舒芬太尼减轻大鼠肝缺血再灌注损伤的机制研究

Mechanism of sufentanil attenuating hepatic ischemia-reperfusion injury in rats

目的 探讨舒芬太尼预处理减轻大鼠肝缺血再灌注损伤(HIRI)的作用机制。方法 48只雄性SD大鼠按照随机数字表法分为:假手术组(S组)、HIRI组(IR组)、舒芬太尼预处理+HIRI组(SF组)、转化生长因子β_(1)(TGF-β_(1))抑制剂+舒芬太尼预处理+HIRI组(SB组)、TGF-β_(1)激动剂+舒芬太尼预处理+HIRI组(SRI组)和二甲基亚砜(DMSO)+HIRI组(DMSO组),每组8只,于造模完成后4 h取材。HE染色观察肝组织的形态变化;收集腹主动脉血测定各组丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平;制备10%肝组织匀浆,测定各组丙二醛(MDA)及超氧化物歧化酶(SOD)水平;TUNEL法检测肝细胞凋亡率;Western blot法检测肝组织TGF-β_(1)及Smad2/3、p-Smad2/3、p38、p-p38蛋白表达水平。结果 与S组相比,其余各组损伤程度加重,ALT、AST、MDA水平及肝细胞凋亡率升高(P<0.05),SOD水平降低(P<0.05),IR组、SF组、SRI组中TGF-β_(1)、p-Smad2/3、p-p38蛋白表达水平升高(P<0.05),SB组中TGF-β_(1)、p-p38蛋白表达水平升高(P<0.05)。与IR组比较,SF组及SRI组损伤程度减轻,ALT、AST、MDA水平降低,肝细胞凋亡率降低,p-p38蛋白表达水平均降低(P<0.05),SOD水平升高,TGF-β_(1)、p-Smad2/3蛋白表达水平均升高(P<0.05);SB组损伤程度加重,ALT、AST、MDA水平,肝细胞凋亡率,p-p38蛋白表达水平均升高(P<0.05),SOD水平和TGF-β_(1)、p-Smad2/3蛋白表达水平均降低(P<0.05)。与SF组比较,SB组损伤程度加重,ALT、AST、MDA、肝细胞凋亡率及p-p38蛋白表达水均升高(P<0.05),SOD、TGF-β_(1)、p-Smad2/3蛋白表达水平均降低(P<0.05);而SRI组损伤程度减轻,ALT、AST、肝细胞凋亡率、p-p38蛋白表达水均降低(P<0.05),SOD、TGF-β_(1)、p-Smad2/3蛋白表达水平均升高(P<0.05)。结论 舒芬太尼预处理可能通过激活TGF-β/Smad信号通路和抑制p38 MAPK磷酸化,减轻肝细胞凋亡和氧化应激,从而发挥对大鼠HIRI的保护作用。

Objective To investigate the mechanism of sufentanil preconditioning in reducing hepatic ischemiareperfusion injury(HIRI) in rats.Methods A total of 48 SD male rats were randomly divided into the Sham operation group(S group),the HIRI group(IR group),the sufentanil pretreatment+HIRI group(SF group),the TGF-β_(1) inhibitor+sufentanil pretreatment+HIRI group(SB group),the TGF-β_(1) agonist+sufentanil pretreatment+HIRI group(SRI group) and the dimethyl sulfoxide(DMSO)+HIRI group(DMSO group),with 8 rats in each group.The samples were taken 4 hours after the completion of modeling.Then,HE staining was performed to observe the morphological changes of liver tissue.The abdominal aortic blood samples were collected,and the supernatant was collected after centrifugation to measure levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in each group.10% liver tissue homogenate was prepared to measure levels of malondialdehyde(MDA) and superoxide dismutase(SOD) in each group.The apoptosis rate of hepatocytes was detected by TUNEL method.Western blot method was used to detect the protein expression levels of TGF-β_(1),Smad2/3,p-Smad2/3,p38 and p-p38 in liver tissue.Results Compared with the S group,the damage degree was aggravated in the other groups,and levels of ALT,AST,MDA and the hepatocyte apoptosis rate were increased(P <0.05),while the SOD level was decreased(P<0.05).The protein expression levels of TGF-β_(1),p-Smad2/3 and p-p38 were increased in the IR group,the SF group and the SRI group(P <0.05),and the protein expression levels of TGF-β_(1) and pp38 were increased in the SB group(P<0.05).Compared with the IR group,the damage degree was reduced in the SF group and the SRI group,and levels of ALT,AST,MDA,the apoptosis rate of hepatocytes and expression level of p-p38 protein were decreased(P<0.05).Levels of SOD,TGF-β_(1) and p-Smad2/3 protein were increased(P<0.05),the damage degree was increased in the SB group,and levels of ALT,AST,MDA,apoptosis rate of hepatocytes and the expression level of p-p38 protein were increased(P<0.05).Levels of SOD,TGF-β_(1) and p-Smad2/3 protein were decreased(P<0.05).Compared with SF group,the damage degree of SB group was aggravated,levels of ALT,AST and MDA,the apoptosis rate of hepatocytes,the expression level of p-p38 protein were all increased(P <0.05),and SOD,TGF-β_(1),p-Smad2/3 protein expression levels were decreased(P <0.05).The damage degree of SRI group was reduced,levels of ALT,AST,the apoptosis rate of hepatocytes and the expression level of p-p38 protein were decreased(P<0.05),while levels of SOD,TGF-β_(1) and p-Smad2/3 protein were increased(P <0.05).Conclusion Sufentanil pretreatment may inhibit the phosphorylation of p38 MAPK by activating the TGF-β/Smad signaling pathway,reducing hepatocyte apoptosis and oxidative stress,thus playing a protective role on HIRI in rats.