丹参通络解毒汤通过调控Beclin-1对缺氧/复氧CMECs的保护作用及机制

Protective effect and mechanism of Danshen Tongluo Jiedu Decection on hypoxic and reoxygenated CMECs by regulating Beclin-1

目的:探讨丹参通络解毒汤(DSTLJD)通过调控Beclin-1对缺氧/复氧(H/R)大鼠心肌微血管内皮细胞(CMECs)的保护作用及机制。方法:体外培养CMECs,构建H/R细胞模型,通过慢病毒转染过表达和沉默Beclin-1。将细胞随机分为pcDNA-Beclin-1组(过表达Beclin-1载体组)、control-pcDNA+DSTLJD组(过表达空载体+丹参通络解毒汤组)、pcDNA-Beclin-1+DSTLJD组(过表达Beclin-1载体+丹参通络解毒汤组)、Beclin-1-siRNA组(沉默Beclin-1载体组)、control-siRNA+DSTLJD组(沉默空载体+丹参通络解毒汤组)、Beclin-1-siRNA+DSTLJD组(沉默Beclin-1载体+丹参通络解毒汤组)。Western Blot法检测丝氨酸/精氨酸蛋白激酶1(SRPK1)、丝氨酸/精氨酸富集剪接因子1(SRSF1)、内皮型一氧化氮合酶(eNOS)及自噬相关蛋白(Atg5)蛋白表达,ELISA法检测超氧化物歧化酶(SOD)、丙二醛(MDA)表达,RT-PCR法检测肺癌转移相关转录本1(MALAT1)、SRPK1、SRSF1 mRNA表达。结果:与control-pcDNA+DSTLJD组比较,pcDNA-Beclin-1+DSTLJD组ATG5蛋白表达显著升高(P<0.01),eNOS蛋白表达显著下降(P<0.01),SOD水平下降(P<0.05),MDA水平升高(P<0.05);与pcDNA-Beclin-1组比较,pcDNA-Beclin-1+DSTLJD组ATG5蛋白表达下降(P<0.05),eNOS蛋白表达显著升高(P<0.01),SRPK1、SRSF1蛋白及mRNA表达均显著下降(P<0.01),MALAT1 mRNA表达显著下降(P<0.01),SOD水平升高(P<0.05),MDA水平下降(P<0.05);与control-siRNA+DSTLJD组比较,Beclin-1-siRNA+DSTLJD组ATG5蛋白表达显著下降(P<0.01),eNOS蛋白表达升高(P<0.05),SOD水平升高(P<0.05),MDA水平下降(P<0.05);与Beclin-1-siRNA组比较,Beclin-1-siRNA+DSTLJD组ATG5蛋白表达下降(P<0.05),eNOS蛋白表达升高(P<0.05),SRPK1、SRSF1蛋白及mRNA表达均下降(P<0.01,P<0.05),MALAT1 mRNA表达显著下降(P<0.01),SOD水平升高(P<0.05),MDA水平下降(P<0.05)。结论:丹参通络解毒汤可能通过抑制MALAT1-Beclin-1轴,降低ATG5蛋白,增强eNOS蛋白,上调SOD,下调MDA保护心肌微血管内皮细胞。

Objective:To investigate the protective effect and mechanism of Dansheng Tongluo Jiedu Decoction(DSTLJD)on cardiac microvascular endothelial cells(CMECs)by regulating Beclin-1 in rats with hypoxia/reoxygenation(H/R).Methods:CMECs were cultured in vitro to construct H/R cell model,and Beclin-1 was overexpressed and silenced by lentivirus transfection.The cells were randomly divided into pcDNA-Beclin-1 group(over expressing Beclin-1 vector group),control-pcDNA+DSTLJD group(over expressing empty vector+Danshen Tongluo Jiedu Decoction group),and pcDNA-Beclin-1+DSTLJD group(over expressing Beclin-1 vector+Danshen Tongluo Jiedu Decoction group),Beclin-1-siRNA group(Beclin-1 carrier silencing group),control-siRNA+DSTLJD group(empty carrier silencing+Danshen Tongluo Jiedu Decoction group),Beclin-1-siRNA+DSTLJD group(Beclin-1 carrier silencing+Danshen Tongluo Jiedu Decoction group).The protein expressions of SRPK1,SRSF1,eNOS and ATG5 were detected by Western Blot,SOD and MDA were detected by ELISA,and the mRNA expressions of MALAT1,SRPK1 and SRSF1 were detected by PT-PCR.Results:Compared with control-pcDNA+DSTLJD group,ATG5 protein expression in pcDNA-Beclin-1+DSTLJD group was significantly increased(P<0.01),eNOS protein expression decreased significantly(P<0.01),SOD level decreased(P<0.05),MDA level increased(P<0.05);Compared with pcDNA-Beclin-1 group,ATG5 protein expression decreased in pcDNA-Beclin-1+DSTLJD group(P<0.05),eNOS protein expression was significantly increased(P<0.01),protein and mRNA expressions of SRPK1 and SRSF1 were significantly decreased(P<0.01),MALAT1 mRNA expression was significantly decreased(P<0.01),SOD level increased(P<0.05),MDA level decreased(P<0.05);Compared with control-siRNA+DSTLJD group,ATG5 protein expression in Beclin1-siRNA+DSTLJD group was significantly decreased(P<0.01),eNOS protein expression increased(P<0.05),SOD level increased(P<0.05),MDA level decreased(P<0.05);Compared with Beclin-1-siRNA group,the expression of ATG5 protein in Beclin-1-siRNA+DSTLJD group decreased(P<0.05),eNOS protein expression increased(P<0.05),protein and mRNA expressions of SRPK1 and SRSF1 were decreased(P<0.01,P<0.05),MALAT1 mRNA expression was significantly decreased(P<0.01),SOD level increased(P<0.05),MDA level decreased(P<0.05).Conclusion:Dansheng Tongluo Jiedu Decoction may protect myocardial microvascular endothelial cells by inhibiting MALAT1-Beclin-1 axis,reducing ATG5 protein,enhancing eNOS protein,up-regulating SOD and down-regulating MDA.