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摘要：
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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10 μg/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62.5 μmol/L、125 μmol/L
和250 μmol/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，125 μmol/L组和250 μmol/L组细胞增殖率分别
为（66.7 ± 8.6）%和（45.6 ± 3.0）%。处理48 h，125 μmol/L组和250 μmol/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62.5 μmol/L组、125 μmol/L组、250 μmol/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125 μmol/L组、250 μmol/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
松素对肝星状细胞具有抑制增殖、促进凋亡、抑制激活的作用，推测其可能通过调控
多项基因表达，主要调控因子之间相互作用信号通路、卟啉与叶绿素代谢信号通路、
Hedgehog信号通路和PPARγ信号通路发挥作用。
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Abstract: 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 62.5 μmol/L, 125 μmol/L and
250 μmol/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 of α-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 125 μmol/L group and taxifolin 250 μmol/L group were (66.7 ± 8.6)% and (45.6 ±
3.0)%, respectively; at 48 h, the cell proliferation rates of taxifolin 125 μmol/L group and
taxifolin 250 μmol/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 62.5 μmol/L,
taxifolin 125 μmol/L group and taxifolin 250 μmol/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 of α-SMA, Collagen1A1, Collagen3A1
gene and α-SMA and Collagen1A1 protein were inhibited by 125 μmol/L and 250 μ mol/L
taxifolin (all P ＜ 0.05). Differential gene expression were characterized by downregulation
of genes related to liver fibrosis, such as oxidized low-density lipoprotein receptor1 (Olr1),
Plexin A4 (Plxna4), Spondin 1 (Spon1), Agrn and upregulation of genes related to antioxidant
activity and cell death, such as heme oxygenase 1 (Hmox1) etc. Conclusions Taxifolin could
inhibit HSC-T6 proliferation, promote apoptosis and inhibit cell activation through the
interaction signal pathway between influencing factors, porphyrin and chlorophyll metabolism
signal pathway, Hedgehog signal pathway and PPARγ signal pathway.
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