摘要
旨在通过单细胞、转录组学测序和体内外实验,探讨巨噬细胞极化在扶正化瘀片(FZHY)治疗肝纤维化中的作用。从GEO数据库中获取肝纤维化相关芯片、转录组数据集及单细胞测序数据集,筛选差异基因;运用GeneCards、DisGeNET、NCBI、PharmgKB、TTD和OMIM 6个数据库获取肝纤维化相关基因;从GeneCards数据库中获取巨噬细胞极化相关基因;将上述3个基因集取交集,构建蛋白-蛋白互作(protein-protein interaction,PPI)网络,使用Cytoscape软件筛选核心蛋白,并通过单细胞测序对核心蛋白的表达模式进行可视化。使用四氯化碳(carbon tetrachloride,CCl_(4))构建的肝纤维化小鼠模型,采用苏木精-伊红(hematoxylin-eosin,HE)染色、马松(Masson)染色对肝组织病理形态学进行观察;免疫组织化学染色检测α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)和转化生长因子(transforming growth factor,TGF)-β1蛋白的表达;比色法检测血清丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST);酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测血清中炎症因子水平;蛋白质印记(Western blot,WB)检测肝组织中α-SMA、TGF-β1、CD86、血小板反应蛋白(thrombospondin,THBS)1的表达情况。使用LPS刺激RAW264.7细胞构建M1巨噬细胞极化模型,通过CCK-8法检测细胞活力,WB检测细胞中CD86、THBS1的蛋白表达情况和实时荧光定量逆转录聚合酶链反应(real-time fluorescent quantitative reverse transcription polymerase chain reaction,RT-qPCR)法检测肿瘤坏死因子(tumor necrosis factor,TNF)-α、白细胞介素(interleukin,IL)-1β的mRNA表达水平。结果显示,共获得26个潜在调节肝纤维化巨噬细胞极化相关的基因,并筛选出THBS1、LUM、FBLN5等10个肝纤维化巨噬细胞极化相关的核心蛋白。单细胞数据分析,显示排名最靠前的THBS1可能由M1巨噬细胞表达。动物实验表明,FZHY可以减少CC
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 intersected 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 tetrachloride(CCl_4).Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological morphology of liver tissues.The expressions of α-smooth muscle actin(α-SMA) and transforming growth factor-β1(TGF-β1) were detected by immunohistochemistry.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 gene
作者
陆华冠
彭望霞
江丽红
姚强
谢新
雷昌
黄丹
刘建军
LU Hua-guan;PENG Wang-xia;JIANG Li-hong;YAO Qiang;XIE Xin;LEI Chang;HUANG Dan;LIU Jian-jun(Science and Technology Innovation Center/State Key Laboratory Cultivation Base of Chinese Medicine Powder and Innovative Drugs,Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第10期2597-2606,共10页
China Journal of Chinese Materia Medica
基金
湖南省教育厅重点项目(18A228)
湖南中医药大学校级研究生创新课题(2022CX95)。
