Melatonin通过影响Bcl-2/Bax比率抑制再灌注后大鼠肝细胞凋亡

Inhibition of hepatocellular apoptosis by Melatonin with ischemia reperfusion injury in rats through ratio of Bcl-2/Bax

目的探讨再灌注损伤对大鼠肝细胞凋亡的影响,Mel对大鼠肝再灌注细胞凋亡的影响作用及其机制。方法150只健康雄性Wistar大鼠(质量190￣210g,6￣7周龄),随机分为褪黑素处理组(Mel)、酒精溶媒对照组(Alc)和生理盐水对照组(NS)。建立肝缺血再灌注损伤模型,缺血均为60min,之后每组分别按再灌注后0.5、1、6、12及24h采集标本。M组(20mg/kg)于缺血前30min腹腔注射melatonin;A组采取与Mel组相同浓度的酒精液,N组则注射同比例的生理盐水。测定血清天门冬酸氨基转移酶(AST)进行测定;对肝组织进行Bcl-2及Bax免疫组化染色;并在此基础上进行透射电镜观察肝细胞核及细胞器。结果Mel组在再灌注后的6及12h时点AST均显著低于Alc及NS对照组(aP<0.05),且Alc组与NS组相比差异无显著性。Mel组在再灌注后各时点的Bcl-2染色的阳性细胞率显著高于Alc及NS对照组(aP<0.05),且各时点内Alc组与NS组相比差异无显著性。Mel组在再灌注后1,6,12及24h时点的Bax染色的阳性细胞率显著低于Alc及NS对照组(cP<0.05),且各时点内Alc组与NS组相比差异无显著性。Mel组再灌注后6,12及24h的Bcl-2/Bax值显著高于Alc组和NS组(aP<0.05),且上述每时点内的Alcohol组与N.S.组相比差异无显著性。电镜观察结果在Alcohol组及N.S对照组发现了普遍的肝细胞凋亡现象,相同时点Mel组则未发现肝细胞凋亡的发生。结论外源性Mel可以抑制再灌注后血清天门冬酸氨基转移酶(AST),增强肝细胞Bcl-2蛋白的表达,抑制Bcl-2蛋白的表达,通过提升Bcl-2/Bax值来抑制再灌注后肝细胞凋亡的发生。

[Objective] To investigate the effect of ischemia repoffusion （I/R） injury on hepatocellular apoptosis and the effect of melatonin （Mel） on hepatocellular apoptosis in liver ischemia reperfusion （I/R） injury in rats. [Methods] 150 male Wistar rats （190-210 g, 6, 7weeks age） were divided into three groups at random： Mel exposure group, alcohol solvent control group and saline control group. The left branches of portal vein, hepatic artery,hepatic duct were blocked up for 60 min and then opened to establish liver I/R I models in rats. In each group, samples were collected in 0.5, 1, 6, 12, and 24 h after reperfusion respectively. 20 mg/kg of Mel was injected peritoneal in rats 30 rain before experimentation in Mel exposure group. The duplicate concentration of alcohol and the same volume of saline were injected in control groups as substitution. Serum alanine AST by auto biochemical analyzer, and immunohistochemical straining of Bcl-2 and Bax were determined with optical microscope, observation of liver tissue under electron microscope in rats. [Results] The level of AST measured in 6, 12 h time after reperfusion in Mel group was totally significantly higher than that in alcohol and saline control groups corresponding （P 〈0.05）. The expression level of Bcl-2 （%） measured at various time after reperfusion in Mel group was totally significantly higher than that in alcohol and saline control groups corresponding （P 〈0.05）, the express level of Bax（%） measured in 1, 6, 12, 24 h time after reperfusion in Mel group was totally significantly lower than that in alcohol and saline control groups corresponding （P 〈0.05）, and the ratio of Bcl-2/Bax in 6, 12, 24 h time after reperfusion in Mel group was totally significantly higher than that in alcohol and saline control groups corresponding （P 〈0.05）. Further subcellular observation under electron microscope indicated that exotic Mel restrained apeptosis in hepatic cells 6 h, 12 h, 24 h after repeffusion in alcohol and saline control groups. [Conclusion] Exotic Mel inhibited the activities of AST; increased the expression of Bcl-2 in liver reperfusion tissue; decreased the expression of Bax in liver reperfusion tissue. So it can improve the hepatic function after reperfusion through the effect of inhibition of apeptosis by improving the ratio of Bcl-2/Bax.