| Diabetes mellitus(DM)is a metabolic disorder whose incidence and mortality rate is increasing year by year,seriously endangering the health of humans and animals,among which type-2 diabetes mellitus(T2DM)is the primary type of disease.Insulin resistance(IR)is often present in the early stage of T2 DM,which gradually progresses to decreased insulin secretion from pancreatic β-cells until β-cell apoptosis.Abnormal lipid accumulation is the main cause of islet β-cell apoptosis,often called lipotoxicity.Therefore,in preventing and treating T2 DM,how can we effectively inhibit lipotoxicity-induced islet β-cell apoptosis becomes a key target.Nowadays,most of the drugs for the treatment of T2 DM are mainly western drugs,and long-term administration has certain damage to the organism,while natural product pharmaceuticals can circumvent the defects caused by long-term administration of western drugs.Rubusoside(Rub),as a natural product extracted from the sweet leaves of Rubus suavissimus S.Lee(Rubus),has been proven to have hypoglycemic,hypolipidemic and anti-inflammatory effects,and has the advantages of low toxic side effects and rich biological activity.At present,because the hypoglycemic mechanism of Rub has not been clarified,this study investigates its protective effect on lipotoxicity-induced pancreatic β-cell apoptosis and the related mechanism of action to provide valuable targets for the development of natural drugs and the prevention and treatment of T2 DM.In this study,mouse islet cells Min6 were selected as the research object.The changes of damage to Min6 cells by different concentrations of palmitic acid(PA)and the safe concentration range of Rub were determined by MTT colorimetry.At the same time,the different degrees of Min6 cell injury induced by 0.1,0.2,and 0.3 mmol/L PA were detected by q RT-PCR method.The above experiments determined the final drug concentration.The effect of Rub on the morphology of lipotoxically damaged cells was visualized and compared with light microscopy.The effect of Rub on the rate of lipotoxicity-induced apoptosis was detected by flow cytometry using Annexin V-FITC/PI double-staining.The level of glucose-stimulated insulin secretion in Min6 cells induced by lipotoxicity was investigated using ELISA.The effect of Rub on the expression of Caspase 9 and Cytochrome c(Cyto-c)in lipotoxicity-induced Min6 cells was detected by immunofluorescence assay.q RT-PCR were used to detect the expression of Bax,Bcl-2,Caspase3,INS-1,IRS-1,PI3 K and Akt in Min6 cells induced by lipotoxicity.Finally,the effect of Rub on the expression levels of Bax,Bcl-2,Cleaved-Caspase-9/Caspase 9,Cleaved-Caspase-3/Caspase3,IRS-1,PI3 K and p-Akt/Akt in lipotoxicity-induced Min6 cells was examined by Western Blot.The results are shown in:(1)0.1mmol/L PA could reduce the survival rate of Min6 cells,induce their apoptosis and reduce the expression level of insulin gene INS-1,indicating that an in vitro lipotoxic T2 DM model was successfully constructed for subsequent experiments;12.5～200μmol/L Rub had no toxic damage to Min6 cells and beneficial for cell proliferation,so 12.5,50,100 and 200μmol/L Rub were selected for later experimental validation.(2)Compared with the control group,the Min6 cells in the model group showed volume was shrunk,synapses were shortened,the connection to the surrounding cells disappeared,and presented a nearly "circular" state;the rate of early apoptosis and total apoptosis were increased(P<0.01);the m RNA expression of Bcl-2/Bax was decreased,while the m RNA expression of Caspase 3 was increased(P<0.01);the protein expression of Cyto-c,Cleared-Casease 9,Caspase9,Cleared-Casease 3,and Caspase 3 were increased(P<0.01).Compared with the model group,after Rub intervention,different degrees of improvement in cell morphology;the rate of early apoptosis and total apoptosis were significantly decreased(P<0.01);the m RNA expression of Bcl-2/Bax was increased(P<0.01),while the m RNA expression of Caspase 3 was decreased(P<0.05 or P<0.01);the protein expression of Cyto-c,Cleared-Casease 9,Caspase 9,Cleared-Casease 3,and Caspase 3 were downregulated(P<0.05 or P<0.01),and the above results were concentration dependent.(3)Compared with the control group,m RNA expression of INS-1 and cellular insulin secretion level after high glucose stimulation were significantly reduced in the model group(P<0.01);m RNA expression of IRS-1,PI3 K and Akt were significantly decreased(P<0.01);protein expression of IRS-1,PI3 K and p-Akt/Akt were significantly decreased(P<0.01).Compared with the model group,the above results were reversed by Rub(P<0.05 or P<0.01).In summary,the conclusion is as follows:(1)Rub could significantly alleviate the apoptosis of Min6 cells induced by Lipotoxicity,and the molecular mechanism may be related to the mitochondrial pathway of apoptosis.(2)Rub could improve the insulin secretion function and enhance insulin secretion level of Min6 cells by inhibiting their apoptosis.(3)The protective effect of Rub on lipotoxic Min6 cells may be correlated with the IRS-1/PI3K/Akt pathway. |
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