α-KG Activates Hepatic Stellate Cells In Advanced Liver Fibrosis Through P300/Smad2/3 Signaling Pathway

Objective:To explore the ability of serum metabolites to distinguish between SO-2(mild-moderate)and S3-4(advanced liver fibrosis)of chronic hepatitis B liver fibrosis,and its key metabolite α-ketoglutarate(The relevant mechanism of a-KG)on the activation of hepatic stellate cells.Methods:A collection of 157 patients with chronic hepatitis B liver fibrosis under liver biopsy and 80 healthy controls(HC),including 106 cases in the test group and 52 cases in the verification group.The diagnostic criteria of viral hepatitis B are based on the"Guidelines for Prevention and Treatment of Chronic Hepatitis B(2015)".Serum and liver tissue were collected and analyzed by gas chromatography-mass spectrometry(GC-MS).Import the original data table into SIMCA-P software for statistical analysis.The unsupervised principal component analysis(PCA)is used to draw the score chart,and the orthogonal partial least squares discriminant analysis model(OPLS-DA)is constructed,and the accuracy of the analysis model is tested by permutation.The variable projection importance value(VIP)>1 in the OPLS-DA model was combined with univariate statistical analysis P<0.05 to jointly screen different metabolites.The receiver operating curve(ROC)and the area under the ROC curve(AUC)were used to evaluate the diagnostic value.And compared with traditional serological indicators Aspartate aminotransferase-to-platelet ratio index(APRI)and Fibrosis 4 Score(FIB-4).MetaboAnalyst 2.0 software was used to screen metabolite pathways.The α-KG analog dimethyl α-ketoglutarate(DMKG)was used to treat hepatic stellate cells.CCK-8 colorimetric method detects cell proliferation activity.The cross-well migration test detects cell migration.Immunohistochemistry(IHC)analysis of liver in patients with liver fibrosis.And use Western blot to detect the expression of P300,Ac-P300,α-SMA and type I collagen;P300siRNA or C646 to detect the possible mechanism of hepatic stellate cells(HSC)activation.Results:In the test set,PCA analysis was not enough to distinguish S0,S1-2,S3-4 and HC,S0-1 and S2-4 could not be distinguished,while SO-2 and S3-4 showed a certain degree of discrimination.In the three-dimensional(3-D)PCA score chart,SO-2 and S3-4 are also clearly distinguished.OPLS-DA model,analysis shows that there is a clear distinction between SO-2 and S3-4.Use VIP>1.0 and P<0.05 to screen metabolites.A total of 20 metabolites were screened out in the test group.After the verification group was screened,1,5-anhydroglucitol,2-hydroxyglutaric acid,adipic acid,α-ketoglutarate(α-KG),small asparagine 4,benzyl alcohol,gluconic acid,pentane 12 metabolites including diacid,linoleic acid,4-hydroxybutyric acid,arachidonic acid and ibuprofen.The combination of 12 serum metabolites has a high accuracy rate for distinguishing SO-2 and S3-4 phases.The area under the combined ROC curve is 0.961,which is higher than that of APRI and FIB-4.According to MetaboAnalyst 2.0 analysis,the metabolic pathways that significantly change liver fibrosis include linoleic acid metabolism,tricarboxylic acid(TCA)cycle,and arachidonic acid metabolism.Most of them revolve around the key metabolite α-KG.Further analyze the relevant functional mechanism research of the key metabolite α-KG in promoting the activation of hepatic stellate cells in vivo.Using transforming growth factor-β as a positive control,it was found that DKMG can promote the activation of HSC in vivo.Immunohistochemistry showed that the expression of P300 and Ac-P300 cells in the liver tissues of S3-4 stage was significantly higher than that of SO-2 stage.Both DMKG and transforming growth factor-β could enhance the ac-P300 protein level of Lx2 cells.α-KG-induced hepatic stellate cell activation was down-regulated by siRNA-mediated P300 or C646-mediated P300 inhibition,indicating that α-KG-mediated hepatic stellate cell activation requires P300.P300 inactivation inhibits α-KG-induced Smad2/3 intranuclear binding.Studies have shown that α-KG promotes the expression and activity of p300,and promotes its binding with Smad2/3 in the nucleus,resulting in the expression of α-SMA and type Ⅰ collagen.Conclusion:The combination of 12 serum metabolites has a higher ability to distinguish between patients with chronic hepatitis B liver fibrosis SO-2(mild-moderate)and S3-4(advanced).The key metabolite of this metabolic combination,α-KG,induces hepatic stellate cell activation through the P300/Smad2/3 signaling pathway.