The Role And Mechanism Of DMKG In The Activation Of HSC And Liver Fibrosis

Hepatic fibrosis is a complex wound healing response to liver injury from various chronic insults,including viruses,drugs,alcoholism,autoimmune hepatitis,and metabolic diseases.Fibrosis of the liver is characterized by excessive deposition of extracellular matrix(ECM),predominantly collagen-1.Excessive deposition of ECM disrupts normal liver architecture,and if left untreated,progresses to cirrhosis.Liver cirrhosis lacks effective treatment methods and has a higher mortality rate.Thus,the regulation of liver fibrosis is very important for chronic liver disease and gradually becomes research hotspot.Activated hepatic stellate cells(HSC)are the principal cell type promoting synthesis and deposition of ECM in response to liver injury.In normal livers,HSC are found within the perisinusoidal space of Disse in a quiescent state marked by abundant lipid droplets containing predominantly,fatty acids,triglyceride and retinyl esters.But upon liver injury,HSC transdifferentiate into myofibroblast-like cells characterized by loss of lipid droplets,expression of a-SMA and ECM,and increased proliferation and contractility.HSC are crucially involved in the promotion of hepatic fibrosis.Thus,inhibition the activation of HSC is critical for the resolution of fibrosis.Macroautophagy(hereafter called autophagy)is a highly conservative cellular process in eukaryotes for segregation and degradation of cytoplasmic constituents by autophagosomes and their digestion by lysosomes.It mediates the removal of dysfunctional or damaged cytoplasmic constituents,which are digested and recycled for cellular metabolic needs.HSC activation and ECM synthesis depend on a continuous supply of energy.Previous studies confirmed that the autophagy of HSC supplies the cells with ATP by processing cytoplasmic lipid droplets.Moreover,inhibition of autophagy could block the activation of HSC.However,most agents used to block autophagy confer major toxic side effects and so their application is limited.How to efficiently suppress autophagy in vivo without major side effects has become of central importance.α-ketoglutarate is a naturally occurring substance found in cells,which has high safety.Some researches suggested that the level ofa-ketoglutarate was involved in the regulation of autophagy,though the conclusions and mechanisms were not consistent.Previously,there have been no studies investigating the effect ofa-ketoglutarate levels on the autophagy or activation of HSC.DMKG is a generally used cell-permeable analog of a-ketoglutarate,which can be easily cleaved intracellularly to increase cytoplasmic levels of a-ketoglutarate.The present study investigated the role of DMKG in HSC activation and liver fibrosis.And we also examined whether autophagy was involved in the mechanism.Furthermore,the mechanism of how DMKG affects autophagy of HSC was explored.PART Ⅰ:THE ROLE OF DMKG IN THE ACTIVATION OF HSC IN VITROObjectives:MTT assay was used to select safe concentrations of DMKG that has no effect on the survival of both liver cells and activated HSC in 24 hours.HSC were maintained in the culture medium for 24 h with the addition of DMKG at the safe concentrations before real-time RT-PCR and western-blot were performed to evaluate the activation of HSC.Methods:1.HSC-T6 and BRL-3A cell lines were used in the experiments of this part in vitro.HSC-T6 is an immortalized HSC cell line with activated phenotype and BRL-3A is a kind of liver cell line.Both cells were conventionally cultured with Dulbecco’s modified Eagle medium(DMEM,Gibco)supplemented with 10%fetal bovine serum.2.Both HSC-T6 and BRL-3A cells were treated with DMKG at gradient concentrations(0-32 mM)for 24 h.The effect of DMKG on the viability of both cells was assessed by MTT assay.The results were quantitatively analyzed.3.According to result of MTT assay,concentrations of DMKG that had no significant impact on survival of HSC-T6 or BRL-3A cells were selected in the real time RT-PCR and western-blot test.Total RNA was isolated from cells using Trizol from HSC-T6 cells untreated or treated with DMKG at the selected concentrations.Real-time RT-PCR was performed to assess the mRNA lev els of a-SMA and collagen-1,both of which are markers for HSC activation.Comparative quantitative analysis was performed.4.Total protein was isolated from cells using RIPA buffer from HSC-T6 cells untreated or treated with DMKG at the selected concentrations.Western-blot assay was performed to assess the expression of a-SMA and collagen-1,both of which are markers for HSC activation.Comparative quantitative analysis was performed.Results:1.DMKG at the concentrations of 1-18 mM had no significant effect on the viability of BRL-3A cells.DMKG at the concentrations of 20-32 mM can significantly suppress the survival of BRL-3A cells in a dose dependent manner.2.DMKG at the concentrations of 1-16 mM had no significant effect on the viability of HSC-T6 cells.DMKG at the concentrations of 18-32 mM can significantly suppress the survival of HSC-T6 cells in a dose dependent manner.3.HSC-T6 cells were more sensitive than BRL-3A cells to the citotoxic effect of DMKG at the concentrations of 18-32mM.4.DMKG at 4 mM decreased the level of a-SMA mRNA.Treatment of HSC-T6 with 1 mM-and 4mM-DMKG led to a down-regulation of collagen-1 mRNA.5.1 mM-and 4 mM-DMKG inhibited the expression of a-SMA and collagen-1 significantly in HSC.Conclusions:1.These results suggest that DMKG at low concentrations is low cytotoxicity for survival of hepatocytes and HSC.Hepatocytes showed better tolerance of chemical toxicity associated with higher concentrations of DMKG than HSC.2.DMKG at the concentrations that didn’t decrease cell viability could inhibit the activation of HSC in vitro.PART Ⅱ:THE ROLE OF DMKG IN CCL4-INDUCED LIVER FIBROSISObjectives:The well-established carbon tetrachloride(CCl4)-induced liver fibrosis model of Wistar rats was used for DMKG treatment in vivo.Then liver fibrosis were evaluated.Methods:1.20 Wistar rats(6 weeks old)was intraperitoneally injected(i.p)with CCl4(0.5 ml/kg)diluted with olive oil(50%CCl4 in olive oil,vol/vol)twice a week(in Tuesday and Thursday)for 9 weeks.2.Rats were randomly divided into four groups(n=five each group):control,DMKG,CCl4-induced liver fibrosis model,and CCl4/DMKG co-treated groups.For the therapeutic administration of DMKG,200 mg/kg/rat dissolved in 2 ml phosphate-buffered saline(PBS)was given i.p twice a week(in Wednesday and Friday)for 9 weeks.Animals that received an equal volume of olive oil and PBS were used as controls.The livers were isolated and blood samples from the left ventricles were collected.3.Histochemistry:Fresh rat livers were fixed with formalin and then embedded in paraffin wax.5 μm paraffin sections were de-waxed in xylene,rehydrated,and stained for Sirius red staining and Masson’s trichrome staining.Sirius red staining and Masson’s trichrome staining were performed for the evaluation of liver fibrosis.Polarized light was used to capture images of sirius red(Collagen is shown in bright red when the background becomes dark.).Quantitative analysis of Sirius red-positive areas were calculated with the following formula:fibrotic area/(total area-vascular lumen area)×100%.4.Hepatic hydroxyproline content was colorimetrically quantified to show collagen deposition of the livers.Fresh liver tissue was homogenized and hydrolyzed with HCl at 110℃.The samples were neutralized in NaOH and colorized with Ehrlich’s reagent.Absorbance of samples and standards were measured at 570 nm.Quantification was done using the standard curve.5.Serum biochemistry:Serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected using standardized kits(Nanjing Jiancheng Bioengineering Institute)according to the manufacturer’s instructions.Results:1.Collagen deposition decreased significantly in the CCl4/DMKG co-treated group compared with the CCl4-induced fibrosis group as evaluated by the Masson’s trichrome staining and the Sirius Red staining.Quantitative analysis showed a 44%down-regulation of Sirius Red-stained fibrotic areas.2.Hydroxyproline content of the livers was significantly decreased in the CCl4/DMKG co-treated group compared to the CCl4-induced fibrosis group.3.In the test for ALT and AST,we observed no significant difference in liver function between the DMKG/CCl4 co-treated group and the CCl4 fibrotic model.No significant difference was found between the DMKG-treated group and the control group,neither.Conclusions:1.DMKG reduces CCl4-induced liver fibrosis in vivo.2.DMKG treatment does not induced or aggravated hepatocytes injury in vivo.PART Ⅲ:MECHANISM OF HOW DMKG INHIBITS HSC ACTIVATION AND LIVER FIBROSISObjectives:In this part,we examined whether autophagy was involved in the mechanism of how DMKG inhibit the activation of HSC and liver fibrosis.The mechanism of how DMKG affects autophagy of HSC was also explored.Methods:1.Double immunofluorescent staining:a-SMA is a marker for the activation of HSC while hepatocytes do not express a-SMA.LC3B and Beclin-1 both are markers for autophagy.5 μm sections were de-paraffinized and boiled for antigen retrieval.The sections were incubated with the appropriate primary antibodies(mouse anti-a-SMA and rabbit anti-LC3B or Beclin-1)overnight.Then,the sections were incubated with their respective secondary antibodies(antibody with red fluorescence for a-SMA,antibody with green fluorescence for LC3B and Beclin-1).Images were acquired using a fluorescence microscope.Fluorescence intensity and co-localization were measured and quantified by software.2.Observation of autophagosomes with transmission electron microscopy(TEM)is the "gold standard:in detecting autophagy.HSC-T6 cells were treated with DMKG.Then the cells were fixed and the ultrathin sections were cut before TEM was performed to assess the autophagy level.3.3MA is a classical autophagy inhibitor.Total protein was isolated from HSC-T6 cells treated with DMKG or 3MA.Western-blot assay was performed to assess the expression of markers for HSC activation(a-SMA and collagen-1)and markers for autophagy(LC3B-Ⅱ/Ⅰ and Beclin-1).By comparing the effects of DMKG and 3MA on the autophagy and activation of HSC-T6 we identified the role of DMKG in the activation of HSC.4.HSC-T6 cells were treated with DMKG.Oil red O staining for HSC was performed to show the lipid droplets in the cells and quantification of Oil red O stained areas was performed.5.HSC-T6 cells were treated with DMKG.ATP was measured using a commercially available firefly luciferase-based ATP assay kit(Beyotime)as per the manufacturer’s instructions.6.Activation of mTORC1 leads to the inhibition of autophagy.S6 is a characterized substrate of mTORC1.Phosphorylation of S6 can be regarded as a mark reflecting the degree of mTORC1 activation.Total protein was isolated from from HSC-T6 cells untreated or treated with DMKG.Western-blot assay was performed to assess the expression of total S6 and phosphorylated S6.Comparative quantitative analysis was performed.7.DMKG increased the cytoplasmic level of a-ketoglutarate,which is then converted to AcCoA.Some studies found that AcCoA could facilitates acetylation of EP300 while some other studies indicated that autophagy could be suppressed when some autophagy-related proteins are acetylated by EP300.Lipoic acid(LA)is an alternative AcCoA-replenishing agent to DMKG and c646 is an inhibitor that competes for AcCoA.We investigated the autophagic levels of HSC-T6 with the addition of c646 and LA together with DMKG to identify the mechanism of how DMKG inhibits the autophagy of HSC.Results:1.Using double immunofluorescent staining,we found that fluorescent signals of markers for autophagy and marker for the activation of HSC were all increased in fibrotic livers compared to the control group.However,signals were decreased in fibrotic livers treated with DMKG.Co-localization analysis showed a positive association between the autophagy and activation of HSC.2.As observed via TEM,abundant autophagosomes were present in HSC without DMKG treatment.Quantification clearly revealed a marked reduction in the number of autophagosomes in cells treated with DMKG.3.The expression of markers for autophagy were notably attenuated in both the DMKG-treated group and the 3MA treated group.More importantly,the expression of markers for the activation of HSC decreased along with autophagic levels in both treatments.4.Previous studies have shown that the lost lipid droplets in HSC are processed via autophagy to generate ATP,which fuels HSC activation.We found that DMKG could attenuate the depletion of LDs and the subsequent ATP production.5.No significant difference was observed in the phosphorylation of S6 in HSC-T6 cells between DMKG-treated group and untreated group.That is to say,DMKG inhibit the autophagy of HSC in a mTORC1-independent way.6.The autophagy of HSC was inhibited by LA as well as DMKG,and c646 could reverse the autophagic-inhibitory effects of DMKG and LA.Conclusions:1.DMKG inhibits HSC activation and liver fibrosis through the inhibition of autophagy in HSC.2.DMKG inhibits the autophagy of HSC via AcCoA and EP300.