| Mesona chinensis Benth is an annual herb of the family Labiatae,which can be used for both medicinal and food purposes and is found in many southern provinces of China.Polysaccharide is one of the main active components of Mesona chinensis Benth,and studies have shown that Mesona chinensis Benth polysaccharide(MBP)has biological activities such as antioxidant,immune regulator and hypoglycemic,but there are few reports on the improvement effect of MBP on colitis and its mechanism.In this paper,MBP was used to investigate its protective effects on intestinal damage in colitis.By constructing an in vitro model of dextran sodium sulfate(DSS)-induced inflammatory injury in Caco-2 cells,the effects of MBP intervention on apoptosis,oxidative stress status and intestinal barrier integrity were initially investigated;by establishing a mouse model of DSS-induced ulcerative colitis in vivo,the effects of MBP intervention on pathological indicators,intestinal barrier permeability,MAPK/NF-κB related intestinal inflammation,intestinal flora and its metabolites short-chain fatty acids(SCFAs),and hepatic oxidative stress levels,to reveal the mitigating effects and molecular mechanisms of MBP on DSS-induced Caco-2 cell injury and colitis in mice.The main findings obtained in this paper are as follows:1.A model of DSS-induced inflammatory injury in Caco-2 cells and a monolayer cell model were established to study the effects of MBP on intestinal cell status and intestinal barrier.The results of cell viability assay and LDH assay showed that DSS had a strong toxic effect on Caco-2 cells,while MBP intervention enhanced the viability of Caco-2 cells and reduced the production of LDH.Secondly,DSS dysregulated the oxidative stress status of cells,while MBP intervention could improve the dysregulation by enhancing SOD activity,reducing ROS content,decreasing the secretion of inflammatory factors TNF-α and IL-6 and decreasing COX-2 protein expression.In addition,cell cycle,apoptosis and protein blotting experiments showed that MBP intervention could normalize the cell growth cycle and reduce apoptosis caused by DSS through Caspase-3/Bcl-2/Bax pathway.Further,by constructing a Caco-2 monolayer cell model,it was found that MBP intervention could enhance the integrity of the Caco-2 monolayer cell barrier,increase the TEER value,decrease the fluorescent yellow infiltration rate,and promote the expression of tight junction proteins(TJs)such as ZO-1,Occludin,and Claudin.In summary,the results tentatively showed that MBP could alleviate DSS-induced intestinal cell injury and restore the intestinal barrier function simulated in vitro,providing a reference for the subsequent in vivo experiments.2.A model of DSS-induced ulcerative colitis in mice was established to study the effects of MBP on the pathological features and intestinal inflammatory indexes in mice with colitis.Compared with normal mice,mice with colitis showed obvious pathological features such as shortening of the colon,weight loss,blood in the stool and diarrhea,and the analysis of the section results showed that the structure of the intestinal crypts and villi were destroyed and excessive infiltration of inflammatory cells occurred,while MBP intervention significantly increased the disease activity index(DAI),alleviated the shortening of the colon,reduced myeloperoxidase(MPO)activity,effectively protecting the structural integrity of the intestine.Meanwhile,DSS induced the secretion of inflammatory factors in colonic tissues to become disturbed,while MBP intervention back-regulated the production of inflammatory factors and effectively improved the level of intestinal inflammation by inhibiting the overexpression of proteins related to TLR4/MAPK/NF-κB signaling pathway.The best effect was observed in the MD group.In summary,the results showed that MBP intervention could effectively alleviate the inflammatory state of the intestine in mice with colitis,which may be related to the regulation of TLR4/MAPK/NF-κB signaling pathway to restore the levels of intestinal inflammatory factors.3.Based on the mouse model of ulcerative colitis,the effects of MBP on the intestinal barrier and liver oxidative stress status of mice with colitis were investigated.The results of protein blotting and immunofluorescence experiments showed that MBP intervention significantly increased the expression of intestinal TJs protein and mucin MUC-2,elevated the ratio of apoptosis-related protein Bcl-2/BAX,and enhanced the integrity of the intestinal barrier in colitis mice.It also restored the serum levels of ET and LBP,which in turn allowed the hepatic oxidative stress state to return to equilibrium.The results showed that MBP intervention could restore the integrity of the intestinal barrier and the oxidative dysregulation of the liver in mice with colitis.4.Based on a mouse model of ulcerative colitis,the effect of MBP on the intestinal flora and its metabolites,SCFAs,in mice with colitis was investigated.The effects of MBP on intestinal flora and its metabolites SCFAs,in mice with colitis was examined.The results showed that MBP intervention significantly promoted the production of SCFAs,especially acetate and propionate,in the intestinal tract of mice with colitis.16 S r RNA high-throughput sequencing showed that DSS induction caused severe dysbiosis in the intestinal flora of mice,as evidenced by a significantly higher ratio of Firmicutes to Bacteroidetes and a lower diversity.The MBP intervention restored the structural changes of intestinal flora in mice with colitis,and reduced the relative abundance of many pathogenic bacteria such as Clostridiaceae＿Clostridium,Helicobacter and Prevotella,while the relative abundance of Lactobacillus and Coprococcus in the MD group increased significantly.The relative abundance of beneficial bacteria such as Bifidobacterium and Oscillospira increased significantly in the HD group,while the results of the diversity of the flora in the HD group were similar to those of the normal group.the results of LEf Se analysis showed that the dominant species in the MC group were Turicibacter,Acinetobacter and Clostridium;while the marker bacteria in the LD,MD and HD groups were Coprococcus,Akkermansia and Sphingomonas,Blautia and Dysgonomonas,respectively.In summary,this study firstly established DSS-induced inflammatory injury model of Caco-2 cells and monolayer cell model in vitro,and initially verified that MBP could alleviate the damage caused by DSS on intestinal cells and cell barrier.Based on this,we also established an in vivo model of DSS-induced ulcerative colitis in mice,and demonstrated that MBP can alleviate the effects of colitis in mice from various perspectives,including intestinal inflammation,intestinal barrier,intestinal flora and its metabolites,and explored the mechanism of action.The results of this study can provide scientific guidance and experimental basis for the use of natural plant polysaccharides in the treatment of colitis,and also help to develop the added value of Mesona chinensis Benth. |
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