摘要
目的:探究高糖高脂(HGHF)环境下矢车菊素-3-O-葡萄糖苷(C3G)对胰岛β细胞损伤的影响及作用机制。方法:采用含10、20、30、40、50μmoL/L C3G的培养液处理胰岛β细胞24h,以未经处理的胰岛β细胞作为对照组,CCK-8法检测各处理组细胞活力;使用含25mmoL/L葡萄糖与0.5mmoL/L棕榈酸的培养液建立HGHF环境,并添加含10、20、30、40、50μmoL/L C3G处理胰岛β细胞24h,以未经处理的胰岛β细胞作为对照组,25mmoL/L葡萄糖与0.5mmoL/L棕榈酸诱导的胰岛β细胞作为HGHF组,CCK-8法检测各处理组细胞活力。实验分为对照组、HGHF组、HGHF+C3G组、HGHF+C3G+3-MA组,进行相应处理后,CCK-8法检测各组细胞活力,DCFH-DA荧光探针检测各组细胞活性氧(ROS)水平,比色法检测各组细胞丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)水平,ELISA法测定各组细胞胰岛素分泌情况,实时荧光定量PCR(qRT-PCR)法检测各组细胞中自噬相关基因5(Atg5)、微管相关蛋白轻链3(LC3)、Beclin-1的mRNA表达水平,蛋白质免疫印记(Western blot)测定各组细胞中LC3-Ⅱ/LC3-Ⅰ、Beclin-1及Bcl-2关联X蛋白(Bax)、B细胞淋巴瘤2(Bcl-2)、活化型半胱天冬酶-3(Cleaved Caspase-3)的蛋白表达水平。结果:与对照组比较,不同浓度(10、20、30、40、50μmoL/L)C3G组胰岛β细胞活力升高(P<0.05),HGHF组胰岛β细胞活力下降(P<0.05);与HGHF组比较,在HGHF环境下加入20、30、40、50μmoL/L C3G能够显著提高胰岛β细胞活力(P<0.05)。与HGHF组比较,HGHF+C3G组细胞活力升高(P<0.05),ROS含量和MDA含量下降(P<0.05),SOD活性和GSH-Px含量上升(P<0.05),胰岛素分泌水平增加(P<0.05),Atg5、LC3、Beclin-1的mRNA相对表达量上调(P<0.05),LC3-Ⅱ/LC3-Ⅰ比值和Beclin-1蛋白相对表达量上调(P<0.05),Bax、Cleaved Caspase-3的蛋白相对表达量下调且Bcl-2蛋白相对表达量上调(P<0.05)。而在HGHF环境下添加C3G处理的同时使用自噬抑制剂3-甲基腺嘌呤(3-MA)作用,C3G对HGHF环境下胰岛β细胞的保护作用消失。结论:C3G对HGHF环境下的胰岛β细胞起到保护作用,其能够提高细胞活力,抑制氧化应激损伤,该作用可能与促进自噬相关。
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 pancreaticβcells were treated with C3G(10,20,30,40,50μmol/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(10,20,30,40,50μmol/L)was added to treat the pancreaticβcells 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 corresponding 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 dismutase(SOD),and glutathione(GSH)in each group were detected by colorimetric assay.The insulin secretion 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 expression 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(10,20,30,40,50μmol/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 20,30,40,50μmol/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 increased(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-Ⅰratio and Beclin-1 protein relative expression levels were up-regulated(P<0.05),the Bax and Cleaved Caspase-3 protein relative expression 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 autophagy inhibitor,was also used,the protective effect of C3G on pancreaticβcells in HGHF environment disappeared.Conclusion:C3G has a protective effect on pancreaticβcells in HGHF environment.It can improve cell viability,inhibit oxidative stress damage,and this effect may be related to the promotion of autophagy.
作者
李恒
张翠兰
LI Heng;ZHANG Cuilan(Hainan Western Central Hospital,Hainan Danzhou 571700,China)
出处
《河北医学》
CAS
2024年第2期182-188,共7页
Hebei Medicine
基金
海南省卫生健康行业科研项目,(编号:22A200171)。
