三七皂甙Rg1、Rb1对肝纤维化大鼠线粒体DNA三磷酸腺苷酶6、8亚基的作用机制研究

Study on the mitochondrial mtDNA ATPase6、8 gene mutation and on the function mechanism of panax notoginseng saponins Rg1,Rb1 for hepatic fibrosis rats

目的探讨三七皂甙(panax notoginseng saponins,PnS)Rg1、PnS Rb1干预下,肝纤维化大鼠线粒体DNA三磷酸腺苷(adenine triphosphate,ATP)酶6亚基、ATP酶8亚基的基因突变和ATP含量变化。方法 96只Wistar大鼠分为对照组、四氯化碳(carbon tetrachloride,CCl4)肝纤维化大鼠模型组、PnS Rg1组、PnS Rb1组各24只。除对照组外,其余3组用5%CCl4橄榄油按5 ml/kg灌胃制作肝纤维化大鼠模型。PnS Rg1组在每次CCl4灌胃同时腹腔注射PnSRg1(5 mg/kg)。PnS Rb1组在每次CCl4灌胃同时腹腔注射Rb1(5 mg/kg)。用生物发光法测定各组大鼠实验前及实验开始2、4、6周末时线粒体内ATP含量并比较。提取肝细胞线粒体总RNA,逆转录为互补脱氧核糖核酸(complementary DNA,cDNA),用基因重组、测序,通过GenBank与线粒体DNA已知序列进行比较,研究PnS Rg1、PnS Rb1干预6周后线粒体DNA的ATP酶6亚基7904-8584区域和ATP酶8亚基7743-7946区域基因突变情况。结果 (1)与对照组相比,模型组大鼠线粒体DNA ATP酶6亚基7904-8584区域有3处突变:C8294G、T8505G、G8584A。用Fisher确切概率法分析,突变增加有显著性意义(P<0.01)。PnS Rg1组、PnS Rb1组与对照组相比突变增加没有显著性意义(P>0.05)。(2)与对照组相比,ATP酶8亚基7743-7946区域有2处突变:A7797non、T7863C。用Fisher确切概率法分析,突变增加有显著性意义(P<0.01)。PnS Rg1、Rb1组与对照组相比突变增加没有显著性意义(P>0.05)。(3)各组肝纤维化大鼠线粒体ATP含量与CCl4处理时间的相关性分析,用pearson's检验r=-0.935,呈负相关。(4)肝纤维化大鼠线粒体内ATP含量与ATP酶6亚基突变的相关性分析,用pearson's检验r=-0.943,呈负相关;肝纤维化大鼠线粒体内ATP含量与ATP酶8亚基突变的相关性分析,用pearson's检验r=-0.961,呈负相关。结论 PnS Rg1、Rb1可以显著抑制线粒体ATP含量下降,可以显著抑制线粒体DNA ATP酶6亚基、ATP酶8亚基突变,从而推断PnS Rg1、Rb1可能通过减少线粒体DNA ATP酶6、8亚基突变而抑制线粒体ATP含量下降,从而延缓肝纤维化发生发展。

Objective To explore the changes of the mitochondrial DNA （ mt DNA ） adenine triphospholibase（ATPase）6 gene , ATPase 8 gene mutation under the intervention of panax notoginseng saponins（PnS）Rg1, PnS Rb1 for hepatic fibrosis rats which may have theoretical sense in the prevention and cure hepatic fibrosis. Methods 96 Wistar rats divided into contrast group, carbon tetrachloride （CCl4） group, liver fibrosis rats model group, PnS Rg1 group and PnS Rb1group, with 24 rats in each＆amp;nbsp;group. Except contrast group, other three groups were reproduced by repeated injection 5% CCl4 solution in rats into liver fibrosis rats model. PnS Rg1 group were also injected 5 ml/kg, PnS Rb1 group were injected 5 ml/kg, and at the same time given 5% CCl4 solution. Extract RNA of hepatic mitochondrial and reverse transcription complementary DNA （cDNA）. Gene-recommended and sequencing have been researched. Then further study the PnS Rg1 group, PnS Rb1 group ATPase 6 gene 7904-8584 region and ATPase 8 gene 7743-7946 region gene mutation after 6 weeks. Biological fluorescent method detect ATP content. Re-sults （1）Compare with the contrast group, gene analysis of mtDNA ATPase 6 in hepatic fibrosis of model group rats showed that there were three mutation sites：C8294G, T8505G, C8584A. Using Fisher＇s exact test to analyze, mutations have increased significantly（P〈0. 01）. Compare with the contrast group, PnS Rg1 group、PnS Rb1 group mutation increasing does not have marked sense （ P 〉0. 05 ） . （ 2 ） Compare with the contrast group , the gene analysis of mtDNA ATPase 8 were two mutation sites： A7797non, T7863C. Using Fisher＇s exact test to analyze, mutations have increased significantly（P〈0. 01）. （3） Liv-er fibrosis rats mitochondrial DNA ATP of each group with CCl4 manage time correlation analysis, undergo-ing pearson＇s test r= -0. 935 negative correlation. （4） Liver fibrosis rats mitochondrial DNA ATP of each group with ATPase6 gene mutation has correlation analysis, undergoing pearson＇s test r= -0. 943 negative correlation. Liver fibrosis rats mitochondrial DNA ATP of each group with ATPase8 gene mutation has cor-relation analysis, undergoing pearson＇s test r= -0. 961 negative correlation. Conclusion The mutation of mtDNA ATPase 6 gene and ATPase 8 gene may be responsible for hepatic fibrosis development. PnS Rg1, PnS Rb1 were protective function remarkably for the mitochondrial of hepatic fibrosis rats through inhibition of mtDNA ATPase 6 gene, ATPase 8 gene mutation rate. Therefore delay hepatic fibrosis development.