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Integration of Network Pharmacology, Transcriptomics and Single-cell Sequencing to Explore the Effect of Rougan Keli in Alleviating Liver Cirrhosis

ABSTRACT: Background: The progression of liver cirrhosis leads to severe complications, significantly threatening the survival and prognosis of patients. Rou gan ke li (Rgkl), a herbal formula derived from classical prescriptions, has used clinically over two decades and has good efficacy. However, its molecular mechanisms and active components remain undefined. Purpose: Exploring the molecular mechanisms of Rgkl in alleviating liver cirrhosis. Methods: CCl4-induced liver cirrhosis mice models were established. Liver stiffness and intrahepatic blood flow velocity were assessed using imaging. Serum ALT, AST, HA, and histopathology were analyzed. Hepatic stellate cells (HSCs) activation, liver sinusoidal endothelial cells (LSECs) fenestration, and angiogenesis were evaluated using immunohistochemistry and scanning electron microscopy. UPLC-Q-TOF-MS/MS and network pharmacology identified active components. Transcriptomics and single-cell sequencing identified key targets and pathways, validated via WB, immunofluorescence, and molecular docking. Results: Rgkl significantly reduced Liver stiffness and collagen deposition while increasing intrahepatic blood flow velocity in cirrhotic mice. Serum ALT, AST, and HA were markedly decreased. Rgkl inhibited α-SMA expression in HSC and downregulated pathological angiogenesis by reducing VEGF and CD34 expression. Additionally, Rgkl enhanced eNOS expression and preserved sinusoidal fenestration in LSEC. Furthermore, Rgkl ameliorated liver cirrhosis by modulating LSEC metabolic functions via the CD36/PPAR/CPT-1 pathway and suppressing HSC activation through the RhoA/ROCK/YAP and PI3K/AKT/NF-κB pathways. Eighteen active components, such as Levistilide A and Quercetin, were strongly correlated with the amelioration of liver cirrhosis. Conclusions: Rgkl significantly attenuated hepatic injury and fibrosis. Mechanistically, Rgkl modulated LSEC lipid metabolism and phenotypic regulation, and suppressed HSC contraction and activation. Key active components contributing to these effects included Paeonilactone C, Levistilide A, and Quercetin.

ORGANISM(S): Mus musculus

PROVIDER: GSE305510 | GEO | 2025/11/18

REPOSITORIES: GEO

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Project description:Liver sinusoidal endothelial cells (LSEC) are highly specialized within the hepatic vascular niche, controlling liver function and disease pathogenesis by angiocrine signaling. Recently, we identified GATA4 as a major transcription factor controlling LSEC development and protecting against liver fibrosis. As the transcription factor c-Maf was strongly downregulated in Gata4-deficient LSEC, we hypothesized that c-Maf might be an important downstream effector of GATA4 in LSEC differentiation and liver fibrogenesis. Clec4g-iCre/Maf fl/fl mice (Maf-LSEC-KO) mice with LSEC-specific Maf deficiency were generated and liver tissue was analyzed by histology, immunofluorescence, and in situ-hybridization at the age of three months. LSECs were isolated for RNA-, ATAC-seq, and single-cell RNA-seq (scRNA-seq) analysis. The expression of MAF and its targets were analyzed in published human scRNA-seq data. Endothelial Maf deficiency resulted in perisinusoidal liver fibrosis without affecting metabolic liver zonation, accompanied by a switch from sinusoidal to continuous endothelial differentiation. Furthermore, endothelial Maf deficiency caused hepatic endothelial proliferation and expression of profibrotic angiocrine factors such as Pdgfb, Igfbp5, Sparcl-1, and Flrt2. scRNA-seq revealed replacement of zonated LSEC subpopulations by capillarized, proliferative, sprouting and secretory endothelial cell subset promoting liver fibrogenesis and angiogenesis. This fundamental dysregulation of LSEC gene expression and differentiation was caused by changes in chromatin accessibility and transcription factor network alterations at promoter and enhancer regions following loss of Maf. Notably, endothelial MAF expression was also significantly reduced in human liver cirrhosis patients. Hepatic endothelial c-Maf protects against metabolic dysfunction-associated steatohepatitis-like liver fibrosis and regulates endothelial differentiation and zonation by controlling chromatin opening. Notably, our findings may open up new avenues to develop angiotargeted strategies for hepatic disease prevention and liver repair.

Project description:Liver sinusoidal endothelial cells (LSEC) are highly specialized within the hepatic vascular niche, controlling liver function and disease pathogenesis by angiocrine signaling. Recently, we identified GATA4 as a major transcription factor controlling LSEC development and protecting against liver fibrosis. As the transcription factor c-Maf was strongly downregulated in Gata4-deficient LSEC, we hypothesized that c-Maf might be an important downstream effector of GATA4 in LSEC differentiation and liver fibrogenesis. Clec4g-iCre/Maffl/fl mice (MafLSEC-KO) mice with LSEC-specific Maf deficiency were generated and liver tissue was analyzed by histology, immunofluorescence, and in situ-hybridization at the age of three months. LSECs were isolated for RNA-, ATAC-seq, and single-cell RNA-seq (scRNA-seq) analysis. The expression of MAF and its targets were analyzed in published human scRNA-seq data. Endothelial Maf deficiency resulted in perisinusoidal liver fibrosis without affecting metabolic liver zonation, accompanied by a switch from sinusoidal to continuous endothelial differentiation. Furthermore, endothelial Maf deficiency caused hepatic endothelial proliferation and expression of profibrotic angiocrine factors such as Pdgfb, Igfbp5, Sparcl-1, and Flrt2. scRNA-seq revealed replacement of zonated LSEC subpopulations by capillarized, proliferative, sprouting and secretory endothelial cell subsets promoting liver fibrogenesis and angiogenesis. This fundamental dysregulation of LSEC gene expression and differentiation was caused by changes in chromatin accessibility and transcription factor network alterations at promoter and enhancer regions following loss of Maf. Notably, endothelial MAF expression was also significantly reduced in human liver cirrhosis patients. Hepatic endothelial c-Maf protects against metabolic dysfunction-associated steatohepatitis-like liver fibrosis and regulates endothelial differentiation and zonation by controlling chromatin opening. Notably, our findings may open up new avenues to develop angiotargeted strategies for hepatic disease prevention and liver repair.

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Integration of Network Pharmacology, Transcriptomics and Single-cell Sequencing to Explore the Effect of Rougan Keli in Alleviating Liver Cirrhosis

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