基于肝损伤级联反应关系的黄药子水煎液致肝损伤机制研究

Study on the mechanism of liver injure induced by the decoction of Dioscorea bulbifera based on hepatic cascade reaction

目的:基于肝损伤级联反应关系观察黄药子水煎液(decoction of Dioscorea bulbifera,DDB)连续给药对小鼠肝脏的影响,探讨其致肝损伤的早期作用机制。方法:黄药子水煎液多次灌胃给予小鼠,测定不同给药剂量和不同给药时间下黄药子水煎液对小鼠生化指标、细胞膜指标、氧化与抗氧化指标、线粒体指标、病理组织的影响,并使用Western蛋白印迹法检测小鼠肝组织细胞色素P450(CYP)2E1表达。结果:黄药子水煎液剂量-毒性试验中,较空白对照组2g/kg剂量组小鼠钠钾ATP酶(Na^+-K^+-ATP)、钙镁ATP酶(Ca^(2+)-Mg^(2+)-ATP)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽S转移酶(GST)及总巯基(T-SH)均有统计学差异;当剂量增加到20g/kg,小鼠Na^+-K^+-ATP、Ca^(2+)-Mg^(2+)-ATP、MDA、SOD、GST及T-SH均呈显著变化,SDH明显降低。黄药子水煎液时间-毒性试验中,与各自空白对照组比较,小鼠灌胃给予20g/kg剂量的黄药子水煎液,各给药组小鼠丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)均升高,同时黄药子水煎液可显著增强20天给药组及30天给药组CYP2E1表达。10天给药组病理组织学检查可见个别小鼠肝细胞轻微嗜酸变性;20天给药组病理组织学检查可见肝细胞点状或小灶性坏死外,还可见Na^+-K^+-ATP酶活性明显降低;30天给药组小鼠病理组织学检查结果除与20天给药组结果相同外,还可见大量小鼠肝细胞坏死,Na^+-K^+-ATP、Ca^(2+)-Mg^(2+)-ATP酶活性显著降低,同时MDA、SOD、GST、T-SH及SDH均有明显改变。结论:小鼠长期给予DDB可使肝脏发生损伤,损伤程度具有一定的时间和剂量依赖性,其对肝脏的时间-毒性关系为首先降低细胞膜Na^+-K^+-ATP、Ca^(2+)-Mg^(2+)-ATP活性,上调CYP450,进一步破坏氧化与抗氧化系统或线粒体功能,黄药子水煎液致肝损伤早期作用机制可能为损伤肝细胞膜。

Objective： to observe the affection of liver induced by the decoction of Dioscorea bulbifera（DDB） in mice after long-term administration, and that explore its mechanism of liver injury which based on hepatic cascade reaction. Methods： mice were multiple continuously intragas-triced DDB in different doses and times, then spectively detect the change of the mice , s biochemical indicator, Cell membrane index, Oxidation and anti-oxidant index, mitochondria index and pathological change . the protein expression of cytochrome P450（CYP） 2El in the liver was analyzed by Western bloting. Results ： compared with the control group, Na^＋ -K^＋ -ATPase, Ca^2＋ -Mg^2＋ -ATPase, metahne dicarboxylic （MDA） ,superoxide dismutase （SOD） ,glutathione S-transferase（GST） and total sulihydryl（T-SH） level have a statistics difference（ p 〈 0. 05） in the mice which were intragastriced with DDB at doses of 2g/kg in dose-toxicity experiment. When the dose increased to 20g/kg, Na^＋ - K^＋ -ATP, Ca^2＋ -Mg^2＋ -ATP, MDA, SOD, GST and T-SH Show significant changes （ P 〈 0.01 ）, and succinate dehydrogenase （SDH） level have a obvious decreament（ p 〈 0.05 ）. DDB were multiple continuously intragastriced to mice at doses of 20g/kg in the time-toxicity experiment, compared with the control group of themselves,level of aspartate transaminase （AST） and alanine transaminase （ALT） were increased in all groups. At the same time, the protein expression of CYP2E1 was significantly enhanced by thirtieth days group and thirtieth day group of DDB （ P 〈 0.05, P 〈 0.01 ）. the detection of pathology changes of medication administration team after giving DDB for the tenth days involve liver cells, eosino denaturation which was little or slight, both the twentieth days and the thirtieth days,mice could find the punctiform or focal nec- rosis, hepatic sinusoid extension and hyperemia, meanwhile the DDB of the thirtieth days,mice liver appears necrosis. All the twentieth days and thirtieth days groups of Na^＋ -K^＋ -ATP enzyme and Ca^2＋ -Mg^＋ -ATP enzyme have a statistics difference （ P 〈 0.05 ）, and it is clear that SOD,GST,MDA,SDH,HYP in mice,liver thirtieth days groups of DDB present significant difference（ P 〈0.01 ） ,T-SH also present a statistics difference. Conclusion： DDB can cause liver damage in mice after long-term administration, and its liver damage have a certain dose-time relationships, analysis that its time dose relation of liver injured was likely concerned with the decrease of the Na^＋-K^＋ -ATP, Ca^2＋ -Mg^2＋ -ATP enzyme activity firstly. Second, up-regulation of the function of CYP450 followed by initial injury was observed. Subsequently, up-regulation of CYP450 would destroy oxidant and anti-oxidant system and mitochondria dysfunction. The initial mechanism of liver injure induced by DDB is likely relate to membrane.