Study On Mechanisms Of Punicalagin On Ameliorating High-fat Induced Pancreatic Cell Injuries

Punicalagin(PU),a polyphenol compound is abundant in pomegranate juice,with strong antioxidant and anti-inflammatory activity.Pancreatic?-cells are the main insulin-secreting functional cells,which are the key to maintain homeostasis of blood glucose level and healthy glucose and lipid metabolism.It has been suggested that the damages of pancreatic cells induced by unhealthy high fat diet or obesity are major causes to trigger the pathogenesis of diabetes mellitus.In this study,the role of punicalagin in anti-oxidation,anti-endoplasmic reticulum stress and anti-pyrotosis as well as its inherent molecular regulatory mechanism on high free fatty acid(FFA)stimulated pancreatic?-cell were identified to provide experimental evidence for the protective activity of dietary polyphenol punicalagin on islet cells and provide theoretical background for promoting development of related new functional foods.The research content of this thesis is mainly divided into three parts:(1)Punicalagin inhibits oxidative stress induced by high lipid in pancreatic?-cellThe survival rate of pancreatic RINm5F?-cells were used in MTT assay to.To explore the regulation of punicalagin on oxidative stress induced by FFA in islet cells.Various concentrations of punicalagin(6.25?M,12.5?M,25?M and 50?M)could inhibit the decline of cell viability caused by FFA,and 12.5?M had the best protective effect.The results showed that punicalagin significantly inhibited the production of ROS(reactive oxygen species),MDA(malondialdehyde)and GSSG(glutathione)in pancreatic?-cells induced by FFA.In addition,punicalagin can increase the levels of reduced GSH(glutathione)and antioxidant enzymes,such as CAT(catalase),SOD(superoxide dismutase)and GPx(glutathione peroxidase),reduced by FFA.These results suggested that punicalagin could enhance the activity of antioxidant enzymes,scavenge ROS and reduce the degree of lipid peroxidation,thereby alleviate the damage of oxidative stress on pancreatic beta cells.(2)Punicalagin inhibited ERS(endoplasmic reticulum stress)of pancreatic?cells induced by FFAWestern blot,flow cytometry,and RT-PCR experiment results showed that punicalagin significantly reduced the expression of ERS marker proteins GRP78,p-PERk,p-e IF2?,cleaved ATF6,cleaved caspase12 and CHOP and reduced mitochondrial Ca²⁺overload,thereby alleviated FFA-induced ERS in pancreatic?-cells.RNA interference assay showed that CHOP plays an important role in FFA-induced ERS and apoptosis.Silencing CHOP gene can reduce mitochondrial Ca²⁺overload,down-regulate the expression of ERS marker protein,and alleviate ERS,decreased cleaved-caspase3 and cleaved-PARP,and ultimately inhibited pancreatic?-cells apoptosis.In addition,knockdown of CHOP gene can enhance the protective effect of punicalagin on pancreatic?-cells.(3)Punicalagin inhibited the high lipid-induced pyrotosis of pancreatic?-cellsWe demonstrated anti-inflammatory activity of punicalagin and the anti-FFA-induced pyrotosis of pancreatic?-cells through the regulating of pro-inflammatory factor gene expression,enzyme activity detection and micro RNA expression.RT-PCR results showed that in FFA treated cell apoptosis marker genes,NLRP3,TXNIP,caspase1,IL-1?,IL-18,and GSDMD m RNA expression levels were significantly increased.After pretreatment with punicalagin,the up-regulation effect of FFA on these pro-inflammatory genes like TNF-?,COX-2 and iNOS were significantly decreased,the activity of iNOS enzyme and the level of NO in FFA stimulated cells were reduced likewise.These results indicated that punicalagin exerted anti-inflammatory activity by inhibiting proinflammatory factors and regulating iNOS/NO pathway.Of note,we found that FFA treatment resulted in down-regulation of mi R-200a expression,while punicalagin pretreatment significantly increased mi R-200a expression.Because TXNIP is a direct mi R-200a targeting gene,punicalagin can regulate the TXNIP/NLRP3pathway through mi R-200a to ameliorate islet?-cell pyrotosis.In conclusion,as a natural product of polyphenols,punicalagin has multiple protective effects on pancreatic?-cells,including antioxidant,anti-ERS,and anti-pyrotosis.The results of the molecular mechanism showed that punicalagin not only scavenged oxidative free radicals in cells,but also regulated antioxidative enzyme to maintain the redox balance of pancreatic?-cells.In addition,punicalagin can regulate related signaling pathways to effectively mitigate the damage of FFA induced ERS and pyrotosis in islet?-cells.In particular,the cascade regulation between mi R-220a/TXNIP/NLRP3 is the key mechanism by which punicalagin alleviates the pyrotosis of pancreatic?-cells.