基于对衰老大鼠心脏抗氧化保护作用及Keap1-Nrf2-HO-1通路探讨川芎临床“久服”用药策略

Based on the antioxidant protective effect of Ligusticum chuanxiong hort on aging rats and the mechanism of Keap1-Nrf2-HO-1 pathway to discuss the clinical“long-term use”drug strategy of Ligusticum chuanxiong hort

目的通过构建衰老合并血瘀大鼠模型,基于抗氧化通路探讨川芎小剂量下久服对于延缓衰老的药效及作用机制,从而探讨川芎的临床用药策略。方法SD大鼠66只,分为空白组、模型组、维生素E组、川芎2 g组、川芎4 g组、川芎配伍人参2 g组;除空白组外各组腹腔注射D-半乳糖(300 mg/kg)连续42天,同时继续造模并给药14天,第15天冰水浴20分钟,连续14天,最后一天注射0.1%肾上腺素0.8 mg/kg。WST-1法检测血清及心脏组织中超氧化物歧化酶(superoxide dismutase,SOD)、比色法检测谷胱甘肽过氧化物酶活性(glutathione peroxidase,GSH-PX)、总抗氧化能力(total antioxidant capacity,T-AOC),TBA法脂质过氧化产物丙二醛(malondialdehyde,MDA),ELISA检测蛋白质羰基化合物(protein carbonyl,PC)含量,检测心脏组织Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶含量。蛋白免疫印迹法(Western Blot)测定检测心脏组织核因子E2相关因子2(nuclearfactor erythroidderived 2-like 2,Nrf2)蛋白、血红素加氧酶1(heme oxygenase 1,HO-1)蛋白、Kelch样ECH相关蛋白1(recombinant kelch like ECH associated protein 1,Keap1)表达量。结果与空白组相比,模型组体质量下降(P<0.05),血清及心脏组织SOD、GSH-PX、T-AOC含量降低(P<0.01),MDA、PC含量升高(P<0.01),心脏组织中Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶含量降低(P<0.01),Nrf2、HO-1表达量降低(P<0.05);与模型组相比,维生素E组能升高大鼠体质量、血清及心脏组织SOD、GSH-PX、T-AOC含量,降低MDA、PC含量,升高心脏组织中Na^(+)-K^(+)-ATP酶(P<0.05);川芎2 g、4 g升高血清GSH-PX、T-AOC含量,且川芎4 g提高心脏GSH-PX,降低血清及心脏MDA含量,增加心脏组织中Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶含量(P<0.05),增加Nrf2、HO-1蛋白表达量(P<0.05)。川芎配伍人参配伍后进一步升高血清及心脏SOD、GSH-PX含量,降低血清MDA、PC含量,增加心脏组织中Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶含量(P<0.01),增加Nrf2、HO-1蛋白表达量(P<0.05)。结论对衰老合并血瘀大鼠模型,川芎小剂量久服即可发挥抗氧化作用;川芎与人参合用后川芎剂量减少可以起到协同增效作用,可通过Keap1-Nrf2-HO-1抗氧化通路对心脏起到保护作用。

Objective By constructing a rat model of aging and blood stasis,the efficacy and mechanism of action of long-term administration in small doses of Ligusticum chuanxiong for anti-aging were discussed based on antioxidant pathway,and the clinical drug strategy of Ligusticum chuanxiong was discussed.Methods 66 SD rats were divided into the blank group,the model group,the vitamin E group,the Ligusticum chuanxiong 2 g group,the Ligusticum chuanxiong 4 g group,the Ligusticum chuanxiong-Panax ginseng 2 g group.Except the blank group,all the rats in each group were injected with D-galactose(300 mg/kg)peritoneally for 42 days,while continuing to mold and administer for 14 days,the 15th ice water bath for 20 min for 14 days,and the last day of injection of 0.1%epinephrine 0.8 mg/kg.To observe the general state and body mass of rats,WST-1 method was used to detect the level of superoxide dismutase(superoxide dismutase,SOD)in serum and heart tissues,colorimetric method was used to detect the level of glutathione peroxidase activity(glutathione peroxidase,GSH-PX),total antioxidant capacity(total antioxidant capacity,T-AOC),TBA method lipid peroxidation product malondialdehyde(malondialdehyde,MDA),ELISA was used to detect protein carbonyl compound(protein carbonyl,PC)content,cardiac tissue Na^(+)-K^(+)-ATPase,Ca^(2+)-Mg^(2+)-ATPase content.Western Blot method was used to detect protein expression of cardiac tissue nuclear factor E2-related factor 2(Nrf2),heme oxygenase 1(HO-1)protein,and kelch-like ECH-associated protein 1(Keap1).Results Compared with the blank group,the body mass of the model group was decreased(P<0.05),the contents of SOD,GSH-PX and T-AOC in serum and heart tissue method was used todecreased(P<0.01),the contents of MDA and PC increased(P<0.001),the contents of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase in heart tissue were decreased(P<0.01),and the expressions of Nrf2 and HO-1 decreased(P<0.05);compared with the model group,the body mass in the vitamin E group was increased,the contents of SOD,GSH-PX,T-AOC in serum and heart tissue,the contents of MDA and PC were decreased,and the Na^(+)-K^(+)-ATP ase in heart tissues(P<0.05)were increased;the content of GSH-PX in serum and T-AOC were increased in the Ligusticum chuanxiong 2 g and 4 g group,and the content of GSH-PX in Ligusticum chuanxiong 4 g group were increased in heart,the contents of MDA in serum and heart were decreased,the contents of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase were increased in heart tissue(P<0.05),and the expression of Nrf2 and HO-1 protein(P<0.05)were increased.The compatibility of Ligusticum chuanxiong with Panax ginseng further increased the contents of SOD and GSH-PX in serum and heart,decreased the contents of MDA and PC in serum,increased the contents of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase in heart tissue(P<0.01),and increased the expression of Nrf2 and HO-1 protein(P<0.05).Conclusion For rat models of aging and blood stasis,small doses of Ligusticum chuanxiong can exert antioxidant effects by taking it for a long time;The reduction of the dose of Ligusticum chuanxiong after the combination of Ligusticum chuanxiong and ginseng can play a synergistic effect,and can protect the heart through the Keap1-Nrf2-HO-1 antioxidant pathway.