利拉鲁肽对高糖诱导的H9c2心肌细胞焦亡的保护作用

Protective effect of liraglutide on pyroptosis of H9c2 cardiomyocytes induced by high glucose

目的研究利拉鲁肽(LRG)对高糖诱导的H9c2心肌细胞焦亡的保护作用和机制。方法分别用不同剂量的LRG和高糖处理细胞48 h,挑选出LRG最佳干预剂量。将H9c2细胞分为6组:对照组、高糖组和低、中、高剂量LRG(LRG-L、LRG-M、LRG-H)组、LRG-H+自噬抑制剂3-甲基腺嘌呤(3-MA)组。用细胞计数试剂盒-8(CCK-8)法检测细胞存活率;用流式细胞术检测细胞焦亡情况;用酶联免疫吸附(ELISA)法检测白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)水平;用实时荧光定量聚合酶链反应法检测Nod样受体蛋白3(NLRP3)炎症体相关基因表达;用蛋白质印迹法检测NLRP3炎症体、自噬、沉默信息调节因子1/腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白(Sirt1/AMPK/mTOR)信号通路相关蛋白表达。结果对照组、高糖组、LRG-L组、LRG-M组、LRG-H组、LRG-H+3-MA组细胞焦亡率分别为(1.62±0.74)%、(15.30±0.98)%、(12.68±0.66)%、(9.70±0.67)%、(7.24±0.83)%、(13.66±0.70)%;IL-1β水平分别为(22.33±4.76)、(74.51±5.56)、(61.33±6.00)、(48.94±4.20)、(37.62±4.03)、(64.10±6.07)ng·L^(-1);NLRP3蛋白表达量分别为1.01±0.11、6.44±0.48、5.31±0.21、4.16±0.20、3.18±0.46、5.26±0.43;Beclin-1蛋白表达量分别为1.01±0.11、0.25±0.04、0.44±0.03、0.63±0.03、0.84±0.08、0.40±0.07。上述指标,高糖组与对照组比较,差异均有统计学意义(均P<0.01);LRG-L组、LRG-M组、LRG-H组与高糖组比较,差异均有统计学意义(P<0.05或P<0.01);LRG-H+3-MA组和LRG-H组比较,差异均有统计学意义(均P<0.01)。LRG各剂量组Sirt1、p-AMPK/AMPK蛋白表达显著升高,p-mTOR/mTOR蛋白表达显著降低(P<0.05或P<0.01)。结论LRG通过调控Sirt1/AMPK/mTOR信号通路增强细胞自噬,抑制NLRP3炎症体活化,从而减轻高糖诱导的心肌细胞焦亡。

Objective To explore the protective effect and mechanism of liraglutide(LRG)on high glucose-induced H9c2 cardiomyocytes pyroptosis.Methods The H9c2 cells were treated with different concentrations of LRG and high glucose for 48 h.After the optimal concentration of LRG were selected,the H9c2 cells were divided into six groups:control group,high glucose group,low,medium and high dose LRG(LRG-L,LRG-M and LRG-H)group,LRG-H+autophagy inhibitor trimethyladenine(3-MA)group.The cell viability was evaluated by cell counting kit-8(CCK-8)assay.The pyroptosis of H9c2 cells was detected by flow cytometry.The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)were detected by the enzyme linked immunosorbent assay(ELISA).The mRNA expression of Nod-like receptor protein 3(NLRP3)inflammasome was detected by real-time quantitative polymerase chain reaction.The protein expression of NLRP3 inflammasome,autophagy and related to silencing information regulator 1/adenosine monophosphate-activated protein kinase/mammalian target of rapamycin(Sirt1/AMPK/m TOR)signaling pathway was detected by Western blot.Results The pyroptotic rate of H9c2 cells in control group,high glucose group,LRG-L group,LRG-M group,LRG-H group and LRG-H+3-MA group were(1.62±0.74)%,(15.30±0.98)%,(12.68±0.66)%,(9.70±0.67)%,(7.24±0.83)%,(13.66±0.70)%;IL-1βlevels were(22.33±4.76),(74.51±5.56),(61.33±6.00),(48.94±4.20),(37.62±4.03),(64.10±6.07)ng·L^(-1);NLRP3 protein expression levels were 1.01±0.11,6.44±0.48,5.31±0.21,4.16±0.20,3.18±0.46,5.26±0.43;Beclin-1 protein expression levels were 1.01±0.11,0.25±0.04,0.44±0.03,0.63±0.03,0.84±0.08,0.40±0.07.Compared between high glucose group and control group,the difference was significantly(all P<0.01);compared between LRG-L group,LRG-M group,LRG-H group and high glucose group,the difference was significantly(P<0.05 or P<0.01);compared between LRG-H+3-MA group and LRG-H group,the difference was significantly(all P<0.01).In addition,the protein expression of Sirt1 and p-AMPK/AMPK in LRG intervention group increased;the protein expression of p-m TOR/m TOR decreased(P<0.05 or P<0.01).Conclusion LRG promotes cell autophagy by modulating(Sirt1/AMPK/m TOR)signaling pathway,inhibits NLRP3 activation,and attenuates high glucose-induced H9c2 cardiomyocytes pyroptosis.