摘要
目的观察凉膈散对创伤失血性休克大鼠肝脏细胞凋亡的影响，并探讨其机制。方法健康雄性Wistar大鼠90只，随机分为正常组，模型1、3、6、24h组和凉膈散1、3、6、24h组，每组10只。采用骨折后经颈动脉放血制备创伤失血性休克大鼠模型。凉膈散组于制模前连续3d灌胃凉膈散10ml／kg；模型组给予等量生理盐水；正常组不予任何处理。取血，采用速率法测定血浆天冬氨酸转氨酶（AST）浓度；取肝组织，用原位末端缺刻标记法（TUNEL）检测凋亡细胞，用免疫组化法检测天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）蛋白表达，用逆转录-聚合酶链反应（RT-PCR）检测Bax、Bcl一2、Fas、FasL的基因表达。结果模型组1、3、6、24h血AST（U／L）呈上升趋势，24h达峰值（647．36±60．02），与正常组（66．69±19．95）比较差异有统计学意义（均P〈0．05）；凉膈散组各时间点血AST（194．24±29．53、306．01±83．85、388．36±92．71、451．48±99．70）均较模型组显著降低（均P〈0．05）。正常组未见凋亡细胞；模型组1、6、24h时有散在凋亡细胞，3h时中央静脉周围肝脏实质细胞出现大量凋亡（个：20．60±0．47）；凉膈散组3h时只发现肝窦内淋巴细胞凋亡（个：14．70±1．02），较模型组显著降低（P〈0．05）。模型组1、3、6和24hcaspase-3蛋白表达（×10^3，A值）均较正常组（8．96±1．22）显著升高，3h达峰值（49．71±0．89）；凉膈散组各时间点caspase-3表达（17．81±0．48、34．48±1.09、28.52±1．14、31．76±1．39）较模型组显著下降（均P〈O．05）。模型组各时间点凋亡相关基因表达均较正常组升高，且Bax、Bcl-2、Fas均于3h达峰值（1．98±0．07、0．87±0．07、0．25±0．02），FasL于6h达峰值（0．57±0．02）；凉膈散组在3h（0．40±0．03）和6h（0．18±0．03）能显著降低Bax基因表达（均P〈0．05），对Bcl-2基因表达无明显影响（均P〉0．05），且未检测到Fas和FasL基因表达。结论凉膈散能够抑制创伤失血性休克大鼠肝脏caspase一3蛋白表达及Bax、Fas、FasL基因表达，但对肝脏Bcl-2表达无明显影响；能显著抑制肝脏实质细胞凋亡，促进肝窦内出现淋巴细胞凋亡，从而避免炎性细胞过度活化造成对组织的损伤。
Objective To observe the effect of Liangge San （凉膈散） on apoptosis of liver cells in rats with traumatic hemorrhagic shock and to discuss the mechanism. Methods Ninety healthy male Wistar rats were divided randomly into nine groups ： normal group, model 1-, 3-, 6-, 24-hour groups and Liangge San 1-, 3-, 6-, 24-hour groups （each, n=10）. By bloodletting from carotid artery after bone fracture, the rat model of traumatic hemorrhagic shock was established. The rats in Liangge San groups were infused into the stomach with Liangge San （10 ml/kg） once a day for 3 days before the model establishment, the rats in model groups were infused with equal amount of 0. 9% normal saline （NS） and no treatment was given in normal group. The plasma was collected to measure aspartate aminotransferase （AST） concentrations with Rate method. The liver was harvested to detect the cell apoptosis with the terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling （TUNEL） assay, the expression of caspase-3 protein by the techniques of immunohistochemistry, and the mRNA expressions of Bax, Bcl-2, Fas, FasL with reverse transcriptase- polymerase chain reaction （RT-PCR）. Results AST （U/L） in model 1-, 3-, 6-, 24-hour groups was increased gradually, and peaked at 24 hours （647.36±60.02） with statistically significant difference compared with that of the normal group （66.69±19.95, all P〈0.05）. The levels of AST in Liangge San groups at all time points （194.24 ± 29.53, 306.01 ± 83.85, 388.36 ± 92.71, 451.48± 99.70） were obviously lower than those in the model groups （all P〈0.05）. No apoptosis was found in normal group; in model 1-, 6-, 24-hour groups there were scattered apoptotic cells, and in the 3-hour model group, the apoptosis in a large amount was around the central vein （20.60± 0.47）; the count of apoptotic lymphocytes in the sinusoid in Liangge San 3-hour group （14.70±1.02） was less significantly compared with the model 3-hour group （P〈0. 05）. The levels of caspase-3 protein （×10^3, A value） in model 1-, 3-, 6-, 24-hour groups were increased markedly, peaked at 3 hours （49.71 ± 0.89） compared with those of the normal group （8.96 ± 1. 22）; compared with model groups at the corresponding time points, the levels of caspase-3 protein in Liangge San 1-, 3-, 6- and 24-hour groups （17.81 ± 0.48, 34.48 ± 1.09, 28.52 ± 1.14, 31.76 ± 1.39） were remarkably decreased （all P〈0. 05）. The levels of apoptotic genes in model groups were increased compared with those in normal group, the levels of Bax, Bcl-2, Fas mRNA expressions were peaked at 3 hours （1.98±0.07, 0. 87±0.07, 0.25±0.02）, while the level of FasL was peaked at 6 hours （0. 57± 0. 02） ; the level of Bax mRNA expression was obviously reduced in Liangge San 3-hour （0.40±0.03） and 6-hour （0.18±0. 03） groups （both P〈 0.05 ）, and Liangge San had no effect on the level of Bcl-2 mRNA expression （all P〈0.05）; the mRNA expression of Fas and FasL in Liangge San groups were not seen. Conclusion Liangge San can inhibit the mRNA expressions of Bax, Fas, FasL as well as caspase-3 protein, but has no significant effect on Bcl-2 in the liver of rats with traumatic hemorrhagic shock. It obviously inhibits hepatic parenehymal cell apoptosis and promotes the appearance of apoptosis of lymphoeytes in the sinusoid, thus the liver damage caused by excessive activation of inflammatory cetts can be avoided.
出处
《中国中西医结合急救杂志》
CAS
北大核心
2011年第2期82-85,I0001,共5页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
天津市医药卫生科研基金资助项目（05KZE17）
关键词
凉膈散
休克
创伤失血性
肝脏
凋亡
Liangge San
Traumatic hemorrhagic shock
Liver
Apoptosis
