误服误用含吡咯里西啶生物碱(pyrrolizidine alkaloid，PA)的中草药是导致我国临床肝窦阻塞综合征(hepatic sinusoidal obstruction syndrome，HSOS)的主要原因，利尿剂是目前临床上治疗PA致HSOS的常用药物之一。泽泻作为传统利尿中药，但其利尿机制仍未完全明确，且尚未有从利尿角度阐释泽泻改善PA致HSOS的报道。基于课题组前期研究，本研究以泽泻三萜23-乙酰泽泻醇B(alisol B 23-acetate，AB23A)为代表性药物，评价其对毒性PA千里光碱致小鼠急性肝损伤的保护作用，并进一步考察AB23A对模型小鼠水液失衡的影响。所有动物实验经上海中医药大学实验动物伦理委员会批准(批准号PZSHUTCM220808017)，符合实验动物伦理相关规范。小鼠单次灌胃千里光碱(50 mg·kg-1)造模(SEN组)，并设AB23A(40 mg·kg-1)干预组(AB23A+SEN组)、溶剂对照组(Ctrl组)和AB23A对照组(AB23A组)。结果表明，AB23A可明显降低千里光碱致急性肝损伤小鼠血清肝功能生化指标水平，缓解肝细胞凝固性坏死、肝窦淤血等病理状态;此外，AB23A还可改善小鼠血清肾功能指标，改善千里光碱造成的尿液排泄减少、机体电解质紊乱，并降低千里光碱及其代谢产物的含量。进一步测定小鼠水平衡通路相关蛋白和mRNA的表达，AB23A可明显下调千里光碱模型组小鼠水通路蛋白2(aquaporin-2，AQP2)、血管紧张素Ⅱ 1型受体的蛋白和mRNA表达水平，抑制AQP2向顶端质膜运输，并降低血清血管紧张素Ⅱ含量。体外研究进一步证实AB23A可调节肾脏内髓集合管细胞AQP2的表达。本研究表明，AB23A可调节千里光碱致急性肝损伤小鼠肾脏髓质AQP2，影响其对水的重吸收，改善水液平衡。研究结果进一步加深了对泽泻传统利尿活性的认识，为临床利用泽泻治疗PA致HSOS提供实验基础和理论依据。
Effects of alisol B 23-acetate on water-liquid balance in mice with senecionine-induced acute liver injury
Misuse of pyrrolizidine alkaloid(PA)-containing herbs is the major cause of hepatic sinusoidal obstruction syndrome(HSOS)in China.And diuretics are among the most commonly used medications for the treatment of PA-induced HSOS in clinical practice.As a traditional diuretic in traditional Chinese medicine,the diuretic mechanism of Alismatis Rhizoma(AR)has not been fully clarified,and there is no report on AR ameliorating PA-induced HSOS from a diuretic point of view.Therefore,this study aims to investigate the therapeutic potential of alisol B 23-acetate(AB23A)against acute liver injury induced by senecionine(a representative toxic PA)in mice,and to further elucidate its effect on impaired water-liquid balance in mice exposed to PA.All experiments were approved by the Animal Research Committee of Shanghai University of Traditional Chinese Medicine(Registration number:PZSHUTCM220808017).Animal welfare and the animal experimental protocols were strictly consistent with related ethics regulations of Shanghai University of Traditional Chinese Medicine.Model of mice was induced by a single oral exposure of senecioine(50 mg·kg-1)(SEN group),and AB23A(40 mg·kg-1)intervention group(AB23A+SEN group),solvent control group(Ctrl group)and AB23A control group(AB23A group)were set up.The results showed that AB23A could significantly attenuate the levels of serum biochemical indices of liver functions in senecioine-induced acute liver injury mice,as evident by alleviated hepatocyte necrosis and hepatic sinusoidal stasis.AB23A also improved kidney function of mice exposed to senecionine,fascinated urinary excretion and repaired electrolyte disorders,as well as decreased content of senecioine metabolites.Further,the protein and mRNA expression of genes related to the water balance pathway were measured.AB23A could significantly down-regulate the elevated protein and mRNA expression levels of aquaporin 2(AQP2)and angiotensin Ⅱ type 1 receptor,and inhibit the transport of AQP2 to the apical plasma membrane induced by senecionine exposure.AB23A also significantly decreased serum levels of angiotensin Ⅱ.In vitro studies further confirmed that AB23A regulates AQP2 expression in renal inner medullary collecting duct cells 3(IMCD3).These data indicate that AB23A regulates the expression of AQP2 in renal medulla,thereby affecting its water reabsorption in mice with senecionine-induced acute liver injury.This work achieves a better understanding of the diuretic effect of AR,and provides experimental foundation and theoretical basis for the treatment of PA-induced acute liver injury by AR in clinics.
alisol B 23-acetatepyrrolizidine alkaloidaquaporin 2diureticwater-liquid balance
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