凉血化瘀方对肝衰竭小鼠肝细胞凋亡和相关基因作用机制的初步研究

PRELIMINARY STUDY ON MECHANISM OF COOLING BLOOD TO REMOVE BLOOD STASIS PRESCRIPTION ON HEPATOCYTIC APOPTOSIS AND EXPRESSION OF RELATED GENES ON RATS WITH HEPATIC FAILURE

目的:研究凉血化瘀方对内毒素脂多糖(Lipoplysaccaride,LPS)诱导的肝细胞凋亡的干预效应,以及对半胱天冬酶-3(caspase-3)mRNA、Fas相关死亡区蛋白(FADD)mRNA表达的影响,探讨凉血化瘀方防治肝衰竭的作用机制。方法:腹腔注射LPS于D-氨基半乳糖(D-galactosamine,GalN)致敏的小鼠,造成暴发性肝衰竭模型,用脱氧核苷酸转移酶介导的缺口原位末端标记技术(TUNEL法)、HE染色观察肝细胞凋亡和病理损伤情况及药物的干预作用。用逆转录-聚合酶链反应(RT-PCR)半定量分析caspase-3mRNA、FADDmRNA表达情况,同时观察药物的作用。结果:造模6小时模型组有大量凋亡阳性细胞表达,治疗组则明显减少(P<0.01)。至24小时肝细胞损害以坏死为主,其程度与细胞凋亡相一致。造模3小时肝细胞caspase-3mRNA明显表达,治疗组83%,模型组141%,6小时两组表达水平相近。FADDmRNA表达在造模3小时模型组和治疗组肝组织中分别为110%、100%,至6小时模型组的表达增加(124%)而治疗组的表达下调(81%)。结论:凉血化瘀方能延缓内毒素肝损伤肝细胞caspase-3mRNA表达和下调FADDmRNA表达并抑制其凋亡效应。凉血化瘀方对肝衰竭的治疗作用可能与其阻断LPS诱导的肝细胞凋亡信号转导,从而抑制肝细胞凋亡有关。

Objective: To study the effect of cooling blood to remove blood stasis prescription on hepatocytic apoptosis induced by LPS and the influence on mRNA expression of caspase-3 and Fas associated death domain protein(FADD), to explore the mechanism of this prescription in treating hepatic failure(HF).Methods: The models of HF were induced by injecting LPS to mice sensited by D-galactosamine, the condition of hepatocytic apoptosis, the pathological condition and the effect of Chinese herbs were examined by TUNEL,HE staining and transmission electron microscopy.Results: A lot of positive apoptotic cells were expressed in the model mice at 6h after induced by LPS, and that was significantly reduced in the treatment mice. The expression of caspase-3mRNA was obvious in the model at3h, 83%in the treatment group, and 141%in the model group, the expression of the two groups were very close at 6h. The expressions of FADD mRNA at 3h in the model and treatment groups were 110%,100% respectively, the expression was increased in model at 6h, that in the treatment was decreased.Conclusion: The effect of cooling blood to remove stasis prescription on HF is possibly related to its inhibiting on signal transduction of hepatocytic apopotosis induced by LPS, which can delay the expression of caspase-3mRNA and reduced the expression of FADD mRNA.