黄芪汤含药血清对血管内皮生长因子诱导的大鼠肝窦内皮细胞增殖、迁移和成管能力的影响及其作用机制

王浩艺; 麦静愔; 平键; 成扬

doi:10.3969/j.issn.1001-5256.2022.10.015

黄芪汤含药血清对血管内皮生长因子诱导的大鼠肝窦内皮细胞增殖、迁移和成管能力的影响及其作用机制

DOI: 10.3969/j.issn.1001-5256.2022.10.015

王浩艺¹,
麦静愔²,
平键^{1, 3},
成扬^{1, 3, 4, , ,}

1.
上海中医药大学附属曙光医院肝病研究所，上海 201203
2.
上海市光华中西医结合医院治未病科，上海 200052
3.
上海中医药大学肝肾疾病病证教育部重点实验室，上海 201203
4.
上海市中医临床重点实验室，上海 201203

基金项目:

国家自然科学基金 (81774098);

上海市浦东新区名中医工作室建设项目 (PWRzm2020-14)

伦理学声明：本研究方案于2019年4月28日经由上海中医药大学实验动物伦理委员会审批，批号：PZSHUTCM 2019041835，符合实验室动物管理与使用准则。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：王浩艺参与课题设计，收集数据，资料分析，撰写论文；麦静愔、平键参与课题设计，收集数据，修改论文；成扬负责课题设计，拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
成扬, drchengyang@126.com

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出版历程
- 收稿日期: 2022-02-24
- 录用日期: 2022-03-26
- 出版日期: 2022-10-20

Effect of serum containing Huangqi decoction on the proliferation, migration, and tubulogenesis of rat liver sinusoidal endothelial cells induced by vascular endothelial growth factor and its mechanism of action

Haoyi WANG¹,
Jingyin MAI²,
Jian PING^{1, 3},
Yang CHENG^{1, 3, 4
, ,
,}

1.
Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2.
Department of Preventive Treatment of Diseases, Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai 200052, China
3.
Key Laboratory of Liver and Kidney Diseases, Ministry of Education, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
4.
Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China

Research funding:

National Natural Science Foundation of China (81774098);

The Project of Shanghai Pudong New Area Famous Chinese Medicine Studio Construction (PWRzm2020-14)

More Information

Corresponding author: CHENG Yang, drchengyang@126.com(ORCID: 0000-0001-7984-930)

摘要

摘要: 目的基于蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路探讨黄芪汤含药血清对血管内皮生长因子(VEGF)诱导的大鼠肝窦内皮细胞(LSEC)增殖、迁移和成管能力的影响。方法制备黄芪汤含药血清，体外分离、培养SD大鼠LSEC，将大鼠LSEC随机分为空白组，VEGF组，血清对照组，低、中、高剂量黄芪汤含药血清组，利用噻唑蓝(MTT)比色法、Transwell实验、成管实验分别检测各组LSEC增殖、迁移和成管能力，通过Western Blot法检测血小板-内皮细胞黏附分子-1(CD31)、内皮素-1(ET-1)、内皮性一氧化氮合酶(eNOS) 和AKT/mTOR信号通路中AKT、磷酸化(p)-AKT、mTOR、磷酸化(p)-mTOR蛋白表达水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用Tukey’s检验。结果与空白组相比，VEGF组、血清对照组能够促进大鼠LSEC增殖、迁移和血管新生(P值均＜0.05)；Western Blot结果显示，CD31、ET-1、eNOS和AKT/mTOR信号通路相关蛋白表达显著升高(P值均＜0.05)。VEGF组与血清对照组比较，上述指标差异均无统计学意义(P值均＞0.05)。与血清组比较，黄芪汤中、高剂量组能显著抑制VEGF诱导的大鼠LSEC增殖、迁移和血管新生(P值均＜0.05)；Western Blot结果显示，CD31、ET-1、eNOS和AKT/mTOR信号通路相关蛋白表达均显著降低(P值均＜0.05)。结论较高剂量黄芪汤含药血清能抑制VEGF诱导的大鼠LSEC增殖、迁移和成管能力，其作用机制可能与调控AKT/mTOR信号通路有关。
- 肝硬化 /
- 黄芪 /
- 肝窦内皮细胞 /
- 大鼠, Sprague-Dawley
Abstract: Objective To investigate the effect of serum containing Huangqi decoction on the proliferation, migration, and tubulogenesis of rat liver sinusoidal endothelial cells (LSECs) induced by vascular endothelial growth factor (VEGF) and its mechanism of action based on the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. Methods The serum containing Huangqi decoction was prepared, and rat LSECs were isolated and cultured in vitro. Then rat LSECs were randomly divided into blank group, VEGF group, serum control group, and low-, middle-, and high-dose serum containing Huangqi decoction groups. MTT colorimetry, Transwell assay, and tubulogenesis assay were used to measure the proliferation, migration, and tubulogenesis abilities of LSECs in each group, and Western Blot was used to measure the protein expression levels of platelet endothelial cell adhesion molecule-1 (CD31), endothelin-1 (ET-1), and endothelial nitric oxide synthase (eNOS), as well as AKT, phosphorylated-AKT (p-AKT), mTOR, and phosphorylated mTOR (p-mTOR) in the AKT/mTOR signaling pathway. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Tukey's test was used for further comparison between two groups. Results Compared with the blank group, the VEGF group and the serum control group had significantly promoted proliferation, migration, and angiogenesis of rat LSECs (all P < 0.05), and Western Blot showed significant increases in the expression levels of CD31, ET-1, eNOS, and AKT/mTOR signaling pathway-related proteins (all P < 0.05). There were no significant differences in the above indices between the VEGF group and the serum control group (all P > 0.05). Compared with the serum group, the middle- and high-dose serum containing Huangqi decoction groups had significantly inhibited proliferation, migration, and angiogenesis of rat LSECs induced by VEGF (all P < 0.05), and Western Blot showed significant reductions in the expression levels of CD31, ET-1, eNOS, and AKT/mTOR signaling pathway-related proteins (all P < 0.01). Conclusion A relatively high dose of serum containing Huangqi decoction can significantly inhibit the proliferation, migration, and tubulogenesis of rat LSECs induced by VEGF, possibly by regulating the AKT/mTOR signaling pathway.
- Liver Cirrhosis /
- Astragalus membranaceus /
- Liver Sinusoidal Endothelial Cells /
- Rats, Sprague-Dawley

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图 1 不同剂量黄芪汤含药血清对大鼠LSEC迁移的影响(结晶紫染色，×100)

注：a，空白组；b，VEGF组；c，血清组；d，低剂量组；e，中剂量组；f，高剂量组。

Figure 1. Effects of different doses of serum containing Huangqi Decoction on LSEC migration (crystal violet staining, ×100)

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图 2 不同剂量黄芪汤含药血清对大鼠LSEC成管能力的影响(×100)

注：a，空白组；b，VEGF组；c，血清组；d，低剂量组；e，中剂量组；f，高剂量组。

Figure 2. Effects of different doses of serum containing Huangqi Decoction on LSEC tubulogenesis abilities (×100)

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图 3 各组大鼠LSEC中CD31、eNOS、ET-1、GAPDH蛋白表达

Figure 3. Expression of CD31, eNOS, ET-1 and GAPDH proteins in LSEC in each group

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图 4 各组大鼠LSEC中AKT/mTOR信号通路相关蛋白表达

Figure 4. Expression of AKT/mTOR signaling pathway related proteins in LSEC in each group

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表 1 各组大鼠LESC活力比较

Table 1. Comparison of LESC cell activity in each group

组别	细胞活力(%)
空白组	100.00±12.05
VEGF组	156.35±16.89¹⁾
血清组	165.22±18.52¹⁾
低剂量组	173.52±21.56¹⁾
中剂量组	131.21±15.67²⁾
高剂量组	111.52±12.36²⁾³⁾
F值	7.317
P值	0.002
注：与空白组比较，1)P＜0.05；与血清组比较，2)P＜0.05；与中剂量组比较，3)P＜0.05。

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表 2 各组大鼠LESC迁移细胞数目比较

Table 2. Comparison of LESC migration cell numbers in each group

组别	迁移细胞数(个)
空白组	35.64±3.65
VEGF组	153.63±16.59¹⁾
血清组	155.24±15.22¹⁾
低剂量组	371.25±41.26²⁾
中剂量组	85.64±10.37²⁾
高剂量组	81.23±9.57²⁾
F值	83.199
P值	＜0.001
注：与空白组比较，1)P＜0.05；与血清组比较，2)P＜0.05。

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表 3 各组大鼠LESC成管能力比较

Table 3. Comparison of LESC tubulogenesis abilities in each group

组别	节点(个)	交叉点(个)	血管长度(%)
空白组	126.00±11.14	33.33±7.02	100.00±12.63
VEGF组	985.67±93.93¹⁾	266.00±26.85¹⁾	421.56±68.53¹⁾
血清组	922.34±71.01¹⁾	263.33±9.29¹⁾	475.63±59.67¹⁾
低剂量组	1537.67±84.33²⁾³⁾	431.67±10.50²⁾³⁾	864.55±124.35²⁾³⁾
中剂量组	424.34±51.00²⁾³⁾⁴⁾	115.67±9.61²⁾³⁾⁴⁾	225.64±42.36²⁾³⁾⁴⁾
高剂量组	205.34±49.00^2)3)4)5)	60.00±14.11^2)3)4)5)	185.66±25.67²⁾³⁾⁴⁾
F值	204.507	332.945	46.289
P值	＜0.001	＜0.001	＜0.001
注：与空白组比较，1)P＜0.05；与VEGF组比较，2)P＜0.05；与血清组比较，3)P＜0.05；与低剂量组比较，4)P＜0.05；与中剂量组比较，5)P＜0.05。

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表 4 各组大鼠LESC中CD31、eNOS、ET-1蛋白表达比较

Table 4. Comparison of CD31, eNOS and ET-1 protein expression in LESC in each group

组别	CD31	eNOS	ET-1
空白组	0.201±0.025	0.053±0.006	0.048±0.005
VEGF组	1.385±0.139¹⁾	0.465±0.047¹⁾	1.052±0.106¹⁾
血清组	1.362±0.142¹⁾	0.452±0.046¹⁾	1.163±0.116¹⁾
低剂量组	1.353±0.136	0.461±0.048	1.583±0.237²⁾³⁾
中剂量组	0.575±0.073²⁾³⁾⁴⁾	0.227±0.027²⁾³⁾⁴⁾	0.685±0.069²⁾³⁾⁴⁾
高剂量组	0.067±0.012^2)3)4)5)	0.153±0.015^2)3)4)5)	0.206±0.021^2)3)4)5)
F值	89.514	56.273	60.760
P值	＜0.001	＜0.001	＜0.001
注：与空白组比较，1)P＜0.05；与VEGF组比较，2)P＜0.05；与血清组比较，3)P＜0.05；与低剂量组比较，4)P＜0.05；与中剂量组比较，5)P＜0.05。

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表 5 各组大鼠LESC中AKT/mTOR信号通路蛋白表达比较

Table 5. Comparison of AKT/mTOR signaling pathway protein expression in LESC in each group

组别	p-AKT/AKT	p-mTOR/mTOR
空白组	0.257±0.025	0.852±0.027
VEGF组	0.865±0.139¹⁾	1.229±0.013¹⁾
血清组	0.834±0.142¹⁾	1.227±0.021¹⁾
低剂量组	1.425±0.136¹⁾³⁾	1.197±0.012¹⁾³⁾
中剂量组	0.268±0.073²⁾³⁾⁴⁾	1.065±0.025²⁾³⁾⁴⁾
高剂量组	0.054±0.012^2)3)4)5)	0.696±0.024^2)3)4)5)
F值	52.884	337.037
P值	＜0.001	＜0.001
注：与空白组比较，1)P＜0.05；与VEGF组比较，2)P＜0.05；与血清组比较，3)P＜0.05；与低剂量组比较，4)P＜0.05；与中剂量组比较，5)P＜0.05。

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