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黄芪甲苷Ⅳ对四氯化碳诱导的肝纤维化小鼠模型的影响及其作用机制
DOI: 10.12449/JCH260316
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：朱宛春进行实验和文章撰写；邱嘉昊、崔钰、张伊婧负责数据收集和统计分析；尚志负责技术支持和文章修改；黄凌鹰、高月求指导撰写文章并最后定稿。
Effect of astragaloside Ⅳ on a mouse model of carbon tetrachloride-induced liver fibrosis and its mechanism
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摘要:
目的 明确黄芪甲苷Ⅳ（AS-Ⅳ）在体内外的肝脏保护和抗肝纤维化作用，并探讨其在抗肝纤维化中的作用机制。 方法 动物实验：将C57BL/6J小鼠分为对照组、模型组、AS-Ⅳ低剂量组（20 mg/kg）和AS-Ⅳ高剂量组（80 mg/kg）。通过腹腔注射四氯化碳6周构建肝纤维化模型，从第3周开始，AS-Ⅳ组分别予以AS-Ⅳ 20 mg/kg、80 mg/kg灌胃，给药4周后检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平，以及血清透明质酸（HA）、层粘连蛋白（LN）、Ⅲ型前胶原（PⅢNP）及Ⅳ型胶原（Col-Ⅳ）水平；苏木精-伊红染色、天狼星红染色和马松染色观察肝组织病理及胶原沉积情况；定量逆转录聚合酶链反应检测肝组织Acta2、Col1a1、Col3a1的mRNA表达；Western Blot法检测肝组织α-平滑肌肌动蛋白（α-SMA）、Ⅲ型胶原蛋白（Col-Ⅲ）、磷酸化磷脂酰肌醇3-激酶（pPI3K）/PI3K、磷酸化蛋白激酶B（pAkt）/Akt蛋白表达水平；运用转录组学技术对小鼠肝组织进行测序分析，筛选差异基因并进行生物信息学分析。细胞实验：采用转化生长因子β（TGF-β）诱导LX-2细胞活化，进一步使用PI3K抑制剂LY294002以及PI3K激动剂740 Y-P对细胞进行干预，设对照组、模型组、AS-Ⅳ组、LY294002组、AS-Ⅳ+740 Y-P组，36 h后收集细胞。检测LX-2细胞α-SMA、Col-Ⅲ、pPI3K/PI3K和pAkt/Akt蛋白表达的变化，以及Acta2、Col1a1和Col3a1的mRNA相对表达量。计量资料多组间比较采用单因素方差分析，进一步两两比较使用LSD-t检验。 结果 动物实验：与模型组相比，AS-Ⅳ治疗组小鼠血清ALT、AST、HA、LN、PⅢNP和Col-Ⅳ水平均显著降低（P值均<0.01）；肝组织Acta2、Col1a1和Col3a1的mRNA表达均显著降低（P值均<0.05）；与模型组相比，治疗组小鼠肝组织α-SMA、Col-Ⅲ、pPI3K和pAkt（Ser473）的蛋白表达均显著减少（P值均<0.05）。细胞实验：与对照组相比，TGF-β诱导后模型组细胞α-SMA、Col-Ⅲ、pPI3K和pAkt蛋白表达水平显著升高（P值均<0.05）；与模型组比较，AS-Ⅳ组细胞α-SMA、Col-Ⅲ、pPI3K和pAkt（Ser473）蛋白表达水平显著降低（P值均<0.05），LY294002组细胞pPI3K蛋白表达，以及Acta2、Col1a1和Col3a1的mRNA相对表达量亦显著降低（P值均<0.05）。而予740 Y-P干预后，与AS-Ⅳ组相比，pPI3K蛋白表达及Acta2、Col1a1和Col3a1的mRNA相对表达量均明显升高（P值均<0.05）。 结论 AS-Ⅳ通过抑制肝星状细胞活化从而改善肝纤维化，其作用机制可能与抑制PI3K/Akt信号通路有关。 -
关键词:
- 肝纤维化 /
- 黄芪甲苷Ⅳ /
- 小鼠， 近交C57BL /
- 肝星状细胞
Abstract:Objective To investigate the liver-protecting and anti-liver fibrosis effects of astragaloside Ⅳ （AS-Ⅳ） in vitro and in vivo， as well as its mechanism of action in intervention against liver fibrosis. Methods In the animal experiment， C57BL/6J mice were divided into control group， model group， low-dose AS-Ⅳ （20 mg/kg） group， and high-dose AS-Ⅳ （80 mg/kg） group. The mice were given intraperitoneal injection of carbon tetrachloride for 6 weeks to induce liver fibrosis， and since week 3 of injection， the mice in the low-dose AS-Ⅳ group and the high-dose AS-Ⅳ group were given AS-Ⅳ by gavage at a dose of 20 mg/kg and 80 mg/kg， respectively. The serum levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were measured after 4 weeks of administration， as well as the serum levels of hyaluronic acid （HA）， laminin （LN）， procollagen Ⅲ N-terminal peptide （PⅢNP）， and collagen type Ⅳ （Col-Ⅳ）. HE staining， picrosirius red staining， and Masson staining were used to observe liver histopathology and collagen deposition； RT-qPCR was used to measure the mRNA expression levels of Acta2， Col1a1， and Col3a1 in liver tissue， and Western blot was used to measure the protein expression levels of α-smooth muscle actin （α-SMA）， collagen type Ⅲ （Col-Ⅲ）， phosphatidylinositol 3-kinase （PI3K）， phosphorylated PI3K （pPI3K）， protein kinase B （Akt）， and phosphorylated AKT （p-Akt） in liver tissue； transcriptome sequencing was performed for liver tissue to identify differentially expressed genes and perform a bioinformatics analysis. In the cell experiment， transforming growth factor-β （TGF-β） was used to induce the activation of LX-2 cells， and the PI3K inhibitor LY294002 and the PI3K activator 740 Y-P were used for intervention. The cells were divided into control group， model group， AS-Ⅳ group， LY294002 group， and AS-Ⅳ+740 Y-P group， and the cells were harvested after 36 hours of intervention. Changes in the protein expression levels of α-SMA， Col-Ⅲ， pPI3K/PI3K， and pAkt/Akt in LX-2 cells were measured， as well as changes in the relative mRNA expression levels of Acta2， Col1a1， and Col3a1. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results In the animal experiment， compared with the model group， the AS-Ⅳ treatment group had significant reductions in the serum levels of ALT， AST， HA， LN， PⅢNP， and Col-Ⅳ （all P<0.01）， the mRNA expression levels of Acta2， Col1a1， and Col3a1 in liver tissue （all P<0.05）， and the protein expression levels of α-SMA， Col-Ⅲ， pPI3K， and pAkt （Ser473） in liver tissue （all P<0.05）. In the cell experiment， compared with the control group， the model group had significant increases in the protein expression levels of α-SMA， Col-Ⅲ， pPI3K， and pAkt （Ser473） after TGF-β induction （all P<0.05）； compared with the model group， the AS-Ⅳ group had significant reductions in the protein expression levels of α-SMA， Col-Ⅲ， pPI3K， and pAkt （Ser473） （all P<0.05）， and both the AS-Ⅳ group and the LY294002 group had significant reductions in the protein expression level of pPI3K and the relative mRNA expression levels of Acta2， Col1a1， and Col3a1 （all P<0.05）. Compared with the AS-Ⅳ group， there were significant increases in the protein expression level of pPI3K and the relative mRNA expression levels of Acta2， col1a1， and Col3a1 after 740 Y-P intervention （all P<0.05）. Conclusion AS-Ⅳ can inhibit hepatic stellate cell activation and improve liver fibrosis， possibly by inhibiting the PI3K/Akt signaling pathway. -
Key words:
- Hepatic Fibrosis /
- Astragaloside Ⅳ /
- Mice， Inbred C57BL /
- Hepatic Stellate Cells
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注： a，血清ALT、AST水平；b，HE染色（×400）。ALT，丙氨酸氨基转移酶；AST，天冬氨酸氨基转移酶；AS-Ⅳ，黄芪甲苷Ⅳ；HE，苏木精-伊红。
图 1 AS-Ⅳ改善CCl4诱导的小鼠肝脏炎症
Figure 1. AS-Ⅳ attenuates inflammation in mice treated with CCl4
注： a，染色结果；b，小鼠血清HA、LN、PⅢNP、Col-Ⅳ水平。AS-Ⅳ，黄芪甲苷Ⅳ；HA，透明质酸；LN，层粘连蛋白；Col-Ⅳ，Ⅳ型胶原；PⅢNP，Ⅲ型前胶原。
图 2 AS-Ⅳ改善小鼠肝脏胶原沉积
Figure 2. AS-Ⅳ attenuates hepatic collagen deposition in mice
注： a，α-SMA、Col-Ⅲ的蛋白表达；b，Acta2、Col1a1、Col3a1的mRNA表达。AS-Ⅳ，黄芪甲苷Ⅳ；Col-Ⅲ，Ⅲ型胶原蛋白；α-SMA，α-平滑肌肌动蛋白；HSC，肝星状细胞。
图 3 AS-Ⅳ抑制小鼠肝脏HSC活化
Figure 3. AS-Ⅳ attenuates the activation of HSC in the mouse liver
注： a，差异表达基因火山图；b，差异表达基因聚类分析热图。
图 4 小鼠肝脏转录组测序分析
Figure 4. Mouse liver transcriptome sequencing analysis
注： a～c，生物过程、分子功能及细胞组分的GO富集分析；d，KEGG通路富集结果。GTP，鸟苷三磷酸；ATP，腺嘌呤核苷三磷酸；PI3K/Akt，磷脂酰肌醇3-激酶/蛋白激酶B；EGFR，表皮生长因子受体；ECM，细胞外基质。
图 5 功能富集分析和信号通路分析
Figure 5. Functional annotation and pathway enrichment analysis of differentially expressed genes
注： PI3K，磷脂酰肌醇3-激酶；pPI3K，磷酸化PI3K；Akt，蛋白激酶B；pAkt，磷酸化Akt；AS-Ⅳ，黄芪甲苷Ⅳ。
图 6 AS-Ⅳ对小鼠肝脏PI3K/Akt通路活化的影响
Figure 6. Effect of AS-Ⅳ on the activation of the PI3K/Akt pathway in the mouse liver
注： a，LX-2细胞中α-SMA、Col-Ⅲ的蛋白表达；b，LX-2细胞中PI3K、pPI3K、Akt、pAkt的蛋白表达；c，CCK-8检测AS-Ⅳ对LX-2细胞存活率的影响。AS-Ⅳ，黄芪甲苷Ⅳ；Col-Ⅲ，Ⅲ型胶原蛋白；α-SMA，α-平滑肌肌动蛋白；PI3K，磷脂酰肌醇3-激酶；pPI3K，磷酸化PI3K；Akt，蛋白激酶B；pAkt，磷酸化Akt；HSC，肝星状细胞；CCK-8，细胞计数试剂盒-8。
图 7 AS-Ⅳ对HSC激活的影响
Figure 7. Effect of AS-Ⅳ on HSC activation
注： a、c，LX-2细胞中PI3K、pPI3K的蛋白表达；b、d，LX-2细胞中Acta2、Col1a1、Col3a1的mRNA表达。AS-Ⅳ，黄芪甲苷Ⅳ；HSC，肝星状细胞；PI3K，磷脂酰肌醇3-激酶；pPI3K，磷酸化PI3K；Akt，蛋白激酶B。
图 8 AS-Ⅳ通过调控PI3K/Akt通路抑制HSC激活
Figure 8. AS-Ⅳ activates hepatic stellate cells through the PI3K/Akt pathway
表 1 引物序列
Table 1. Primer sequences
基因 正向（5'-3'） 反向（5'-3'） Col3a1 小鼠肝组织 CTGTAACATGGAAACTGGGGAAA CCATAGCTGAACTGAAAACCACC 人LX-2细胞 CGTGGTAGCCCTGGTGAGAGAG TGGAGAACCGCTGGGACCTG Col1a1 小鼠肝组织 CAGGCTGGTGTGATGGGATT CGTTCTCCGCTCTCTCCAAA 人LX-2细胞 GAGGGCCAAGACGAAGACATC CAGATCACGTCATCGCACAAC Acta2 小鼠肝组织 TATCCCCGGGACTAAGACGG CTGTAGTCCCCCACTACCAC 人LX-2细胞 CTCTGGACGCACAACTGGCATC CCCATCAGGCAACTCGTAACTCTTC GAPDH 小鼠肝组织 AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA 人LX-2细胞 GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG 
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