| Objective:Diabetes mellitus(DM),as the third largest non-communicable diseases after cardiovascular diseases and tumors,has seriously threatened the life and health of patients.Therefore,elucidating the pathogenesis of DM has become a hot spot in current medical research.Pancreatic ? cell damage caused by lipotoxicity plays a central role in the pathogenesis of DM.However,the involved mechanism of lipotoxicity in ? cell damage remains obscure.Thus,it is particularly important to screen natural drugs that antagonize lipotoxicity.Acacetin,a natural flavonoid originally isolated from vascular plants,has many pharmacological activities such as anti-oxidative,anti-inflammatory and hypoglycemic effects.Acacetin has broad prospects in the treatment of DM.The present study aims to explore the protective effect of acacetin on free fatty acid(FFA)-induced pancreatic ?-cell damages and to elucidate the potential molecular mechanism,which could provide the theoretical basis for the treatment of DM.Methods:The rat pancreatic RINm5F ? cells were cultured and divided into the following groups:control group,acacetin group,FFA group,acacetin+FFA group.The cell viability was assessed by the MTT assay.Intracellular ROS generation was detected by the DCF-DA method.The mRNA transcription,protein expression,and enzyme activities of antioxidant enzymes were analyzed by the real-time PCR,western blot and colorimetric assay kits,respectively.The apoptotic sub-G1 population was detected by flow cytometry after propidium iodide(PI)staining.The protein levels of cleaved poly ADP-ribose polymerase(PARP)and caspase-3 were determined by western blot analysis.The mitochondrial calcium(Ca2+)levels were measured by flow cytometry and fluorescence microscope after Rhod-2 staining.The expression of glucose-regulated protein 78(GRP78),phosphorylation of protein kinase-like ER kinase(PERK),phosphorylation of eukaryotic initiation factor 2?(eIF2?),cleaved activating transcription factor 6(ATF6),cleaved capsase-12,and CCAAT/enhancer-binding protein homologous protein(CHOP)was determined by western blot analysis.X-box binding protein 1(XBP1)mRNA splicing was detected by the reverse transcription PCR.After cells were transfected with Control siRNA or CHOP siRNA,the cell viability of the transfected cells was assessed by the MTT assay.The mitochondrial Ca2+ levels were measured after Rhod-2 staining.The apoptotic sub-Gi population and the expression of apoptosis related proteins were analyzed by the PI method and western blot,respectively.Results:FFA significantly decreased cell viability in a dose-dependent manner.At 0.35 mmol/L of FFA,the cell viability of RINm5F was nearly 50%.Acacetin at different concentrations(10-25 ?mol/L)exhibited little cytotoxic effect on RIN m5F cells.On the other hand pretreatment with acacetin protected cells from FFA-induced cell death,and acacetin at 15?mol/L showed the highest efficiency on protection of RINm5F cells.Acacetin inhibited FFA-induced increase of intracellular ROS and reduction in the,capacity of antioxidant enzymes.Notably,exposure to FFA markedly induced cell apoptosi's indicated by an increase in the apoptotic sub-G1 population and the up-regulation of cleaved PARP and cleaved caspase-3,whereas pretreatment with acacetin signifcantly reversed this phenomenon.In addition,acacetin relieved FFA-induced mitochondrial Ca2+overload in RIN m5F cells.Moreover,acacetin treatment also attenuated FFA-induced endoplasm ic reticulum stress(ERS)evidenced by the down-regulation of ERS-related proteins including GRP78,p-PERK,p-eIF2a,cleaved ATF6,cleaved caspase-12 and CHOP,as well as reduction of XBP1 splicing.Furthermore,silencing of CHOP express,ion partly alleviated FFA-induced apoptosis and augmented the cytoprotective effect of acacetin.Conclusion:Our findings demonstrated that acacetin could attenuate the FFA-induced cell damage in pancreatic ?-cells via mitigation of oxidative stress and endoplasmic reticulum stress,which was parti,ally associated with CHOP-mediated apoptotic cell death. |
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