| Objective: In recent years,there has been a significant increase in reports of liver injury induced by polygonum multiflorum,a traditional Chinese medicine commonly used in clinic.However,its hepatotoxic components and liver injury mechanism have not yet been fully elucidated.In this study,the effects of emodin and2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucopyranoside(TSG),two active constituents in polygonum multiflorum,on bile acid metabolism and hepatocyte apoptosis in vivo and in vitro were investigated at the animal and cellular levels.Methods: 1.Animal level: 84 specific pathogen free male ICR mice were kept in the laboratory for 3 days to acclimate,and then were randomly divided into 12 groups(n=7)following 7,14 and 28 day treatment of vehicle,100 mg/kg emodin,100mg/kg emodin + 150 mg/kg TSG and 100 mg/kg emodin + 750 mg/kg TSG,respectively.After daily weighing,the corresponding drugs were administered orally according to their respective weight.ICR mice were sacrificed 24 hours after the last administration,blood and liver were collected.The changes of liver biochemical indexes and histopathology,the differences in bile acid composition and content as well as the expression levels of m RNA and protein related to bile acid metabolism and hepatocyte apoptosis were investigated.2.Cellular level: Human normal liver cells HL-7702(L-02)were used as for hepatotoxicity study in vitro.Based on the cell proliferation inhibition evaluation experiment(CCK-8 assay),the concentration of emodin(10 M and 30 M for low and high concentrations,respectively),the co-administration dose ratio of TSG(2 times and 8 times)and the incubation time after administration(24 h)were determined.By means of optical microscope,flow cytometry,immunofluorescence staining,quantitative real-time PCR and western blotting,the effects of different concentrations of emodin and TSG on the morphology,proliferation inhibition or apoptosis rate,mitochondrial membrane potential(MMP)and the expression levels of m RNA and protein related to bile acid metabolism and hepatocyte apoptosis were investigated.Results: 1.Animal level:(1)Behavioral observation: After 7 day treatment,ICR mice in each group had smooth and glossy hair,synchronized weight increasing rate and normal diet and behavior.However,the urine color of the emodin group,the mixed low-dose group and the mixed high-dose group gradually darkened(from light yellow to light red)compared with the control group.After 14 day treatment,ICR mice in each group had smooth and glossy hair and normal diet and behavior,the differences of urine color were still significant and the weight increasing rat e began to show differences(control group>mixed low-dose group>emodin group>mixed high-dose group).After 28 day treatment,ICR mice in each group had normal diet and behavior,the differences of urine color and weight increasing rate were significant(mi xed low-dose group≥control group>emodin group≥mixed high-dose group).Except for the ICR mice in the control group,whose hair was smooth and glossy,the hair of other groups were fluffy and dull.In addition,compared with the control group,ICR mice in the other groups were manic and more sensitive to external stimuli.(2)Biochemical indicators and histopathological examination: With the prolongation of administration time,liver injury of each administration group aggravated(including the increase of ALT and AST,the decrease of liver coefficient,etc.),but the mixed low-dose group showed certain protective effect on liver when compared with the emodin group and the mixed high-dose group.Interestingly,in terms of indexes of cholestasis,the intrahepatic cholestasis in each drug experimental group aggravated first and then alleviated.H&E and TUNEL staining showed liver injury and hepatocyte apoptosis were significant time-dependent,and the injury was aggravated when TSG was co-administered with emodin,but there was no significant dose-dependent.(3)Bile acid composition changes: Based on the results of principal component analysis(PCA),the drug intervention failed to cause significant changes in bile acid composition at 7 days after administration,and the bile acid composition of each group changed significantly at 14 days after administration,while the bile acid composition of each group recovered after 28 day treatment.Based on the results of orthogonal partial least squares discriminant analysis(OPLS-DA),we found that two bile acids,namely α-muricholic acid(α-MCA)and hyocholic acid(HCA),demonstrated significant differences in different times and patterns of administration,and might be used as a potential bio-marker for this research.(4)Expression levels of m RNAs and proteins(liver tissue): With the prolongation of administration time,the expression levels of m RNAs and proteins related to bile acid metabolism,including FXR,SHP,BSEP,MRP2,and MRP3,were decreased first and then increased.From the perspective of the overall regulation of bile acid homeostasis,when the low-dose of TSG was co-administrated,emodin-induced disorder of bile acid was recovered,while the high-dose TSG co-administrated showed exactly opposite effect.In terms of apoptosis,the time-dependent down-regulation of BCL-2 and the time-dependent up-regulation of Bax were observed in each drug experimental group.At 7 days after administration,the cleaved-caspase-3 in each group was not significant,while at 14 days and 28 days after administration,cleaved-caspase-3 increased in each drug experimental group and cleaved-PARP-1 which did not occur at 7 days of administration had appeared.It was worth noting that the mixed low-dose group delayed the process of emodin-induced hepatocyte apoptosis in the early stage of administration,but aggravated the apoptosis in the late stage of administration(mixed high-dose group>mixed low-dose group>emodin group>control group).2.Cellular level:(1)Cell morphological observation and immunofluorescence staining: The results showed that when the low-concentration emodin(10 μM)was given,the low/high multiple of TSG inhibited the emodin-induced apoptosis,and when the high-concentration emodin(30 μM)was given,the low/high concentration of TSG aggravated the apoptosis induced by emodin.(2)Apoptosis rate and MMP: The result showed that the combination of low multiple TSG and low-concentration emodin decreased the apoptosis rate and reversed the decrease of MMP caused by emodin.Although the high concentration of TSG also increased the MMP,the apoptosis rate was increased.The late stage of apoptosis was observed after treatment with high-concentration emodin,and the apoptosis rate and MMP were positively correlated with the concentration of TSG.(3)Expression levels of m RNAs and proteins(L-02): TSG did not play a role in the direct regulation of FXR and BSEP,but it could up-regulate the expression levels of SHP,MRP2 and MRP3.When the low/high-concentration emodin was co-administrated,the expression levels of CYP7A1 and NTCP were down/up-regulated in a concentration-dependent manner,respectively.From the perspective of the overall regulation of bile acid homeostasis,the combination of TSG and low-concentration emodin concentration dependently reduced the synthesis and uptake of bile acid and increased the efflux of bile acid,while the combination of TSG and high-concentration emodin was exactly opposite.In terms of apoptosis,TSG could up-regulate the expression level of BCL-2 and concentration dependently down/up-regulate the expression level of BAX when the low/high-concentration emodin was co-administrated.Cleaved-caspase-3 was observed in the combination of high concentration of TSG regardless of the concentration of emodin,and the experimental groups with high-concentration emodin showed distinct cleaved-caspase-3 immunoblotting.Conclusions: Drug-induced liver injury caused by polygonum multiflorum is related to the relative contents of TSG and emodin.The disorder of bile acid metabolism caused by emodin aggravates emodin-induced hepatocyte apoptosis.FXR,a key nuclear receptor for bile acid synthesis,transport and metabolism,and its downstream regulator SHP may play the key role in this damage process.This research provides an explanation for the "processing and detoxification" of shengshouwu,and also provides a theoretical basis for the different pharmacological effects of polygonum multiflorum extract and its monomer component emodin in vivo and in vitro. |
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