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目的:观察清热化瘀中药复方——清肝方对急性肝衰竭(acute liver failure,ALF)大鼠高迁移率族蛋白B1(HMGB1)表达的影响,从mRNA水平探讨其抗ALF的机制。方法:采用D-氨基半乳糖(D-GalN)单次腹腔注射构建ALF大鼠模型,125只SD大鼠以是否接受造模和药物干预随机分为空白组、模型组、复方甘草酸苷组和清肝方组,每组再以36,96 h 2个时间点继续随机分为1,2两个亚组,共8组,其中亚组1用于造模后36 h取血及肝组织标本,亚组2用于96 h后观察大鼠的生存率。用全自动生化分析法检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)和总胆红素(TBiL);全自动血凝分析法检测血浆凝血酶原时间(PT);常规HE染色作肝组织病理学观察。实时荧光定量(RT-PCR)检测HMGB1 mRNA的表达变化,以管家基因β-actin为对照,2-ΔΔCt法计算目的基因相对表达量。结果:模型组、复方甘草酸苷组及清肝方组估计平均生存时间分别为64.6,71.9,83.3 h;log-rank检验提示清肝方组累积生存率高于模型组(P<0.05)。空白组、清肝方组和复方甘草酸苷组肝组织HMGB1 RNA/β-actin值均显著降低(0.006±0.003,0.067±0.033,0.112±0.027 vs 0.245±0.153,均P<0.01),清肝方组低于复方甘草酸苷组(P<0.01)。与模型组相比,空白组、清肝方组和复方甘草酸苷组在血清ALT,AST,TBiL和血浆PT水平方面与模型组相比均明显下降(P<0.01),清肝方组低于复方甘草酸苷组(P<0.01)。清肝方组和复方甘草酸苷组肝组织损害程度积分与模型组相比明显下降(1.84±0.13,2.85±0.20 vs 3.56±0.24,均P<0.01),清热化瘀复方组低于复方甘草酸苷组(P<0.01)。结论:清肝方可有效改善D-GalN诱导的大鼠的肝功能、凝血功能及肝脏病理,降低死亡率,其发挥作用的分子机制与抑制HMBG1的表达有关。
Objective: To observe the effect of qinggan decoction (TCM recipe for clearing heat and removing blood stasis) on expression of HMGB1 in rats with acute liver failure (ALF), and to explore its anti-ALF mechanism from mRNA level. Methods: The ALF rat model was established by intraperitoneal injection of D-galactosamine (D-GalN). 125 SD rats were randomly divided into blank group, model group, compound glycyrrhizin group And Qinggan Fang group. Each group was divided into two subgroups of 1 and 2 at 36 and 96 hours respectively. A total of 8 groups were used, of which subgroup 1 was used to take blood and liver tissue at 36 hours after modeling Specimens, subgroup 2 were used to observe the survival rate of rats after 96 h. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBiL) were detected by automatic biochemical analysis. Plasma prothrombin time (PT) HE staining for liver histopathology. The expression of HMGB1 mRNA was detected by real-time fluorescence quantitative PCR (RT-PCR). The relative expression level of target gene was calculated by 2-ΔΔCt method using the β-actin gene as the control. Results: The average survival time of model group, compound glycyrrhizin group and Qingganfang group were 64.6, 71.9 and 83.3 h, respectively. The log-rank test indicated that the cumulative survival rate of Qingganfang group was higher than that of model group (P <0.05). The serum levels of HMGB1 RNA / β-actin in the blank group, Qingganfang group and compound glycyrrhizin group were significantly decreased (0.006 ± 0.003,0.067 ± 0.033,0.112 ± 0.027 vs 0.245 ± 0.153, both P <0.01) The square group was lower than compound glycyrrhizin group (P <0.01). Compared with the model group, the blank group, Qingganfang group and compound glycyrrhizin group had significantly lower serum ALT, AST, TBiL and plasma PT levels than the model group (P <0.01) In compound glycyrrhizin group (P <0.01). Compared with the model group, the scores of liver damage in Qingganfang group and compound glycyrrhizin group were significantly decreased (1.84 ± 0.13, 2.85 ± 0.20 vs 3.56 ± 0.24, all P <0.01) Glucoside group (P <0.01). Conclusion: Qinggan Formula can effectively improve D-GalN-induced liver function, coagulation and liver pathology, reduce mortality, the molecular mechanism of its role in inhibiting HMBG1 expression.