Berberine Improves Diabetic β Cell Damage Through MiR-204/SIRT1 Signaling Pathway

In recent years,the incidence of diabetes in China and even the world has increased year by year,and type 2 diabetes accounts for more than 90% of diabetic patients.It is well known that the pathogenesis of type 2 diabetes mainly includes insulin resistance and beta cell dysfunction.Therefore,research on the mechanism of occurrence of β-cell dysfunction and the development of therapeutic drugs targeting this are of great significance for the prevention and treatment of diabetes.SIRT1 is a NAD+-dependent protein deacetylase.It has been found to be involved in the regulation of various physiological functions of islet β cells,and is closely related to the function of islet β cells and type 2 diabetes,but its upstream regulatory factors need to be further explored.Micro RNAs are a class of non-coding small-molecule single-stranded RNAs that have the effect of inhibiting downstream target genes.Micro RNAs can play an important role in the pathogenesis of type 2 diabetes as an important regulatory factor.However,in terms of beta cell function,it is unclear whether any micro RNA can be involved in the regulation by targeting SIRT1.However,we predicted by Targetscan that mi R-204-5p is an islet-enriched mi RNA that is a micro RNA with a highly complementary binding site to the 3’UTR end of SIRT1.Studies have reported that mi R-204-5p is elevated in type 2 diabetic islets and is associated with beta cell damage.Studies have confirmed that mi R-204-5p can target the inhibition of SIRT1 expression in the cornea,but in islet tissue Whether mi R-204 can affect beta cell function by regulating the expression of SIRT1 is still unknown.Therefore,we hypothesized that mi R-204-5p may be an upstream regulatory target of SIRT1 in pancreatic islets and thus participate in the occurrence of islet β-cell function damage.Drugs targeting the mi R-204/SIRT1 signaling pathway are expected to improve diabetic islet beta cell damage.Our research group has carried out a series of studies on the hypoglycemic effect of berberine.It has been proved that berberine has a therapeutic effect on diabetes.Recent studies have shown that the expression of mi R-204-5p is decreased in islets of diabetic mice after berberine administration.Whether berberine can act on mi R-204-5p and thereby regulate beta cell function is still unclear.In summary,we hypothesized that berberine may reverse the β-cell dysfunction in diabetes by inhibiting the mi R-204/SIRT1 signaling pathway and play a role in the treatment of diabetes.This study intends to establish type 2 diabetes mellitus mouse models via the combined use of high-fat diet and multiple administration of low-dose streptozotocin(STZ),a palmitic acid-induced MIN6 cell injury model,a mi R-204 combined with SIRT1 overexpressing MIN6 cell model to observe the effect of mi R-204 on β-cell damage in high-fat and diabetic states,to verify our hypothesis,to clarify the important role of mi R-204/SIRT1 pathway in the protection of β-cells by berberine,and provide a new target for the pathogenesis of type 2 diabetes and anti-diabetic drug therapy.The research content of this thesis is as follows:I.To explore that mi R-204 is a key regulator of pancreatic islet β cell function damage in type 2 diabetes.In this part of the experiment,C57BL/6J mice were given a high-fat diet combined with multiple low-dose intraperitoneal injections of streptozotocin(STZ)to establish a mouse model of type 2 diabetes,and palmitic acid-induced establishment of MIN6 islet β at the cellular level.The cell function damage model was used to observe the changes of mi R-204 level,islet cell apoptosis and insulin secretion in diabetic state and high fat induction in vitro.The changes of signaling pathways related to apoptosis and secretion were examined to explore the role of mi R-204 in the mechanism of β cell function damage.The results showed that compared with the normal control group,fasting blood glucose,fasting insulin,high-density lipoprotein,low-density lipoprotein and triglyceride and total cholesterol were significantly increased in the type 2 diabetes model group,and the islet area was decreased.Changes occur to irregularities,insulin synthesis decreases,and mi R-204 levels in islet tissue increase.In the palmitic acid-induced MIN6 islet β cell injury model group,the palmitic acid-treated model group caused significant apoptosis of MIN6 cells compared with the normal control group,and the basal accumulated insulin and ATP content in the supernatant decreased,mi R-The expression of 204 was significantly increased,and the protein expression level of Caspase-3 was increased.The palmitic acid treatment combined with the mi R-204 silencing group showed the opposite result.The above results indicate that mi R-204 is a key regulator of damage caused by islet β-cell function in diabetic state.II.To clarify the mechanism of action of mi R-204 to induce β cell dysfunction through SIRT1.In this part of the experiment,combined with the upper part of the cell and animal model,the m RNA and protein levels of SIRT1 were detected.At the same time,the islet β cell overexpression mi R-204 model and the mi R-204 and SIRT1 overexpression model were established by transfection to observe the islet β.Cell apoptosis and insulin secretion,The expression levels of mi R-204 and SIRT1 and the changes in signaling pathways related to apoptosis and secretion were examined.The results showed that the m RNA level and protein expression of SIRT1 were decreased in the model group of type 2 diabetes mellitus and palmitic acid treatment.The silencing of mi R-204 at the cell level increased the m RNA level and protein expression of SIRT1.When overexpressing mi R-204 in MIN6 cells,the apoptosis increased,the basal cumulative insulin content and ATP content in the supernatant decreased,the m RNA and protein levels of SIRT1 decreased,and the protein expression level of Caspase-3 increased,while in SIRT1.Overexpression can reverse the reversion of islet beta cell damage caused by mi R204.These results suggest that high expression of mi R-204 increases apoptosis and decreases basal insulin secretion.The mechanism may be through inhibition of SIRT1 m RNA expression.On the one hand,mi R-204 inhibits islet β cell apoptosis by inhibiting SIRT1/Caspase-3 pathway,and inhibits insulin secretion by inhibiting SIRT1/UCP2/ATP insulin synthesis and secretion pathway.Thereby impairing islet beta cell function.III.To verify the mechanism of berberine protection of β-cell damage induced by palmitic acid through the mi R-204/SIRT1 pathway.In this part of the experiment,the animal model of type 2 diabetes and the model of islet β-cell function injury were established according to the above experimental methods,and berberine was given on the basis of the model to observe the changes of model animals and injured cells given berberine,to verify the protective effect of berberine on islet β cell function damage.The results showed that the berberine treatment group can reverse the islet area reduction,insulin synthesis,mi R-204 content,and the decrease of SIRT1 m RNA and protein levels in the islet tissue of the type 2 diabetes model group,suggesting that berberine is involved in diabetic islet tissue.Beta cell function has a protective effect.The results of the cell experiments showed consistent with the overall level results,which again proved that berberine has a protective effect on diabetic islet β-cell function.At the same time,in the cell experiment,mi R-204 was transfected on the berberine treatment group,which reversed the protective effect of berberine on the apoptosis and insulin secretion of MIN6 cells,and further confirmed the protective mechanism of berberine on islet cells,which is related to its inhibition of mi R-204 content.In summary,this study clarified that: 1.mi R-204 can damage islet β-cell function,which may be through inhibiting the expression of SIRT1 m RNA,thereby increasing islet β-cell apoptosis and inhibiting insulin secretion on the other hand,indicated that mi R-204 may be an important target for diabetic insulin β cell function damage;2.The therapeutic effect of berberine on diabetes may be through the regulation of mi R-204/SIRT1 pathway,improving islet β-cell function,providing new targets and new ideas for diabetes prevention and treatment.