Comparative Study On The Liver And Bile Duct Injury Effects Of Tripterygium Wilfordii Glycosides And Their Main Components And Their Mechanis

Tripterygium wilfordii multiglycoside(GTW),known as ’traditional chinese herbal hormones’,is the main fat-soluble component isolated from Tripterygium wilfordii Hook f.(TWHF),which exerts a broad spectrum of biological activities,including anti-inflammation,anti-tumor growth and immunosuppression.Recently,it has been reported that GTW exposure resulted in injury of various organs,including kidney,spleen and heart,especially liver,in animals and even in human.Several ingredients,such as triptonide,wilforlide A,triptolide(TP)and celastrol(CEL)are identified in GTW,and TP and CEL are the representative components.TP exerts several biological activities including anti-inflammation and immunosuppression,and CEL has been listed as one of ’five natural products of most likely to be developed into drugs’ by Cell journal.Considering the insufficient evidence of the effects of GTW,TP and CEL,it is urgent to explore their effects on liver and hepatic target cells.Taken together,this study illustrated the effects of GTW,TP and CEL on liver,and further unravel the mechanisms by network pharmacological analysis and molecular biology experiments in vivo and in vitro.Objective:(1)To evaluate and compare the effects of GTW,TP and CEL on liver in vivo.(2)To investigate hepatic target cells of GTW and compare the effects of GTW,TP and CEL on biliary epithelial cell in vitro.(3)To explore the mechanisms of liver injury caused by GTW,TP and CEL via network pharmacological analysis and a variety of molecular biology experiments in vivo and in vitro.Methods:(1)Male and female BALB/c mice,8 weeks old,were provided with standard chow and tap water ad libitum.Mice were gavage with GTW(150 mg/kg,200 mg/kg),equivalent doses of TP(12 μg/kg,16 μg/kg)and equivalent doses of CEL(37.5 μg/kg,50 μg/kg)for 24 h.Mice were sacrificed and the liver were immobilized with 4%formaldehyde and embedded in paraffin.Tissues were cut into 4.5 μm sections and stained with hematoxylin and eosin(H&E).Serum levels of ALT,AST.MDA and SOD levels were determined following the manufacturer’s instructions of corresponding kits.The mRNA levels of IL-6,IL-1β,TNF-α,HNF-4a,ALB and cytokeratin 19(CK-19)were detected by q-PCR assays.(2)Firstly,MPH,KC,HIBEC and LX-2 cells were identified by checking the mRNA expression of several cell markers,including HNF-4α,F4/80,CK-19 and α-SMA,and the IC50 values of GTW on MPH,KC,HIBEC and LX-2 were detected by CCK-8 experiment.After identifying the hepatic target cells,the effects of GTW,TP and CEL on cell morphology was observed by microscope and viability was detected by CCK-8 experiment.Cell scratch experiment was further used to detect the effects of GTW,TP and CEL on cell migration,and flow cytometry was used to detect the effects of GTW,TP and CEL on cell cycle and cell apoptosis.(3)Targets of ’GTW’ and ’bile duct injury’ were obtained from TCMSP,BATMAN-TCM,GeneCards and OMIM,respectively,and the overlapping targets were obtained through R software.The PPI network of overlapping targets was constructed by STRING,and GO analysis and KEGG analysis were performed by R software.The mRNA levels of Caspase 3,Bax,Bcl2,Ki67,c-Myc,and CTGF were detected by q-PCR assays.The protein levels of Caspase 3,Bax,Bcl2,p-ERK,ERK,p-AKT,AKT were detected by Western blot assays.Results:(1)The ratios of liver weight over body weight of mice were increased after GTW and TP treatment.Serum levels of ALT,AST and ALP were markedly increased in GTW and TP treatment groups.Histological analysis further revealed the inflammatory cell infiltration in the portal area of the liver,indicating the bile duct injury of mice after GTW and TP treatment.GTW and TP significantly stimulated oxidative stress in mice,as illustrated by increased hepatic MDA and reduced SOD content.In addition,the q-PCR data confirmed that GTW and TP increased the inflammatory responses and subsequently resulted in liver injury.Interestingly,hepatic target cells of GTW,TP and CEL were biliary epithelial cells,which was confirmed by the decreased mRNA levels of CK-19 and unaltered mRNA levels of HNF-4α.(2)The CCK-8 data confirmed that hepatic target cells of GTW were biliary epithelial cells,and GTW,equivalent doses of TP and 40 times equivalent doses of CEL significantly induced biliary epithelial cells injury.Our results showed that GTW,TP and CEL inhibited cell proliferation and migration,impacted cell morphology,arrested G0/G1 phase and S phase in the cell cycle and induced cell apoptosis.(3)Comparing the potential targets of GTW with 228 candidate targets relating to bile duct injury,there was an overlap containing 35 targets,such as Caspase 3,Bax,ERK and AKT.Consistently,q-PCR and Western blot results showed that GTW,TP and CEL stimulated cell apoptosis,increased the levels of Caspase 3 and Bax and decreased Bcl2 level by inhibiting phosphorylation of ERK and AKT.Conclusion:(1)The effects of GTW,TP and CEL on liver were evaluated and compared with the equivalent dose in vivo,which provide insights into the GTW-induced liver injury.(2)Biliary epithelial cells were identified as hepatic target cells of GTW.GTW,TP and CEL significantly stimulated biliary epithelial cells injury,which was illustrated by the influences on cell proliferation,cell morphology,cell migration,cell cycle and cell apoptosis.(3)The network pharmacological results showed that there was an overlap containing 35 targets.As demonstrated by molecular biological experiments,GTW,TP and CEL significantly stimulated biliary epithelial cell injury.Overall,these results provided novel insights into the pharmacological mechanisms of the liver injury and bile duct injury caused by GTW.