摘要
目的 探讨黄柏碱对高糖(HG)诱导的晶状体上皮细胞(LEC)自噬和凋亡的影响及机制.方法 将HLEB3细胞随机分为对照组(A组)、HG组(B组)、黄柏碱组(C组)、Compound C(AMPK抑制剂)组(D组).应用CCK-8法检测细胞活力,流式细胞术测定细胞凋亡,活性氧荧光探针(DCFH-DA)法检测细胞内活性氧(ROS)含量,ELISA试剂盒检测细胞内丙二醛(MDA)和超氧化物歧化酶(SOD)水平,Western blot法检测Bcl-2、Bax、Beclin-1、LC3-Ⅱ、LC3-Ⅰ、p-AMPK、AMPK、p-mTOR、mTOR 蛋白表达.结果 5 mg/L 为黄柏碱对 HLEB3 细胞最佳作用浓度.与A组比较,B组细胞活力、自噬体数量以及SOD、Beclin-1、LC3-Ⅱ/LC3-Ⅰ、Bcl-2、p-AMPK/AMPK水平显著降低(F=30.857～213.971,P＜0.001),细胞凋亡率及ROS、MDA、Bax、p-mTOR/mTOR水平显著升高(F=19.111～156.780,P＜0.001);与 B 组相比,C 组细胞活力、自噬体数量以及 SOD、Beclin-1、LC3-Ⅱ/LC3-Ⅰ、Bcl-2、p-AMPK/AMPK水平显著升高(P＜0.05),细胞凋亡率及ROS、MDA、Bax、p-mTOR/mTOR水平显著降低(P＜0.05);Compound C可逆转黄柏碱对HLEB3细胞自噬和凋亡的改善作用(P＜0.05).结论 黄柏碱能减少HG诱导的LEC氧化应激和凋亡,促进细胞自噬,其作用机制可能与调节AMPK/mTOR信号通路有关.
Abstract
Objective To investigate the effect of phellodendrine on the autophagy and apoptosis of lens epithelial cells(LECs)induced by high glucose(HG)and its mechanism.Methods HLEB3 cells were randomly divided into control group(group A),HG group(group B),phellodendrine group(group C),and Compound C(AMPK inhibitor)group(group D).CCK-8 assay was used to measure cell viability;flow cytometry was used to measure cell apoptosis;the DCFH-DA probe was used to measure the content of reactive oxygen species(ROS)in cells;ELISA kits were used to measure the levels of malondialdehyde(MDA)and superoxide dismutase(SOD);Western blot was used to measure the protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2 related X gene(Bax),Beclin-1,microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅰ),AMP-activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(mTOR),and phosphorylated mTOR(p-mTOR).Results The results showed that 5 mg/L phellodendrine was the best concentration for the culture of HLEB3 cells.Compared with group A,group B had significant reductions in cell viabi-lity,the number of autophagosomes,and the levels of SOD,Beclin-1,LC3-Ⅱ/LC3-Ⅰ,Bcl-2,and p-AMPK/AMPK(F=30.857-213.971,P＜0.001)and significant increases in cell apoptosis rate and the levels of ROS,MDA,Bax,and p-mTOR/mTOR(F=19.111-156.780,P＜0.001).Compared with group B,group C had significant increases in cell viability,the number of autophagosomes,and the levels of SOD,Beclin-1,LC3-Ⅱ/LC3-Ⅰ,Bcl-2,and p-AMPK/AMPK(P＜0.05)and significant re-ductions in cell apoptosis rate and the levels of ROS,MDA,Bax,and p-mTOR/mTOR(P＜0.05).The AMPK inhibitor Com-pound C could reverse the effect of phellodendrine in improving the autophagy and apoptosis of HLEB3 cells(P＜0.05).Conclu-sion Phellodendrine can reduce the oxidative stress and apoptosis of LEC induced by HG and promote autophagy,possibly by re-gulating the AMPK/mTOR signaling pathway.
维普
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