目的 观察丹参预处理对肝脏缺血再灌注损伤后磷酸化真核生物翻译起始因子2α(p-eIF-2α)和caspasel2表达的影响.方法 将80只Wistar大鼠按随机数字表法分成正常对照组5只,假手术组、缺血再灌注组和丹参预处理组各25只,后3组大鼠再根据缺血再灌注时限(0、3、12、24和72 h)分为5个亚组,每组5只.采用动脉夹钳夹肝蒂45 min后,使血液复流的方法制备大鼠缺血再灌注损伤模型.正常对照组大鼠不做处理；假手术组仅解剖肝门不钳夹肝蒂；丹参预处理组大鼠在肝血流阻断前30 min经尾静脉注入丹参注射液6 ml/kg；假手术组和缺血再灌注组大鼠则在肝血流阻断前30 min经尾静脉注入等量生理盐水.采用Western blot检测各组大鼠肝组织中p-eIF-20和caspase12蛋白的表达,HE染色观察各组大鼠同期肝组织的病理形态学变化.多组间比较采用单因素方差分析,组间比较方差齐采用LSD法,方差不齐采用Games-Howell法.结果 正常对照组大鼠肝组织中p-eIF-2α蛋白的相对表达量为0.296±0.038,假手术组在缺血再灌注0、3、12、24和72 h分别为0.304±0.048、0.298±0.038、0.272±0.042、0.266±0.076和0.296±0.043,两组比较,差异无统计学意(P＞0.05).缺血再灌注组大鼠肝组织中p-eIF-2α蛋白的相对表达量在缺血再灌注0h为0.310±0.034,与正常对照组和假手术组同时相点比较,差异无统计学意义(F=0.15,P＞0.05)；在缺血再灌注3、12、24 h,p-eIF-2α蛋白的相对表达量分别为0.386±0.021、0.710±0.034和0.474 ±0.017,与正常对照组和假手术组同时相点比较,差异有统计学意义(F=11.90,211.52,25.15,P＜0.05)；至缺血再灌注72 h,p-eIF-2α蛋白的相对表达量为0.336±0.043,基本回落至正常对照组和假手术组同时相点水平(F=1.57,P＞0.05).丹参预处理组大鼠肝组织中p-eIF-2α蛋白的相对表达量在缺血再灌注0、12、24和72 h分别为0.278 ±0.044、0.800±0.079、1.056±0.125、0.736±0.087和0.442 ±0.047,丹参预处理组在缺血再灌注3、12、24、72 h明显高于缺血再灌注组同时相点水平(P＜0.05).正常对照组大鼠肝组织中caspase12蛋白的相对表达量为0.983±0.003,假手术组在缺血再灌注0、3、12、24和72 h分别为0.974±0.004、0.983±0.005、0.985±0.003、0.981±0.004和0.978±0.004,两组比较,差异无统计学意义(P＞0.05).缺血再灌注组大鼠肝组织中caspase12蛋白的相对表达量在缺血再灌注0h开始上升为1.018 ±0.076,但与正常对照组和假手术组同时相点比较,差异无统计学意义(F=1.43,P＞0.05)；在缺血再灌注3h明显升高为2.056±0.067,12h达到峰值为2.804±0.050,24 h仍处于相对高水平为1.882 ±0.037,72 h有所下降为1.282±0.066,与正常对照组和假手术组同时相点比较,差异均有统计学意义(F=1290.69,6490.84,2898.71,103.61,P＜0.05).丹参预处理组肝组织中caspase12蛋白的相对表达量在缺血再灌注0、3、12、24、72 h分别为0.998±0.056、1.442±0.066、1.990±0.068、1.364±0.056和0.962±0.054,缺血再灌注3、12、24、72 h均明显低于缺血再灌注组同时相点(P＜0.05).病理形态学检测结果显示:正常对照组和假手术组大鼠肝组织肝小叶结构基本完整,肝细胞连续成索,胞核大而圆；肝缺血再灌注组大鼠肝组织可见肝小叶变形,细胞体积变小,细胞核浓缩,部分出现片状坏死；丹参预处理组大鼠肝细胞肿胀,出现轻微的点状坏死.结论 丹参可能通过上调p-eIF-20的水平稳定内质网内环境,减轻经内质网细胞凋亡途径中caspase12的表达,在肝脏缺血再灌注损伤期发挥肝脏保护作用.%Objective To investigate the influences of Radix salvia miltiorrhiza (RSM) preconditioning on the expressions of phosphorylated eukaryotic initiation factor 2α (p-eIF-2ot) and caspase12 in hepatic ischemia reperfusion in rats.Methods Eighty Wistar rats were randomly divided into the control group (5 rats),sham operation group (25 rats),ischemia reperfusion (IR) group (25 rats) and RSM pretreated group (25 rats).Rats in the sham operation group,IR group and RSM pretreated group were subdivided into 5 groups at different time intervals (0,3,12,24 and 72 hours).Mter midline laparotomy,all structures in the hepatic portal were clamped for 45 minutes followed by different periods of reperfusion.Rats in the control group did not receive any treatment; rats in the sham operation group only received anatomy of the hepatic portal without clamping; rats in the RSM pretreated group received RSM by intravenous injection 30 minutes before ischemia at a dose of 6 ml/kg.Rats in the sham operation group and the IR group received a dose of normal saline as RSM pretreated group.The protein expressions of p-eIF-2α and caspase12 in the hepatic tissues of each group were detected by the Western blot,and the pathological changes of hepatic tissues in each group were detected by hematoxylin and eosin staining.All data were analyzed using the analysis of variance,LSD test or Games-Howell method.Results The relative expression of p-eIF-2α in the control group was 0.296 ± 0.038,and the relative expressions of p-eIF-2α in the sham operation group at different time points were 0.304 ± 0.048,0.298 ± 0.038,0.272 ± 0.042,0.266 ± 0.076 and 0.296 ± 0.043,with no significant difference between the 2 groups (P ＞ 0.05).The relative expression of p-eIF-2α in the IR group at 0 hour was 0.310 ± 0.034,which had no significant difference compared with the control group and the sham operation group (F =0.15,P ＞0.05).The relative expressions of p-eIF-2α in the IR group at 3,12,24 hours were 0.386 ± 0.021,0.710 ± 0.034,0.474 ± 0.017,which had no significant difference compared with the control group and the sham operation group (F =11.90,211.52,25.15,P ＜ 0.05).The relative expression of p-eIF-2α in the IR group at 72 hours was 0.336 ± 0.043,which was back to the level of the control group and the sham operation group (F =1.57,P ＞ 0.05).The relative expressions of p-eIF-2α in the RSM pretreated group at different time intervals were 0.278 ± 0.044,0.800 ± 0.079,1.056 ± 0.125,0.736 ±0.087 and 0.442 ± 0.047,which were significantly lower than the group at 3,12,24,72 hours (P ＜ 0.05).The relative expression of caspase12 in the control group was 0.983 ± 0.003,and the relative expressions of caspase12 in the sham operation group at different time intervals were 0.974 ± 0.004,0.983 ± 0.005,0.985 ± 0.003,0.981 ± 0.004 and 0.978 ± 0.004,with no significant difference between the 2 groups (P ＞ 0.05).The relative expression of caspase12 in the IR group at 0 hour was increased to 1.018 ± 0.076,while it had no significant difference compared with the control group and the sham operation group (F =1.43,P ＞ 0.05).The relative expressions of caspase12 in the IR group began to rise at 3 hours (2.056 ±0.067),and peaked at the 12 hours (2.804 ± 0.050),still at a relative higher level at 24 hours (1.882 ± 0.037),and began to decrease at 72 hours (1.282 ± 0.066),it had significant difference compared with the control group and the sham operation group (F=1290.69,6490.84,2898.71,103.61,P ＜ 0.05).The relative expressions of caspase12 in the RSM pretreated group at different time intervals were 0.998 ± 0.056,1.442 ± 0.066,1.990 ± 0.068,1.364 ± 0.056and 0.962 ±0.054.The relative expressions of caspase12 in the RSM pretreated group at 3,12,24,72 hours were significantly lower than the IR group (P ＜ 0.05).The results of pathomorphological examination showed that the hepatic lobules were integrated and the nucleuses were large and round in the control group an d the sham operation group; deformation of the hepatic lobule,smaller cell volume,nuclear condensation and necrosis were observed in the IR group; cell swelling and slight spotty necrosis were detected in the RSM pretreated group.Conclusions RSM could protect liver from damages during IR through up-regulating the expression of p-eIF-2α,reducing the apoptosis induced by endoplasmic reticulum stress.
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