大鼠肝脏冷缺血再灌注损伤时姜黄素后处理对肝细胞凋亡的影响

Effects of curcumin post-conditioning on hepatocyte apoptosis in rats with liver cold ischemia/reperfusion injury

背景:姜黄素预处理可减轻肢体缺血再灌注对肝脏的损伤,但姜黄素后处理对肝脏冷缺血再灌注损伤是否有保护作用及其机制目前研究甚少。目的:探讨大鼠肝脏冷缺血再灌注损伤时姜黄素后处理对肝细胞凋亡的影响。方法:选取成年雄性SD大鼠80只,采用随机数字表法将其分成4组(n=20):假手术组、冷缺血再灌注组、姜黄素后处理组、地塞米松组。使肝脏血流处于完全阻断状态,随后以脾静脉作为流入道和右肾上腺静脉作为流出道注入0℃复方乳酸林格液,冷灌注30min;停止冷灌注后,结扎近端脾静脉和右肾上腺静脉,切除脾脏,随即恢复肝脏血流,完成制作冷缺血再灌注模型。在大鼠冷缺血30min后,姜黄素后处理组经尾静脉注射姜黄素60 mg/kg,地塞米松组尾静脉注射地塞米松0.5 mg/kg,其他组以等量的生理盐水替代。再灌注6 h时经下腔静脉取血,检测血清天门冬氨酸氨基转移酶、丙氨酸转移酶水平,随后处死大鼠,取肝组织检测丙二醛水平;采用苏木精-伊红染色观察肝脏病理变化;Hoechst33258染色法检测肝细胞凋亡指数;Westernblot检测肝组织Bcl-2和Bax蛋白表达;RT-PCR检测肝组织促细胞凋亡基因Caspase-9m RNA表达;ELISA检测肝组织肿瘤坏死因子α及白细胞介素1β水平。结果与结论:①与假手术组比较,冷缺血再灌注组天门冬氨酸氨基转移酶、丙氨酸转移酶、丙二醛和凋亡指数明显升高(P<0.05);苏木精-伊红染色切片可见肝血窦内有大量炎性细胞浸润,肝细胞嗜酸性变,胞浆内疏松化,肝细胞呈气球样变,偶可见斑片状坏死,散在点状坏死灶;Bcl-2表达下降,Bax表达明显升高(P<0.05);Caspase-9 mRNA表达、肿瘤坏死因子α及白细胞介素1β水平明显升高(P<0.05);②与冷缺血再灌注组比较,姜黄素后处理组天门冬氨酸氨基转移酶、丙氨酸转移酶、丙二醛和凋亡指数明显下降(P<0.05);苏木精-伊红染色可见肝血窦内炎性浸润明显减轻,胞浆嗜酸性变和气球样变的肝细胞明显减少,但偶可见少量散在的点状坏死;Bcl-2表达升高,Bax表达明显下降(P<0.05);Caspase-9 mRNA表达、肿瘤坏死因子α及白细胞介素1β水平明显下降(P<0.05);③姜黄素后处理组上述各指标与地塞米松组比较差异无显著性意义(P>0.05);④综上所述,姜黄素后处理可减轻大鼠肝脏冷缺血再灌注损伤,其作用机制可能通过上调Bcl-2/Bax比值,抑制凋亡启动子Caspase-9mRNA的表达,减少炎性因子肿瘤坏死因子α和白细胞介素1β的释放,发挥抗凋亡的肝保护作用。

BACKGROUND:Curcumin pre-conditioning can alleviate liver injury induced by limb ischemia/reperfusion(I/R),but whether curcumin post-conditioning has protective effect against liver cold I/R injury and its mechanism are still poorly studied.OBJECTIVE:To investigate the effects of curcumin post-conditioning on hepatocyte apoptosis in rats with liver cold I/R injury in rats.METHODS:Eighty adult male Sprague-Dawley rats were randomly divided into four groups(n=20 per group)by using a random number table:sham group,I/R group,curcumin post-conditioning group(I/R+Cur group),and dexamethasone group(I/R+Dex group).The liver blood flow was completely blocked.Then the splenic vein and the adrenal vein were used as the inflow and outflow tracts to inject 0 oC compound Ringer lactate solution followed by cold perfusion for 30 minutes.After stopping cold perfusion,the proximal splenic vein and the right adrenal vein were ligated to remove the spleen,and then the blood flow in the liver restored.The cold I/R model was successfully established.After 30 minutes of cold ischemia,60 mg/kg curcumin was injected into the rat tail vein in the I/R+Cur group,0.5 mg/kg dexamethasone was injected into the rat tail vein in the I/R+Dex group,and the same amount of saline was injected in the other groups.Blood sample was taken from the carotid artery at 6 hours after reperfusion.Serum levels of aspartate aminotransferase and alanine transferase were detected.Then the rats were sacrificed to detect malondialdehyde level in liver tissue,observe liver pathological changes by hematoxylin-eosin staining,measure hepatocyte apoptosis index by Hoechst 33258 staining,detect expression of Bcl-2 and Bax protein by western blot,expression of caspase-9 mRNA by RT-PCR,and levels of tumor necrosis factor-αand interleukin-1βby ELISA.RESULTS AND CONCLUSION:Compared with the sham group,aspartate aminotransferase,alanine transferase,malondialdehyde levels and apoptosis index in the I/R group increased significantly(P<0.05).Hematoxylin-eosin staining showed a large number of inflammatory cells infiltrated in the hepatic sinusoids,eosinophilic changes of hepatocytes,cytoplasmic loosening,balloon-like changes of hepatocytes,occasional patchy necrosis,and scattered punctate necrosis foci in the I/R group.The I/R group had significantly decreased expression of Bcl-2,and increased expression of Bax(P<0.05),Caspase-9 mRNA,tumor necrosis factor-αand interleukin-1β(P<0.05).Compared with the I/R group,aspartate aminotransferase,alanine transferase,malondialdehyde levels and apoptosis index in the I/R+Cur group decreased significantly(P<0.05).Hematoxylin-eosin staining showed that inflammatory infiltration in the hepatic sinusoids decreased significantly,eosinophilic and balloon-like hepatocytes decreased significantly,but occasionally a small amount of scattered punctate necrosis was observed in the I/R+Cur group.There was significantly increased Bcl-2 expression,and significantly decreased Bax(P<0.05),Caspase-9 mRNA,tumor necrosis factor-αand interleukin-1β(P<0.05).No significant differences were observed between I/R+Cur group and I/R+Dex group(P>0.05).In a word,curcumin-post conditioning can alleviate liver injury induced by cold I/R in rats.Its mechanism may be related to down-regulation of Bcl-2/Bax ratio,inhibition of caspase-9 mRNA expression,and reduction of the release of tumor necrosis factor-αand interleukin-1β,therefore playing an anti-apoptotic role in liver protection.