Effects Of Oxymatrine Ameliorating Ulcerative Colitis Through RhoA/ROCK Signaling Pathway

?Objective?Ulcerative Colitis(UC)is a kind of chronic non-specific inflammation involving colorectal mucosa.A large number of studies have shown that factors such as immune system dysfunction and intestinal mucosal barrier dysfunction play a vital role in the occurrence and development of ulcerative colitis.ROCK belongs to Rho A downstream target effector molecules that are relatively well studied at present,which can reconstruct the cytoskeleton structure by phosphorylating the downstream myosin light chain(MLC)and participate in the destruction of tight junction(TJ)of intestinal mucosa,and also act on NF-? Bp65 to damage the function of intestinal mucosa barrier.In addition,Rho A/ROCK signaling pathway is involved in immune response of cells,and is closely related to the occurrence and development of various human diseases,but the specific pathogenesis in ulcerative colitis remains elusive.The purpose of this study was to explore whether oxymatrine could alleviate UC by acting on Rho A/ROCK signaling pathway in DSS-induced acute ulcerative colitis mice,as well as the specific mechanism.This study not only explored the possible mechanism of ulcerative colitis,but also explored a new treatment for ulcerative colitis.?Methods?48 Balb/c mice(18-22g)were randomly divided into the following 6 groups:control group?model group?low?medium and high-dose oxymatrine group?inhibitor group,8 mice in each group.In addition to the normal group,the rest groups were given 3%DSS for free drinking for one week on the 8th day,and on the first day of modeling,each treatment group was given intraperitoneal injection of the corresponding dose of the drug,the normal group and the model group was intraperitoneally injected with an equal dose of PBS for 7 days.During the modeling period,the weight,diarrhea and hematochezia of the mice were recorded every day for DAI score.All mice were sacrificed on the 15 th day,and colon tissue,mesenteric lymph nodes and spleen were taken.Paraffin-embedded sections of the diseased colon tissues were taken and stained with HE,and the pathological changes of the colon of mice in each group were observed under the microscope.The ultrastructural changes of colon tissues were observed by transmission electron microscopy.The expression of Zo-1 and Occludin in colon tissues of mice was detected by immunohistochemistry.Western blot was used to detect the expressions of Zo-1,Occludin,ROCK-1,ROCK-2,NF-?Bp65,p-MLC,MLC,p-MYPT-1,MYPT-1,ROR?t and FOXP3 in colon tissues of mice.The expressions of ROCK-1,ROCK-2,IL-17 A,IL-10,ROR?t and FOXP3 in colon tissues of mice were detected by fluorescence quantitative PCR.The expression of IL-17 A,IL-21 and IL-10 in colon tissues was determined by ELISA.Flow cytometry was used to detect the expression of Th17 cells and Treg cells in mouse mesenteric lymph nodes,spleen.?Results?After DSS modeling,from the third day after DSS drinking,all the mice in each group had different degrees of mental malaise,and their diet and activity were significantly decreased,with loose stools,soft stools and mucous blood stools,and their body weight was significantly reduced.Compared with the model group,the DAI scores of the low,medium and high doses of oxymatrine and the inhibitor group decreased significantly(P <0.01).The normal group was observed under TEM: the glands were arranged neatly,the cells were closely connected,and no obvious changes in organelles were observed.In the model group,the intestinal epithelial cells had sparse surface microvilli and irregular morphology,and the intercellular junctions were significantly widened,goblet cells were significantly reduced,organelles were swollen,and vacuoles were observed in the cytoplasm.Compared with the model group,the other treatment groups had different degrees of improvement.Compared with the normal group,the protein contents of ROCK-1,ROCK-2,NF-?Bp65,p-MLC,p-MYPT-1,ROR?t,the m RNA level of ROCK-1,ROCK-2,IL-17 A,the contents of IL-17 A,IL-21 cytokines,and the proportion of Th17 cells in spleen and mesenteric lymph nodes were significantly increased in the model group(p < 0.05).Compared with the model group,the contents of the above indicators in the oxymatrine dose groups and the inhibitor group were significantly reduced(P<0.05);Compared with the normal group,the ZO-1,Occludin,FOXP3 protein content,FOXP3,IL-10 m RNA level,IL-10 cytokine content,and the proportion of Treg cells in the spleen and mesenteric lymph nodes were significantly decreased in the model group(P<0.05).Compared with the model group,the content of above indicators in the oxymatrine dose groups and the inhibitor group was significantly increased(P<0.05).?Conclusion?1.Rho A/ROCK signaling pathway is involved in the development of inflammation in mice with ulcerative colitis induced by DSS;2.Oxymatrine can alleviate DSS-induced ulcerative colitis;3.By inhibiting Rho A/ROCK signaling pathway,oxymatrine plays a role in inhibiting inflammation,improving intestinal mucosa barrier,inhibiting Th17 cell differentiation and promoting Treg cell differentiation to alleviate DSS-induced ulcerative colitis.