Mechanism of Fuzheng Huayu Tablets against hepatic fibrosis based on transcriptome and single-cell sequencing
This study aimed to investigate the role of macrophage polarization in the treatment of liver fibrosis by Fuzheng Huayu Tablets(FZHY)through single-cell,transcriptome sequencing and in vitro and in vivo experiments.Liver fibrosis-related datasets,transcriptomic datasets,and single-cell sequencing datasets were obtained from the Gene Expression Omnibus(GEO)database to screen differential genes.Liver fibrosis-related genes were obtained from GeneCards,DisGeNET,NCBI,PharmgKB,TTD and OMIM databases.Macrophage polarization-related genes were obtained from the GeneCards database.The above three gene sets were intersec-ted to construct a protein-protein interaction(PPI)network.Cytoscape software was used to screen core proteins,and the expression pattern of core proteins was visualized by single-cell sequencing.A mouse model of liver fibrosis was constructed using carbon tetra-chloride(CCl4).Hematoxylin-eosin(HE)staining and Masson staining were used to observe the pathological morphology of liver tis-sues.The expressions of α-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1)were detected by immunohisto-chemistry.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by colorimetry.The le-vels of inflammatory factors in serum were detected by the enzyme-linked immunosorbent assay(ELISA).Furthermore,the expressions of α-SMA,TGF-β1,cluster of differentiation 86(CD86)and thrombospondin 1(THBS1)in liver tissues were detected by Western blot(WB).Lipopolysaccharide(LPS)was used to stimulate RAW264.7 cells to construct the M1 macrophage polarization model.The cell counting kit-8(CCK-8)method was used to detect cell viability.WB was used to detect the protein expressions of CD86 and THBS1 in cells,and the messenger ribonucleic acid(mRNA)expression levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-1β by real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR).The results showed that a total of 26 potential genes related to the polarization of liver fibrosis macrophages were obtained,and 10 core proteins related to the po-larization of liver fibrosis macrophages such as THBS1,lumican(LUM)and fibulin-5(FBLN5)were screened.Single-cell data analy-sis indicated that THBS1,ranking highest,may be expressed by M1 macrophages.Animal experiments demonstrated that FZHY re-duced inflammatory cell infiltration and collagen deposition in CCl4-induced mouse liver,relieved liver injury and inflammation levels,and inhibited the expressions of α-SMA,TGF-β1,CD86,and THBS1 proteins.Cell experiments revealed that FZHY significantly re-duced intracellular expression of CD86 and THBS1 proteins and mRNA levels of TNF-α and IL-1β.In conclusion,FZHY may amelio-rate liver fibrosis by inhibiting THBS1 protein expression,suppressing M1 macrophage polarization,and reducing inflammation.
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