摘要
目的探讨蛋白激酶Cβ2(protein kinase Cβ2,PKCβ2)在高糖与缺氧/复氧(hypoxia/reoxygenation,HR)诱导的心肌细胞焦亡中的作用。方法将培养的H9c2心肌细胞按随机数字表法分为5组(每组6孔):低糖组(LG组)、低糖缺氧/复氧组(LG+HR组)、高糖组(HG组)、高糖缺氧/复氧组(HG+HR组)、高糖缺氧/复氧+PKCβ2抑制剂CGP53353(1μmol/L)治疗组(HG+HR+CGP组)。采用三气培养箱(5%CO_(2)、94%N_(2)、1%O_(2))缺氧4 h,正常氧浓度复氧2 h构建细胞HR模型,细胞计数试剂盒(cell counting kit‑8,CCK8)检测细胞存活率,ELISA法检测乳酸脱氢酶(lactate dehydrogenase,LDH)水平,Western blot法检测细胞PKCβ2及焦亡相关蛋白Nod样受体蛋白‑3(Nod‑like receptor pyrin domain3,NLRP3)、IL‑1β、半胱氨酸蛋白酶‑1(caspase‑1)表达水平,JC‑1染色评估H9c2细胞线粒体膜电位水平。结果与LG组比较,HG组、LG+HR组、HG+HR组细胞存活率明显降低而LDH释放水平明显增加(P<0.05),同时伴随PKCβ2活化及NLRP3、IL‑1β、caspase‑1表达上调(P<0.05);与HG组比较,HG+HR组LDH释放水平增加,细胞存活率明显降低(P<0.05),JC‑1单体细胞百分比及磷酸化PKCβ2[phospho‑PKCβ2(Ser660),P‑PKCβ2]蛋白、NLRP3、IL‑1β、caspase‑1表达上调(P<0.05);与LG+HR组比较,HG+HR组LDH释放水平增加,细胞存活率明显降低(P<0.05),P‑PKCβ2表达明显增加,NLRP3、IL‑1β、caspase‑1表达上调(P<0.05)。与HG+HR组比较,HG+HR+CGP组细胞存活率明显增加,LDH释放水平、JC‑1单体细胞百分比、P‑PKCβ2、NLRP3、IL‑1β、caspase‑1表达下调(P<0.05)。结论选择性抑制PKCβ2过度活化可减轻心肌细胞高糖HR性损伤,其机制可能与降低细胞焦亡水平及减轻线粒体损伤有关。
Objective To investigate the effects of protein kinase Cβ2(PKCβ2)in the pyroptosis of cardiac myocytes induced by hypoxia/reoxygenation(HR).Methods H9c2 myocardial cells were cultured and divided into the five groups according to the random number table method(n=6):a low‑glucose(LG)group,a low‑glucose and hypoxia reoxygenation(LG+HR)group,a high‑glucose(HG)group,a high‑glucose and hypoxia reoxygenation(HG+HR)group and a hyperglycemic reoxygenation and PKCβ2 inhibitor CGP53353(1μmol/L)(HG+HR+CGP)group.A cell model of HR was established through hypoxia in a three‑gas incubator(5%CO_(2),94%N_(2),and 1%O_(2))for 4 h followed by reoxygenation at a normal oxygen concentration for 2 h.The cell viability was evaluated by cell counting kit 8(CCK8)and the level of lactate dehydrogenase(LDH)was determined by enzyme‑linked immunosorbent assay(ELISA).The levels of PKCβ2,Nod‑like receptor pyrin domain3(NLRP3),interleukin‑1β(IL‑1β)and caspase‑1 were detected by Western blot,while the mitochondrial membrane potential of H9c2 cells was evaluated by JC‑1 staining.Results Compared with the LG group,the HG group,the LG+HR group and the HG+HR group showed significantly decreased cell survival rates but increased LDH release(P<0.05),as well as up‑regulation in phosphorylated PKCβ2 and the expression of NLRP3,IL‑1βand caspase‑1(P<0.05).Compared with the HG group,the HG+HR group showed increased LDH release and a decreased cell survival rate(P<0.05),as well as up‑regulation in JC‑1 cell percentage and the expression of phosphorylated PKCβ2[phospho‑PKCβ2(Ser660),P‑PKCβ2]protein,NL‑RP3,IL‑1β,and caspase‑1(P<0.05).Compared with the LG+HR group,the HG+HR group showed increased LDH release and a de‑creased cell survival rate(P<0.05),as well as significantly increased expression of P‑PKCβ2 and up‑regulated expression of NLRP3,IL‑1βand caspase‑1(P<0.05).Compared with the HG+HR group,the HG+HR+CGP group presented a significantly increased survival rate and decreased LDH release,as well as down‑regulation in JC‑1 cell percentage and the expression of P‑PKCβ2,NLRP3,IL‑1βand caspase‑1(P<0.05).Conclusions Selective inhibition of PKCβ2 overactivation can relieve hyperglycemic HR injury in cardio‑myocytes,which may be related to relieving pyroptosis and inhibiting mitochondrial damage.
作者
周璐
王雅枫
张岳甫
苏娃婷
夏中元
雷少青
Zhou Lu;Wang Yafeng;Zhang Yuefu;Su Wating;Xia Zhongyuan;Lei Shaoqing(Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,China;Department of Anesthesiology,Union Hos-pital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China)
出处
《国际麻醉学与复苏杂志》
CAS
2022年第3期225-230,共6页
International Journal of Anesthesiology and Resuscitation
基金
国家自然科学基金(81772049)。
