Preliminary Exploration Of The Hepatotoxic Components And Mechanisms Of Polygonum Multiflorum Based On The Accumulation Of Components In Vivo And The Dysfunction Of Transporters In The Live

Polygonum multiflorum(HSW),an extensively used traditional Chinese medicine(TCM),originated from the tuberous root of Polygonum multiflorum Thunb.,which has been used in modern medicine due to its anti-aging,antilipemic and protecting liver,anti-inflammatory,anti-osteoporosis and anti-cancer effects.Since its "Affinal Drug and Diet"effect,HSW was widely recommended by medical experts to nourishing liver and kidney,which has been perceived to be safe.However,the hepatoxic reports induced by HSW and its preparation are increasing in recent years.Moreover,China Food and Drug Administration(CFDA)and other drug administration departments have repeatedly issued safety notices on HSW and its preparation.For this reason,the hepatotoxicity induced by HSW had aroused more and more attention from researchers.However,the hepatoxic mechanism and components induced by HSW remains totally unclear so far.Therefore,the hepatoxic components and mechanisms need to be intensively studied and clarified.HSW included various components,like anthraquinones,stilbene glycosides and tannins.At present,the researches for hepatoxic components in HSW were mainly targeted at anthraquinones and stilbene glycoside.In addition,a few studies had shown that the hepatotoxicity also could be induced by tannins.It was clear that there was obvious contradiction between these researches.Moreover,various TCM,such as Rhubarb,Semen Cassiae and Polygonum cuspidatum,contain higher anthraquinones,which had rare hepatoxic reports,so it is further indicates why it is so difficult to determine hepatoxic substances of HSW.Additionally,another problem was that many studies was primarily based on hepatocellular experiments in vitro,which lacked understanding of the accumulation and exposure of components in vivo.As we know,the accumulation of drugs in tissue is an important prerequisite for the toxicity of tissues,especially in the case of long-term administration.Meanwhile,the interaction between different components is another factor that also need to be considered.Therefore,the study for the distribution and accumulation of components in biosamples in vivo is of great significance for exploring the hepatotoxicity of HSW.At present,it was generally believed that hepatoxic mechanism induced by HSW was related to the following factors,including oxidative stress(OS),functional changes of enzymes or transporters in liver,and patients with immunodeficiency or idiosyncratic reaction.Although the researches took many factors and perspective into consideration,the conclusion was full of contradictions.Studies on the hepatoxic mechanism induced by HSW via dysfunction of transporters have only paid attention to individual transporters,while a comprehensive work from the perspective of uptake and efflux transporters is still important,so the hepatoxic mechanism need to be further investigated.Bile acid and bilirubin are important constituents of bile,but excessive accumulation of endogenous micromolecule such as unconjugated bilirubin and bile acids in liver could lead to liver injury.Bile acid and bilirubin can be efficiently transported into hepatocytes by the uptake transporters locating in the basal side of hepatocytes.Subsequently,these components can be excreted to bile through the efflux transporters in the basolateral side.Therefore,the dysfunction of bile acid or bilirubin-related transporters would lead to cholestasis,bilirubin accumulation and liver injury.Because of the clinical symptoms of hepatoxicity induced by HSW showed hepatoxic symptoms such as jaundice,abnormal liver function,and acute hepatitis,which were similar to the symptoms of transporters dysfunction.It was suggested that hepatotoxicity caused by HSW might be related to dysfunction of bile acid or bilirubin-related transporters.Moreover,oxidative stress would induce an increase in reactive oxygen species(ROS),which might affect the function of ABC efflux transporters.Once such effect was down-regulated,ROS or drugs would be accumulated in tissues or cells,which might even induce cell damage.However,it has not been reported whether there was a connection between hepatotoxicity induced by HSW and the dysfunction of ABC efflux transporters.In summary,it can be seen that functional changes of the transporters in liver have a greater impact on liver function,so a comprehensive study on the function of uptake and efflux transporters in liver is of great significance to explore the hepatoxic mechanism induced by HSW.In addition,Kidney is an important excretory organ,and plays a key role in excretion of drugs,endogenous substances and toxic substances.Renal tubular secretion and reabsorption are usually regulated by various transporters.Researches showed that long-term or high dose use of HSW and its components might induce kidney damage.However,little has been known concerning the correlation of renal tubular secretion and reabsorption-related transporters with HSW-induced renal damage by now.In other words,although there were many studies on the hepatoxic components and mechanisms caused by HSW,no exact conclusion has been reached.Meanwhile the hepatotoxicity and nephrotoxicity of HSW was generally more prone to occur in long-term and high-dose administration.Therefore,the accumulation characteristics of components in liver,kidney,plasma and bile after oral administration with HSW for 42 days in rats was explored,including qualitative identification and quantitative determination,which aimed to found the possible hepatoxic components of HSW.Meanwhile,the function of bile acid,bilirubin and ABC-related intake transporters(Oatp1a1,Oatp1b2,Ntcp,Oat1,Oat2,and Oct2)and efflux transporters(Mrp2,Mrp3,Bsep.Bcrp,and P-gp)were comprehensively investigated to reveal the hepatotoxicity and nephrotoxicity mechanism induced by HSW.1.Quantitative and quantitative analysis of the accumulation of the components and its metabolite in vivo of rats after long-term oral administration of HSWFirstly,Two HSW extracts,including water extract-PMW(15 g·kg-1,20 mL·kg-1)and ethanol extract-PME(12 g·kg-1,20 mL·kg-1),were prepared to give to SD rats by orally administration for 42 days.Secondly,an UPLC-LTQ-Orbitrap MS method was applied to qualitatively identify the components and metabolites of HSW in liver,kidney,plasma and bile.As a result,24 compounds,including emodin(EM),emodin-8-O-β-glucoside(EMG),2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside(TSG),other anthraquinones and their metabolites,were identified or tentatively identified.The components and metabolites of HSW in biosamples were mainly TSG and its metabolites,EM and its metabolites.The glucuronides/sulfates of TSG were the major forms in all biosamples.While the oxidates/glucuronides of EM and other anthraquinones(like rhein)were the major forms in all biosamples.In addition,the relative abundance of TSG,EM and EMG in all biosamples were higher than other components,so the content of the three components were determined.Lastly,the established UPLC-MS/MS analysis method was used to simultaneously quantitative analysis of EM,EMG and TSG in biosamples.As a result,the accumulative characteristic and content of EM,EMG and TSG were different between biosamples,while the accumulation of TSG was more extensive,which indicated that after long-term induction the accumulation of TSG in biosample more than EM and EMG.However,the components that mainly accumulate in liver and kidney were TSG and EM.Therefore,we surmise that hepatotoxicity and nephrotoxicity induced by HSW may be related to the content of TSG and EM.2.The hepatoxic mechanism induced by HSW in rats through the function of transporters in liver and kidneyFirstly,plasma biochemical indexes(ALT,AST,TBA,ALP and TBIL levels)and pathological examination(liver and kidney)after oral administration of HSW for 42 days were adopted in this study to verify HSW-induced hepatotoxicity and nephrotoxicity.In comparison,only ALT and ALP levels in PMW group had been notably up-regulated(P<0.05),while ALT,TBA and ALP levels in PME group were also apparently up-regulated(P<0.01).These findings indicate that both extracts of HSW induce hepatotoxicity in rats,among which,PME induce cholestasis.Combine pathological examination and physiological indexes of rats,the results indicated that both extracts of HSW would induce mild liver and kidney injury,and the rats in PME group were more serious.Secondly,the function of bilirubin,bile acid and ABC-associated transporters in liver and kidney of rats were detected via the activity and mRNA expression of the transporters.The activity results indicated that PME displays a notable inhibitory effect on Oatplal and Oatp1b2,but PMW had almost no effect on them.Meanwhile,both extracts had obvious effects on activating Mrp2.However,mRNA expression results showed that PME can distinctly down-regulate mRNA expression of the efflux transporters(Bsep,Ntcp,Bcrp and P-gp).While the mRNA expression of the uptake transporters(Oatplal and Oatp1b2)and efflux transporters(Mrp3)in the basal side of hepatocytes were up-regulated or no change.In other words,the efflux transporters in the basal side of hepatocytes were almost inhibited.Therefore,PME would lead to oxidative stress or the accumulation of bilirubin and bile acids in liver,which might even induce hepatotoxicity.Although PMW has different degrees of regulation on transporters in liver,its inhibition or activation has no obvious tendency.Therefore,it cannot be speculated whether PMW could cause oxidative stress or accumulation of bilirubin and bile acids in liver.In addition,in kidney both PME and PMW could significantly up or down-regulated the mRNA expression of uptake transporters(Oatl,Oatl and Oct2).Moreover,PME could outstandingly down-regulated the mRNA expression of efflux transporters(P-gp and Mrp2),while PMW could only markedly down-regulate levels of Mrp2.Therefore,the inhibitory effect of PME on efflux transporters was higher than that of PMW,so the toxic components were more likely to accumulate in rats of PME group,which was consistent with the effects of PME and PMW on liver.In conclusion,we speculated that TSG and EM might play a synergistic role in hepatotoxicity induced by HSW.Meanwhile,the hepatotoxicity of PME was more severe than PMW,which was related to the content of TSG and EM.The functional change of multiple transporters in liver and kidney was one of the important hepatotoxicity and nephrotoxicity mechanism of HSW.