Effect Of Weichang’an Pill On Immune Balance Of Ulcerative Colitis Model Mice

BackgroundUlcerative colitis(UC),as a chronic persistent intestinal inflammatory disease,has high recurrence.Most of the patients’ lesions are limited to the mucosa and submucosa,which can involve the rectum and sigmoid colon,causing diarrhea,mucus abscess and bloody stool and other symptoms,resulting in great pain.At present,the pathogenesis of UC in not completely clear,and the immune pathogenic factors have been widely recognized.Most UC patients show varying degrees of immune balance disorder.Regulatory action is the main function of Treg and Th17 cells.Starting from the overall concept,TCM has systematic and unique views on the cause of disease,pathogenesis and diagnosis and treatment of UC.Through syndrome differentiation and treatment,it can alleviate and so that the patient’s symptoms can be effectively controlled.Relieved the quality of life and reduce the recurrence of UC,which shows a good prospect.Whether experimental research or clinical research,TCM is an effective means of UC.The previous experimental research of the team also showed that the experimental effect of Weichang’ an Pill on UC model mice was significant,but the regulatory effect Weichang’ an Pill on Treg、Th17 cells and their cytokines and transcription factors is not known.ObjectiveBy observing the general condition,DAI score,colonic tissue morphology,HE staining results of colonic tissue,T cell subsets Treg and Th17 levels of mesenteric lymph nodes,serum cell anti-inflammatory factors(TGF-β1、IL-10)and proinflammatory factors(IL-1β、IL-6、IL-17)levels,colonic tissue transcription factors(p-NF-κBp65、stat3、p-stat3、RORyt、FOXP3)protein content,to explore the mechanism of Weichang’ an Pill on UC model mice.MethodsThere were 32 c57b1/6 mice divided into normal group、model group、mesalazine group and Weichang’ an Pill group.The UC model was induced by 2.5%DSS aqueous solution and administered by gavage for 10 days.Observe their general condition of mice and calculate DAI scores;The colonic tissues of mice in the four groups were observed,and to observe the effect of Weichang’ an Pill on colonic histomorphology of model mice;the expression levels of Treg and Th17 in mesenteric.Lymph nodes tests were performed on each of the groups and compared by flow cytometry;the expression level of serum cytokine TGF-β1、IL-10、IL-1β、IL-6、IL-17 in each group was detected and compared by ELISA;the protein contents of p-NF-κBp65、stat3 and p-stat3 in the colon of mice in each group were detected by Western Blot;the content of RORγt and FOXP3 protein in colon tissue of mice in each group was detected by fluorescence quantitative PCR.To investigate the therapeutic effect of Weichang’ an Pill on UC model mice and the mechanism of immune balance.Results(1)General condition and DAI score of mice:After DSS induction,except for the normal group,the body weight、mental state、diet and activity of mice decreased compared with those before modeling.The hair of mice is sparse and dull.Mice had increased defecation volume、thin stool、bloody stool and other symptoms.The symptoms gradually worsened during modeling.After stopping modeling and giving intragastric administration,the original symptoms have improved to a certain extent.The weight gain of mice in mesalazine group and Weichang’ an Pill group was lower than the normal group,higher than the control group(P<0.05).The difference of DAI scores in mesalazine group and Weichang’ an Pill group before and after intervention was significantly higher(P<0.05).(2)Colon morphology was observed by HE staining:Under light microscope,the normal group had complete arrangement and structure of colonic mucosal epithelium and glands,clear recess structure,no atrophy and deformation,and no inflammatory cell infiltration.In the model group,the epithelial structure of colonic mucosa was incomplete,the crypts were atrophic and deformed,which contain more inflammatory cells.The pathological morphology of mesalazine group and Weichang’ an Pill group compared with the control group,the drug group better indicators.(3)The expression levels of Treg and Th17 morphological changes were observed in the samples from each group,using a test called Flow cytometry:Compared with the control group,the results of the other groups showed that Treg/Th17 accounted for a relatively high percentage of cells with a higher percentage(P<0.05),percentage of Th17 cells decreased(P<0.05).There was no statistical significant in comparison between mesalazine group and Weichang’ an Pill group(P>0.05).(4)The levels of serum anti-inflammatory factors(TGF-β1、IL-10)and proinflammatory factors(IL-1β、IL-6、IL-17)of mice in each group were detected by ELISA:Compared with the model group,the level of serum TGF-β1 in mesalazine group and Weichang’ an Pill group increased(P<0.05),serum IL-10 increased in mesalazine group(P<0.05),serum IL-10 of mice in Weichang’ an Pill group also increased,but there was no statistical significance(P>0.05).(5)The protein contents of p-NF-κBp65、stat3 and p-stat3 in colon of mice in each group were detected for proteins:Compared the results of two groups,the contents of stat3 and p-stat3 protein in colon tissue of mesalazine group and Weichang’ an Pill group decreased significantly(P<0.05).However,there was no significant difference in the content of p-NF-κBp65 protein between Weichang’ an Pill group and control group(P>0.05).(6)The content of RORyt and FOXP3 the amount of protein in the different groups were detected by fluorescence quantitative PCR:Compared with the model group,the content of RORyt protein in colon tissue of mesalazine group and Weichang’ an Pill group decreased obviously(P<0.05),percentage FOXP3 protein in colon tissue of these two groups increased obviously(P<0.05).There was no difference in the contents of RORyt and FOXP3 protein between mesalazine group and Weichang’ an Pill group(P>0.05).ConclusionWeichang’ an Pill is effective in treating UC model mice,which can improve the general situation and DAI score of UC mice.Weichang’ an Pill can improve the pathological morphology of intestinal mucosa in mice.It can inhibit pro-inflammatory cell Th 17 and up regulate anti-inflammatory cell Treg,can effectively inhibit proinflammatory cytokines(IL-1β、IL-6、IL-17)and increase anti-inflammatory cytokines(TGF-β1、IL-10)in mice serum,can down regulate the protein content of proinflammatory transcription factor(p-NF-κBp65、stat3、p-stat3、RORγt)and up regulate the protein content of anti-inflammatory transcription factor FOXP3 in mice colonic mucosa.Weichang’ an Pill can adjust the immune imbalance of UC model mice and achieve the purpose of treating diseases.