小檗碱靶向HDAC/H3K9ac/KLF4抑制棕榈酸诱导的HepG2脂肪变性体外实验研究  

Berberine suppresses hepatic steatosis by activation of KLF4 via targeting HDAC/H3K9ac in palmitic acid-induced HepG2 cells in vitro

作　　者：李经纬 王子璇 王梦雨 黄瑞贤 信丰智 范建高 汪保灿 Li Jingwei;Wang Zixuan;Wang Mengyu(Department of Gastroenterology,Xinhua Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200092,China)

机构地区：[1]上海交通大学附属新华医院消化内科,上海市200092

出　　处：《实用肝脏病杂志》2021年第6期790-794,共5页Journal of Practical Hepatology

基　　金：上海市新华医院基础研究计划项目(编号:17YJJ27)。

摘　　要：目的探讨小檗碱(BBR)调控HDAC/H3K9ac/KLF4对棕榈酸(PA)诱导的非酒精性脂肪性肝病(NAFLD)细胞模型的保护作用及其可能的机制。方法应用PA诱导HepG2细胞构建NAFLD细胞模型,采用油红O染色法测定细胞脂肪变,采用Western blot法检测细胞HDAC1、H3K9ac、KLF4、SREBP-1c和PPARγ表达,使用染色质免疫沉淀技术(ChIP)测定KLF4启动子区H3K9ac水平,采用ELISA检测细胞培养上清细胞因子,采用SiRNA技术沉默KLF4基因验证BBR靶向HDAC/H3K9ac/KLF4对PA诱导的HepG2细胞脂肪变的保护作用。结果与模型组比,BBR处理使PA诱导的HepG2细胞脂质沉积显著改善,培养上清TNF-α、IL-1β和IL-6水平分别为(514.7±46.4)pg/ml、(241.5±37.7)pg/ml和(362.7±19.9)pg/ml,均显著低于模型组【分别为(1162.0±110.8)pg/ml、(635.8±73.4)pg/ml和(1110.0±85.1)pg/ml,P<0.001】;与模型组比,BBR干预组HDAC1蛋白表达降低了19.0%,而H3K9ac和KLF4蛋白表达增加了1.53倍和1.52倍,KLF4启动子区H3K9ac水平增加了3.97倍,脂质合成相关基因SREBP-1c蛋白表达降低了49.1%,脂质分解相关基因PPARγ蛋白表达增加了1.84倍;与空质粒转染组比,转染真核表达质粒细胞HDAC1蛋白表达水平增加了2.02倍,而H3K9ac蛋白表达水平降低了57.1%;与SiRNA-NC转染组比,转染SiRNA-KLF4的HepG2细胞KLF4蛋白表达水平显著下降(P<0.01),而细胞脂质沉积显著增加,培养上清TNF-α、IL-1β和IL-6水平分别为(887.9±89.9)pg/ml、(471.9±38.4)pg/ml和(793.1±59.3)pg/ml,均显著高于SiRNA-NC转染组【分别为(534.2±46.1)pg/ml、(260.3±26.9)pg/ml和(372.0±30.6)pg/ml,P<0.01】,脂质合成相关基因SREBP-1c蛋白表达增加了1.77倍,而脂质分解相关基因PPARγ蛋白表达降低了41.6%。结论本研究结果提示BBR可能通过调节PA诱导的HepG2细胞HDAC/H3K9AC表达,激活了KLF4,抑制了细胞内炎症反应和脂质沉积,为临床干预NAFLD提供了新的思路。Objective This experiment aimed to investigate the protective effects and mechanisms of berberine(BBR)on hepatic steatosis by activation of KLF4 via targeting HDAC/H3K9ac in palmitic acid(PA)-induced HepG2 cells in vitro.Methods HepG2 cells were induced by PA to construct hepatocyte steatosis model.The fat deposition was determined by oil red O staining.The cellular HDAC1,H3K9ac,KLF4,SREBP-1c and PPARγ expression was assayed by Western blot.The H3K9Ac in the KLF4 promoter region was determined by chromatin immunoprecipitation(ChIP).The supernatant cytokine levels were identified by ELISA kits.The KLF4 gene was silenced by siRNA to verify the protective effects of BBR by targeting HDAC/H3K9Ac/KLF4 in PA-induced HepG2 cells.Results The BBR treatment significantly improved lipid deposition in PA-induced HepG2 cells compared to that in model cells,and the supernatant TNF-α,IL-1β and IL-6 levels were(514.7±46.4)pg/ml,(241.5±37.7)pg/ml and(362.7±19.9)pg/ml,significantly lower than[(1162.0±110.8)pg/ml,(635.8±73.4)pg/ml and(1110.0±85.1)pg/ml,respectively,P<0.001]in the model;the expression of HDAC1 protein in BBR-intervened group decreased by 19.0%,while the expression of H3K9ac and KLF4 protein increased by 1.53 and 1.52 times,the level of H3K9ac in KLF4 promoter region increased by 3.97 times,and the expression of lipid synthesis related gene SREBP-1c protein decreased by 49.1%,the expression of lipid decomposition related gene PPARγprotein increased 1.84 times compared to in the model cells;compared with empty plasmid transfection,the expression level of HDAC1 protein in cells transfected with eukaryotic plasmids was increased by 2.02 times,and the expression level of H3K9Ac protein was decreased by 57.1%;compared with SiRNA-NC transfection,the KLF4 protein expression level in HepG2 cells after SiRNA-KLF4 transfection was significantly decreased(P<0.01),lipid deposition was significantly increased,the levels of TNF-α,IL-1βand IL-6 were(887.9±89.9)pg/ml,(471.9±38.4)pg/ml and(793.1±59.3)pg/ml,significantl

关 键 词：HepG2细胞 小檗碱 组蛋白去乙酰化酶 组蛋白H3第9位赖氨酸乙酰化 Krüppel样因子4 体外 

分 类 号：R285.5[医药卫生—中药学]