Effects And Mechanisms Of Isovitexin On Liver Fibrosis: Based On MiR-21 Mediated-PI3K/Akt And Glutathione Metabolic Pathways

ObjectivesDue to the lack of specific treatment drugs,the treatment effect of liver fibrosis is poor.This study used transcriptomics and metabolomics as the means and combined with cell and animal experiments to explore the effects and mechanisms of Isovitexin（IVT）against liver fibrosis,aiming to provide new ideas for the treatment in hepatic fibrosis.MethodsPart Ⅰ:The pharmacodynamical research of IVT on liver fibrosis（1）The effects of IVT on liver fibrosis in vivoC57BL/6J mice were injected with CCl₄ intraperitoneally for 10 weeks to induce liver fibrosis,followed by IVT administration for 6 weeks.The pathological damage and collagen deposition were assessed with H&E and Sirius staining,respectively;serum biochemical levels including AST,ALT and ALB were analyzed by automatic biochemical analyzer;also,the liver fibrosis biomarkers（HA,LN,PCⅢand Col-IV）,inflammatory indicators（TNF-α,IL-6and IL-10）,and oxidative stress markers（ROS,SOD and CAT）were detected by commercially available kits.Besides,the expressions of Col-Ⅰ,Col-Ⅲandα-SMA were examined by WB.（2）The effects of IVT on liver fibrosis in vitroHepatic stellate cells（LX2 and JS-1 cells）were treated with PDGF for 2 h to induce cell activation,followed by IVT treatment for 24 h.Cell proliferation was detected by MTT assay and colony formation;cell apoptosis was assessed by AO/EB staining and flow cytometry;the m RNA and protein expression levels of Col-Ⅰ,Col-Ⅲandα-SMA were determined by qPCR and WB,respectively.PartⅡ:The effects of IVT on the transcriptome and metabolome in mice with hepatic fibrosis（1）Transcriptomics sequencingThe animal model was the same as the partⅠ,and the mouse liver tissues were sequenced by transcriptomic.Quality control statistics and base mass distribution were applied for evaluating the quality of transcriptome samples.PCA analysis,heatmap and volcano map were used to visualize differentially expressed genes.Besides,pathway enrichment analysis was conducted to clarify pathways annotated by differentially expressed genes.（2）Metabolomics analysisMetabolomics analysis of hepatic tissues was performed by Ultra-performance liquid chromatography equipped with quadrupole time-of-flight mass spectroscopy（UPLC-Xevo G2-XS QTof,Waters,USA）.PCA and Hotelling’s T2 assay of QC samples were used to evaluate instrument stability of metabolomic analysis.PCA analysis,VIP and volcano map of on-board samples were applied to visualize differentially metabolites.In addition,pathway enrichment analysis was utilized to illustrate pathways enriched by differentially metabolites.（3）The combined analysis of transcriptomics and metabolomicsThe correlation analysis and pathway enrichment assay were applied to conduct the overall evaluation of the differentially expressed genes and differentially metabolites.PartⅢ:The effects of IVT on the PI3K/Akt signaling pathway and GSH metabolic pathway（1）The effects of IVT on the PI3K/Akt signaling pathwayThe treatments of mouse and hepatic stellate cells（LX2 and JS-1 cells）were the same as the partⅠ.The effects of IVT on m RNA and protein expression levels of PTEN,PI3K,Akt and m TOR in mouse liver tissues and hepatic stellate cells were measured by qPCR and WB,respectively.（2）The effects of IVT on autophagyThe effects of IVT on autophagosomes in mouse liver tissues and autophagic flux in hepatic stellate cells were assessed by transmission electron microscopy and laser scanning confocal microscope,respectively;meanwhile,the effects of IVT on the m RNA and protein expression levels of autophagy-related molecules were detected by qPCR and WB,respectively.（3）The effects of autophagy on the hepatic stellate cell activation regulated by IVTAutophagy inhibitor 3MA was added to the experiment.The apoptosis,proliferation and protein expression levels of collagen deposition-associated molecules including Col-Ⅰ,Col-Ⅲandα-SMA in hepatic stellate cells were measured by flow cytometry,colony formation assay and WB assay,respectively.（4）The effects of the PI3K/Akt signaling pathway on the autophagy and hepatic stellate cell activation modulated by IVTPI3K activator 740 Y-P and PTEN inhibitor VO-Ohpic were introduced in the experiment.The protein expression levels of autophagy-related molecules（Atg5,Atg7,Beclin-1,P62 and LC3）and collagen deposition-related molecules（Col-Ⅰ,Col-Ⅲandα-SMA）in hepatic stellate cells were assessed by WB assay.（5）The effects of IVT on the GSH metabolic pathwayThe effects of IVT on GSH,GSSG and GSH/GSSG in mouse liver tissues and hepatic stellate cells were detected by commercially available kits.PartⅣ:The effects of IVT on miR-21（1）The effects of IVT on miR-21The animal and cell models were the same as the partⅠ.The effects of IVT on miR-21 in mouse liver tissues and hepatic stellate cells were measured by qPCR.（2）The effects of miR-21 on the PI3K/Akt signaling pathway,autophagy and hepatic stellate cell activation regulated by IVTDual luciferase reporter assay was applied to explore the binding effects of miR-21 and PTEN.Meanwhile,miR-21 mimic was introduced in the experiment;the protein expression levels of the key molecules of the PI3K/Akt signaling pathway,autophagy-associated molecules,and collagen deposition-related molecules in hepatic stellate cells were assessed by WB assay.（3）The effects of miR-21 on the GSH metabolic pathway modulated by IVTmiR-21 mimic was added in the experiment.The GSH and GSSG levels,and GSH/GSSG ratio in hepatic stellate cells were measured by the commercially available kit.（4）The effects of IVT on m6AThe effects of IVT on pri-miR-21 in mouse liver tissues and hepatic stellate cells were measured by qPCR.And the levels of the total m6A and m6A enrichment of pri-miR-21 were detected by m6A kit and Me RIP,respectively.ResultsPart Ⅰ:The pharmacodynamical research of IVT on liver fibrosis（1）IVT ameliorated liver fibrosis in vivoIVT significantly ameliorated the hepatic pathological damage and collagen deposition in mice.Meanwhile,IVT dramatically reduced the activities of AST,ALT and ALB in mice serum（P<0.05 or P<0.01）.IVT also mitigated the levels of liver fibrosis biomarkers containing HA,LN,PC III and Col-IV in mouse liver tissues（P<0.05 or P<0.01）.Moreover,IVT led to a decrease of the contents in inflammatory markers including TNF-α,IL-6 and IL-10 in mouse liver tissues（P<0.05 or P<0.01）.In addition,IVT could effectively reverse the increase in ROS content and the decrease in SOD and CAT activities caused by CCl₄（P<0.05or P<0.01）.Besides,IVT obviously inhibited the protein expressions of Col-Ⅰ,Col-Ⅲandα-SMA（P<0.05 or P<0.01）.These results indicated that in the CCl₄-induced mice model of liver fibrosis,IVT significantly alleviated hepatic fibrosis.（2）IVT inhibited hepatic stellate cell activation in vitroIVT obviously reduced cell viability and colony-forming numbers（P<0.05or P<0.01）,and increased apoptosis rate of LX2 and JS-1 cells（P<0.05 or P<0.01）.Simultaneously,IVT dramatically inhibited the m RNA and protein expression levels of Col-Ⅰ,Col-Ⅲandα-SMA in LX2 and JS-1 cells（P<0.05 or P<0.01）.These results demonstrated that in the PDGF-stimulated liver fibrosis cell model,IVT significantly inhibited hepatic stellate cell activation.PartⅡ:The effects of IVT on the transcriptome and metabolome in mice with hepatic fibrosis（1）The effects of IVT on the transcriptomeThe results of quality control statistics and base mass distribution showed good quality of RNA samples.The data of PCA analysis,heatmap and volcano map visualized many differentially expressed genes between the model group and the IVT high-dose group.Additionally,the results of pathway enrichment analysis showed that the differentially expressed genes were mainly annotated in the PI3K/Akt signaling pathway and metabolism pathway.These results suggested that the PI3K/Akt signaling pathway and metabolism pathway might be the main targets of IVT against liver fibrosis.（2）The effects of IVT on the metabolomeThe results of PCA analysis and Hotelling’s T2 assay of QC samples revealed that the experimental condition with high stability and repeatability could be used for the next detection.The data of PCA analysis,VIP and volcano map of sequenced samples revealed many differential metabolites between the model group and IVT high-dose group.And the results of pathway enrichment analysis showed that the differential metabolites were principally clustered in the glutathione（GSH）metabolic pathway.These results inferred that the GSH metabolic pathway might be the pivotal metabolic process of IVT against hepatic fibrosis.（3）The integrated analysis of transcriptomics and metabolomicsThe correlation between the differentially expressed genes and differential metabolites was high,as evidenced by the combined analysis of transcriptomics and metabolomics.The metabolic pathway analysis between transcriptome and metabolome revealed that the GSH metabolic pathway was also the key metabolic pathway of IVT,which was consistent with the results of a separate analysis of the metabolome.These results deduced that the GSH metabolic pathway might be the chief metabolic course of IVT against liver fibrosis.PartⅢ:The effects of IVT on the PI3K/Akt signaling pathway and GSH metabolic pathway（1）IVT inhibited the PI3K/Akt signaling pathwayIVT significantly increased PTEN m RNA level and decreased PI3K,Akt and m TOR m RNA levels in mouse liver tissues,LX2 and JS-1 cells（P<0.05 or P<0.01）.Additionally,IVT dramatically promoted p-PTEN expression and reduced the levels of p-PI3K,p-Akt and p-m TOR（P<0.05 or P<0.01）.These results indicated that IVT suppressed the PI3K/Akt signaling pathway.（2）IVT activated autophagyIVT obviously increased autophagosomes in mouse liver tissues and autophagic flux in LX2 and JS-1 cells（P<0.05 or P<0.01）.Simultaneously,IVT effectively caused an increase in the m RNA and protein expression levels of autophagy-related molecules including Atg5,Atg7,Beclin-1,P62 and LC3 in mouse liver tissues and LX2 and JS-1 cells（P<0.05 or P<0.01）.These results demonstrated that IVT could not only promote the initiation of autophagy but also facilitate the process of autophagic flow,thereby activating autophagy.（3）IVT executed the regulation of hepatic stellate cell activation based on autophagy3MA led to a reversion of the increased apoptosis rates,and the decreased colony numbers and protein expressions of collagen deposition-related molecules including Col-I,Col-III,andα-SMA in LX2 and JS-1 cells induced by IVT（P<0.05 or P<0.01）.These results indicated that the effects of IVT on hepatic stellate cell activation depended on autophagy.（4）IVT exerted a significant regulatory effect on autophagy and hepatic stellate cell activation based on the PI3K/Akt signaling pathway740 Y-P and VO-Ohpic caused a restoration of the promoted protein expressions of autophagy-related molecules including Atg5,Atg7,Beclin-1,P62and LC3,and the reduced protein levels of collagen deposition-related molecules containing Col-I,Col-III,andα-SMA in LX2 and JS-1 cells induced by IVT（P<0.05 or P<0.01）.These results demonstrated that the effects of IVT on autophagy and hepatic stellate cell activation relied on the PI3K/Akt signaling pathway.（5）IVT activated the GSH metabolic pathwayIVT effectively augmented GSH level and GSH/GSSG,and mitigated GSSG level in mouse liver tissues and LX2 and JS-1 cells（P<0.05 or P<0.01）.These results illustrated that IVT activated the GSH metabolic pathway.PartⅣ:The effects of IVT on miR-21（1）IVT mitigated miR-21IVT significantly decreased miR-21 level in mouse liver tissues and LX2 and JS-1 cells（P<0.05 or P<0.01）,indicating that IVT inhibited miR-21.（2）IVT conducted the regulation of the PI3K/Akt signaling pathway,autophagy and hepatic stellate cell activation based on miR-21The results of dual luciferase reporter assay showed the direct binding sites between miR-21 and PTEN.Meanwhile,miR-21 mimic led to a reversion of the increased p-PTEN level and the decreased protein expressions of p-PI3K,p-Akt and p-m TOR in LX2 and JS-1 cells induced by IVT（P<0.05 or P<0.01）;simultaneously,miR-21 mimic caused a restoration of the promoted protein expressions of autophagy-related molecules including Atg5,Atg7,Beclin-1,P62and LC3,and the reduced protein levels of collagen deposition-related molecules including Col-I,Col-III,andα-SMA both in liver tissues and cells induced by IVT（P<0.05 or P<0.01）.These results demonstrated that the effects of IVT on the PI3K/Akt signaling pathway,autophagy and hepatic stellate cell activation depended on miR-21.（3）IVT activated the GSH metabolic pathway via regulation of miR-21miR-21 mimic brought about a reversion of the increased GSH level and GSH/GSSG ratio,and the decreased GSSG level in LX2 and JS-1 cells induced by IVT（P<0.05 or P<0.01）.These results indicated that the effects of IVT on the GSH metabolic pathway relied on miR-21.（4）IVT reduced m6A levelIVT notably enhanced pri-miR-21 level both in liver tissues and cells（P<0.05 or P<0.01）.Meanwhile,IVT significantly mitigated total m6A and m6A enrichment of pri-miR-21（P<0.05 or P<0.01）.These results demonstrated that IVT might regulate pri-miR-21 through m6A modification,thereby affecting the maturation of miR-21.ConclusionsThis study illustrates that IVT may alter m6A modification to modulate miR-21.And IVT regulates the PI3K/Akt signaling pathway and GSH metabolic pathway by reducing miR-21 level,by which it exerts a strong ability to inhibit collagen deposition and hepatic stellate cell activation,ultimately ameliorating hepatic fibrosis.