Transcriptomics-based Analysis of the Effects of Different Liver Injury-inducing Factors on Hepatic Stellate Cells
OBJECTIVE To explore the effects and potential mechanisms of different liver fibrosis stimuli including transforming growth factor-β1(TGF-β1),acetaminophen(APAP),and lipopolysaccharide(LPS)on the hepatic stellate cells(HSCs)LX-2.METHODS The experiment was divided into a control group and experimental groups that were treated with TGF-β1(50 ng·mL-1),APAP(20 mmol·L-1),and LPS(100 ng·mL-1)for 24 h.Cell samples were collected from each group.Used transcriptome sequencing to analyze the changes in expression of markers such as proliferation,apoptosis,inflammation,fibrosis and performed RT-PCR experimental verification.Potential mechanisms were explored using functional enrichment analysis and protein interaction networks.Data statistical analysis was performed using one-way ANOVA for multiple group comparisons and LSD-t test for pairwise comparisons.RESULTS TGF-β1 could effectively initiate the activation process of LX-2,express classical fibrosis marker genes actin alpha 2(ACTA2),collagen type Ⅰ alpha l(COL1A1)and fibronectin(FN1);LPS could activate the expression of ACTA2,but genes related to the deposition of extracellular matrix(ECM)of these myofibroblasts(MFBs)such as COL1A1 and FN1 were downregulated;APAP couldn't activate the expression of ACTA2 and COL1A1,but could only affect the increase in FN1 expression(P＜0.001).The reliability of the sequencing data was verified by RT-PCR(P＜0.05).Functional enrichment analysis found that TGF-β1 and LPS might participate in the regulation of inflammation and liver fibrosis through P13K-Akt signaling pathway and cytokine-cytokine receptor interaction signaling pathway.TGF-β1 might also participate in fibrosis and glucose metabolism through ECM receptor interaction and AGE-RAGE signaling pathway in diabetes complications.LPS might participate in the occurrence of inflammatory reactions through tumor necrosis factor signaling pathway,complement and coagulation cascades signaling pathway.APAP might mainly participate in metabolic processes such as cell proliferation and apoptosis by regulating protein polysaccharides in aging,apoptosis,cancer and MAPK signaling pathways.Key regulatory genes under different stimulation conditions were found by protein interaction network analysis and experimentally verified.Among them,ITGB3,ITGAV and FN1 were located at the core position under TGF-β1 stimulation;PIK3R1,FOS and CDC42 were located at the core position under APAP stimulation;ITGAM,CXCL8,CCL2 and IFIH1 were located at the core position under LPS stimulation.CONCLUSION TGF-β1 and LPS trigger a certain degree of inflammatory response,TGF-β1 is associated with liver fibrosis,while LPS may cause collagen degradation in fibrosis,APAP may lead to apoptosis and necrosis of LX-2.The selection of models based on different stimuli plays an important role in the research of disease mechanisms,target discovery and drug development.
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