CAJ | 학술논문

目的了解N-乙酰半胱氨酸(NAC)对内质网氧化应激介导的肝细胞凋亡的阻抑作用.探讨其治疗肝细胞损伤的作用机制.方法用毒胡萝卜素(TC)诱导HepG2细胞,建立内质网应激凋亡模型,用NAC进行干预.噻唑蓝(MTT)、流式细胞仪、DNA梯形电泳检测凋亡率及活性氧(ROS),Western印迹检测葡萄糖调节蛋白(GRP)78、Caspase-12酶原及ADP聚合酶(PARP)表达.多样本间均数采用方差分析.结果用2μmol/L TG诱导HepG2细胞0、24、36和48 h,随诱导时间延长,细胞活力逐渐下降;GRP 78、Caspase-12酶原及PARP表达增高;凋亡率逐渐增加.分别是0.7%±0.5%、27.6%±6.3%、29.7%±3.03%和47.9%±3.5%(P<0.05);ROS产生逐渐增加,分别是14.0%±0.5%、36.1%±300%、38.2%±6.0%和48.3%±12.4%(P<0.05);诱导36、48 h后细胞出现典型DNA梯形条带.10 mmol/L、20 mmol/L NAC分别与2/μmol/L TG共同温育HepG2细胞后发现,NAC可明显提高细胞活力;抑制GRP 78、Caspase-12酶原及PARP表达,细胞凋亡率分别降至14.0%±1.3%和11.0%±0.3%;细胞内ROS的产生减至34.7%±0.8%与31.5%±2.9%.结论 TG作为一种内质网特异的钙离子ATP酶抑制剂,能触发HepG2细胞内质网氧化应激凋亡;而NAC作为巯基合成的前体.直接抑制氧自由基反应,阻断内质网氧化应激介导的细胞凋亡,减轻肝细胞损伤,达到临床治疗肝功能衰竭的作用.
목적 료해N-을선반광안산(NAC)대내질망양화응격개도적간세포조망적조억작용.탐토기치료간세포손상적작용궤제.방법 용독호라복소(TC)유도HepG2세포,건립내질망응격조망모형,용NAC진행간예.새서람(MTT)、류식세포의、DNA제형전영검측조망솔급활성양(ROS),Western인적검측포도당조절단백(GRP)78、Caspase-12매원급ADP취합매(PARP)표체.다양본간균수채용방차분석.결과 용2μmol/L TG유도HepG2세포0、24、36화48 h,수유도시간연장,세포활력축점하강;GRP 78、Caspase-12매원급PARP표체증고;조망솔축점증가.분별시0.7%±0.5%、27.6%±6.3%、29.7%±3.03%화47.9%±3.5%(P<0.05);ROS산생축점증가,분별시14.0%±0.5%、36.1%±300%、38.2%±6.0%화48.3%±12.4%(P<0.05);유도36、48 h후세포출현전형DNA제형조대.10 mmol/L、20 mmol/L NAC분별여2/μmol/L TG공동온육HepG2세포후발현,NAC가명현제고세포활력;억제GRP 78、Caspase-12매원급PARP표체,세포조망솔분별강지14.0%±1.3%화11.0%±0.3%;세포내ROS적산생감지34.7%±0.8%여31.5%±2.9%.결론 TG작위일충내질망특이적개리자ATP매억제제,능촉발HepG2세포내질망양화응격조망;이NAC작위구기합성적전체.직접억제양자유기반응,조단내질망양화응격개도적세포조망,감경간세포손상,체도림상치료간공능쇠갈적작용.
Objective To analyze the effect of N-acetyl-L-cysteine(NAC)on endoplasmic reticulum stress mediated HepG2 cells apoptosis and evaluate the role of NAC in the treatment of liver injury.Methods HepG2 cells were treated with thapsigargin(TG)to establish the model of oxidative endoplasmic reticulum stress mediated apoptosis,and NAC was used to intervene in apoptosis.To evaluate the apoptosis,various methods such as MTT assay,flow cytometry,DNA ladder and Western blot were performed.Results After treated with 2 μmol/L TG for 0,24,36 and 48 hours,the vitality of HepG2 cells decreased.The ratio of apoptotic cells increased along with the prolonged treatment duration of TG(0.7%±0.5%,27.6%±6.3%,29.7%±3.3%,47.9%±3.5% respectively,P<0.05),and the production of reactive oxygen species(ROS)also increased in time-dependent manner(14.0%±0.5%,36.1%±3.0%,38.2%±6.0%,48.3%±12.4%,P<0.05).The HepG2 cells showed typical morphologic change of endoplasmic retieulum stress induced by 2 μmol/L TG after 36 h and 48 h.DNA ladder was observed at the same concentration and time point correspondingly.Endoplasmic reticulum stress mediated-apoptosis was confirmed by Western blot.Both 10 mmol/L and 20 mmol/L NAC could protect ceils from apoptosis.The ratio of apoptotic cells decreased to 14.0%±1.3% and 11.0%±0.3%,respectively.The production of ROS decreased to 34.7%±0.8% and 31.5%±2.9%,respectively.The effect was related to the concentration of NAC.Conclusions As a Ca2+-adenosine triphoshatase inhibitor,TG may disrupt intracellular calcium homeostasis,which can induce endoplasmie reticulum stress and apoptosis.NAC,the precursor of the synthesis of-SH,can directly inhibit the ROS reaction and alleviate liver damage,which may play a role in the treatment of liver failure.