目的:研究炒苦参(SAA)对马兜铃酸I(AAI)诱导的肾毒性和DNA加合物的保护作用,并阐明确保细辛安全使用的潜在机制。
方法:体外,通过慢病毒转导构建过表达Flag标记的多药耐药相关蛋白3(MRP3)的HEK293T细胞,并利用自行构建的荧光筛选系统验证了细辛10对临床常见药材对MRP3活性的抑制作用。mRNA,蛋白质表达,在分化的HepaRG细胞中测量NAD(P)H醌脱氢酶1(NQO1)和细胞色素P4501A2(CYP1A2)的酶活性水平。使用细胞计数试剂盒-8测定法检测抑制AAI代谢物转运后的肝细胞毒性。在体内,C57BL/6小鼠按随机数字表法随机分为5组,包括:对照(1%碳酸氢钠),AAI(10mg/kg),炒SAA(1.75g/kg)和AAI+炒SAA(1.75和8.75g/kg)组,每组6只小鼠。连续灌胃给药7天后,评估肝脏和肾脏损害,同时测定肝脏代谢酶NQO1和CYP1A2的蛋白表达和酶活性。
结果:体内,1.75g/kgSAA和10mg/kgAAI的组合抑制AAI诱导的肾毒性和减少26.7%的dA-ALI形成,这些解毒作用呈剂量依赖性(P<0.01)。机械上,SAA在体外抑制MRP3转运,体内NQO1表达下调,增加CYP1A2的表达和酶活性在体外和体内,(P<0.05或P<0.01)。值得注意的是,SAA还在整个解毒过程中降低了AAI诱导的肝毒性,肝加合物数量减少41.3%(P<0.01)。
结论:Stir-friedSAA是抑制AAI诱导的肝和肾损害的新型候选药物。其保护机制可能与转运蛋白和代谢酶的调节密切相关。
OBJECTIVE: To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma.
METHODS: In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously.
RESULTS: In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01).
CONCLUSIONS: Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes.