从内源性大麻素受体2的激活探究异丙酚预处理缓解缺血再灌注损伤的保护机制

Activation of endocannabinoid receptor 2 explores the protective mechanism of propofol preconditioning to alleviate ischemia-reperfuSion injury

目的从内源性大麻素受体2的激活探究异丙酚预处理缓解缺血再灌注损伤的保护机制。方法利用低氧培养箱构建心肌损伤模型。阐明异丙酚调节对体外和体内心脏内源性大麻素信号传导的影响:将心肌细胞(体外)或大鼠(体内)分成4组:对照(假),异丙酚,H/R(I/R)和异丙酚+H/R(I/R),每组6只大鼠。阐明CB1R和CB2R信号通路在异丙酚诱导的I/R损伤心肌保护中的作用:分为7组:对照组、I/R组、异丙酚+I/R组、AM251+I/R组、AM251+异丙酚+I/R组、AM630+1/R组、AM630+异丙酚+I/R组,每组6只大鼠。LC-MS/MS测定内源性大麻素浓度;使用商业FAAH抑制剂筛选测定试剂盒(Cayman Chemicals,Ann Arbor,MI,USA)测定FAAH活性;在流式细胞术中使用Annexin-V/PI双染法检测细胞凋亡;ELISA测定血清心肌肌钙蛋白I(cTnI)和MPO浓度。结果Post hoc Bonferroni测试发现I/R(P<0.001)和异丙酚调理I/R(P<0.001)增加血清AEA浓度。异丙酚孵育10min后,异丙酚和异丙酚+H/R组的细胞培养基AEA浓度增加。在对照组和异丙酚组缺氧结束或相应于缺氧结束的时间点,异丙酚中AEA浓度增加。与对照相比,异丙酚调节和H/R增加了CB1R mRNA转录。与对照相比,在异丙酚和H/R组CB2R mRNA水平增加(P<0.05)。与对照相比,异丙酚调节和H/R均增加CB1R蛋白质水平。异丙酚调节和H/R也增强了CB2R蛋白质翻译。与对照相比,异丙酚与H/R组合增加CB1R和CB2R蛋白水平。URB和VDM11预处理均可抑制H/R诱导的细胞凋亡(P<0.001)。与I/R相比,异丙酚调节降低血清cTnI浓度,而AM630增加血清cTnI。与假手术组相比,I/R引起血清MDA和MPO浓度升高。与I/R相比,异丙酚调理降低了血清MDA和MPO浓度。与I/R相比,单独的AM251和AM251与异丙酚调节相结合降低血清MDA浓度。AM251和AM251联合异丙酚调节(P<0.001)组的血清MPO浓度有类似的效果。结论异丙酚调节赋予对心肌I/R损伤的心脏保护作用主要通过增强内源性大麻素释放和随后激活CB2受体信号传导机制。

Objective To investigate the protective mechanism of propofol preconditioning to relieve ischemia-reperfusion injury from the activation of endocannabinoid receptor 2.Methods The model of myocardial injury was established by hypoxia incubator.To elucidate the effect of propofol regulation on endocardial cannabinoid signal transduction in vitro and in vivo:cardiomyocytes(in vitro)or rats(in vivo)were divided into four groups:control(false),propofol,H/R(I/R)and propofol+H/R(I/R),six rats in each group.To clarify the role of CB1R and CB2R signaling pathway in the protection of I/R injury induced by propofol:7 groups:control group,I/R group,propofol+I/R group,AM251+I/R group,AM251+propofol+I/R group,AM630+I/R group,AM630+propofol+I/R group;6 rats in each group.LC-MS/MS was used to determine the concentration of endogenous cannabinoid;commercial FAAH inhibitor screening kit(Cayman chemicals,Ann Arbor,MI,USA)was used to determine the activity of FAAH;annexin-V/PI double staining method was used to detect apoptosis in flow cytometry;serum cardiac troponin I(cTnI)and MPO concentration were measured by ELISA.Results The Post hoc Bonferroni test found that I/R(P<O.OOl)and propofol conditioning I/R(P<0.001)increased serum AEA concentration.After incubation with propofol for 10 minutes,the concentration of cell medium AEA in propofol and propofol+H/R groups increased.The concentration of AEA in propofol increased in the control group and the propofol group at or corresponding to the end of hypoxia.Propofol regulation and H/R increased CB1R mRNA transcription compared to control.Compared with the control group,the mRNA level of CB2R increased in propofol and H/R groups(P<0.05).Both propofol regulation and H/R increased CB1R protein levels compared to controls.Propofol regulation and H/R also enhance CB2R protein translation.Propofol combined with H/R increased CB1R and CB2R protein levels compared to controls.Both URB and YDM11 preconditioning inhibited H/r-induced apoptosis(P<0.001).Compared with I/R,propofol regulated a decrease in serum cTnI concentration,while AM630 increased serum cTnI.Compared with the sham group,I/R caused elevated serum MDA and MPO concentrations.Compared with I/R,propofol conditioning reduced serum MDA and MPO concentrations.Compared with I/R,AM251 and AM251 alone combined with propofol regulation reduced serum MDA concentrations.AM251 and AM251 combined with propofol had a similar effect on serum MPO concentrations in the group(P<0.001).Conclusion The cardioprotective effect of propofol regulation on myocardial I/R injury is mainly through enhanced endocannabinoid release and subsequent activation of the CB2 receptor signaling mechanism.