养血柔肝丸对多因素诱导的肝纤维化大鼠模型TGFβ1/Smad信号通路的影响

柴焱; 张涛; 范引科; 惠爱武; 赵林涛; 李芳; 张小丽

doi:10.3969/j.issn.1001-5256.2022.09.018

养血柔肝丸对多因素诱导的肝纤维化大鼠模型TGFβ1/Smad信号通路的影响

DOI: 10.3969/j.issn.1001-5256.2022.09.018

柴焱¹,
张涛²,
范引科³,
惠爱武³,
赵林涛³,
李芳³,
张小丽^3, , ,

1.
宝鸡市中医医院临床药学室，陕西宝鸡 721000
2.
清华德人西安幸福制药有限公司，西安 710043
3.
陕西省中医药研究院中药研究所，西安 710003

基金项目:

陕西省科技计划-重点产业链项目 (2020ZDLSF05-06)

伦理学声明：本研究方案于2020年6月19日经由陕西省中医药研究院伦理委员会审批，批号：(2020)伦审意见第(512)号，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：柴焱负责数据整理，撰写论文；张涛、范引科、惠爱武、赵林涛、李芳负责实验操作，数据分析，病理分析，并指导修改文章；张小丽负责课题设计，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:
张小丽，380136809@qq.com

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出版历程
- 收稿日期: 2022-04-24
- 录用日期: 2022-05-30
- 出版日期: 2022-09-20

Effect of Yangxue Rougan pills on the TGF-β1/Smad signaling pathway in a rat model of liver fibrosis induced by multiple factors

1.
Department of Clinical Pharmacy, Baoji Hospital of Traditional Chinese Medicine, Baoji, Shaanxi 721000, China
2.
Tsinghua Deren Xi'an Happiness Pharmaceutical Co., Ltd., Xi'an 710043, China
3.
Institute of Traditional Chinese Medicine, Shaanxi Academy of Traditional Chinese Medicine, Xi'an 710003, China

Research funding:

Science and Technology Plan of Shaanxi Province-Key Industry Chain Project (2020ZDLSF05-06)

More Information

Corresponding author: ZHANG Xiaoli, 380136809@qq.com(ORCID: 0000-0003-2405-5244)

摘要

摘要: 目的观察养血柔肝丸对多因素诱导所致的大鼠肝纤维化的影响, 探讨养血柔肝丸治疗肝纤维化的作用机制。方法 50只雄性大鼠随机分成空白对照组、多因素模型组、养血柔肝丸高剂量组、养血柔肝丸中剂量组、养血柔肝丸低剂量组和扶正化瘀胶囊组，共6组。空白对照组给予正常饮水和正常饲料。剩余各组大鼠给予改良高脂低蛋白饲料，5%食用酒精，并且大鼠皮下注射含有40%四氯化碳的橄榄油溶液和腹腔注射猪血清0.5 mL/只，2次/周，连续12周。从第7周开始，给予养血柔肝丸，分为高剂量组(9.5 g/kg)、中剂量组(4.75 g/kg)、低剂量组(2.38 g/kg)，扶正化瘀胶囊组(0.75 g/kg)，空白对照组、多因素模型组每日灌胃等体积量蒸馏水，连续6周，于第12周处理大鼠。采用HE染色及Masson染色病理观察大鼠肝纤维化程度; PCR和Western blot检测大鼠肝TGFβ1、Smad3、Smad7的表达情况。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Dunnett-t检验法。结果多因素模型组与空白对照组相比可见肝小叶结构严重破坏，纤维化明显增强，形成不同大小的假小叶; 而养血柔肝丸各剂量组较多因素模型组肝纤维化程度明显好转，以养血柔肝丸高、中剂量组治疗趋势最显著。多因素模型组大鼠肝组织TGFβ1、Smad3的表达与空白对照组比较有明显提升(P值均＜0.05)，Smad7的表达则明显下降(P＜0.05)，而养血柔肝丸各剂量组TGFβ1的表达与多因素模型组比较有所降低(P值均＜0.05)，Smad7的表达则明显升高(P值均＜0.05)；养血柔肝丸高、中剂量组Smad3的表达与多因素模型组比较有所降低, 差异有统计学意义(P值均＜0.05)。结论养血柔肝丸可明显抑制大鼠肝纤维化，使大鼠肝组织中TGFβ1、Smad3的表达下调，Smad7的表达上调，TGFβ1/Smad信号通路是养血柔肝丸改善肝纤维化的作用机制之一。
- 肝硬化 /
- 转化生长因子β1 /
- Smad蛋白质类 /
- 药理作用分子作用机制
Abstract: Objective To investigate the effect of Yangxue Rougan pills on a rat model of liver fibrosis induced by multiple factors and the mechanism of action of Yangxue Rougan pills in the treatment of liver fibrosis. Methods A total of 50 male rats were randomly divided into blank control group, multi-factor model group, Fuzheng Huayu capsule group, and high-, middle-, and low-dose Yangxue Rougan pill groups. The rats in the blank control group were given normal water and feed, and those in the other groups were given modified high-fat low-protein diet and 5% alcohol, as well as subcutaneous injection of olive oil solution containing 40% carbon tetrachloride and intraperitoneal injection of pig serum 0.5 mL per rat, twice a week for 12 consecutive weeks. Since week 7, the rats in the high-, middle-, and low-dose Yangxue Rougan pill groups were given Yangxue Rougan pills at a dose of 9.5, 4.75, and 2.38 g/kg, respectively, those in the Fuzheng Huayu capsule group were given Fuzheng Huayu capsules at a dose of 0.75 g/kg, and those in the blank control group and the multi-factor model group were given an equal volume of distilled water by gavage every day for 6 consecutive weeks. The rats were treated at week 12. HE staining and Masson staining were used to observe the degree of liver fibrosis in rats, and PCR and Western blot were used to measure the expression of TGF-β1, Smad3, and Smad7 in the liver. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Dunnett's t-test was used for further comparison between two groups. Results Compared with the blank control group, the multi-factor model group had a severely damaged lobular structure and a significantly higher degree of liver fibrosis, with the formation of pseudolobules with different sizes; compared with the multi-factor model group, the Yangxue Rougan pill groups had a significant improvement in the degree of liver fibrosis, with the most significant therapeutic effect in the high- and middle-dose Yangxue Rougan pill groups. Compared with the blank control group, the multi-factor model group had significant increases in the expression of TGF-β1 and Smad3 and a significant reduction in the expression of Smad7 in liver tissue (all P < 0.05); compared with the multi-factor model group, the Yangxue Rougan pill groups had a significant reduction in the expression of TGF-β1 and a significant increase in the expression of Smad7 (all P < 0.05); compared with the multi-factor model group, the high- and middle-dose Yangxue Rougan pill groups had a significant reduction in the expression of Smad3 (both P < 0.05). Conclusion Yangxue Rougan pills can significantly inhibit liver fibrosis in rats by downregulating the expression of TGF-β1 and Smad3 and upregulating the expression of Smad7, and therefore, the TGF-β1/Smad signaling pathway is one of the mechanisms of action of Yangxue Rougan pills in improving liver fibrosis.
- Liver Cirrhosis /
- Transforming Growth Factor beta1 /
- Smad Proteins /
- Molecular Mechanisms of Pharmacological Action

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图 1 各组大鼠肝组织细胞HE染色(×100)

注: a，空白对照组; b，扶正化瘀胶囊组; c，多因素模型组; d，养血柔肝丸低剂量组; e，养血柔肝丸中剂量组; f，养血柔肝丸高剂量组；箭头指示病变。

Figure 1. HE staining of liver tissues of rats in each group(×100)

下载: 全尺寸图片幻灯片

图 2 各组大鼠肝组织细胞Masson染色(×100)

注: a，空白对照组; b，扶正化瘀胶囊组; c，多因素模型组; d，养血柔肝丸低剂量组; e，养血柔肝丸中剂量组; f，养血柔肝丸高剂量组；箭头指示病变。

Figure 2. Masson staining of liver tissues of rats in each group(×100)

下载: 全尺寸图片幻灯片

图 3 各组大鼠肝组织细胞TGFβ1、Smad3、Smad7 mRNA表达水平

注: a，空白对照组; b，扶正化瘀胶囊组; c，多因素模型组; d，养血柔肝丸低剂量组; e，养血柔肝丸中剂量组; f，养血柔肝丸高剂量组。

Figure 3. Expression of TGFβ1, Smad 3 and Smad 7 mRNA in liver tissues of the rats in each group

下载: 全尺寸图片幻灯片

图 4 Western blot检测各组大鼠肝组织TGFβ1、Smad3、Smad7表达结果

注: a，空白对照组; b，扶正化瘀胶囊组; c，多因素模型组; d，养血柔肝丸低剂量组; e，养血柔肝丸中剂量组; f，养血柔肝丸高剂量组。

Figure 4. Expression of TGFβ1, Smad 3 and Smad 7 in liver tissues detected by Western blot

下载: 全尺寸图片幻灯片

图 5 各组大鼠肝组织细胞TGFβ1、Smad3、Smad7蛋白表达水平

注: a，空白对照组; b，扶正化瘀胶囊组; c，多因素模型组; d，养血柔肝丸低剂量组; e，养血柔肝丸中剂量组; f，养血柔肝丸高剂量组。

Figure 5. Expression of TGFβ1, Smad 3 and Smad 7 proteins in liver tissues of the rats in each group

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

基因	引物序列(5′-3′)	扩增片段长度(bp)
β-actin	F: CCCATCTATGAGGGTTACGC	150
	R: TTTAATGTCACGCACGATTTC
TGFβ1	F: ATTCCTGGCGTTACCTTGG	120
	R: AGCCCTGTATTCCGTCTCCT
Smad3	F: GAGACATTCCACGCTTCACA	110
	R: GCTGCATTCCGGTTAACATT
Smad7	F: AAGTCAAGAGGCTGTGTTGCTGTG	128
	R: CATCGGGTATCTGGAGTAAGGAGGAG

下载: 导出CSV

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