| Ulcerative colitis(UC)is a kind of chronic non-specific inflammatory bowel disease with unclear pathogenesis,characterized by a long course and easy recurrence.Modern medical research believes that UC is caused by the interaction of environment,heredity,body immunity and many other factors.Clinical symptoms including abdominal pain,diarrhea,pus and blood stool.In recent years,the incidence of the disease is on the rise,seriously affecting the lives of patients.Currently,some drugs used in the clinical treatment of UC are salicylic acid and glucocorticoid hormone drugs,but there are side effects such as allergy.Compared with western medicine,traditional Chinese medicine has some advantages in the treatment and prevention of UC.In recent years,many bioactivities of traditional Chinese medical herbs had been validated.Schisandra A is deemed a major component of lignin components in Schisandra chinensis(Turcz.)Baill.Recent studies have reported that Schisandra A has many beneficial pharmacological activities including anti-inflammatory,anti-oxidation,hepatoprotective effect,and anti-tumor.However,whether Schisandra A has the potential biological activity to improve the symptoms of UC was not been elucidated.Therefore,the study on these mechanisms may contribute to the development of novel and effective treatments for UC.We used two different methods respectively to establish animal model of UC,namely,TNBS /ethanol solution enema inducing UC mice and 5%(W/V)DSS oral solutions induced UC rats.By detecting the expression of protein and gene related to NF-κB/COX-2 pathway and IL-6/STAT3 pathway,the therapeutic effects and related mechanisms of Schisandra A would be clarified preliminarily.Chapter one: Effects of Schisandra A on gastrointestinal motility in rats and micePurpose: The effects of Schisandra A on gastrointestinal motility in different contractile states were studied in vitro and in vivo.Methods:1.The effects of Schisandra A on gastrointestinal motility in healthy mice,diarrhea mice,and hyperfunctional mice induced by neostigmine had been determined,including the effects on intestinal propulsion rate,gastric residue rate,diarrhea index,motilin,gastrin.2.By adding receptor agonist and antagonist into Kreb’s solution or changing the concentration of Ca2+,K+,or Na+,different contraction states of the isolated smooth muscle had been establisbed,and then,the effect of Schisandra A on the contractility of intestinal smooth muscle in rats had been researched.Results:1.Schisandra A could inhibit gastrointestinal motility in mice Schisandra A showed inhibitory effects on intestinal propulsion rate of normal mice,hyperfunctional mice induced by neostigmine,and diarrhea mice induced by folium sennae.It can significantly reduce the diarrhea rate and diarrhea index of diarrhea mice,significantly increase the incubation time of diarrhea,and reduce the serum motilin and gastrin of diarrhea mice.2.Schisandra A could inhibit the contraction of isolated intestinal smooth muscle in rats The results showed that Schisandra A inhibited the contractility of smooth muscle under different contractility states.Conclusion:Schisandra A has obvious inhibitory effect on gastrointestinal motility in mice,has certain antidiarrheal effect,and regulatory effects of the release of gastrointestinal hormones,in addition,Schisandra A can inhibit the isolated smooth muscle contraction.Chapter two: Effects of Schisandra A on TNBS induced UC micePurpose: Using TNBS/ethanol methods to establish UC mouse model,the effects of Schisandra A on UC mice and related mechanism have been researched.Methods:1.Animal grouping,model preparation and drug administration Mice were randomly divided into 6 groups,including blank control group(Normal group) and Model control group(Model group),Schisandra A high,medium and low dose groups(TNBS+80 mg/kg group,TNBS+40 mg/kg group,TNBS+20 mg/kg group),and positive control group(TNBS+SASP group),with 10 mice in each group.5% TNBS was dissolved in anhydrous ethanol to make 2.5% TNBS/50% ethanol solution,which was slowly injected into the colon of mice through the anus,and repeated after 24 hours.Next,corresponding treatments started for 14 consecutive days.2.Effects of Schisandra A on general symptoms and pathological damage in UC mice The effects of general manifestations,defecation rate,fecal moisture content,degree of occlusive blood,DAI,colonic transit time,spleen index,colonic length and quality in mice were observed,and histopathological damage was also evaluated by HE staining.3.Effects of Schisandra A on NF-κB/ COX-2 pathway in colon of UC mice(1)The activity or content of MPO,SOD,MDA,NO and TNOS in colon tissues of mice were detected by Biochemical Kit.(2)The contents of IL-6、IL-10、NF-κB、COX-2、and TNF-α were detected by ELISA.(3)The expressions of NF-κB and COX-2 protein were determined by immunohistochemistry.(4)Western blot analysis of NF-κB and COX-2 protein expression in colon tissues were observed.(5)The expressions of NF-κB and COX-2 m RNA were detected by PCR.Results:1.Schisandra A could alleviate the clinical symptoms of UC mice The results showed that the mice in the Normal group were in good mental states,and the clinical symptoms of each group were similar to those of UC after the model was established.After the administration of Schisandra A and SASP,the mice of TNBS+80mg/kg group and TNBS+80mg/kg group and TNBS+SASP group had gained weight,defecation frequency,defecation rate,fecal moisture content,colon transit time,degree of occlusion and DAI score were significantly improved(P < 0.05).The TNBS+80 mg/kg group and TNBS+SASP group alleviated colonic shortening,and the colonic mass and spleen index tended to be normal(P < 0.05).HE staining showed that the colonic mucosal injury was significantly reduced in the TNBS+80 mg/kg group and the TNBS+SASP group,and the damage score was significantly lower than that in the Model group(P < 0.05).2.Schisandra A could inhibit the level of inflammation-related factors The results showed that compared with Model group,TNBS+80mg/kg group and TNBS+SASP group could reduce MPO activity in colon tissues(P < 0.05),increase SOD activity and decrease MDA activity(P < 0.05),and meanwhile decrease NO content and TNOS activity in colon tissues(P < 0.05).The levels of IL-6、NF-κB、COX-2、TNF-α were also decreased(P < 0.05),while the levels of IL-10 were increased(P < 0.05).3.Schisandra A inhibited the expression of NF-κB、COX-2 proteins Immunohistochemical and western blot results showed that the expressions of COX-2and NF-κB proteins in colon tissues of the TNBS+80 mg/kg group was significantly lower than those of the Model group(P<0.05),indicating that Schisandra A could down-regulate the expressions of NF-κB and COX-2 proteins.4.Schisandra A inhibited the expression of NF-κB and COX-2 m RNA RT-PCR results showed that,the expressions of NF-κB and COX-2 m RNA in colon tissues of the TNBS+80 mg/kg group and the TNBS+SASP group were significantly decreased compared with the Model group(P < 0.05).Indicating that Schisandra A could inhibit NF-κB and COX-2 genes.Conclusion:Schisandra A can alleviate clinical symptoms and inhibit the development of inflammation in TNBS/ethanol-induced UC mice.The therapeutic mechanism might be related to inhibiting the activation of NF-κB/COX-2 signaling pathway.Chapter three:Effect of Schisandra A on DSS induced UC in ratsPurpose:The UC model rats were induced by drinking 5%(W/V)DSS,and then,the therapeutic effect of Schisandra A on UC rats and related mechanism has been researched.Methods:1.Animal grouping,model preparation and drug administration Rats were randomly divided into 6 groups,including normal control group(Normal group)and Model control group(Model group),Schisandra A high,medium and low dose groups(DSS+ 60 mg/kg group,DSS+30 mg/kg group,DSS+15 mg/kg group),and positive control group(DSS+SASP group),8 rats in each group.UC model rats were established by drinking 5% DSS(W/V)for 10 days.Treatment with corresponding drugs continued for 14 days.2.Effects of Schisandra A on general clinical symptoms and pathological damage in UC rats General conditions of rats,DAI,colonic transport time,fecal moisture content,organ index,and colonic appearance were observed and recorded.Morphological changes were evaluated by HE staining.3.Effect of Schisandra A on IL-6/STAT3 pathway in colon of UC rats(1)The MPO activity in colon tissues and serum of rats was detected by biochemical method.(2)The contents of IL-6 and IL-13 in colonic tissue of rats were detected by ELISA.(3)Immunohistochemical detection of IL-6,STAT3 and p-STAT3 protein expression in colonic tissues of rats.(4)The expression of IL-6 and STAT3 m RNA in colonic tissues of rats was detected by PCR.Results:1.Effects of Schisandra Aon general symptoms and pathological damage in UC rats During the modeling period,the rats in the Normal group were in normal mental states,and most of them began to have loose stools after drinking 5 % DSS(W/V)solution on the4 th day,and their body weight began to decrease,and the DAI score of each group significantly increased(P<0.05).Normal group rats were in good condition during administration.Model group rats continued to lose weight,with symptoms similar to clinical UC.After 14 days,the DSS+60mg/kg group and DSS+SASP group returned nearly to normal state,weight gain was obviously ameliorated,and DAI score was significantly reduced(P<0.05).After 14 days of administration,compared with the Model group,the colonic transport time of rats in treated groups except the DSS+15mg/kg increased to varying degrees(P<0.05),and the fecal moisture content decreased significantly(P <0.05).The thymus index,spleen index and colon index of DSS+60mg/kg group and DSS+SASP group all tended to be normal.Compared with the Model group,the colonic tissue gross score decreased significantly,and the difference was statistically significant(P <0.05).The results of HE staining showed that,the colonic mucosa structure of the normal group was intact and the arrangement of glands was complete.In the Model group,a large number of lymphocytes and neutrophils were observed,with unclear mucosal structures and ulcerations in some animals’ colons.In the DSS+60mg/kg group and the DSS+SASP group,the structure of each layer of the colon tissue was clear,the number of inflammatory cell infiltration was decreased,the glands were clearly structured and arranged,and the tissue damage score was significantly lower than that of the Model group(P <0.05).2.Schisandra A inhibited the expression of IL-6 and STAT3 The results of ELISA showed that,compared with the Model group,except the DSS+15mg/kg group,the colonic tissue IL-6 content was significantly decreased and the IL-13 content was significantly increased in all the groups(P <0.05).Immunohistochemical results showed that,compared with the Model group,DSS+SASP group and DSS+60mg/kg group could significantly reduce the positive expression of IL-6 and STAT3 protein and inhibit the activation of the corresponding p-STAT3 protein,with a significant difference(P <0.05).3.Schisandra A inhibited the expression of IL-6 and STAT3 m RNA PCR results showed that,compared with the Normal group,the expressions of IL-6 and STAT3 m RNA in colon tissues of the Model group were significantly increased(P <0.05),while the expressions of DSS+60mg/kg group and DSS+SASP group were significantly decreased(P <0.05).Conclusion:Schisandra A can alleviate the clinical symptoms of UC rats induced by DSS and achieve the therapeutic purpose,possibly by inhibiting the expression of proteins and m RNA related with IL-6 /STAT3 pathway. |
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