整合素α4在美洲大蠊提取物黏糖氨酸抗肝纤维化中的作用

鲁杰; 周义霞; 刘叶; 高雅; 陈科璇; 李顶春; 陈一晖; 刘怀鄂; 王宏图; 李武

doi:10.3969/j.issn.1001-5256.2022.09.016

整合素α4在美洲大蠊提取物黏糖氨酸抗肝纤维化中的作用

DOI: 10.3969/j.issn.1001-5256.2022.09.016

昆明医科大学第一附属医院感染与肝病科，昆明 650032

基金项目:

国家自然科学基金 (82160801);

云南省“万人计划”名医专项 (Yunrenweifa〔2020〕20);

云南省“万人计划”名医专项 (RLMY20200015);

云南省科技厅-应用基础研究重点项目 (2017FE468 (-173));

云南省科技厅-应用基础研究联合专项资金项目 (2018FE001 (-214))

伦理学声明：本研究方案于2021年2月26日由昆明医科大学动物伦理委员会审批通过，批号：2021-206，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：鲁杰负责课题设计，资料分析，撰写论文；周义霞、高雅、陈科璇参与实验设计；李顶春、刘叶、陈一晖拟定写作思路；刘怀鄂、王宏图修改论文；李武指导撰写论文并最后定稿。

详细信息

通信作者:
李武，liwukm@qq.com

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出版历程
- 收稿日期: 2022-01-09
- 录用日期: 2022-03-10
- 出版日期: 2022-09-20

The role of integrin α4 in the anti-liver fibrosis effect of the sticky sugar amino acid extract of Periplaneta americana

Department of Infection and Liver Diseases, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China

Research funding:

National Natural Science Foundation of China (82160801);

Yunnan Province Famous Doctor Project (Yunrenweifa〔2020〕20);

Yunnan Province Famous Doctor Project (RLMY20200015);

Yunnan Provincial Department of Science and Technology-Applied Basic Research Key Project (2017FE468 (-173));

Yunnan Provincial Department of Science and Technology-Applied Basic Research Joint Special Fund Project (2018FE001 (-214))

More Information

Corresponding author: LI Wu, liwukm@qq.com(ORCID: 0000-0002-1222-3629)

摘要

摘要: 目的本研究基于黏糖氨酸(SSAA)在大鼠中的抗肝纤维化作用，研究整合素α4(ITGA4)在肝纤维化中的作用机制。方法大鼠腹腔内注射CCl₄诱导肝纤维化模型，在此基础上使用秋水仙碱或SSAA低、中、高剂量进行干预，以空白对照组、SSAA组作为对照。实验干预12周后，收集大鼠血清、肝脏标本，检测大鼠血清ALT、AST水平，HE染色、天蓝猩红染色观察肝组织病理情况；实时荧光定量PCR检测肝组织ITGA4、整合素β1(ITGB1)、TGFβ1、α-SMA、TIMP2的转录水平；Western Blot检测ITGA4、ITGB1、TGFβ1、α-SMA、MMP2、TIMP1、TIMP2的相对蛋白表达；免疫组化观察TGFβ1、α-SMA蛋白表达。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LSD-t检验。结果 CCl₄模型组中AST、ALT显著升高，秋水仙碱或SSAA低、中、高剂量干预后能降低AST、ALT水平，各组与CCl₄模型组相比差异均有统计学意义(P值均＜0.05)。CCl₄模型组中的HE染色和天蓝猩红染色结果显示肝小叶结构紊乱、胶原纤维增多，秋水仙碱或SSAA低、中、高剂量干预后肝小叶结构得到改善。CCl₄模型组中的ITGA4、TGFβ1、α-SMA和TIMP2各指标转录水平均显著高于其余各组，秋水仙碱或SSAA干预后能降低各因子转录水平，与CCl₄模型组相比差异均有统计学意义(P值均＜0.05)。CCl₄模型组中的ITGA4、TGFβ1、α-SMA、TIMP2和TIMP1蛋白表达高于其余各组，MMP2蛋白表达低于其余各组，在秋水仙碱或SSAA干预后均能抑制ITGA4、TGFβ1、α-SMA、TIMP2、TIMP1表达，促进MMP2表达。免疫组化结果提示CCl₄模型组的TGFβ1、α-SMA的表达显著高于其余各组，秋水仙碱或SSAA干预后均能抑制表达。SSAA高剂量组在降低转氨酶、改善肝小叶结构和抑制致肝纤维化因子蛋白表达上效果最显著。结论肝脏中ITGA4的高表达与肝纤维化的发生相关，与TGFβ1、α-SMA表达升高一致，抑制ITGA4表达可为抗肝纤维化提供更多治疗靶点，同时拓展了SSAA的抗肝纤维化作用机制。
- 肝硬化 /
- 黏糖氨酸 /
- 整合素类 /
- 大鼠, Sprague-Dawley
Abstract: Objective To investigate the mechanism of action of integrin α4 (ITGA4) in liver fibrosis based on the anti-liver fibrosis effect of sticky sugar amino acid (SSAA) in rats. Methods A rat model of liver fibrosis was induced by intraperitoneal injection of CCl₄, and then colchicine and low-, middle-, and high-dose SSAA were used for intervention, with blank control group and SSAA group as control. After 12 weeks of experimental intervention, serum and liver samples were collected to measure the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and HE staining and Sirius Red staining were used to observe the pathological conditions of liver tissue; quantitative real-time PCR was used to measure the transcriptional level of ITGA4, integrin β1 (ITGB1), transforming growth factor-β1 (TGFβ1), alpha-smooth muscle actin (α-SMA), and TIMP2 in liver tissue; Western blot was used to measure the relative protein expression levels of ITGA4, ITGB1, TGFβ1, α-SMA, MMP2, TIMP1, and TIMP2; immunohistochemistry was used to observe the protein expression of TGFβ1 and α-SMA. 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 comparison between two groups. Results There were significant increases in AST and ALT in the CCl₄ model group, and intervention with colchicine or low-, middle-, and high-dose SSAA reduced the levels of AST and ALT, with a significant difference between the CCl₄ model group and the other groups (all P < 0.05). HE staining and Sirius Red staining showed disordered structure of hepatic lobules and an increase in collagen fibers in the CCl₄ model group, and the structure of hepatic lobules was improved after intervention with colchicine or low-, middle-, and high-dose SSAA. The CCl₄ model group had significantly higher transcriptional levels of ITGA4, TGFβ1, α-SMA, and TIMP2 than the other groups, and there were significant reductions in the transcriptional levels of each factor after intervention with colchicine or SSAA, with a significant difference between the CCl₄ model group and the other groups (all P < 0.05). The CCl₄ model group had significantly higher protein expression levels of ITGA4, TGFβ1, α-SMA, TIMP2, and TIMP1 and a significantly lower protein expression level of MMP2 than the other groups, and intervention with colchicine or SSAA inhibited the expression of ITGA4, TGFβ1, α-SMA, TIMP2, and TIMP1 and promoted the expression of MMP2. Immunohistochemistry showed that the CCl₄ model group had significantly higher expression levels of TGFβ1 and α-SMA than the other groups, which was inhibited by intervention with colchicine or SSAA. The high-dose SSAA group had the most significant effect in reducing aminotransferases, improving lobular structure, and inhibiting the protein expression of liver fibrosis factors. Conclusion The high expression of ITGA4 in the liver is associated with the development of liver fibrosis, which is consistent with the increases in the expression of TGFβ1 and α-SMA. Inhibiting the expression of ITGA4 can provide more therapeutic targets for liver fibrosis and expand the anti-liver fibrosis mechanism of SSAA.
- Liver Cirrhosis /
- Mucoglycosine /
- Integrins /
- Rats, Sprague-Dawley

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图 1 各组HE染色结果(×200)

Figure 1. HE staining in each group(×200)

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图 2 各组天狼猩红染色结果(×200)

Figure 2. Sirius red staining in each group(×200)

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图 3 各组相关因子转录水平

注：与空白对照组相比，#P＜0.05；与SSAA组相比，△P＜0.05；与CCl₄模型组相比，*P＜0.05。

Figure 3. Transcription levels of related factors in each group

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图 4 各组相关因子的蛋白表达情况

Figure 4. Protein expression of related factors in each group

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图 5 各组中TGFβ1的免疫组化结果(×200)

Figure 5. Immunohistochemical results of TGFβ1 in each group(×200)

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图 6 各组中α-SMA的免疫组化结果(×200)

Figure 6. Immunohistochemical results of α-SMA in each group(×200)

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表 1 PCR引物

Table 1. PCR primer

基因	引物序列(5′-3′)	长度(bp)
ITGA4	F: ACTTCGCAAGGTTTTGTGCC	20
	R: CCTGTGGGGAGTTGGACATT	20
ITGB1	F: TGTCCTACTGGTCCCGACAT	20
	R: TTTTCACCCGTGTCCCACTT	20
TGFβ1	F: AGGGCTACCATGCCAACTTC	20
	R: CCACGTAGTAGACGATGGGC	20
α-SMA	F: CATCACCAACTGGGACGACA	20
	R: TCCGTTAGCAAGGTCGGATG	20
TIMP2	F: ATGGCAACCCCATCAAGAGG	20
	R: TCTTTCCTCCAACGTCCAGC	20
注：F，正向引物；R，反向引物。

下载: 导出CSV

表 2 各组ALT、AST水平

Table 2. Transaminase levels in each group

组别	ALT(U/L)	AST(U/L)
空白对照组	41.7±8.9³⁾	71.1±4.9³⁾
SSAA组	41.3±10.9³⁾	69.0±10.9³⁾
CCl₄模型组	236.6±131.2	568.3±416.2
秋水仙碱组	91.7±31.2¹⁾²⁾³⁾	139.7±48.3³⁾
SSAA低剂量组	127.9±43.8¹⁾²⁾³⁾	191.6±112.7³⁾
SSAA中剂量组	105.5±39.8^1)2)3)4)	141.8±97.2³⁾
SSAA高剂量组	81.5±36.5³⁾	120.6±48.4³⁾
F值	30.324	12.249
P值	＜0.001	＜0.001
注：与空白对照组相比，1)P＜0.05；与SSAA组相比，2)P＜0.05；与CCl₄模型组相比，3)P＜0.05；与SSAA低剂量组相比，4)P＜0.05。

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