Silibinin Ameliorates Non-alcoholic Steatohepatitis By Regulating CFLAR-JNK Pathway

Objective:Non-alcoholic steatohepatitis(NASH)is a chronic metabolic syndrome,characterized by intracellular excessive fat deposition and inflammation.Although there’s no history of excessive drinking,all changes in liver tissues are similar to alcoholic liver disease.Nowadays,the worldwide prevalence of NASH continues to increase.The pathogenesis of NASH is closely related to liver lipid accumulation,insulin resistance and oxidative stress disorder.Currently,the pathogenesis of NASH is not well understood,and there are no clinically approved drugs that could treat NASH.Treatment of NASH mainly involves lipid-lowering,insulin resistance regulation,antioxidant and anti-inflammatory drugs,with unsatisfactory results.Caspase-8 and fas-associated protein with death domain-like apoptosis regulator(CFLAR)is one of the natural immune regulatory molecules.The latest research suggests that CFLAR could inhibit phosphorylation of c-Jun N-terminal kinase(JNK),and reverse the process of NASH by regulating glucolipid metabolism,insulin resistance and oxidative stress disorder.Silibinin is a flavonolignan extracted from the fruit and seeds of Silybum marianum(L.)Gaertn,reported to be the most effective flavonoid compound against hepatic disease.It can function as hepatoprotective,antioxidant,antineoplastic,regulates glucolipid metabolism and maintains the stability of liver cell membrane.Silibinin can regulate IRS1-PI3K-AKT pathway to ameliorate dyslipidemia and insulin resistance in SD rats fed by high-fat diet and in BRL3 A and HepG2 cells pretreated by palmitic acid(PA).It inhibits NF-κB activity to improve hepatic nitrosative stress in db/db mice fed by methionine-choline deficiency(MCD)diet.Some reports suggest that a silibinin-phospholipid complex can prevent the mitochondrial dysfunction in Wistar rats fed by MCD diet.Clinically,the mixture of silibinin,phosphatidylcholine and vitamin E is applied to treat patients with NASH.At present,it’s not clear about the effect of silibinin on the CFLAR-JNK pathway in the liver of NASH.The purpose of this paper is to study the effect of silibinin on CFLAR-JNK pathway and its downstream key regulatory factors of lipid metabolism,insulin resistance and oxidative stress in hepatocytes in NASH models.Methods:(1)The in vivo models of NASH were established by feeding C57BL/6 mice with MCD diet and intragastric silibinin(10 mg/kg/day,20 mg/kg/day)for 6 weeks.When the processing was completed,pathology slice of hepatic lobule was made for Oil red O,H&E and MT staining and biochemical indicators were detected,including serum levels of ALT and AST on behalf of the degree of liver injury in mice,hepatic TG and TC content in the representative of the degree of liver fat accumulation,hepatic MDA content on behalf of the liver oxidative damage degree,activity of oxygen radical scavenging enzymes such as GSH-Px,CAT and of HO-1,activity of oxygen free radical generating enzymes such as CYP 2E1 and CYP 4A.The relative mRNA expression levels related to lipid accumulation in liver tissues were assessed by RT-q PCR,including Pparα,Fabp5,Cpt1α and Acox related to lipid oxidation,Scd-1,Gpat and Mttp related to lipid transport out of liver,Srebp-1c and Pnpla3 related to lipid synthesis.Western blot was used to detect the relative protein expression levels of CFLAR,p JNK,NRF2,CYP 2E1 and CYP 4A in liver tissues.(2)HepG2 cells were treated with 0.5 mM oleic acid(OA)for 24 h to establish the model and then treated with silibinin(5 μM,20 μM,50 μM,100 μM)for 24 h.After the treatment,the biochemical indexes of NASH(namely,the intracellular contents of TG,ROS,NO and the uptake of 2-NBDG)were measured by the corresponding detection kits.The relative mRNA expression levels of SREBP-1C,PNPLA3 and PPARα related to lipid accumulation were assessed by RT-q PCR in HepG2 cells.Western blot was applied to detect the relative protein expression levels of CFLAR and p JNK and the downstream key regulatory factors of lipid metabolism(SREBP-1C,PNPLA3,PPARα),insulin resistance(PI3K,p AKT)and oxidative stress(NRF2,CYP 2E1,CYP 4A)in HepG2 cells.(3)NCTC-1469 cells were treated with a mixture of 0.25 mM PA and 0.5 mM OA for 24 h to establish the model and then treated with silibinin(50 μM,100 μM)for 24 h.After the treatment,the qualitative analysis of silibinin on lipid accumulation was carried out by Oil red O staining.The biochemical indexes of NASH(namely,the intracellular contents of TG and the uptake of 2-NBDG)were also measured by the corresponding detection kits.Western blot was used to assess the relative protein expression levels of CFLAR and p JNK and the downstream key regulatory factors of insulin resistance(p IRS1)and oxidative stress(NRF2)in NCTC-1469 cells.(4)NCTC-1469 cells were treated with an Ad-sh Cflar for 24 h to establish the model and then treated with silibinin(50 μM,100 μM)for 24 h.After the treatment,the mRNA expression level of Cflar was measured by RT-q PCR.Western blot was used to detect the relative protein expression levels of CFLAR and p JNK and the downstream key regulatory factors of insulin resistance(p IRS1)and oxidative stress(NRF2)in NCTC-1469 cells.Results:(1)Compared with those in the normal group,histological pathological section analysis showed that lipid accumulation,inflammation and balloon in the group of C57BL/6 mice induced by MCD diet were significantly increased in the liver tissue.Treatment with silibinin resulted in a dose-dependent reduction of intracellular lipids,inflammation and balloon.The results of biochemical tests suggested that the activity of serum ALT and AST,the hepatic contents of TG,TC and MDA in the mice fed MCD diet were increased.Silibinin could dose-dependently reduce the activity of ALT and AST in serum and the content of TG,TC and MDA in liver homogenate.The results of activity detection of oxidative stress related enzymes showed inhibition of the enzyme activity of CAT,GSH-Px and HO-1 but promotion of the enzyme activity of CYP 2E1 and CYP 4A for the mice treated with MCD.Silibinin could increase the activity of CAT,GSH-Px and HO-1 in a dose-dependent manner and inhibit the activity of CYP 2E1 and CYP 4A.The results of RT-q PCR indicated that the mRNA expression levels of Pparα,Fabp5,Cpt1α,Acox,Scd-1,Gpat and Mttp were inhibited for the MCD diet treated mice while no changes were found in the mRNA expression levels of Srebp-1c and Pnpla3.Silibinin could improve the mRNA expression of Pparα,Fabp5,Cpt1α,Acox,Scd-1,Gpat and Mttp in a dose-dependent manner and had no significant effect on the mRNA expression of Srebp-1c and Pnpla3.The results of Western blot analysis showed that MCD diet inhibited the protein expression of CFLAR and NRF2 and promoted the protein expression of CYP 2E1,CYP 4A and p JNK.Silibinin could increase the protein expression of CFLAR and NRF2 in a dose-dependent manner and inhibit the protein expression of CYP 2E1,CYP 4A and p JNK.(2)In the OA-induced group of HepG2 cells,compared with those in the blank control group,TG,ROS and NO contents were significantly increased while the uptake of 2-NBDG was inhibited.Silibinin could reduce the intracellular content of TG and NO in a concentration-dependent manner and improve the uptake of 2-NBDG in HepG2 cells.RT-q PCR method suggested that the mRNA expression levels of PNPLA3 and SREBP-1C were up-regulated in the OA treatment group.The mRNA expression of PPARα was down-regulated and the ratio of PPARα/SREBP-1C was reduced.Silibinin could decrease the mRNA expression of PNPLA3 and SREBP-1C in a concentration-dependent manner,up-regulate the mRNA expression of PPARα,and increase the ratio of PPARα/SREBP-1C.Western blot analysis showed that the protein expression levels of p JNK,PNPLA3,SREBP-1C,CYP 2E1 and CYP 4A in the OA treatment group were up-regulated while the protein expression levels of CFLAR,PPARα,PI3 K,p AKT and NRF2 were down-regulated.Silibinin could down-regulate the protein expression of p JNK,PNPLA3,SREBP-1C,CYP 2E1 and CYP 4A and up-regulate the protein expression of CFLAR,PPARα,PI3 K,p AKT and NRF2.(3)In PA and OA induced NCTC-1469 cells,compared with those in the blank control group,the results of Oil red O showed that the content of lipid drops in cells was significantly increased while silibinin could reduce the content of lipid drops in cells in a concentration-dependent manner.The treatment of PA and OA could significantly increase the content of TG in cells and decrease the content of 2-NBDG in NCTC-1469 cells.Silibinin could reduce the intracellular TG content and increase the content of 2-NBDG.Results of Western blot analysis showed that the protein phosphorylation level of JNK was up-regulated,the protein expression levels of CFLAR and NRF2 were down-regulated,and the protein phosphorylation level of IRS1 was down-regulated in the PA and OA treatment group.Silibinin could increase CFLAR and NRF2 protein expression and phosphorylation of IRS1 in a concentration-dependent manner and reduce the phosphorylation of JNK.(4)After NCTC-1469 cells were treated with Ad-sh Cflar for 24 h,the mRNA and protein expression levels of CFLAR were significantly inhibited,which could be relieved by silibinin in a concentration-dependence manner.The results of Western blot analysis showed that in the model group,protein expression levels of NRF2 and p IRS1 were inhibited while phosphorylation of JNK was promoted.Silibinin could reduce the phosphorylation of JNK as well as enhance the protein expression of NRF2 and the phosphorylation of IRS1 in a concentration-dependent manner.Conclusion:In this paper,four models of NASH were adopted to study the effects of silibinin on the CFLAR-JNK pathway,namely:(1)C57BL/6 mice treated with MCD diet,(2)HepG2 cells treated with OA,(3)NCTC-1469 cells treated with PA plus OA,(4)NCTC-1469 cells treated with Ad-sh Cflar.The results showed that silibinin could activate the expression of CFLAR and inhibit the phosphorylation of JNK,then regulate several downstream pathways to ameliorate NASH,including:(1)by promoting lipid oxidation,lipid transport out of liver and inhibiting lipid synthesis to reduce the lipid accumulation,(2)by regulating the IRS1-PI3K-AKT pathway to improve insulin resistance,(3)by regulating the activity of NRF2 and its downstream antioxidant enzyme system and free radical generation enzyme system to improve the oxidative stress disorder.