| Background and Objective:Type 2 diabetes mellitus(T2DM)with a high morbidity worldwide is a kind of chronic metabolic disease,bringing serious economic burden to the society.Therefore,it is urgent to find the safe and effective drugs to treat T2 DM.Recently,numous studies have shown that Berberine(BBR)has multiple functions,including reducing plasma glucose,regulating blood lipid,improving the insulin resistance and inhibiting intestinal glucose absorption.AMP activated protein kinase(AMPK)is considered as one of the most important upstream molecules of BBR exerting the resistance of oxidative stress,while it remains unknown whether BBR activates AMPK expression in cells.Recent studies have indicated that the down-regulation or deletion of phosphatase and tensin homolog(PTEN)can improve the glucose metabolism of insulin target organs and inhibit the occurrence of diabetes in mice.Moreover,the specific deletion of PTEN in the pancreas can inhibit the occurrence of streptozocin-induced diabetes and reduce the apoptosis of β cells.However,the specific mechanism remains unclear.Thus,the aim of this study was to investigate the proliferation,apoptosis,oxidative stress,AMPK,PTEN and Nrf2 expressions change of βTC6 cells via palmic acid(PA)-induced the injure of βTC6 cells.Methods:Part 1: The islet βTC6 cells were cultivated and then treated with different concentrations of PA(0.2,0.4,0.8,1.0 mmol/L)and BBR(0.001,0.01,0.1,1.0,10,100umol/L),respectively.Afterwards,the apoptosis of βTC6 cells were determined by MTT assay.Part 2: The islet βTC6 cells were treated with 1 mmol/L of PA(PA group)and 0.1 mol/L of BBR(BBR group),respectively.Meanwhile,the normal βTC6 cells without any treatment were regarded as the control(Con group).Expression levels of related signal transcription factors AMPK,PTEN and Nrf2 in Con,PA and BBR treatment groups were detected by immunofluorescence and Western blotting.The apoptosis of islet βTC6 cells in each treatment group was detected by flow cytometry.Part 3: The islet βTC6 cells were treated with various treatments and divided into the following groups: Con group,the normal βTC6 cells without any treatment;NC and siRNA group: the βTC6 cells were transfected with the scrambled siRNA and siRNA-PTEN,respectively;PA group: the βTC6 cells were treated with 1 mmol/L of PA;NC+PA group: the βTC6 cells were transfected with the scrambled siRNA and treated with 1 mmol/L of PA;siRNA+PA group: the βTC6 cells were transfected with siRNA-PTEN and treated with 1 mmol/L of PA;PA+BBR group: the βTC6 cells were treated with 1 mmol/L of PA and 0.1 μmol/L BBR;NC+PA+BBR group: the βTC6 cells were transfected with the scrambled siRNA and treated with 1 mmol/L of PA as well as 0.1 μmol/L BBR;siRNA+PA+BBR group: the βTC6 cells were transfected with the siRNA-PTEN and treated with 1 mmol/L of PA as well as 0.1 μmol/L BBR.RT-PCR was applied to detect the silencing efficiency of PTEN.The proliferation of βTC6 cells in each group was estimated by MTT assay.The apoptosis of βTC6 cells in each group was detected by flow cytometry.The expressions of AMPK,PTEN,Nrf2 mRNA and protein in each group were detected by RT-PCR and Western blotting respectively.The contents of caspase3,nitrotyrosine(NT)and IL-6 in each group were detected by ELISA.ROS content in each group was determined by fluorescent probe.Results: Part 1: 1.MTT assay showed that the survival rate of βTC6 cells in the 1.0 mol/L of PA group was the lowest.2.MTT assay showed that the survival rate of βTC6 cells in the 0.1 mol/L of BBR group was the highest.Part 2: 1.Immunofluorescence showed that no significant changes were observed in AMPKa expression between the PA and con groups(P>0.05),while the expression of PTEN and Nrf2 was significantly increased(P<0.05).After the treatment of BBR,there was no significant changes in the AMPKa expression between the PA and BBR groups(P>0.05),while the PTEN and Nrf2 expression was dramatically declined(P<0.05).2.Western blotting showed that no significant changes were observed in AMPKa protein expression between the PA and con groups(P>0.05),while the PTEN and Nrf2 protein expression was obviously increased in the PA group compared with that in the con grou After the treatment of BBR,there was no significant changes in the AMPKa expression between the PA and BBR groups(P>0.05),while the PTEN and Nrf2 protein expression was obviously decreased in the BBR group compared with that in the PA group(P<0.05).3.Flow cytometry revealed that compared with the con group,the apoptosis rate of βTC6 cells was significantly rised in the PA group(P<0.05).Furthermore,the βTC6 cells apoptosis rate was obviously declined in the BBR group compared with that in the PA group(P<0.05).Part 3: 1.RT-PCR and Western blotting results showed that the mRNA and protein levels of PTEN were significantly decreased after transfection of siRNA-1,siRNA-2 and siRNA-3 compared with the NC group(P<0.05).In particular,the most significant silencing efficiency was observed in the transfection of siRNA-1.2.MTT assay presented that there was an upward trend in the survival rate of βTC6 cells treated with PA or BBR after the knockdown of PTEN.3.Flow cytometry and ELISA assay of caspase-3 showed that compared with the Con and NC group,PTEN knockdown had no significant effect on caspase-3 expression and βTC6 cells apoptosis(P>0.05).Compared with the PA and NC+PA groups,the caspase-3 expression was obviously declined after the PTEN knockdown(P<0.05).4.RT-PCR and Western blotting showed that the mRNA and protein expressions of Nrf2 in the siRNA+PA group and the siRNA+PA+BBR group were significantly increased after the knockdown of PTEN(P<0.05).There was no significant changes in the expression of AMPK mRNA and protein(P>0.05).5.Fluorescence probe method presented that the ROS content in the βTC6 cells was dramatically decreased after the knockdown of PTEN.6.ELISA results showed that the NT content in the βTC6 cells was obviously decreased after the knockdown of PTEN(P<0.05).7.Animal experiments showed that there was a downward trend in the plasma glucose of diabetic mice in the BBR compared with that in the Con group and reduce the inflammatory response of diabetic mice.8.ELISA results showed that the IL-6 expression in the DM and MET groups was significantly higher than that in the Con group(P<0.05).However,the IL-6 expression in the BBR group was dramatically lower than that in the DM group(P<0.05).Similarly,compared with the MET group,the IL-6 expression was obviously declined in the BBR+MET group(P<0.05).Conclusion: BBR plays a protective role against βTC6 in islets by down-regulating PTEN.On the one hand,PTEN down-regulation can inhibit the apoptosis of βTC6 cells and promote βTC6 cells proliferation.On the other hand,the inhibition of the PTEN expression promote the expression of Nrf2,thus making the βTC6 cells have the effect of anti-oxidative stress.Besides,BBR treatment relieves the inflammatory response of diabetic mice and reduces the plasma glucose level of diabetic mice,suggesting that BBR has the potential to treat T2 DM. |
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