七氟醚通过STING/AMPK信号通路减轻小鼠肝脏缺血再灌注损伤的研究

Sevoflurane Attenuates Hepatic Ischemia-reperfusion Injury in Mice via STING/AMPK Signaling Pathway

为探讨七氟醚(sevoflurane)麻醉处理对小鼠肝脏缺血再灌注损伤(hepatic ischemia-reperfusion injury, HIRI)的影响及机制,将健康成年C57雄性小鼠随机分为假手术组(Sham组)、缺血再灌注+戊巴比妥钠组(HIRI+Pent组)、缺血再灌注+七氟醚组(HIRI+Sevo组),每组10只;经腹腔手术构建小鼠70%肝脏HIRI模型,肝脏缺血60 min,再灌注3 h后,将小鼠处死取材。取肝组织行苏木精-伊红(hematoxylin-eosin, HE)染色,观察肝组织病理损伤,同时采用免疫荧光染色检测髓过氧化物酶(myeloperoxidase, MPO)含量,并采用TUNEL检测组织细胞凋亡;通过酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)、聚合酶链反应(polymerase chain reaction, PCR)检测肝组织炎症相关因子的表达;通过Western-blot检测肝组织干扰素基因刺激因子(stimulator of interferon genes, STING)、AMP活化蛋白激酶(AMP-activated protein kinase, AMPK)等相关蛋白质的表达;采用生化分析仪检测血清丙氨酸转氨酶(alanine transaminase, ALT)、天冬氨酸转氨酶(aspartate transaminase, AST)和乳酸脱氢酶(lactate dehydrogenase, LDH)水平;分别采用硫代巴比妥酸(thiobarbituric acid, TBA)比色法、黄嘌呤氧化酶法及二硫代二硝基苯甲酸法测定还原型谷胱甘肽(glutathione, GSH)、脂质过氧化物丙二醛(malondialdehyde, MDA)和超氧化物歧化酶(superoxide dismutase, SOD)含量。结果显示,与Sham组比较,缺血再灌注后,HIRI+Pent组小鼠血清ALT、AST明显升高,肝脏组织可观察到大片梗死区域,细胞凋亡明显增加, STING、核因子κB (nuclear factor-κB, NF-κB)表达升高,同时AMPK下降明显,炎症因子肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)、白细胞介素(interleukin, IL)-1β、IL-6、IL-18明显升高, MPO表达明显增加,氧化应激标志物GSH、MDA及SOD水平均出现明显差异,而在HIRI+Sevo组中,上述变化程度均得到明显缓解,且差异具有统计显著性(P<0.05)。结果表明,七氟醚可部分通过STING/AMPK信号通路减轻小鼠肝脏缺血再灌注损伤。

To explore the effects and mechanisms of sevoflurane anesthesia on hepatic ischemia-reperfusion injury(HIRI)in mice,healthy adult male C57 mice were randomly divided into three groups,including sham group(Sham),HIRI+pentobarbital sodium group(HIRI+Pent),and HIRI+sevoflurane group(HIRI+Sevo),10 in each group.The mouse model of partial(70%)HIRI was constructed by laparotomy.After liver ischemia for 60 min and reperfusion for 3 h,mice were sacrificed for sample collection.The pathological damage of liver tissue was observed with hematoxylin-eosin(HE)staining,the content of myeloperoxidase(MPO)was detected by immunofluorescence staining,and apoptosis in tissue was detected using the terminal deoxynucleotidyl transferase(TdT)-mediated dUTP-biotin nick end labeling(TUNEL)method.The expression of inflammation-related factors was detected by both enzyme-linked immunosorbent assay(ELISA)and polymerase chain reaction(PCR).The expression of stimulator of interferon genes(STING),AMP-activated protein kinase(AMPK)and other related proteins was detected by Western-blot.Serum alanine transaminase(ALT),aspartate transaminase(AST)and lactate dehydrogenase(LDH)were detected by biochemical analyzer.Meanwhile,the contents of reduced glutathione(GSH),lipid peroxide malondialdehyde(MDA)and superoxide dismutase(SOD)were measured by thiobarbituric acid(TBA)colorimetry,xanthine oxidase method,and dithio-dinitrobenzoic acid method,respectively.The results showed that,compared with mice in Sham group,after ischemia-reperfusion,mice in HIRI+Pent group had significantly increased serum ALT and AST levels,large infarcted area in liver tissue,significantly enhanced apoptosis elevated STING and nuclear factor-κB(NF-κB)expression levels,significantly decreased AMPK expression,significantly increased levels of inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,IL-18,and MPO,and significantly different levels of the oxidative stress markers GSH,MDA and SOD.However,after ischemia-reperfusion,mice in HIRI+Sevo group exhibited alleviated degrees of all the above changes,with statistical significance(P<0.05).The study demonstrated that sevoflurane can partially reduce HIRI in mice through STING/AMPK signaling pathway.