机构地区:[1]首都医科大学附属北京友谊医院医疗保健中心七区,北京100050 [2]首都医科大学附属北京佑安医院北京肝病研究所,北京100069
出 处:《中国肝脏病杂志(电子版)》2023年第3期34-42,共9页Chinese Journal of Liver Diseases:Electronic Version
基 金:北京市属医学科研院所公益发展改革试点项目(京医研2021-10)。
摘 要:目的探索花旗松素对肝星状细胞的激活作用,通过转录组测序表征基因表达谱,分析潜在的作用机制。方法在10μg/L转化生长因子β1(transforming growth factor-β1,TGF-β1)诱导下,体外培养大鼠肝星状细胞HSC-T6,分别给予62.5μmol/L、125μmol/L和250μmol/L花旗松素处理,在24 h、48 h分别加入CCK8试剂,450 nm波长测定吸光度,计算不同浓度和时间下的细胞增殖率。在24 h通过AnnexinV/7AAD染色,流式细胞检测细胞凋亡。通过荧光定量聚合酶链式反应(polymerase chain reaction,PCR)法检测α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)、Ⅰ型胶原α1链(Collagen1A1)及Ⅲ型胶原α1链(Collagen3A1)基因mRNA相对表达量。通过Western blot检测α-SMA、Collagen1A1蛋白表达量。通过转录组测序,表征细胞基因表达谱并对差异基因进行基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)功能注释及功能富集分析。结果花旗松素可显著抑制肝星状细胞增殖,处理24 h,125μmol/L组和250μmol/L组细胞增殖率分别为(66.7±8.6)%和(45.6±3.0)%。处理48 h,125μmol/L组和250μmol/L组的细胞增殖率分别为(60.5±2.9)%和(43.9±1.9)%,均显著低于模型组(P均<0.05)。花旗松素可促进细胞凋亡,处理24 h,花旗松素62.5μmol/L组、125μmol/L组、250μmol/L组细胞凋亡率分别为(8.537±0.445)%、(8.85±0.169)%及(14.453±0.577)%,均显著高于模型组(P均<0.05)。处理24 h,花旗松素125μmol/L组、250μmol/L组可抑制α-SMA、Collagen1A1、Collagen3A1基因和α-SMA、Collagen1A1蛋白表达(P均<0.05)。差异基因表达表现为促肝纤维化发生相关基因下调,如氧化型低密度脂蛋白受体1(oxidized low-density lipoprotein receptor 1,Olr1)、人从状蛋白(Plexin A4,Plxna4)、反应蛋白1(Spondin 1,Spon1)、聚集蛋白(Agrn)等,以及细胞抗氧化及死亡相关的基因上调,如血红素加氧酶1(heme oxygenase 1,Hmox1)等。结论花旗松�Objective To investigate the effect of taxifolin on inhibiting the activation of hepatic stellate cells,characterize the gene expression profile through transcriptome sequencing and elucidate the potential mechanisms.Methods In presence of transforming growth factor-β1(TGF-β1),HSC-T6 were cultured in vitro and treated with 62.5μmol/L,125μmol/L and 250μmol/L of taxifolin,CCK 8 reagent was added at 24 h and 48 h,the absorbance was measured at 450 nm,and cell proliferation rate was calculated at different concentrations and times.Cell apoptosis was detected by flow cytometry with AnnexinV/7AAD staining at 24 h.Relative mRNA expression levels of alpha smooth muscle actin(α-SMA),Collagen1A1 and Collagen3A1 were detected by quantitative fluorescence quantitative polymerase chain reaction(PCR).Relative protein expression levels ofα-SMA and Collagen1A1 were detected by Western blot.The gene expression profile was characterized by transcriptome sequencing.The functional annotation and enrichment of differential genes were analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Results Taxifolin can inhibit the proliferation of hepatic stellate cells.At 24 h,the cell proliferation rates of taxifolin 125μmol/L group and taxifolin 250μmol/L group were(66.7±8.6)%and(45.6±3.0)%,respectively;at 48 h,the cell proliferation rates of taxifolin 125μmol/L group and taxifolin 250μmol/L group were(60.5±2.9)%and(43.9±1.9)%,respectively,which were significantly lower than those of model group(all P<0.05).Taxifolin can promote the apoptosis rates of hepatic stellate cell.At 24 h,the apoptosis rates of taxifolin 62.5μmol/L,taxifolin 125μmol/L group and taxifolin 250μmol/L group were(8.537±0.445)%,(8.85±0.169)%and(14.453±0.577)%,respectively,which were significantly higher than those of model group(all P<0.05).At 24 h,the expression ofα-SMA,Collagen1A1,Collagen3A1 gene andα-SMA and Collagen1A1 protein were inhibited by 125μmol/L and 250μmol/L taxifolin(all P<0.05).Differential gene expression
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