摘要
目的评价组蛋白去乙酰化酶3(HDAC3)在原代大鼠心肌细胞高糖缺氧复氧损伤中的作用及其与自噬的关系。方法将1~3 d左右的新生SD乳鼠提取原代心肌细胞,铺板。按随机数字表法分为5组(n=24):对照组(C组,糖浓度5.5 mmol/L)、缺氧复氧组(H/R组)、高糖组(H组,糖浓度30 mmol/L)、高糖缺氧复氧组(HH/R组)和高糖缺氧复氧+HDAC3抑制剂RGFP966组(HH/R+RG组)。采用50%葡萄糖注射液制备高糖培养基(终浓度为30 mmol/L)。H/R组细胞置于低氧环境(5%CO2-0.9%O2-94.1%N2)中培养6 h,再置于常氧环境中复氧2 h制备心肌细胞缺氧复氧模型。HH/R+RG组于缺氧复氧前24 h加入RGFP966,终浓度10μmol/L。于细胞复氧2 h时,采用CCK-8法检测细胞活力;ELISA法检测细胞上清LDH活性;自噬双标腺病毒(mRFP-GFP-LC3)转染下共聚焦显微镜检测细胞自噬水平;Western blot法检测心肌细胞HDAC3、p62、LC3Ⅱ和LC3Ⅰ的表达,计算LC3Ⅱ/LC3Ⅰ比值。结果与C组比较,H/R组和H组心肌细胞活力降低,上清液LDH活性升高,H/R组自噬小体数增加,心肌细胞HDAC3表达上调,p62表达下调,LC3Ⅱ/I比值升高,H组自噬小体数减少,心肌细胞HDAC3和p62表达上调,LC3Ⅱ/I比值降低(P<0.05);与H/R组比较,HH/R组心肌细胞活力降低,上清液LDH活性升高,自噬小体数量降低,HDAC3和p62表达上调,LC3Ⅱ/I比值降低(P<0.05);与H组比较,HH/R组心肌细胞活力降低,上清液LDH活性升高,自噬小体数量增加,HDAC3和p62表达上调,LC3Ⅱ/I比值升高(P<0.05);与HH/R组比较,HH/R+RG组心肌细胞活力升高,上清液LDH活性降低,自噬小体数量增加,HDAC3和p62表达下调,LC3Ⅱ/Ⅰ比值升高(P<0.05)。结论HDAC3表达上调参与了原代心肌细胞高糖缺氧复氧损伤的过程,与降低细胞自噬水平有关。
Objective To evaluate the role of histone deacetylase 3(HDAC3)in high glucose hypoxia/reoxygenation(H/R)injury to primary rat cardiomyocytes and the relationship with autophagy.Methods The primary cardiomyocytes extracted from newborn Sprague-Dawley rats,aged about 1-3 days,were divided into 5 groups(n=24 each)according to the random number table method:control group(C group,glucose concentration 5.5 mmol/L),H/R group,high glucose group(H group,glucose concentration 30 mmol/L),high glucose H/R group(HH/R group),and high glucose H/R+HDAC3 inhibitor RGFP966 group(HH/R+RG group).Fifty percent glucose injection was used to prepare high-glucose medium(final concentration 30 mmol/L).Cells were cultured in a hypoxic environment(5%CO2-0.9%O2-94.1%N2)for 6 h,followed by reoxygenation in a normoxic environment for 2 h to establish the cardiomyocyte H/R model in H/R group.RGFP966 at a final concentration of 10μmol/L was added at 24 h before H/R in HH/R+RG group.At 2 h of reoxygenation,the cell viability was measured using CCK-8 kit,the activity of lactic dehydrogenase(LDH)in the cell supernatant was determined using enzyme-linked immunosorbent assay,the level of autophagy was detected with a confocal microscope after cells were transfected with autophagy double-labeled adenovirus(mRFP-GFP-LC3),and the expression of HDAC3,p62,LC3Ⅱand LC3Ⅰwas detected using Western blot.LC3Ⅱ/LC3Ⅰratio was calculated.Results Compared with group C,the cell viability was significantly decreased,and the activity of LDH in supernatant was increased in H/R and H groups,the number of autophagosomes was significantly increased,the expression of HDAC3 in cardiomyocytes was up-regulated,the expression of p62 was down-regulated,and the LC3Ⅱ/I ratio was increased in group H/R,and the number of autophagosomes was significantly decreased,the expression of HDAC3 and p62 in cardiomyocytes was up-regulated,and the LC3Ⅱ/I ratio was decreased in group H(P<0.05).Compared with group H/R,the cell viability was significantly decreased,the activity of L
作者
邱珍
李璐
赵博
雷少青
夏中元
Qiu Zhen;Li Lu;ZhaoBo;Lei Shaoqing;Xia Zhongyuan(Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2021年第10期1252-1255,共4页
Chinese Journal of Anesthesiology
基金
国家自然科学基金(81970722)。
