氯沙坦钾对棕榈酸所致骨骼肌细胞凋亡的干预作用  

Effects of losartan potassium on the apoptosis of skeletal muscle cell induced by palmitic acid

作　　者：杨明 陈琳[1] 潘凌[1] 李丽 李思雨[1] 彭永德[1] 游利[1] YANG Ming;CHEN Lin;PAN Ling;LI Li;LI Si-yu;PENG Yong-de;YOU Li(Department of Endocrinology and Metabolism,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China)

机构地区：[1]上海交通大学附属第一人民医院内分泌代谢科骨质疏松亚专科

出　　处：《中华骨质疏松和骨矿盐疾病杂志》2019年第6期595-601,共7页Chinese Journal Of Osteoporosis And Bone Mineral Research

基　　金：国家自然科学基金面上项目(81570797)

摘　　要：目的从细胞水平探索氯沙坦钾对骨骼肌损伤的保护作用及其机制。方法用2%马血清诱导分化L6成肌细胞;CCK-8试剂盒及Western Blot确定合适的氯沙坦细胞处理浓度。实验分为4组:对照组,二甲基亚砜组,棕榈酸(1 mmol/L)组以及棕榈酸(1 mmol/L)+氯沙坦钾(50μmol/L)组。Western Blot检测凋亡相关蛋白(cleaved caspase-3、cleaved PARP、Bcl-2、Bax)、自噬相关蛋白(LC3、Beclin 1),实时荧光定量PCR (quantitative real-time PCR,q PCR)检测P53和P21 m RNA表达水平。结果显微镜下观察及Western Blot检测myogenin蛋白表达增加,证明骨骼肌细胞诱导分化成功。CCK-8试剂盒检测示氯沙坦钾浓度为50、100、200、400μmol/L时L6成肌细胞活力分别为0.87±0.087、1.11±0.037、1.21±0.099、1.30±0.068,其中50μmol/L时细胞活力最大,且对cleaved caspase-3蛋白表达抑制最明显。与未作处理的对照组比较,qPCR显示棕榈酸处理组骨骼肌细胞P53、P21 m RNA表达量分别从0.36±0.03、0.50±0.05上调至0.68±0.08、1.07±0.02 (P<0.05),cleaved caspase3、cleaved PARP蛋白表达均增加,LC3Ⅱ/LC3Ⅰ及Beclin 1蛋白表达减低;与棕榈酸单独处理组比较,氯沙坦钾与棕榈酸共处理组P53、P21 m RNA表达量分别从0.68±0.08、1.07±0.02下调至0.44±0.01、0.67±0.03 (P<0.05),cleaved caspase-3、cleaved PARP蛋白表达减低,LC3Ⅱ/LC3Ⅰ及Beclin1蛋白无显著改变。结论氯沙坦钾通过抑制cleaved caspase3及其底物cleaved PARP表达抑制细胞凋亡,但这种保护效应并不是通过自噬途径实现的。Objective To explore the protective effects of losartan potassium on skeletal muscle injury and its mechanisms at the cellular level.Methods L6 myoblasts were differentiation by 2% horse serum.The appropriate concentration of losartan was determined by CCK-8 kit and Western Blot.There were 4 groups: control group,DMSO group,palmitic acid(1 mmol/L) group and palmitic acid(1 mmol/L) + losartan potassium(50 μmol/L) group.Western Blot was used to detect the apoptosis-related proteins(cleaved caspase-3,cleaved PARP,Bcl-2,Bax),and autophagy-associated proteins(LC3,Beclin 1);quantitative real-time PCR(q PCR) was used to detect the levels of P53 and P21 m RNA expression.Results Microscopic observation and Western Blot showed the increased expression of myogenin protein,which indicated that L6 myoblasts were differentiation successfully.The CCK-8 kit assay showed that the cell viability was0.87±0.087,1.11±0.037,1.21± 0.099 and 1.30± 0.068 when the concentration of losartan potassium was 50,100,200,and 400 μmol/L,respectively.The cell viability was the highest at 50 μmol/L and the most inhibition on cleaved caspase-3 protein expression.After palmitic acid treatment,compared with the untreated control group,q PCR showed that,the expression of P53,P21 m RNA was up-regulated from 0.36 ± 0.03,0.50 ± 0.05 to 0.68 ± 0.08,1.07 ± 0.02(P<0.05),cleaved caspase-3,cleaved PARP protein expression increased,LC3Ⅱ/LC3Ⅰ and Beclin 1 protein expression decreased.Compared with palmitic acid alone treatment,the expression of P53 and P21 m RNA were down-regulated from 0.68±0.08,1.07±0.02 to 0.44±0.01,0.67±0.03 in losartan potassium treated group(P<0.05),the expressions of cleaved caspase-3,cleaved PARP proteins were decreased,and LC3Ⅱ/LC3Ⅰ and Beclin1 proteins were not changed.Conclusion Losartan potassium may reduce apoptosis by inhibiting cleaved caspase-3 and its substrate cleaved PARP protein expression,but this protective effect is not achieved by autophagy.

关 键 词：肌少症 氯沙坦钾 凋亡 自噬 

分 类 号：R681[医药卫生—骨科学]