The Mechanism Research On The Anti-Liver Fibrosis Of Emodin Via Induced Hepatic Stellate Cell Senescence By Regulating Glutaminolysis

BackgroundLiver fibrosis is a dynamic that is highly integrated molecules,cells and tissues injury repairing process,which drives the excessive accumulation of extracellular matrix(ECM)components.The activation of hepatic stellate cells(HSC)plays an important role in maintaining the secretion of ECM.Targeted inhibition of HSC activation has become an important anti-liver fibrosis strategy.Among them,inducing HSC senescence is an important strategy to inhibit its activation.Besides,recent studies have shown that glutaminolysis reprogramming occurred during the activation of HSC.After HSCs was activated,their bioenergy and biosynthesis requirements increase dramatically.Inhibiting glutaminolysis effectively inhibits their activation.Glutamine is hydrolyzed to glutamate under the catalysis of glutaminase(GLS),and finally converted to α-ketoglutarate(α-kg).The α-kg participates in the tricarboxylic acid cycle(TCA)as an important intermediate,replenishing sufficient ATP and key metabolic intermediates for cell proliferation,which were for the biosynthesis of nucleic acids,amino acid and lipids.Therefore,it is of great significance to explore the relationship between glutaminolysis and activation and senescence of HSCs.More and more evidences show that the natural active compound emodin has extensive pharmacological activity in the treatment of liver fibrosis,but its molecular mechanism remains to be explored.Therefore,we explore how emodin can interferes with liver fibrosis by regulating glutaminolysis to inducing senescence of the HSCs and its mechanism.Methods1.The research in vitroTreating the HSC-LX2 cells with different concentrations of emodin for 24h,the viability of HSC-LX2 cells was detected by MTT experiment.Three effective concentrations were selected for subsequent experiments.Western blot,real-time PCR,immunofluorescence staining,etc.were used to detect the effect of emodin or glutaminase inhibitor CB-839 on HSC-LX2 activation marker proteins α-SMA and COL1α,and senescence marker proteins p16,p21,and Hmgal.Furthermore,the effects of emodin or CB839 on senescence of HSC-LX2 were confirmed by β-galactosidase staining and flow cytometry techniques.Besides,the effects of emodin on glutaminolysis was evaluated by the analysis of related indicators of glutaminolysis.On the other hand,we explore the effect of emodin and Nur77 agonist Csn-B on Nur77 nuclear translocation.Then,it was detected the relevant indicators of glutaminolysis,activation and senescence of HSCs by using Nur77 siRNA transfecting HSC-LX2,and treating with emodin.It was aim to clarify the importance of Nur77 in emodin regulating the fate of HSC-LX2 cells.Real time PCR and western blot were used to assess the expression of GLS1 after treated with emodin or silencing Nur77 or DNA methylation inhibitor 5’-AZA,and use methylation-specific PCR analysis to confirm its appearance genetic regulation mechanism.Finally,the online network PPA was used to predict the interaction of Nur77 with DNA methylase,and verify it by co-immunoprecipitation(CO-IP)experiments.2.The exploration in vivoFirstly,it was aimed to confirm the effects of emodin and emodin-VA liposomes on liver injury and liver fibrosis.The 48 ICR mice were randomly divided into 8 groups(n=6):normal control group,emodin treatment group,VA liposome group,emodin-VA liposome group,CCl4 group,CCl4+emodin treatment Group,CCl4+emodin-VA liposome treatment group.The liver tissue structure and collagen deposition were detected by histological HE staining,Masson staining,Sirius Red staining.Besides,it was detected that the serum liver injury indexes AST,ALT,ALP,TBIL,LDH and liver fibrosis indicators HYP,HA,LN,PC-Ⅲ,Ⅳ-C by the kits.The activation of HSCs cells was detected by α-SMA immunohistochemistry,real-time,and western blot.Secondly,it was aimed to confirm the key role of Nur77 in emodin in regulating glutaminolysis HSCs to induce them senescence in vivo.The 30 ICR mice were randomly divided into 5 groups(n=6):NC shRNA lentiviral group,CCl4+NC shRNA lentiviral group,CCl4+NC shRNA lentiviral+emodin-VA liposome treatment group,CCl4+Nur77 shRNA lentiviral group,CC14+Nur77 shRNA lentiviral group+emodin-VA liposome treatment group.The liver tissue structure and collagen deposition were detected by histological HE staining,Masson staining,Sirius red staining.Serum AST,ALT,ALP,TBIL,LDH and HYP,HA,LN,PC-Ⅲ,Ⅳ-C were detected by corresponding kits.The activation of HSCs was detected by α-SMA immunohistochemistry,real-time PCR,and western blot.In addition,the content of ATP and glutamate in liver tissue was detected by the kits,to verify the target role of Nur77 in improving liver injury and liver fibrosis by emodin.The expression of glutaminase and its co-localization with α-SMA were detected by tissue immunofluorescence double staining to verify the role of Nur77 in the regulation of glutaminolysis by emodin.Finally,the expression of cell senescence marker proteins p16,p21,Hmgal and their co-localization with α-SMA were detected by real-time,western blot,and tissue immunofluorescence double staining,to verify of the key role of Nur77 in emodin-induced senescence of HSCs.ResultsEmodin inhibited HSCs glutaminolysis and activation and induce them senescence in vivo and in vitro,which showed the similar results when treating with glutaminase inhibitor CB-839.Further research found that emodin can promote nuclear translocation of the energy regulating molecule Nur77,which showed the similar results when treating with Nur77’s agonist Csn-B,indicating that emodin activated Nur77.Interestingly,knockout Nur77 via Nur77 siRNA transfection in vitro or Nur77 shRNA lentiviral in vivo offset the inhibitory effect of emodin.It shows that Nur77 was the essence for emodin in the intervention of liver fibrosis.We further found that emodin inhibited the protein expression of GLS1,but Nur77 siRNA treatment reversed the effect of emodin.After treating HSCs with the DNA methylation inhibitor 5’-AZA,the expression of GLS1 was down-regulated.Methylation-specific PCR detection showed that the methylation level of GLS1 increased after treating with emodin,and the methylation level of GLS1 decreased after transfection HSCs with Nur77 siRNA,which counteracted the effect of emodin.Through the online website PPA prediction,it is found that Nur77 has a higher binding affinity with DNA methyltransferase DNMT3b,and the result of co-immunoprecipitation verifies the prediction result.In addition,the results of in vivo studies showed that compared with the administration of emodin solution,the administration of emodin-VA liposomes had better hepatoprotective effects,and better improvement of the liver fibrosis caused by CCl4 without toxic effect on liver tissues.ConclusionsEmodin can inhibit the activation of HSCs and induced them senescence in vivo and in vitro.Emodin blocked glutaminolysis by promoting the methylation of GLS1 via activating the Nur77 to interact with DNA methyltransferase DNMT3b.Compared with the administration of emodin solution,the administration of emodin-VA liposomes has a better hepatoprotective effect and better improvement of the liver fibrosis caused by CCl4 without toxic effect on liver tissue.Our study revealed that emodin induces HSCs glutaminolysis-related senescence for anti-liver fibrosis.The emodin-VA liposomes administration reduces toxicity of the direct intraperitoneal administration of emodin solution,providing a certain theoretical and experimental basis for the application of emodin against liver fibrosis.