丹酚酸B调节SIRT1信号途径抑制H_(2)O_(2)诱导的NLRP3炎症小体激活

Salvianolic Acid B Attenuates H_(2)O_(2)-induced NLRP3 Inflammasome Activation via Regulating SIRT1 Signaling Pathway

目的基于沉默信息调节因子1(SIRT1)/NOD样受体蛋白3(NLRP3)炎症小体途径探讨丹酚酸B(Sal B)抗氧化应激致细胞损伤的作用机制,以期阐明丹酚酸B抗心肌缺血的作用机制。方法采用体外构建H_(2)O_(2)诱导的氧化应激细胞模型,将H9c2细胞分为6组,分别为正常组、模型组(600μmol·L^(-1)H_(2)O_(2)刺激)、H_(2)O_(2)+丹酚酸B(5、10、20μmol·L^(-1))组和H_(2)O_(2)+丹酚酸B(20μmol·L^(-1))+EX527(10μmol·L^(-1))组。采用MTT法测定细胞活性;Hoechst染色法测定细胞凋亡;比色法及ELISA法分别测定乳酸脱氢酶(LDH)、白细胞介素(IL)-1β水平;采用DCFH-DA荧光探针检测细胞内活性氧(ROS)水平;采用JC-1荧光探针测定线粒体膜电位;Western Blot法测定H9c2细胞中NLRP3、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)、凋亡相关斑点样蛋白(ASC),以及SIRT1信号通路相关的SIRT1、磷酸化AMP蛋白活化激酶α(p-AMPKα)、AMP蛋白活化激酶α(AMPKα)、过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)蛋白表达。结果与正常组比较,模型组细胞活力及线粒体膜电位明显降低(P<0.01),细胞凋亡程度及细胞内ROS、LDH和炎症因子IL-1β水平明显升高(P<0.01);与NLRP3炎症小体相关的NLRP3、Caspase-1和ASC蛋白表达显著上调(P<0.01),SIRT1、p-AMPKα/AMPKα和PGC-1α蛋白表达明显下调(P<0.01)。与模型组比较,丹酚酸B组的细胞活力以及线粒体膜电位显著升高(P<0.05,P<0.01),细胞凋亡程度及细胞内ROS、LDH及炎症因子IL-1β释放水平明显降低(P<0.01)。丹酚酸B能够明显上调SIRT1信号通路中SIRT1、p-AMPKα/AMPKα、PGC-1α蛋白表达(P<0.05,P<0.01),下调NLRP3炎症小体相关的NLRP3、Caspase-1和ASC蛋白表达(P<0.05,P<0.01),且呈一定量效关系。在应用SIRT1特异性抑制剂EX527干预后,丹酚酸B抑制H_(2)O_(2)诱导的NLRP3炎症小体激活作用被明显逆转。结论丹酚酸B可能通过激活SIRT1/AMPK/PGC-1α信号通路,抑制氧化应激诱导的NLRP3炎症小体的激活,继而发挥抗心肌缺血作用。

Objective To investigate whether salvianolic acid B(Sal B)inhibits the activation of NOD like receptor protein 3(NLRP3) inflammasome is closely related to the regulation of silent information regulator 1(SIRT1)signaling pathway.MethodsOxidative stress injury was constructed in vitro with hydrogen peroxide(H_(2)O_(2)).H9c2cells were divided into the control group,H_(2)O_(2) group,different doses(5,10,20μmol·L^(-1))of salvianolic acid B groups and EX527(10μmol·L^(-1))group.MTT assay was performed to detect the cell viability.Cells were stained with Hoechst to observe the apoptotic morphological changes.The levels of LDH,IL-1βwere detected respectively by colorimetry and ELISA.The level of intracellular reactive oxygen species (ROS) was detected by DCFH-DA fluorescent probe,and mitochondrial membrane potential(MMP)was determined using the fluorescent dye JC-1.Western Blot was used to detect nucleode binding oligomeric domain-like receptor protein 3(NLRP3),apoptosis related speckle protein(ASC),Caspase-1 and the expression levels of related proteins in SIRT1/AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γcoactivator 1α(PGC-1α) pathway.Results Compared with the normal control group,cell viability and MMP of cells in model group were significantly decreased(P<0.01),the percentage of apoptotic cells and levels of ROS,LDH and IL-1βincreased significantly(P<0.01);and the levels of NLRP3 inflammasome related proteins including NLRP3,Caspase-1 and ASC were significantly up-regulated(P<0.01).After pretreatment by different concentrations of Sal B for 24 h on H9c2 cells,compared with the model group,the cell viability and MMP showed a dose-dependent increase(P<0.05,P<0.01).The percentage of apoptotic cells and release of LDH,ROS and IL-1βwere decreased after being pretreated with different concentrations of Sal B(P<0.01).SIRT1,p-AMPKα/AMPKα,PGC-1αproteins expression increased after pretreatment with Sal B(P<0.05,P<0.01),and protein expression levels of NLRP3,ASC,and Caspase-1were down-regulated in a dose-dependent manner(P<0.05,P<0.01).The effect of inhibiting the activation of NLRP3 inflammasomes induced by H_(2)O_(2) can be reversed by SIRT1 specific inhibitor EX527.ConclusionThese results indicate that SIRT1 regulates NLRP3 inflammasome-mediated inflammatory response is an important mechanism of H_(2)O_(2)-induced myocardial ischemia.Pretreatment with Sal B could inhibit the NLRP3 inflammasome activation and alleviating myocardial inflammatory injury by up-regulating SIRT1/AMPK/PGC-1α signaling pathway.