组蛋白去乙酰化酶3(HDAC3)抑制剂通过抑制核因子kB减轻乙醇诱导的人肠上皮Caco-2细胞损伤

Histone deacetylase 3 inhibitor alleviates alcohol-induced disruption of intestinal epithelial barrier via inhibiting nuclear factor kB

目的研究组蛋白去乙酰化酶3(HDAC3)在酒精诱导的肠上皮细胞炎症和通透性中的作用和机制。方法培养Caco-2细胞达到分化,采用(10、25、50、100.200)mmol/L乙醇对其进行处理,通过廛哇蓝(MTT法)检测乙醇对细胞活性的影响,实时定量PCR检测Caco-2细胞HDAC3 mRNA水平,Western blot法检测其蛋白水平,从中筛选出适宜的乙醇浓度。将分化的Caco-2细胞分为对照组、乙醇组(50 mmol/L乙醇处理60 min)和RGFP966预处理组(乙醇处理前用2 p.mol/L HDAC3抑制剂RGFP966预处理1 h)。EUSA检测培养的细胞上清液中肿瘤坏死因子α(TNF-α)的含量,电阻仪检测跨上皮电阻值(TER),Western blot法检测细胞中闭合蛋白(occludin)、密封蛋白1(claudin-1)以及磷酸化的核因子kBp65(p-NF-i<Bp65)、核因子kB抑制蛋白(IkB)的蛋白水平。结果(10、25、50)mmol/L乙醇对细胞活力无明显影响,这些浓度的乙醇处理后HDAC3 mRNA和蛋白表达均升高,且具有剂量依赖性。与对照组相比,乙醇处理组细胞TNF-α含量和p-NF-KBp65蛋白水平增加,TER值与occludin、claudin-1和IkB蛋白水平均降低;与乙醇处理组相比,RGFP966预处理组细胞TNF-α含量和p-NF-KBp65蛋白水平均降低,TER值与occludin、claudin-1和IkB蛋白水平均增加。结论抑制HDAC3能够减弱乙醇诱导的肠上皮细胞炎症和通透性,可能是通过抑制NF-kB的活化发挥作用的。

Objective To investigate the role and mechanism of histone deacetylase 3(HDAC3)in alcohol-induced inflammation and permeability of intestinal epithelial cells.Methods To select the proper concentration of alcohol,differentiated Caco-2 cells were treated with different concentrations(10,25,50,100 and 200 mmol/L)of alcohol,and then cell viability was assayed by MTT assay;the mRNA and protein levels of HDAC3 were analyzed by real-time PCR and Western blot analysis.Differentiated Caco-2 cells were divided into three groups:control group,alcohol group(treatment with 50 mmol/L alcohol for 60 minutes),and alcohol combined with HDAC3 inhibitor group(pretreatment with 2 pmol/L RGFP9661 hour before alcohol).ELISA was performed to detect tumor necrosis factorα(TNF-α)level in cell supernatant.Transepithelial electrical resista nee(TER)was measured using a resista nee meter.Western blot analysis was used to determine the protein levels releva nt to tight ju nction(occludi n and claudin-1)and NF-kB activatio n(IkB and phosphorylated NF-KBp65).Results Alcohol at 10,25 and 50 mmol/L did not affect cell viability.The mRNA and protein expression levels of HDAC3 increased in a dose-dependent manner after alcohol treatment at these concentrations.Compared with the control group,TNF-αand phosphorylated NF-kBp65 levels in creased,whereas TER and protein levels of occludin,claudin-1 and IkB decreased in the alcohol group.Compared with the alcohol group,TNF-αand phosphorylated NF-KBp65 levels were reduced,while TER and protein levels of occludin,claudin-1 and IkB were elevated in the alcohol combined with HDAC3 inhibitor group.Conclusion HDAC3 in hibiti on can attenuate alcohol-induced in flammation and permeability of intestinal epithelial cells,which may be related to the inactivation of NF-kB.