was thoroughly understood from the perspective of ion channels,so as to enrich the understanding of the efficacy of Wuji Wan,and to complete the efficacy evaluation and pharmacological research on the treatment of IBD by Wuji Wan.Finally,the liquid-mass combination technology was used to further explain the material basis of the drug effect of Wuji Wan,so as to provide laboratory evidence support for increasing the indication of Wuji Wan for the treatment of IBD,further in-depth pharmacological mechanism and the material basis of the drug effect in vivo,and at the same time provide new methods and new ideas for the research and treatment of IBD.Methods and contents1.The content of 5 components in Wuji Wan was determined by HPLCHPLC was used to determine the content of main components of commercially available Wuji Wan,and to evaluate the content of components of commercially available Wuji Wan,so as to ensure the quality of experimental drugs.2.Efficacy evaluation of Wuji Wan on rats with IBD(1)The rat model of IBD induced by TNBS-ethanol enema was established.24 h later,the patients were treated with Chinese herbal compound(Wuji Wan),IBD clinical drugs such as 5-aminosalicylate(sulfasalazine)and glucocorticoid(prednisolone)for 8 consecutive days.The normal group and TNBS model group were given the same amount of drinking water every day.Indexes of animal disease activity(body weight,fecal morphology,and fecal occult blood)were recorded on a daily basis.(2)The overall colonic injury of rats was observed and scored;Observed the pathological injury and inflammatory infiltration of colon tissue,and calculated the ratio of colon weight to length.3.Pharmacological mechanism exploration of Wuji Wan on IBD based on "ion channel and PPARγ"(1)Serum detection:The serum levels of TNF-α,IL-1β,IL-10 and MPO were determined by enzyme-linked immunosorbent assay.(2)Determination of colon tissues:The relative contents of Foxp3,RORγt and Tbet mRNA in colon tissues were determined by RT-PCR.The protein expression levels of PPARγ,NF-κB p65,Th1,Th17 and the transcription factors T-bet,RORγt and Foxp3 in Treg cells were determined by WB.(3)Ion channel measurement:In order to reflect the pharmacodynamic effect of the drug in vivo,plasma ultrafiltrate of Wuji Wan was used as the test drug,and the influence of ion channel high-throughput detector ICR8000 on the potassium ion plasma channel activity of IEC-6 cells was measured.4.Based on UPLC-Q-TOF-MS/MS technology to explore the pharmacodynamic material basis of Wuji WanUPLC-Q-TOF-MS/MS technology was used to analyze and identify the extracts of Wuji Wan,rat drug-containing plasma and plasma ultrafiltrate,further elucidate the possible material basis of Wuji Wan in the treatment of IBD,and speculate the target and signal pathway of Wuji Wan in the treatment of IBD through network pharmacology.To provide reference for the comprehensive interpretation of the pharmacodynamic material basis of Wuji Wan and in vivo study of traditional Chinese medicine compound.Results1.The content of 5 components in Wuji Wan was determined by HPLCTo establish a reasonable HPLC analysis method for Wuji Wan:using 0.1%phosphoric acid-acetonitrile,gradient elution,paeoniflorin,berberine,palmatine,Evodiamine and Rutaecarpine in Wuji Wan were better isolated,and ensured the quality of tested drugs.2.Efficacy evaluation of Wuji Wan on rats with inflammatory bowel disease(IBD)TNBS-ethanol enema-induced IBD model was established successfully.After modeling,the rats showed weight loss,blood in stool or occult blood in stool,and loose and unformed feces,indicating successful modeling.During continuous administration for 8 days,the disease activity index of rats decreased significantly,and the hematochezia and occult blood of rats were better relieved.Wuji Wan can reduce colon injury;It can improve the colonic length-to-weight ratio of IBD rats.Decreased spleen index;Reduce inflammatory infiltration.Compared with the other two traditional drugs,it has better efficacy in improving body weight.3.Pharmacological mechanism of Wuji Wan on IBD rats based on "ion channel and PPARγ"(1)Cytokines:The contents of pro-inflammatory factor(TNF-α,IL-1β)and antiinflammatory factor(IL-10)in serum of rats in each group were detected.It was found that the levels of TNF-α,IL-1β,were decreased and the levels of IL-10 were increased in Wuji Wan group,sulfamazine group and prednisolone acetate group,and the inflammatory damage was alleviated.Reduce serum MPO content.(2)Molecular level:In rat colon tissues,mRNA levels of Foxp3,RORγt and Tbet were increased due to immune activation.Compared with the control group,Wuji Wan group can increase the protein expressions of PPARγ and Foxp3,and decrease the protein expressions of NF-κB p65,RORγt and T-bet in the colon tissue of rats.The effect of Wuji Wan on the reduction of NF-κB p65 and the enhancement of FOXP3 was stronger than that of sulfanazine and prednisolone acetate.(3)Ion channels:The blood ultrafiltrate of Wuji Wan affected the K channel activity of IEC-6 cells at different time periods,and could enhance the K channel activity in the first 4h,suggesting that Berberine and plasma ultrafiltrate of Wuji Wan could enhance the K+channel activity of IEC-6 cells,promote the internal flow of K+ions,inhibit the efflux of potassium ions,and Improve diarrhea caused by IBD.4.Based on UPLC-Q-TOF-MS/MS and network pharmacology,the pharmacodynamic material basis of Wuji Wan was discussedUPLC-Q-TOF-MS/MS technique was used to analyze and identify Wuji Wan,drug-containing plasma and ultrafiltrate of plasma in rats.Results 26 identical compounds were identified in Wuji Wan preparation and rat drug-containing plasma,and 15 identical compounds were identified in rat drug-containing plasma and plasma ultrafiltrate.Based on these compounds,46 potential targets were obtained by network pharmacological method.It is concluded that Wuji Wan can act on PTGS,NCOA2,RXRA and other targets through magnolianine,berberine,etc.,and play a pharmacodynamic role by regulating calcium ion,VEGF and other signaling pathway.ConclusionThrough this research,we draw the following conclusions:1.The content of 5 components in Wuji Wan was determined by HPLC to ensure the quality of the experimental drug,which provided a reference for the quality control of Wuji Wan.2.In the overall efficacy study,the rat model of IBD was successfully established,and Wuji Wan can improve the weight and blood in stool of IBD rats,reduce DAI score,improve the weight and length of colon,reduce inflammatory infiltration of colon tissue,and reduce spleen index.3.Study on pharmacodynamic mechanism of PPARγ:By increasing the expression of PPARγ or enhancing its activity level,Wuji Wan can reduce the expression of inflammatory factors,enhance the expression of anti-inflammatory factors,inhibit the phosphorylation of NF-κB,and maintain the physiological function of colon tissue in IBD rats.Moreover,it regulates T cell differentiation,promotes the expression of related factors in Treg cells,reduces Th1-mediated inflammatory response,and plays a protective role in intestinal tract.4.Ion channel study:Plasma ultrafiltrate obtained from metabolism of Wuji Wan can affect potassium channel activity of IEC-6 cells at different time periods,suggesting that Wuji Wan can inhibit potassium efflux and improve colonic motion.5.Based on UPLC-Q-TOF-MS/MS technology,the methanol extract of Wuji Wan,plasma samples and plasma ultrafiltration samples of rats after administration were detected and analyzed.39 compounds were identified in Wuji Wan preparation and 40 in medicated plasma,among which 26 contained the same compounds.Plasma ultrafiltrate analysis identified a total of 15 components that were the same as medicated plasma,and explored the material basis of the effect of Wuji Wan.In this paper,a new HPLC method was established to determine the content of five components in Wuji Wan,so as to provide reference for quality control and ensure the quality of Wuji Wan.It is suggested that Wuji Wan has therapeutic effect on IBD disease,which may promote the differentiation of T cells and inhibit inflammatory factors by influencing PPARγ.In addition,regulating the activity of related ion channels and exerting pharmacological effects provide a new mechanism for the pharmacodynamic treatment of Wuji Wan on IBD.The liquid-mass combined administration was used to further analyze the components of Wuji Wan in vivo to provide support for the study on the efficacy of Wuji Wan in vivo and the overall efficacy of TCM compounds.Network pharmacology was used to explore the potential treatment possibilities of Wuji Wan,which provided experimental basis for increasing the indications of Wuji Wan in the treatment of IBD,expanded the efficacy of Wuji Wan in the treatment of inflammatory bowel disease,and also provided new methods and new ideas for the drug development and treatment of IBD.It also has suggestive significance for the treatment of digestive diseases with colonic inflammation injury as common pathological feature.