The Protective Effects And Synergetic Mechanism Of Longdan Xiegan Formula Against Pyrrolizidine Alkaloid-induced Liver Injury

1.Background and Objective Pyrrolizidine alkaloids(PAs)are a kind of widespread phytotoxins that are highly hepatotoxic.Consuming large amounts of plants containing PAs,such as Gynura japonica,can cause hepatic sinusoidal obstruction syndrome(HSOS),which is clinically characterized by abdominal distention,hepatalgia symptom,ascites,jaundice,hepatomegaly,and cholestasis.There is no effective treatment for PA-induced HSOS in clinics.And the current clinical treatment mainly adopts symptomatic treatment,which partially contributes to the high mortality rate and poor prognosis of PA-induced HSOS at late stage.Therefore,it is in urgent need of developing effective therapeutic means.Longdan Xiegan formula(LDXG)is firstly recorded in Yi Fang Ji Jie and has effect of clearing away the liver and gallbladder fire.Our previous studies have shown that Longdan Xiegan decoction has a protective effect against hepatic injury caused by the PA compounds Clivorine and Isoline.Therefore,an acute HSOS model was established by oral administration of total alkaloids(TA)made from G.japonica and this research will focus on constructing the ’component-target-pathway’ network to elucidate the synergetic effect of LDXG against PA-induced hepatotoxicity.The present study may point to the possibility of utilizing LDXG for protection against PA-induced liver injury,and provide the basis for the research and development of drugs for the treatment of HSOS caused by G.japonica as well clinically.2.Methods(1)To evaluate the protective efficacy of LDXG and herbs in the formula against PAinduced liver injury:(1)Mice were pretreated with different doses of LDXG.The efficacy of the LDXG against PA-induced liver injury was evaluated by the serum biochemical indexes,liver pathological sections,and contents of pyrrole-protein adducts in serum,i.e.,the marker for PA-induced toxicity.(2)Different modified formula were prepared and applied for the evaluation of the protection effect against PA-induced liber injury in mice to uncover the herbs in the formula that antagonize PA-induced hepatotoxicity.(2)To explore the synergetic effects and pharmacodynamic mechanism of LDXG against PA-induced liver injury:(1)The potential mechanism of LDXG against PA-induced liver injury was studied by the network pharmacology approach.(2)Key molecules in pathways related to oxidative stress and bile acid homeostasis were examined.(3)Contents of bile acids in different bio-samples of mice,including serum,liver,gallbladder,ileum,and feces,were analyzed by LC-MS.(4)Most of PAs are metabolic activated by CYP3A4 to initiate their toxicity.Therefore,the contents of PAs and their metabolites in different bio-samples of mice,i.e.,urine,feces and serum,were measured.Furthermore,the inhibitory effects of the main active ingredients of the formula on the enzyme activity of CYP3A4,a key enzyme for PAs metabolism,were evaluated by using the human liver microsomal system.(5) Cluster analysis,principal component analysis,partial least squares discriminant analysis,and correlation analysis were performed on the clinical biochemical indicators of liver function,gene expression level,bile acid content,and metabolism of PAs,and preliminary discussion on the synergetic effects of LDXG against pyrrolizidine alkaloidinduced hepatotoxicity.3.Results(1)LDXG improve PA-induced liver injury in mice in a dose-dependent manner.Gentianae Radix et Rhizoma,Scutellariae Radix,and Plantaginis Semen,as well as their combination can obviously protect mice from PA-induced liver injury.Thus,the three drugs might be the main pharmacodynamic material against PA-induced liver injury in the formula.(2)The pharmacodynamic mechanism of LDXG against PA-induced liver injury involves regulating oxidative stress,increasing the synthesis of hepatic glutathione(GSH),regulating bile acid homeostasis via increasing the efflux of bile acids and reducing intrahepatic bile stasis,and fascinating the clearance of PAs and their nitrogen oxides as well as GSH conjugates,and inhibiting the metabolic activation of PAs.4.ConclusionLDXG can protect mice from liver injury caused by PAs,and Gentianae Radix et Rhizoma,Scutellariae Radix,and Plantaginis Semen are the main pharmacodynamic material basis that antagonize PA-induced hepatotoxicity.The mechanism is related to the regulation of the antioxidant system,upregulation of liver GSH levels,modulation of bile acid homeostasis,and influence the metabolism of PAs.And there are synergetic pharmacodynamic effects among Gentianae Radix et Rhizoma,Scutellariae Radix,and Plantaginis Semen on PA-induced liver injury in mice.