| BackgroundType 2 diabetes mellitus(T2DM),known as non-insulin dependent diabetes or adult diabetes,which accounts for about 90% of all diabetes cases.T2DM has become one of the major chronic metabolic diseases and public health problems affecting the health of Chinese people.According to the latest data released by the International Diabetes Federation in 2021,537 million people have diabetes in 2021 and this number is projected to reach 642 million by 2040.T2DM has become the sixth largest disability factors which not only brings great pain to patients,but also cause a heavy burden to social economy.The development of T2DM is affected by complex risk factors including age,genetics,lifestyle and eating habits.Dietary factors are closely related to the occurrence of T2DM,and high-energy and high-fat diets have been found as important inducers of T2DM development.Rational nutrition is a constant intervention for the prevention and treatment of T2DM.The intestinal tract,an important organ connecting the internal and external environment of the human body,which acts as a critical barrier separating intestinal contents and blood circulation.Researches indicated that intestinal dysfunction,including intestinal barrier integrity destruction and intestinal flora disturbance,promote the development of dietary factors-induced T2DM.Intestinal barrier destruction lead the flux of enterogenic pathogens,toxins or food metabolites from the intestinal lumen into the blood circulation results in a variety of diseases,including diabetes,non-alcoholic fatty liver,cardiovascular disease,inflammatory colitis.Researches also showed that intestinal flora dysfunction is closely related to the occurrence of chronic metabolic diseases such as diabetes.Intestinal flora disturbance caused by imbalanced dietary structure(such as high-energy or high-fat diets)leads to the increasing of Gram-negative bacteria and the translocation of intestinal flora through the intestinal barrier.Lipopolysaccharide(LPS),a component of Gram-negative bacteria,which transmigrates into the blood circulation to trigger a chronic low-grade systemic inflammation.Inflammatory factors such as TNF-α inhibit the activation of insulin receptor pathway in metabolism-related target organs,resulting in obesity and insulin resistance.The occurrence and development of T2DM is closely related to intestinal mucosal barrier function and intestinal barrier pathological changes and dysfunction play a key role in the occurrence of T2DM.However,the related mechanism is still not clear.In recent years,the health effects and mechanism of plant food has become a research highlight in the field of nutrition.Some phytochemicals have become as potential treatment options due to the effects on antioxidation and regulating glucose and lipid metabolism,and natural phytochemicals have become optimistic candidates for the prevention and treatment of chronic metabolic diseases.As flavonoid phytochemicals,dihydromyricetin(DHM)has a variety of biological effects with no obvious side effects.DHM has antioxidant,anti-inflammatory,anti-oxidative stress,anti-tumor,antibacterial and other effects.Our previous studies found that DHM can effectively improve fasting blood glucose and insulin resistance and may act as an ideal phytochemical for the prevention and treatment of T2DM.However,the related mechanism has not been fully elucidated.Researches showed that DHM could significantly reduce the expression of inflammatory cytokines in the intestinal tissue,increase the expression of intestinal tight junction protein and adhesion junction protein,and reduce intestinal permeability to improve the intestinal dysfunction of high-intensity exercise mice.DHM also alleviated the colonic inflammation induced by dextran sulfate sodium(DSS)by regulating intestinal flora and bile acid metabolism and regulated intestinal barrier function through changing the richness and diversity of intestinal flora,upregulating the ratio of Firmicutes/Bacteroidetes(F/B)and increasing the relative abundance of Akkermansia muciniphila,and inhibiting NLRP3 inflammasome to reduce Escherichia coli lipopolysaccharide-induced ileal damage.Therefore,DHM may regulate intestinal barrier function to improve T2DM.Previous studies in our laboratory have shown that DHM can regulate hepatocyte lipid metabolism and maintain redox homeostasis through SIRT3,slowing down the progression of non-alcoholic fatty liver disease,suggesting that SIRT3 may be a potential target for DHM to exert its biological effects in vivo.Studies have also shown that a high-fat diet can inhibit mitochondrial SIRT3 expression,leading to increased acetylation modifications of various mitochondrial proteins or enzymes,causing mitochondrial dysfunction.At the same time,increased SIRT3 expression and activity can inhibit oxidative stress,mediate mitochondrial function protection and induce cellular autophagy.Therefore,SIRT3 may play a vital role in regulating intestinal barrier function by DHM.Based on the above research background,this study proposed the following hypothesis: DHM may improve intestinal barrier function and reduce enterogenic chronic inflammation in T2DM mice induced by high-fat diet through SIRT3,thereby improving insulin resistance and prevention and treatment of T2DM.This study aims to reveal the role of DHM in the prevention and treatment of T2DM and its related molecular mechanism around the intestinal epithelial barrier function,and provide a new intervention strategy for the prevention and treatment of T2DM through diet.ObjectiveC57BL/6J mice,wild type 129 mice,SIRT3 gene knockout mice were fed with High-fat diet(HFD),and the intestinal epithelial cells were induced by LPS.The effect of DHM on T2DM and intestinal epithelial barrier damage induced by HFD was investigated,and the mechanism of SIRT3 in the prevention and treatment of T2DM and regulation of intestinal epithelial barrier function was explored.Our research was aimed to provide new molecular targets and dietary nutrition intervention strategies for the prevention and treatment of T2DM.Methods1.C57BL/6J mice were randomly divided into 4 groups: control group(NC),high fat group(HFD),high fat plus dihydromyricetin group(HFD+DHM)and dihydromyricetin group(DHM).T2DM model was established by high-fat feeding for 12 weeks,and DHM was intervened for 12 weeks.The body mass and food intake of mice in each group were recorded.The OGTT,ITT,serological indexes and intestinal pathological changes were detected.The intestinal permeability,inflammatory factors and tight junction protein expression were detected,and the effect of DHM on T2DM and intestinal epithelial barrier function induced by high fat diet was observed.2.SIRT3 KO mice and WT mice were randomly divided into three groups: control group(NC),high fat group(HFD)and high fat plus dihydromyricetin group(HFD+DHM).T2DM model was established by high-fat feeding for 12 weeks,and DHM was intervened for 12 weeks.The body mass and food intake of mice in each group were recorded,and the effects of high-fat diet on intestinal epithelial barrier were evaluated by measuring body mass,fasting blood glucose,OGTT,ITT,intestinal mucosal barrier function,intestinal permeability and intestinal tight junction protein expression,respectively.And the mechanism of SIRT3 in the improvement of T2DM and intestinal epithelial barrier function by DHM was elucidated.3.The stable transfection model of intestinal epithelial cell line Caco-2 was established by SIRT3 lentivirus plasmid transfection.The expression of tight junction protein,inflammatory factor and autophagy-related protein was detected by LPS induction and DHM intervention,and the mechanism of SIRT3 in DHM improving LPSinduced intestinal epithelial cell injury was further verified.Results1.DHM can significantly improve the disorder of glucose and lipid metabolism induced by high-fat diet,delay the occurrence of T2DM,promote the expression of intestinal tight junction protein,reduce intestinal barrier permeability and maintain the integrity of intestinal mucosal barrier.It was found that HFD induced a significant increase in body weight,abnormal glucose and lipid metabolism,increased intestinal permeability,intestinal epithelial structural damage such as shallower intestinal crypt depth and shorter epithelial villi length,increased expression of inflammatory factors,and decreased expression of tight junction protein and SIRT3 in intestinal epithelial cells.The intervention of DHM could significantly improve the abnormal glucose and lipid metabolism and intestinal epithelial barrier damage induced by HFD in T2DM mice.Meanwhile,it was found that SIRT3 may play an important role in the improvement of HFD-induced intestinal epithelial barrier injury by DHM.2.SIRT3 plays an important role in the improvement of T2DM and intestinal epithelial barrier damage induced by high-fat diet by DHM,but the protective effect of DHM is not obvious in SIRT3 KO mice.It has been found that DHM can effectively improve the occurrence of T2DM in WT mice induced by high-fat diet,improve glucose and lipid metabolism and intestinal barrier function,and inhibit inflammatory response in mice,but the effect of DHM is not significant in SIRT3 KO mice.3.DHM may inhibit the increased expression of inflammatory cytokines and decreased expression of tight junction protein in intestinal epithelial cells induced by LPS through SIRT3-mediated mitochondrial autophagy,thus maintaining the integrity of intestinal epithelial barrier.It was found that DHM could significantly inhibit the injury of Caco-2 cells induced by lipopolysaccharide(LPS).In the negative control virus LV-control transfected cell line,DHM significantly improved the increased expression of inflammatory cytokines and decreased expression of tight junction protein induced by LPS,but in Caco-2 cells transfected with LV-SIRT3 lentivirus plasmid.DHM had no significant effect on the increased expression of inflammatory factors and decreased expression of tight junction protein induced by LPS,and the protective effect of DHM on Caco-2 cells was weakened.Westernblot assay was used to detect the expression of autophagy proteins LC3 b and Beclin1 in LV-SIRT3 and LV-control cells treated with DHM.It was suggested that DHM could improve the decreased expression of autophagy-related proteins induced by LPS,but its effect was weakened in LV-SIRT3 transfected cells.ConclusionDHM can significantly improve the disorder of glucose and lipid metabolism and delay the occurrence of T2DM in mice induced by high-fat diet,which may be related to the upregulation of SIRT3 expression and mitochondrial autophagy in intestinal epithelial cells,leading to the improvement of intestinal epithelial barrier function,reduction of intestinal barrier permeability and inflammatory damage,and maintainance of intestinal epithelial barrier integrity and finally reduced endotoxemia.This study not only provides a scientific experimental basis for the prevention of T2DM through dietary nutrition,but also provides new ideas and potential intervention targets for the treatment of T2DM. |
PDF Full Text Request