Effect of Inhibition of Autophagy on Protection of β-Cell Damage by Cyanidin-3-O-Glucoside in High Glucose and High Fat Environment
Objective:To investigate the effects of cyanidin-3-O-glucoside(C3G)on islet beta cell injury and its mechanism in high glucose and high fat(HGHF)environment.Methods:The pancreatic β cells were treated with C3G(10,20,30,40,50 μmol/L)for 24 h,and the cell viability was detected by CCK-8 assay.The HGHF environment was established by using the culture medium containing 25 mmol/L glucose and 0.5 mmol/L palmitate,and C3G(10,20,30,40,50 μmol/L)was added to treat the pancreat-ic β cells for 24 h.The cell viability was detected by CCK-8 assay.The experiment was divided into four groups:control group,HGHF group,HGHF+C3G group,and HGHF+C3G+3-MA group.After the corre-sponding treatment,CCK-8 assay was used to detect the cell viability of each group.DCFH-DA fluorescent probe was used to detect the ROS level of each group.The levels of malondialdehyde(MDA),superoxide dis-mutase(SOD),and glutathione(GSH)in each group were detected by colorimetric assay.The insulin secre-tion of each group was determined by ELISA assay.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the mRNA expression levels of autophagy-related genes 5(Atg5),microtubule-associated protein light chain 3(LC3),and Beclin-1 in each group.Western blot was used to detect the protein expres-sion levels of LC3-Ⅱ/LC3-Ⅰ,Beclin-1,Bcl-2-associated X protein(Bax),B-cell lymphoma 2(Bcl-2),and activated caspase-3(Cleaved Caspase-3)in each group.Results:Compared with the control group,the cell viability of the C3G group(10,20,30,40,50 μmol/L)was increased(P<0.05),and the cell viability of the HGHF group was decreased(P<0.05).Compared with the HGHF group,the addition of 20,30,40,50 μmol/L C3G in HGHF environment could significantly increase the cell viability(P<0.05).Compared with the HGHF group,the cell viability of the HGHF+C3G group was increased(P<0.05),the ROS content and MDA content were decreased(P<0.05),the SOD activity and GSH-Px content were in-creased(P<0.05),the insulin secretion level was increased(P<0.05),the mRNA relative expression levels of Atg5,LC3,and Beclin-1 were up-regulated(P<0.05),the LC3-Ⅱ/LC3-Ⅰ ratio and Beclin-1 protein relative expression levels were up-regulated(P<0.05),the Bax and Cleaved Caspase-3 protein relative ex-pression levels were down-regulated,and Bcl-2 protein relative expression level was up-regulated(P<0.05).However,when C3G was added to the HGHF environment and 3-methyladenine(3-MA),an autoph-agy inhibitor,was also used,the protective effect of C3G on pancreatic β cells in HGHF environment disap-peared.Conclusion:C3G has a protective effect on pancreatic β cells in HGHF environment.It can improve cell viability,inhibit oxidative stress damage,and this effect may be related to the promotion of autophagy.
Islet beta cellsHigh glucose and high fatCyanidin-3-O-glucosideAutophagyApoptosis
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