RNA-seq Reveals The Anti-hepatic Fibrosis Mechanism Of Curcumin Derivative Curc-mPEG454

ObjectivesLiver fibrosis is a complex process of liver damage caused by external inflammation and other stimulating factors,which heals repeatedly and forms scars.When the damage persists,the extracellular matrix（ECM）continues to deposit and further develops into liver cirrhosis or liver cancer,eventually leading to liver failure or death.Many studies have confirmed that curcumin has anti-inflammatory and anti-fibrosis pharmacological activities,but its low bioavailability limits its clinical conversion.Previous studies have shown that Curc-m PEG454,a curcumin derivative modified with short-chain polyethylene glycol,not only increases the blood concentration of curcumin,but also retains the anti-inflammatory activity of curcumin.Therefore,this study aimed to evaluate the anti-fibrosis effect of Curc-m PEG454 on carbon tetrachloride（CCl₄）-induced rat liver fibrosis model,and applied RNA sequencing（RNA-seq）to reveal its potential mechanism.Methods1.140 male Sprague-Dawley（SD）rats weighing 170～210g,the normal and Curc-m PEG454 control group were injected intraperitoneally with olive oil,and the treatment group was injected intraperitoneally with CCl4in olive oil[2:3（v/v）]twice a week at a dose of 0.3 m L/100 g for 10 weeks to construct a liver fibrosis model.Rats from Normal control group（0.9%Na Cl,i.v.）,Curc-m PEG454 control group（100mg/kg,i.v.）,Curc-m PEG454treatment groups（25,50,100mg/kg respectively,i.v.）and curcumin group（400 mg/kg,i.g.）were treated daily during 5-10 weeks.After the experiment,test liver weight,body weight and alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,creatinine（Scr）,hydroxyproline（HYP）,serum hyaluronic acid（HA）,type III Collagen（PCIII）,type IV collagen（IV-C）and laminin（LN）content.2.Primary HSCs were isolated from male SD rats by density gradient centrifugation to evaluate the inhibitory activity of Curc-m PEG454.Rat primary HSCs were treated with 0μM,4μM and 8μM Curc-m PEG454respectively,and the cell cycle was detected by flow cytometry（FACS）.3.RNA-seq was applied to analyze the livers of the normal control group,Curc-m PEG454 control,Curc-m PEG454（25 and 100mg/kg）and curcumin（400mg/kg）treatment groups,in order to verify the expression of hub genes in the significantly enriched KEGG pathway.Finally,we integrate RNA-seq data and recent liver Single-cell RNA sequencing（sc RNA-seq）studies to makes a preliminary exploration on the interactions among scar-related hepatic stellate cells,macrophages,and endothelial cells.Results1.The results of animal experiments show that:compared with the model group,50mg/kg and 100mg/kg Curc-m PEG454 treatment for 6weeks could significantly reduce the increase in transaminase ALT and AST induced by CCl₄,and the incidence of liver cirrhosis decreased from75%of the model group to 37%and 35%,respectively,which was better than 400mg/kg curcumin treated group.2.Cell experiments showed that:Curc-m PEG454（8μM）significantly inhibited the proliferation and activation of primary hepatic stellate cells,induced cell cycle arrest in G2/M phase from 7.79%to 31.22%,and significantly reduced the expression of TGF-β（-92%）,α-SMA（-93%）and Collagen I（-87%）,which inhibited the transformation of hepatic stellate cells to myofibroblasts.3.The liver RNA-seq analysis revealed that:the differential genes（DGEs）caused by Curc-m PEG454 were mainly enriched in the retinol pathway,glutathione pathway and arachidonic acid pathway.Specifically,Curc-m PEG454 significantly reduced the expression of Cyp26a1,Cyp26b1and restored retinoic acid（RA）of liver;Curc-m PEG454 significantly increased Gclc and Gclm to increase liver glutathione（GSH）levels to resist oxidative stress;Curc-m PEG454 reduced liver inflammation by down-regulating the COX2-PEG2 signaling pathway.Further integration of sc RNA-seq data showed that Curc-m PEG454 could effectively inhibit the expansion of scar-related TREM2⁺CD9⁺macrophage subpopulation and PDGFRα⁺PDGFRβ⁺scar-producing fibroblasts in damaged livers,and remodeled the fibrotic niche through regulation ligand-receptor interactions including IL-34,VEGFA,PDGFD and CTGF signaling.ConclusionsOur work suggests that Curc-m PEG454 can improve liver fibrosis by regulating multiple signal pathways related to liver fibrosis.As a multi-target drug,Curc-m PEG454 deserves further attention and research.