咖啡酸苯乙基酯缓解L02细胞脂毒性机制研究

Mechanism of caffeic acid phenethyl ester protects L02 cells from palmitate-induced lipotoxicity in vitro

目的探讨咖啡酸苯乙基酯(CAPE)对棕榈酸酯诱导的L02细胞脂毒性的保护机制。方法取正常肝脏L02细胞和过氧化物酶体增殖激活受体共激活因子1α(PGC1α)敲除细胞,分别分为对照组、棕榈酸酯处理组和CAPE联合棕榈酸酯处理组,后两组分别给予终浓度300mM棕榈酸酯处理7 d或是联合终浓度10μM CAPE处理7 d。分别检测细胞甘油三酯(TG)、上清肿瘤坏死因子α(TNF-α)和白介素6(IL-6)水平,使用流式细胞术检测线粒体活性氧(ROS)水平,采用qPCR定量法检测PGC1α和过氧化物歧化酶2(SOD2)mRNA水平,采用Western-blot法检测PGC1α蛋白表达。结果棕榈酸酯处理组细胞TG含量为(16.92±1.43)mg/g protein,显著高于CAPE联合棕榈酸酯处理组【(10.53±0.81)mg/g protein,P<0.05】;棕榈酸酯处理组细胞上清TNF-α和IL-6水平分别为(117.6±3.72)pg/ml和(67.9±2.7)pg/ml,均显著高于CAPE联合棕榈酸酯处理组【分别为(74.88±3.37)pg/ml和(53.94±2.39)pg/ml,P<0.05】;棕榈酸酯处理组线粒体ROS水平为(1.7±0.06),显著高于CAPE联合棕榈酸酯处理组【(1.36±0.04),P<0.05】;CAPE联合棕榈酸酯处理组SOD2和PGC1αmRNA水平分别为(0.76±0.03)和(0.73±0.04),显著高于棕榈酸酯处理组【(0.55±0.05)和(0.57±0.03),P<0.05】;CAPE联合棕榈酸酯处理组PGC1α蛋白表达水平显著高于棕榈酸酯处理组;在敲除PGC1α后,CAPE联合棕榈酸酯处理组细胞TG含量为(23.73±1.95)mg/g protein,与棕榈酸酯处理组的(25.86±1.02)mg/g protein比,无显著性差异(P>0.05),上清TNF-α和IL-6水平分别为(128.33±4.41)pg/ml和(80.33±3.76)pg/ml,与棕榈酸酯处理组的(145.78±5.79)pg/ml和(87.23±4.85)pg/ml比,无显著性差异(P>0.05);线粒体ROS水平为(1.83±0.25),与棕榈酸酯处理组的(2.05±0.14)比,无显著性差异(P>0.05)。结论通过上调PGC1α信号通路,CAPE对棕榈酸酯诱导的肝脏L02细胞脂毒性具有有效的保护作用。

Objective The experiment aimed to study the protective effects and its mechanism of caffeic acid phenethyl ester(CAPE)on the lipotoxicity of hepatocytes in vitro.Methods Wild type L02 cells and peroxisome proliferator-activated receptor coactivator 1α(PGC1α)knockdown LO2 cells were respectively divided into control,palmitate-and palmitate plus CAPE-intervened groups.In palmitate-intervened group,the cells were cultured at 300 mM palmitate for 7 days,and in palmitate plus CAPE-intervened cells were cultured at 10μM CAPE and 300mM palmitate for 7 days.Cell triglycerides(TG),and tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels in the supernatants were detected.The mitochondrial reactive oxygen species(ROS)was measured by flow cytometry,the PGC1α and superoxide dismutase 2(SOD2)mRNA were detected by qPCR,and the PGC1α proteins were detected by Western blotting.Results The cell TG level in palmitate-intervened group was(16.92±1.43)mg/g protein,significantly higher than[(10.53±0.81)mg/g protein,P<0.05]in palmitate plus CAPE-intervened group;the TNF-αand IL-6 levels in supernatants were(117.6±3.72)pg/ml and(67.9±2.7)pg/ml,both significantly higher than[(74.88±3.37)pg/ml and(53.94±2.39)pg/ml,respectively,P<0.05]in palmitate plus CAPE-intervened group;the ROS level was(1.7±0.06),much higher than[(1.36±0.04),P<0.05]in palmitate plus CAPE-intervened group;the SOD2 and PGC1α mRNA levels in palmitate plus CAPE-intervened group were(0.76±0.03)and(0.73±0.04),significantly higher than[(0.55±0.05)and(0.57±0.03),P<0.05]in palmitate-intervened group;the PGC1α protein expression was more strong than in palmitate-intervened group.After PGC1αknockdown,the cell TG level in palmitate plus CAPE-intervened knockdown L02 cells was(23.73±1.95)mg/g protein,not significantly different compared to(25.86±1.02)mg/g protein in palmitate-intervened cells(P>0.05),supernatant TNF-αand IL-6 levels were(128.33±4.41)pg/ml and(80.33±3.76)pg/ml,not significantly different compared to(145.78±5.79)pg/ml and(87.23±4.85)pg/ml in palmitate-intervened cells(P>0.05);the mitochondrial ROS level was(1.83±0.25),also not significantly different compared to(2.05±0.14)in palmitate-intervened group(P>0.05).Conclusion The CAPE could effectively protect the L02 cells from lipotoxicity,which might be through PGC1α pathway activation.