| ObjectiveBecause of the adverse effects of tripterygium glycosides tablet(TG)in clinical practice,the hepatotoxic effects of TG were investigated based on the abnormal fatty acid-cholesterol metabolism,and the mechanism of hepatotoxic effects of TG and compound glycyrrhizin tablet(CG)to reduce the hepatotoxicity caused by TG.Method1 Hepatotoxicity of tripterygium glycosides tablet in SD rats and attenuating effect of compound glycyrrhizin tablet in combination.SD rats,male,were randomly divided into a control group,TG-L group(TG 189.0 mg·kg-1 with 20 times the clinically equivalent dose),TG-H group(TG 472.5 mg·kg-1 with 50 times the clinically equivalent dose),TG-L+CG group(TG 189.0 mg·kg-1 with CG 20.25 mg·kg-1 with the clinically equivalent dose),and TG-H+CG group(TG 472.5 mg-kg-1 with CG 20.25 mg·kg-1),6 animals in each group.The rats were administered by gavage once a day and examined by autopsy after 3 weeks of continuous administration.The toxic effects of TG on liver injury and the attenuating effects of TG in combination with CG were studied by examining the liver weight and liver/system number,serum biochemical liver injury-related indexes,liver histopathological changes,and ultrastructural changes of hepatocytes.2 Mechanisms of tripterygium glycosides tablet induced hepatotoxicity in rats based on the fatty acid metabolism PPARa pathway and compound glycyrrhizin tablet dosing attenuation.The expression changes of PPARα,ACADM,ACAA1,ACSL1,ACSL4,ACSL5,and CYP4A1 at mRNA level and protein level in rat liver tissues were detected by realtime qPCR,western blot and immunohistochemistry,and the content of A-CoA,an intermediate product of PPARa pathway,was detected by ELISA.3 Study on the mechanism of hepatotoxicity of tripterygium glycosides tablet in rats based on cholesterol metabolism LXR-α pathway and compound glycyrrhizin tablet dosing to reduce toxicity.The expression changes of LXR-α,LDLR,ABCA1,ABCG1,HMG-CoAR,CYP7A1,CYP8B1,and CYP27A1 at mRNA level in rat liver tissues were detected by real-time qPCR.Western blot method was used to further verify the key proteins with changes in transcript level,LXR-α,LDLR,ABCG1,CYP7A1,CYP8B1,CYP27A1,and ELISA to detect the level of HMG-CoAR,an enzyme related to the LXR-α pathway.4 Effect of tripterygium glycosides tablet with compound glycyrrhizin tablet on the metabolic pathway of PPARα and LXR-α in HepG2 cellsHepG2 cells were used as the in vitro test subjects to observe the toxic effects of TG on HepG2 cells,and to detect by real-time qPCR,western blot the intracellular PPARα,ACADM,ACAA1,ACSL1,ACSL4,ACSL5,LXR-α,ABCG1,LDLR CYP7A1,CYP8B1,CYP27A1 at mRNA level and protein level to positively validate the mechanism of attenuation of CG and PPARa agonist fenofibrate(FB)after combined with TG,respectively.Result1 Tripterygium glycosides tablet can cause liver injury in rats,and the characterization indexes mainly point to abnormal lipid metabolism,which has an attenuating effect when used in combination with compound glycyrrhizin tablet.1.1 The serum biochemical results showed that compared with the control group,the low and high-dose groups(TG-L and TG-H)administered for 3 weeks resulted in significant increases in serum triglyceride,LDL-C,CHO and TBIL(P<0.05,P<0.01)and significant decreases in ALB,TP and HDL-C(P<0.05,P<0.01).The high-dose group also resulted in a significant increase in serum ALT(P<0.05).In combination with CG,the serum triglyceride and LDL-C levels were significantly reduced(P<0.05,P<0.01)and HDL-C level was significantly increased(P<0.01)in the matched group compared with the same dose of TG alone;the low-dose matched group also improved the increase of CHO and TBIL levels(P<0.05),while the high-dose matched group also reduced The high-dose combination group also reduced the increase of ALT and the decrease of ALB and TP(P<0.05).1.2 The results of the histopathological examination showed that the TG-H group caused a significant increase in liver weight and liver coefficient,swelling and vacuolar degeneration of hepatocytes,lymphocyte infiltration foci in the confluence area,and lamellar necrotic foci with hemorrhage under the liver peritoneum;the histological structure of rat liver was significantly repaired after CG formulation,and no cell necrosis or degeneration was observed.1.3 The ultrastructural findings showed that the TG-L group caused an increase in intracytoplasmic lipid droplets in the hepatocytes,with myelin-like structures,increased electron density in the mitochondria,and decreased cristae,and the ultrastructural damage in the TG-H group was more severe.group compared with the TG-H group,except for the occasional small amount of myelin-like structures in the cytoplasm,which showed significant improvement.2 Tripterygium glycosides tablet caused liver injury in rats through regulating the fatty acid metabolism PPARa pathway,and the PPARα pathway was associated with the attenuating effect of tripterygium glycosides tablet and compound glycyrrhizin tablet pairing.Real-time qPCR,western blot,and immunohistochemical staining results showed that TG-L and TG-H all caused different degrees of decrease in mRNA levels and protein levels of PPARα,ACADM,ACAA1,CYP4A1 in rat liver tissues(P<0.05,P<0.01);ACSL1,ACSL4,and ACSL5 had different degrees of elevated mRNA levels and protein expression levels(P<0.05,P<0.01).After the combination with CG,the mRNA levels of PPARα,ACAA1,ACADM,and CYP4A1 were increased to different degrees,while the mRNA levels and protein expression levels of ACSL1,ACSL4,and ACSL5 were downregulated when compared with the same dose of TG alone.ELISA results showed that there was no significant change in the A-CoA content of each group.3 Tripterygium glycosides tablet caused liver injury in rats through regulating cholesterol metabolism LXR-α pathway,and the LXR-α pathway was associated with the attenuating effect of tripterygium glycosides tablet and compound glycyrrhizin tablet pairing.Real-time qPCR results showed that compared with the control group,the transcription levels of LXR-α,LDLR,ABCG1,CYP7A1,CYP8B1,and CYP27A1 in the liver tissues of the TG-L and TG-H groups were down-regulated to varying degrees,and the difference between the TG-H group and the control group was significant(P<0.05,P<0.01).After combined treatment with CG,the transcription levels of CYP7A1,CYP8B1,and CYP27A1 were significantly up-regulated compared with the same dose of TG alone group(P<0.05,P<0.01),and the transcription level of LDLR was significantly up-regulated in the TG-H+CG group(P<0.01).Western blot results showed that compared with the control group,the protein expressions of LDLR,CYP7A1,and CYP8B1 in the liver tissues of the TG-L and TG-H groups were significantly down-regulated(P<0.05,P<0.01),and the protein expression of ABCG1 in the TG-L group was significantly down-regulated(P<0.05).The protein expression of LXR-α in the TG-H group was significantly down-regulated(P<0.05).After the combination of CG and TG,the protein expression levels of ABCG1 and LDLR were significantly up-regulated(P<0.05,P<0.01),and the protein expression of LXR-αshowed an increasing trend.The protein expression of CYP7A1 and CYP8B1 in the TG-H+CG group was significantly up-regulated(P<0.05,P<0.01).ELISA results showed that there was no significant change in the content of HMG-CoAR in each group.4 Effect of tripterygium glycosides tablet in combination with compound glycyrrhizin tablet on PPARa and LXR-α metabolic pathways in HepG2 cellsCCK-8 results showed that the IC50 concentration of TG was 220.5567 μg·mL-1,and 15,45,and 135 μg·mL-1(about IC5,<IC10,<IC30,respectively)were further selected as the low,medium,and high(TG-1,TG-m,TG-h)doses of Tripterygium glycosides tablets for cell experiment for subsequent experiments.The dose was chosen as 60 μg·mL-1 for CG and 10 μmol·L-1 for FB.In terms of fatty acid metabolism pathway,Real-time qPCR results showed that compared with the control group,the three TGS-treated groups all caused significant increases in the transcription levels of ACSL5,ACAA1,and ACADM(P<0.01)and a significant reduction in the transcription level of ACSL4(P<0.01).Both TG-m and TGh could significantly reduce the transcription level of PPARa in HepG2 cells(P<0.01),and TG-m could also significantly reduce the transcription level of ACSL1(P<0.01).Compared with the TG-m group,the transcription levels of PPARα,ACAA1,ACADM,and ACSL4 in the TG-m+CG and TG-m+FB groups were significantly increased(P<0.05,P<0.01),while the transcription level of ACSL5 was significantly decreased(P<0.01).Western blot results showed that compared with the control group,the TGm and TG-h groups had significantly decreased protein expression of PPARα,ACAA1,and ACADM in HepG2 cells(P<0.01),and significantly increased protein expression of ACSL5(P<0.01).TG-h treatment also significantly increased the protein expression of ACSL1 and ACSL4 in HepG2 cells(P<0.01).Compared with the TG-h group,the expression of ACSL5 protein in the TG-h+CG and TG-h+FB groups was significantly down-regulated(P<0.05,P<0.01).Compared with the TG-m group,the protein expressions of PPARa and ACADM in the TG-m+CG and TG-m+FB groups were significantly increased(P<0.05,P<0.01),and the protein expression of ACAA1 in the TG-m+FB group was significantly increased(P<0.05).The expression of ACSL5 protein in the TG-m+CG group was significantly decreased(P<0.01).In terms of cholesterol metabolism pathway,compared with the control group,the TG-H group could significantly increase the transcription level of LXR-α in HepG2 cells(P<0.01),while the TG-M and TG-H groups could significantly reduce the transcription level of LDLR(P<0.01),and TG groups could significantly reduce the transcription level of CYP7A1(P<0.01).The transcription level of CYP27A1 was significantly increased(P<0.01).The transcription levels of LDLR and CYP8B1 in the TG-h+CG group were significantly higher than those in the TG-h group(P<0.01).The transcription levels of LDLR,CYP7A1,CYP8B1 and CYP27A1 in the TG-m+CG and TG-m+FB groups were significantly higher than those in the TG-m group(P<0.01).The transcription levels of CYP7A1 in the TG-1+CG and TG-1+FB groups were significantly higher than those in the TG-1 group(P<0.01).Western blot results showed that compared with the control group,both TG-m and TG-h groups could significantly reduce the protein expression of LXR-α,ABCG1,LDLR,CYP7A1,CYP8B1 and CYP27A1 in HepG2 cells(P<0.01).Compared with the TG-h group,the expression of LDLR protein in the TG-h+CG group was significantly increased(P<0.05).Compared with the TG-m group,the protein expressions of ABCG1 and CYP7A1 in the TGm+CG and TG-m+FB groups were significantly increased(P<0.05,P<0.01),and the protein expressions of LDLR and CYP27A1 in the TG-m+CG group were significantly increased(P<0.05,P<0.01).The CYP8B1 protein expression in the TG-m+FB group was significantly increased(P<0.01).Conclusions1 TG caused liver injury in rats at 20 and 50 times the clinically equivalent dose for 3 weeks of continuous administration.TG caused structural changes in liver tissues,hepatic function damage and decreased hepatic synthesis capacity in rats with dose correlation,and CG ameliorated the liver injury caused by TG to some extent.The combination of CG and TG reduced the accumulation of lipid droplets in the cytoplasm of hepatocytes by decreasing the levels of triglycerides,TBIL,CHO and LDL-C and increasing the levels of HDL-C,ALT,ALB and TP,improving disorders of lipid metabolism.2 Based on the study of PPARa pathway of fatty acid metabolism,TG can inhibit the transcription level and protein expression of PPARα,ACADM,ACAA1,and CYP4A1 genes,and increase the transcription level and protein expression of ACSL1,ACSL4 and ACSL5 genes,leading to fatty acid metabolism disorder and lipid deposition in hepatocytes.CG can alleviate the disorder of lipid metabolism by counterregulating the above targets.3.Based on the LXR-α pathway of cholesterol metabolism,inhibition of LXR-α,ABCG1,and LDLR reduced cholesterol reverse transport and endocytosis,and inhibition of CYP7A1 and CYP8B1 reduced cholesterol biotransformation,resulting in cholesterol accumulation in the body.CG can induce LXR-α,ABCG1,and LDLR to promote cholesterol reverse transport and endocytosis and induce CYP7A1,CYP8B1,and CYP27A1 to increase cholesterol biotransformation and reduce cholesterol accumulation in the body.4 TG exposure inhibited HepG2 cell proliferation.It regulates the mRNA level and protein expression of key genes in fatty acid metabolism such as PPARα,ACAA1,ACSL1,ACSL4,ACSL5 and LXR-α,ABCG,LDLR,CYP7A1,CYP8B1,CYP27A1 in cholesterol metabolism.PPARα agonist fenofibrate could reverse-regulate the above genes.CG showed similar effects to the agonist,both of them could reverse-regulate the transcription level and protein expression of PPARα and LXR-α pathway-related genes induced by TG.In summary,this study was validated by in vivo and in vitro assays and found that TG causes abnormal fatty acid-cholesterol metabolism through affecting PPARα and LXR-α pathways,which in turn leads to lipid metabolism disorders and hepatotoxicity;CG has similar effects to PPARα pathway agonists and ameliorates TG-induced lipid metabolism disorders by regulating PPARa and LXR-α-related pathways and alleviates liver injury.Based on the PPARα and LXR-α pathways,this study initially revealed the mechanism of hepatic injury caused by TG and the detoxification mechanism of CG with TG,providing theoretical basis for the safe clinical application of TG. |
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