Study On The Mechanism Of Qing Hua Chang Yin In The Treatment Of Chronic Ulcerative Colitis Based On RNA Sequencing

Objective:The purpose of this study is to investigate the effect of Qing Hua Chang Yin(QHCY)on Dextran sulfate sodium(DSS)-induced chronic ulcerative colitis,and to explore the mechanism of QHCY in the treatment of chronic ulcerative colitis by RNA sequencing.Methods:Male C57BL/6 mice were randomly divided into Normal group(Control),Model group(DSS),Mesalazine group(DSS+Mesalazine),low-dose QHCY group(DSS+QHCY-L),medium-dose QHCY group(DSS+QHCY-M),and high-dose QHCY group(DSS+QHCY-H),with 6 rats in each group.The mice model of chronic ulcerative colitis was induced by 2%DSS.Mice in Control group and DSS group were infused with distilled water,while mice in drug treatment group were administrated with QHCY(0.8 g/kg/d,1.6 g/kg/d,3.2 g/kg/d)or Mesalazine SR Granules(0.2 g/kg/d)for 49 consecutive days.During the experiment,body weight,diarrhea and rectal bleeding and disease activity index(DAI)were recorded.The length of colon was measured.The serum and colon tissues were collected.HE staining was used to observe the histopathological changes of the colon of mice.The Periodic Acid-Schiff staining was used to observe the number of goblet cells in colon tissues of mice.The levels of TNF-α,IL-6 and IL-1β in serum were measured by ELISA.Differentially expressed genes in colon tissues of mice in each group were detected by RNA sequencing.The differentially expressed genes were analyzed by GO and the KEGG database.The results of RNA sequencing analysis were verified: the expression levels of tight junction proteins Occludin,ZO-1 and MUC2 in colon tissues were detected by immunohistochemistry.The TUNEL detection kit was used to detect cell apoptosis in colon tissues.The immunohistochemistry was used to detect the expression levels of apoptosis-related proteins p53,Bax and Bcl-2.The expression levels of PPARγ/NF-κB signaling pathway related proteins PPARγ,p-IκB,IκB,p-NF-κB p65,TNF-α,IL-6 and IL-1β were detected by immunohistochemistry.Results:Compared with mice in the control group,the mice in the DSS group suffered from increase of DAI,weight loss and shortening of colon.Medium and high doses of QHCY and Mesalazine significantly reversed the damage caused by DSS,including increase of DAI,weight loss and shortening of colon;HE staining showed that DSS stimulation resulted in mucosal ulceration,disruption and disarrangement of glands,and infiltration of inflammatory cells.Medium and high doses of QHCY and Mesalazine significantly ameliorated histological damage of colon tissue in the DSS?induced chronic ulcerative colitis mice;The results of Periodic Acid-Schiff staining showed that DSS stimulation reduced the number of goblet cells.Medium and high doses of QHCY and Mesalazine significantly inhibited goblet cell reduction in colon tissue of the DSS?induced chronic ulcerative colitis mice;ELISA results showed that serum levels of TNF-α,IL-6 and IL-1β was increased in DSS group.Medium dose QHCY and Mesalazine significantly inhibited the expression of TNF-α,IL-6,and IL-1β in serum of mice with DSS?induced chronic ulcerative colitis;RNA sequencing results showed that 7599 genes were changed in DSS group compared with Control group.Compared with the DSS+QHCY group,a total of 3831 genes were changed.1166 up-regulated genes induced by DSS intervention were down-regulated after QHCY intervention.425 down-regulated genes induced by DSS intervention were up-regulated after QHCY intervention.GO biological process analysis showed that the two groups of differential genes were enriched to the regulation of inflammatory response,tight junction and regulation of mucus secretion.KEGG pathway analysis showed that the two groups of differential genes were enriched to PPAR signaling pathway,NF-κB signaling pathway,p53 signaling pathway and apoptosis signaling pathway;Immunohistochemical results showed that the expression of tight junction protein Occludin,ZO-1 and MUC2 in colon tissue of mice in DSS group was decreased.Medium dose QHCY and Mesalazine significantly increased Occludin,ZO-1,and MUC2 expression in the DSS?induced chronic ulcerative colitis mice;TUNEL staining showed that the number of apoptosis of colon epithelial cells in the DSS group was significantly increased,while Medium dose QHCY and Mesalazine intervention significantly inhibited the apoptosis of colonic epithelial cells in the DSS?induced chronic ulcerative colitis mice.Immunohistochemical results showed that p53 and Bax expression in the colon tissue of mice in DSS group was significantly increased,and Bcl-2 protein expression was significantly decreased,while Medium dose QHCY and Mesalazine intervention significantly inhibited DSS-induced the increase of p53 and Bax expression and the decrese of Bcl-2 expression.The PPARγ and IκB expression in the colon tissue of mice in DSS group were significantly decreased,and p-IκB,p-NF-κB p65,TNF-α,IL-6 and IL-1β protein expression were significantly increased,while Medium-dose QHCY and Mesalazine intervention significantly inhibited DSS-induced the decrease of PPARγ and IκB expression and the increase of p-IκB,p-NF-κB p65,TNF-α,IL-6 and IL-1β expression.Conclusion:QHCY has a certain therapeutic effect on chronic ulcerative colitis,which can effectively reduce the weight loss,the increase of DAI score,the shortening of colon length and the pathological damage of the colon in mice with chronic ulcerative colitis,repair the intestinal mucosal barrier,and reduce inflammation.The mechanism may be related to activation of PPARγ and inhibition of abnormal activation of NF-κB signaling pathway.The results of this study can not only further enrich the mechanism of QHCY in the prevention and treatment of chronic ulcerative colitis,but also provide further experimental basis for its clinical application.