The Effect Of Kuikeling Modified Prescription On Intestinal Mucosal Barrier Function In Mice With Ulcerative Colitis

Objective:Ulcerative colitis(UC)is an inflammatory bowel disease(IBD)involving the rectum and colonic mucosa.It can be clinically manifested as abdominal pain,diarrhea,mucus pus and blood in the stool,tenesmus and so on.Although the pathogenesis of UC is not completely clear,recent studies have shown that patients with ulcerative colitis exist a series of intestinal mucosal mechanical barriers are damaged,such as increased permeability of the intestinal epithelial barrier,thinning of the mucus colloid layer,decreased mucin and trefoil factor synthesis and secretion by goblet cells,etc.Previous experiments have shown that the traditional Chinese medicine Kuikeling modified prescription(KKL)has a certain preventive and protective effect on the occurrence and development of mouse colitis.It can significantly reduce the mumber and area of colonic ulcers in mice,and improve pathological changes such as colonic mucosa inflammation,congestion and edema.The purpose of this study was to observe the effect of the KKL on the barrier function of intestinal mucosa in colitis model mice,and to further explore the possible therapeutic mechanism of its treatment.Research method:50 male C57BL/6J mice aged 6-7 weeks were randomly divided into 5 groups:normal group(NC),inflammation control group(DSS),kuikeling modified prescription low-dose group(KKL L),kuikeling modified prescription medium-dose group(KKL M),kuikeling modified prescription high-dose group(KKL H).Each group had 10 mice.The NC group drank water freely,and the other groups were given 4.0%DSS solution and free drinking to prepare a mouse colitis model.During this time,the mice in the NC group and DSS group were given normal saline every morning by gavage.And the mice in treatment group were given different concentrations of KKL solution by gavage.Gavage lasts for 7 days.During this period,we observed the general state of the mice daily(such as hair color,perianal condition,fecal traits,mouse mental state,etc.),and recorded changes in daily weight,diet,and water intake.After the mice were fasted for 12 hours,the mice were anesthetized with isoflurane on the 8th day of the experiment,and the eyeballs were removed to collect blood from the mice.The collected mouse blood was allowed to stand at room temperature for 2 hours and then centrifuged to collect the supernatant,and the peripheral blood D-lactic acid and endotoxin levels were detected by ELISA.When the blood was exhausted,the mice were sacrificed by the method of removing the neck.The entire colon of the mouse was dissected and collected,and the length of the terminal colon was measured.Cut the mouse colon from the longitudinal axis,wash away the intestinal feces with saline,and cut a part of the intestine for HE staining and immunohistochemistry to detect the expression of TFF3 and MUC2 proteins in the colon.Fluorescence quantitative PCR method was used to detect the expression of MUC2,TFF3,Claudin-1,Occludin,and Zo-1 mRNA.Western Blot was used to detect the expression of Claudin-1,Occludin,and Zo-1 protein.Results:Compared with the NC group,the weight of the remaining four groups of mice was reduced to varying degrees after modeling(P<0.01);at the same time,four groups of mice showed unformed and bloody stools,colonic mucosa with congestion,shortened intestinal length,and increased DAI scores.The expressions of MUC2,TFF3,Claudin-1,Occludin,and Zo-1 mRNA and protein in the colon mucosa tissue of the modeled mice were lower than those in the normal group,which was statistically significant(P<0.01);the level of D-lactic and endotoxin in serum increased(P<0.01),indicating that the intestinal mucosal barrier function was destroyed.Compared with the DSS group,the weight of the mice,the degree of blood in the stool,the congestion and edema of the colonic mucosa,and the DAI score were improved in varying degrees for the KKL M group and the KKL H group;the expression of MUC2,TFF3,Claudin-1,Occludin,and Zo-1 mRNA and protein in colonic mucosa tissues was increased,which was statistically significant(P<0.05);D-Lac and LPS in serum were down-regulated compared with the DSS group(P<0.05).The KKL L group and the DSS group mice were similar in all aspects,and there was no significant statistical significance.Conclusion:KKL,a traditional Chinese medicine,has a certain effect on the repair of intestinal mucosa in mice with colitis,which may be related to promoting the expression of colonic mucosal tight junction proteins Claudin-1,Occludin,ZO-1 and mucus-related proteins MUC2 and TFF3,thereby promoting the repair of damaged areas.