Role Of Membrane Transporters In Semen Cassiae-mediated Protection Of Mercury Toxicity In Kidney And Aggravation Of ANIT Toxicity In Liver

Semen cassiae?SC?,a widely consumed medicine and drink,possesses a broad range of pharmacological activities,such as anti-hyperlipidemia,hypotensive,lower cholesterol,nephroprotective,and hepatoprotective activities.Nonetheless,safety evaluation of SC has revealed a potential risk of kidney and liver toxicities.A recent research suggested that SC could alter the expression and activities of hepatic microsomal P450 isozymes,suggesting its potential drug-drug interactions.However,little is known about its transporter-based potential interactions with clinical drugs.The overall goal of the present study was to systematically investigate the role of transporters in SC-mediated drug-drug interactions,as well as its effects on kidney and liver.Cell lines overexpressing OAT1,OAT3,organic cation transporter 1?OCT1?,OCT2,organic anion transporting polypeptide 1B1?OATP1B1?,or OATP1B3 were successfully established and validated,and the inhibitors for each transporter were indentified by the initial screening of SC constituents.SC extract was shown to almost abolish the histological alterations induced by HgCl₂ in rat kidneys.The survival rates of HEK-OAT1 and HEK-OAT3 cells after incubating with Cys-Hg were used to determine the protective effects of SC constituents on mercury toxicity in vitro.Only OAT1-overexpressing cells could be protected by the inhibitors of OAT1 identified from SC.Additionally,we also successfully constructed the comparative models of OAT1 and OAT3,which were used to predict and screen the inhibitors of OAT1 and OAT3 from SC constituents.The present study showed for the first time that ANIT-treated mice could be a model for studying the idiosyncratic hepatotoxicity of herbs,such as SC.Mouse studies showed that SC markedly aggravated ANIT-induced cholestatic liver injury in a dose-dependent manner.The potentiating effect of SC on ANIT toxicity was not due to oxidative stress,inflammation or fatty acid ?-oxidation,but mainly due to increased accumulation of BAs.SC interfered with the crosstalk between AMP-activated protein kinase?AMPK?and farnesoid X receptor?Fxr?in the liver,which led to a suppression of bile salt export pump?Bsep?.Fourteen-day treatment of SC in mice confirmed its activation of AMPK and inhibition of Bsep.Moreover,the long-term treatment of SC activated intestinal Fxr-Fgf15 pathway,which was associated with a marked alteration in the intestinal bacterial composition.Compared with SC-treated mice,cotreatment of ANIT and SC increased markedly the major SC constituents,in particular emodin and obtusin,in the liver.Further studies showed that emodin could protect against ANIT-induced cholestasis in mice,mainly through its anti-oxidative and anti-inflammatory activities.Nonetheless,emodin also interfered with AMPK-Fxr crosstalk and inhibited Bsep in livers of mice.In summary:1)The present study identifies the potential SC-drug interactions based on organic anion and cation transporters.2)The present study demonstrates that SC extract could protect against mercury-induced acute kidney injury in rats,which appears to be attributed to the inhibitory effect of SC constituents on OAT1,but not OAT3.3)The present study suggests that inhibitors of OAT1 and OAT3 can be identified from natural products by combining in silico and in vitro screening methods.4)The present study shows for the first time that SC could aggravate the ANIT toxicity in mice,which appears to be due to its interference with AMPK-Fxr crosstalk.5)The present study demonstrates both anti-cholestatic and pro-cholestatic effects of emodin,and identifies emodin as a potentially reponsible ingredient in the hepatotoxicity of emodin-containing herbs.