桃红四物汤对CCl<sub>4</sub>诱导肝纤维化小鼠模型的干预作用及其机制

刘旭凌; 杨广越; 张玮; 孙田甜; 马文婷; 吴柳; 薛冬英; 刘成; 陶乐

doi:10.3969/j.issn.1001-5256.2021.11.016

桃红四物汤对CCl₄诱导肝纤维化小鼠模型的干预作用及其机制

DOI: 10.3969/j.issn.1001-5256.2021.11.016

刘旭凌^{a, b},
杨广越^c,
张玮^c,
孙田甜^c,
马文婷^{a, b},
吴柳^{a, b},
薛冬英^{a, b},
刘成^{a, c},
陶乐^{a, b, ,}

a.
上海中医药大学附属普陀医院肝病实验室，上海 200062
b.
上海中医药大学附属普陀医院感染科，上海 200062
c.
上海中医药大学附属普陀医院中心实验室，上海 200062

基金项目:

国家自然科学基金项目 (81673788);

国家自然科学基金项目 (81873136);

国家自然科学基金项目 (81803898);

国家自然科学基金项目 (81803232);

国家自然科学基金项目 (82004106);

上海市自然基金 (20ZR1450300);

上海市卫生健康委员会卫生行业临床研究专项 (202040149);

上海市普陀区自主创新项目 (ptkwws201817);

上海市普陀区自主创新项目 (ptkwws201904)

详细信息

通信作者:
陶乐，talent901036@163.com

中图分类号: R575.2
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- 被引次数: 0
出版历程
- 收稿日期: 2021-03-09
- 录用日期: 2021-04-21
- 出版日期: 2021-11-20

Therapeutic effect of Taohong Siwu decoction on a mouse model of carbon tetrachloride-induced liver fibrosis and its mechanism

Xuling LIU^{a, b},
Guangyue YANG^c,
Wei ZHANG^c,
Tiantian SUN^c,
Wenting MA^{a, b},
Liu WU^{a, b},
Dongying XUE^{a, b},
Cheng LIU^{a, c},
Le TAO^{a, b
, ,}

a.
Laboratory of Liver Diseases, Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China
b.
Department of Infectious Diseases, Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China
c.
Central Laboratory, Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China

Research funding:

National Natural Science Foundation of China (81673788);

National Natural Science Foundation of China (81873136);

National Natural Science Foundation of China (81803898);

National Natural Science Foundation of China (81803232);

National Natural Science Foundation of China (82004106);

Shanghai National Natural Science Foundation (20ZR1450300);

Shanghai Municipal Health Commission (202040149);

Indenpdent Innovation Project of Shanghai Puotuo District Project (ptkwws201817);

Indenpdent Innovation Project of Shanghai Puotuo District Project (ptkwws201904)

摘要

摘要: 目的探讨桃红四物汤对CCl₄诱导肝纤维化小鼠模型的干预作用及其作用机制。方法 24只雄性C57/BL6小鼠随机分为对照组、模型组和桃红四物汤组，每组8只。模型组和桃红四物汤组小鼠腹腔注射10%CCl₄，第3周开始给予桃红四物汤灌胃给药4周，检测肝功能(ALT、AST)，观测肝组织病理形态学。实时荧光定量PCR检测α-平滑肌肌动蛋白(α-SMA)、透明质酸合成酶-2(HAS-2)、Ⅰ型胶原(Col1)mRNA的表达，蛋白免疫印迹法检测α-SMA、Col1及HAS-2蛋白的表达。分离小鼠原代肝星状细胞(HSC)，应用siRNA沉默HAS-2，检测其对HSC激活的影响。计量资料两组间比较采用t检验，多组间比较采用单因素方差分析，组内进一步两两比较采用SNK或LSD-t检验。结果与正常组相比，模型组血清肝功能指标ALT、AST水平显著升高，桃红四物汤组小鼠血清ALT、AST水平均显著下降(P值均＜0.01)。病理学染色显示：模型组炎症细胞浸润明显，增生的胶原纤维形成纤维间隔；桃红四物汤组胶原纤维间隔较疏松及炎症细胞浸润减轻。与模型组相比，桃红四物汤组α-SMA、Col1 mRNA和蛋白的表达水平均显著降低(P值均＜0.001)。与正常组比较，模型组肝组织中HAS-2 mRNA表达水平显著升高(t=6.14，P＜0.05)，桃红四物汤组HAS-2蛋白表达水平(0.29±0.10)较模型组(1.00±0.12)明显减少(t=70.73，P＜0.001)。siRNA沉默HAS-2，TGFβ处理后(Si HAS-2+TGFβ组)α-SMA、Col1 mRNA表达水平较NC+TGFβ组显著降低(P＜0.01)。结论桃红四物汤通过抑制HAS-2对CCl₄致小鼠纤维化有显著治疗作用。
- 肝硬化 /
- 桃红四物汤 /
- 透明质酸合成酶2 /
- 小鼠, 近交C57BL
Abstract: Objective To investigate the therapeutic effect of Taohong Siwu decoction on a mouse model of carbon tetrachloride (CCl₄)-induced liver fibrosis and its mechanism of action. Methods A total of 24 male C57BL/6 mice were randomly divided into normal group, model group, and Taohong Siwu decoction group, with 8 mice in each group. The mice in the model group and the Taohong Siwu decoction group were given intraperitoneal injection of 10% CCl₄, and Taohong Siwu decoction was given by gavage since week 3 for 4 consecutive weeks. Liver function [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] was measured, and liver pathomorphology was observed. Real-time PCR was used to measure the mRNA expression of α-smooth muscle actin (α-SMA), hyaluronic acid synthase-2 (HAS-2), and collagen type Ⅰ(Col1), and Western blotting was used to measure the protein expression of α-SMA, Col1, and HAS-2. Primary hepatic stellate cells (HSCs) were isolated, and HAS-2 was silenced by siRNA to observe its influence on HSC activation. The t-test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the SNK or least significant difference t-test was used for further comparison between two groups. Results Compared with the normal group, the model group had significant increases in serum liver function parameters (ALT, AST) and the Taohong Siwu decoction group had significant reductions in the serum levels of ALT and AST (all P < 0.01). Pathological staining showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Taohong Siwu decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Compared with the model group, the Taohong Siwu decoction group had significant reductions in the mRNA and protein expression of α-SMA and Col1(all P < 0.001). Compared with the normal group, the model group had a significant increase in the mRNA expression level of HAS-2 in liver tissue (t=6.14, P < 0.05), and compared with the model group, the Taohong Siwu decoction group had a significant reduction in the protein expression level of HAS-2 (0.29±0.10 vs 1.00±0.12, t=70.73, P < 0.001). After HAS-2 was silenced by siRNA, the Si HAS-2+transforming growth factor β (TGFβ) group (treated with TGFβ) had significant reductions in the mRNA expression levels of α-SMA and Col-Ⅰ compared with the NC+TGFβ group (P < 0.01). Conclusion Taohong Siwu decoction exerts a marked therapeutic effect on CCl₄-induced liver fibrosis in mice by inhibiting HAS-2.
- Liver Cirrhosis /
- Taohong Siwu Decoction /
- Hyaluronic Acid Synthase-2 /
- Mice, Inbred C57BL

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图 1 肝组织病理结果(×100)

注：a，HE染色，×100；b，天狼星红染色，×100。

下载: 全尺寸图片幻灯片

图 2 各组小鼠肝组织α-SMA、Col1表达比较

注：a，α-SMA免疫组化结果(×100)；b，α-SMA、Col1免疫印迹结果。

下载: 全尺寸图片幻灯片

图 3 各组小鼠肝组织HAS-2表达比较

注：a，HAS-2免疫组化结果(×100)；b，HAS-2免疫印迹结果。

下载: 全尺寸图片幻灯片

表 1 RT-PCR引物序列

名称	序列
18S RNA	上游引物：5′-GTAACCCGTTGAACCCCATT-3′
	下游引物：5′-CCATCCAATCGGTAGTAGCG-3′
Col1	上游引物：5′-TAGGCCATTGTGTATGCAGC-3′
	下游引物：5′-ACATGTTCAGCTTTGTGGACC-3′
α-SMA	上游引物：5′-GTTCAGTGGTGCCTCTGTCA-3′
	下游引物：5′-ACTGGGACGACATGGAAAAG-3′
HAS-1	上游引物：5′-CTATGCTACCAAGTATACCTCG-3′
	下游引物：5′-TCTCGGAAGTAAGATTTGGAC-3′
HAS-2	上游引物：5′-TGAACAAAACGGTAGCAATCTG-3′
	下游引物：5′-ACTTTAATCCCAGGGTAGGTCAG-3′
HAS-3	上游引物：5′-GATGTCCAAATCCTCAACAAG-3′
	下游引物：5′-CCCACTAATACATTGCACAC-3′

下载: 导出CSV

表 2 各组血清转氨酶水平及病理学改变

指标	正常组(n=8)	模型组(n=8)	桃红四物汤组(n=8)	F值	P值
ALT(U/L)	6.61±1.92	247.33±21.76¹⁾	144.84±15.94²⁾	10.75	＜0.001
AST(U/L)	8.41±3.48	173.44±31.23¹⁾	108.05±18.45²⁾	5.10	＜0.001
炎性细胞面积(%)	2.76±0.98	12.37±1.09¹⁾	6.58±0.72²⁾	12.52	＜0.001
天狼星红面积(%)	1.20±0.35	10.80±2.49¹⁾	5.62±4.31²⁾	5.50	＜0.001
注：1)与正常组比较，P＜0.01;2)与模型组比较，P＜0.01。

下载: 导出CSV

表 3 各组α-SMA、Col1表达水平比较

指标	正常组(n=8)	模型组(n=8)	桃红四物汤组(n=8)	F值	P值
免疫印迹
α-SMA/GADPH	1.00±0.12	10.79±0.47¹⁾	4.34±1.04²⁾	169.96	＜0.001
Col1/GADPH	1.00±0.54	7.56±0.96¹⁾	3.67±1.20²⁾	36.98	＜0.001
RT-PCR
α-SMA mRNA	1.00±0.28	5.50±1.74¹⁾	3.12±0.70²⁾	33.73	＜0.001
Col1 mRNA	1.00±0.44	4.63±1.54¹⁾	2.98±1.29²⁾	18.80	＜0.001
注：1)与正常组比较，P＜0.01;2)与模型组比较，P＜0.01。

下载: 导出CSV

表 4 各组HAS-1、HAS-2、HAS-3mRNA表达情况

指标	正常组	模型组	t值	P值
HAS-1	1.00±0.19	1.22±0.49	1.46	0.247
HAS-2	1.00±0.04	2.59±0.17	6.14	0.027
HAS-3	1.00±0.24	0.97±0.31	0.00	0.962

下载: 导出CSV

表 5 α-SMA、Col1、HAS-2mRNA在原代HSC中的表达水平

指标	对照组	TGFβ处理组	t值	P值
α-SMA mRNA	1.00±0.53	50.59±6.28	-11.165	0.008
Col1 mRNA	1.00±0.63	30.39±1.49	-27.73	0.001
HAS-2 mRNA	1.00±0.47	9.72±0.64	-19.36	0.003

下载: 导出CSV

表 6 基因沉默HAS2检测THSWD对HSC活化的影响

组别	α-SMA mRNA	Col1 mRNA
NC	1.00±0.14	1.00±0.92
NC+TGFβ	52.98±4.89¹⁾	30.28±0.42¹⁾
NC+TGFβ+THSWD	30.31±0.61²⁾	18.33±0.90²⁾
Si HAS-2	0.90±0.077	1.53±0.24
Si HAS-2+TGFβ	25.11±2.51²⁾	19.09±2.35²⁾
Si HAS-2+TGFβ+THSWD	24.44±1.05	20.68±2.08
F值	146.12	151.59
P值	＜0.001	＜0.001
注：NC，空白对照；1)与NC比较，P＜0.01；2)与NC+TGFβ，比较P＜0.01。

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