Mechanism Study Of Gandouling Mitigating Liver Fibrosis In Wilson’s Disease By Regulating The PI3K/AKT/mTOR Pathway To Activate Autophagy

1 ObjectiveIn order to elucidate the molecular mechanism by which Gandouling(GDL)treats liver fibrosis in Wilson’s disease(WD).2 Methods2.1 Exploration of the potential mechanism of GDL in treating liver fibrosis in WD based on network pharmacologyInitially,the active constituents of the primary Chinese herbs found in Gandouling(GDL)were extracted from the TCMSP database,leading to the establishment of a network representing the relationship between drugs and active components.Subsequently,Swiss target prediction and the TCMSP websites were utilized to identify the target proteins associated with these components.Concurrently,liver fibrosis targets were sourced from the Gene Cards and OMIM databases.The common targets shared by both liver fibrosis and GDL active components were subjected to protein interaction analysis using the STRING database.Key targets were identified through the utilization of Cytoscape software,specifically the Gene MANIA,MCODE,and Cyto NCA plugins.Finally,enrichment analysis and pathway network analysis were conducted employing the GENE DENOVO platform.2.2 Mechanism study of GDL mitigating liver fibrosis in Wilson’s disease by regulating the PI3K/AKT/mTOR pathway to activate autophagyTo establish a cellular model of liver fibrosis in WD,LX-2 cells exposed to high levels of copper were utilized.The viability of high-copper-induced LX-2 cells was assessed using the cell counting kit-8(CCK-8)assay to evaluate the effects of serum containing GDL.Optimal concentration and duration of action were determined through these experiments.The expression of α-SMA,Collagen I,Beclin-1,LC3-II,p62,pAKT/AKT,PI3 K,and p-mTOR/mTOR proteins was analyzed using Western blot(WB).Additionally,real-time quantitative PCR(RT-q PCR)was employed to assess the expression of AKT,PI3 K,mTOR,Beclin-1,p62,and LC3 B m RNA in each experimental group.Immunofluorescence(IF)was performed to investigate the expression of Beclin-1,LC3-II,p62,AKT,PI3 K,mTOR,p-AKT,and p-mTOR in each group.The interaction between core proteins and key active components was validated using Discovery Studio software.Acridine Orange(AO)levels were measured through flow cytometry,while mitochondrial damage and autophagosome formation were observed using transmission electron microscopy.3 Results3.1 Potential Mechanisms of GDL in Treating WD-Related Liver Fibrosis Explored by Network Pharmacology3.1.1 Target Prediction ResultsA total of 119 compounds with an oral bioavailability(OB)of at least 30% and drug likeness(DL)of at least 0.18 were extracted from the TCMSP database as potential active ingredients of GDL that meet pharmacokinetic criteria.These compounds were subsequently mapped to 1,152 target genes.To identify target genes associated with liver fibrosis,a search was conducted in the Gene Cards and OMIM databases,resulting in the retrieval of 799 relevant target genes.By comparing the datasets,176 overlapping genes were identified,representing common targets between GDL compounds and liver fibrosis.3.1.2 Protein-Protein Interaction Analysis ResultsThe protein-protein interaction network consisted of 128 nodes and 688 edges,indicating strong interactions among the nodes.Through the utilization of the Cyto NCA plugin,five key target genes were identified as follows: STAT3,SRC,TP53,HSP90AA1,and EP300.3.1.3 Enrichment Analysis ResultsGene Ontology(GO)analysis showed significant enrichment of 2,722 biological process(BP)terms,113 cellular component(CC)terms,and 177 molecular function(MF)terms.Additionally,the KEGG analysis identified 139 enriched pathways,which included key pathways such as the PI3K/AKT,mTOR,and Autophagy pathways.3.1.4 Results of signaling pathway network analysisA network analysis was performed on the significantly enriched pathways mentioned earlier,leading to the formation of a network consisting of 271 nodes and 1060 edges.To further investigate these pathways,the Cyto NCA plugin was utilized to screen the top 30 nodes.Literature research,combined with the analysis results,revealed a substantial correlation between the PI3K/AKT/mTOR signaling pathway,autophagy,and the pathological progression of liver fibrosis.3.2 Mechanism study of GDL mitigating liver fibrosis in Wilson’s disease by regulating the PI3K/AKT/mTOR pathway to activate autophagy3.2.1 Effects of GDL on copper-loaded LX-2 cells viability,α-SMA,and Collagen I protein expressionThe CCK-8 assay results indicated that a concentration of 100 μmol/L and a treatment time of 48 hours were identified as the optimal copper loading concentration and time.Similarly,a concentration of 5 % GDL-containing serum with a treatment time of 48 hours was determined as the optimal concentration and time.The administration of GDL-containing serum demonstrated a significant inhibitory effect on the viability of LX-2 cells induced by high copper.Furthermore,GDL-containing serum significantly suppressed the expression of α-SMA and Collagen I proteins in LX-2 cells induced by high copper.These findings suggest that GDL-containing serum effectively inhibits the activation of LX-2 cells induced by high copper.3.2.2 Effects of GDL on the PI3K/AKT/mTOR signaling pathway in LX-2 cellsThe results obtained from RT-q PCR,Western blotting,and immunofluorescence analyses revealed significant upregulation of PI3 K,AKT,and mTOR expression levels in the model group compared to the control group.Conversely,the GDL-containing serum group,LY294002 group,and RAPA group exhibited notable reductions in the expression levels of PI3 K,AKT,and mTOR compared to the model group.Moreover,the addition of GDL-containing serum further decreased the expression levels of PI3 K,AKT,and mTOR in the LY294002 and RAPA groups.These findings suggest that GDL-containing serum can synergistically interact with LY294002 and RAPA to inhibit the activation of the PI3K/AKT/mTOR signaling pathway.3.2.3 Molecular docking identification of key active components of GDLThe molecular docking analysis revealed that the key active components(MOL000500,MOL000483,MOL007036,MOL000006,and MOL000491)of GDL exhibited higher docking scores with the core targets(PI3KCA,AKT1,and mTOR)compared to D-penicillamine(DPA),which is a first-line clinical treatment drug for WD.These results indicate a stronger affinity of the key active components of GDL towards the core targets,suggesting their potential efficacy in the treatment of WD.3.2.4 Effects of GDL on autophagy markers LC3-II,Beclin-1,p62,and AO in LX-2 cellsThe results obtained from RT-q PCR,WB,and IF analysis demonstrated that,in comparison to the control group,the model group exhibited significantly decreased expression levels of LC3-II and Beclin-1,along with a significant increase in p62 expression.Conversely,the GDL serum-containing group,LY294002 group,and RAPA group displayed significantly increased expression levels of LC3-II and Beclin-1,accompanied by a notable decrease in p62 expression,compared to the model group.Furthermore,upon the addition of GDL serum-containing medium,the expression levels of LC3-II and Beclin-1 were further augmented,while the p62 level was further decreased in the LY294002 and RAPA groups.These findings suggest that GDL serum-containing medium can synergistically interact with LY294002 and RAPA to enhance LC3-II and Beclin-1 expression while inhibiting p62 expression.The flow cytometry results demonstrated a significant decrease in AO level in the model group compared to the control group.In contrast,the GDL serum-containing group,LY294002 group,and RAPA group exhibited a significant increase in AO level compared to the model group.Additionally,the addition of GDL serum-containing medium further elevated AO level in both the LY294002 and RAPA groups.These findings indicate that GDL serum-containing medium can synergize with LY294002 and RAPA to enhance AO expression.3.2.5 Effects of GDL on ultrastructure of LX-2 cellsThe results obtained from transmission electron microscopy showed different levels of mitochondrial damage in the model group,characterized by vacuolar and myelin-like degeneration.However,treatment with GDL serum-containing medium demonstrated an improvement in mitochondrial damage induced by high copper levels in LX-2 cells.Furthermore,the combination of GDL serum-containing medium with LY294002 and RAPA synergistically enhanced the amelioration of mitochondrial damage.3.2.6 GDL can rescue the effects of IGF-1 and MHY1485 on LC3-II,Beclin-1,p62,and AO in LX-2 cellsThe results obtained from RT-q PCR,WB,and IF experiments revealed that the treatment with GDL serum-containing medium was able to rescue the inhibitory effect of IGF-1 and MHY1485 on the autophagy markers LC3-II and Beclin-1 in high copperinduced LX-2 cells.Additionally,the treatment with GDL serum-containing medium reversed the promoting effect of IGF-1 and MHY1485 on p62 expression in the same cells.Furthermore,flow cytometry experiments demonstrated that GDL serumcontaining medium was able to rescue the inhibitory effect of IGF-1 and MHY1485 on the autophagy marker AO in high copper-induced LX-2 cells.3.2.7 The Effects of GDL on the Ultrastructure of LX-2 Cells Induced by IGF-1 and MHY1485The results obtained from transmission electron microscopy revealed that the serum containing GDL had a mitigating effect on the effects of IGF-1 and MHY1485 on mitochondrial damage in LX-2 cells induced by high copper levels.3.2.8 The Effects of GDL on the Viability,α-SMA,and Collagen I Protein of LX-2 Cells induced by IGF-1 and MHY1485The results obtained from the CCK8 assays showed that the serum containing GDL was able to mitigate the effects of IGF-1 and MHY1485 on cells viability in LX-2 cells induced by high copper levels.Additionally,the Western blot experiments revealed that the serum containing GDL could also mitigate the effects of IGF-1 and MHY1485 on α-SMA and Collagen I protein expression in LX-2 cells induced by high copper levels.4 ConclusionGDL demonstrates effective inhibition of the activation of LX-2 cells induced by high copper levels.This inhibitory mechanism is associated with the blockade of the PI3K/AKT/mTOR signaling pathway,promotion of autophagy,and improvement of mitochondrial damage.