土鳖虫调控黏结合蛋白聚糖3改善非酒精性脂肪性肝炎的作用机制

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

doi:10.3969/j.issn.1001-5256.2022.07.012

土鳖虫调控黏结合蛋白聚糖3改善非酒精性脂肪性肝炎的作用机制

DOI: 10.3969/j.issn.1001-5256.2022.07.012

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

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实验中心，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);

上海市普陀区自主创新项目 (ptkwws201817);

上海市普陀区自主创新项目 (ptkwws201904);

上海市普陀区自主创新项目 (ptkwws202223);

上海市普陀区自主创新项目 (ptkwws202112);

上海市第六人民医院医疗集团科研基金 (21-ly-02);

成都中医药大学杏林学者 (YYZX2020117)

伦理学声明：本研究方案于2016年3月8日经上海中医药大学实验动物伦理委员会审批，批号：PTEC-A-2016-4(G)-1。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、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：杨广越负责课题设计，资料分析，撰写论文；张玮、刘旭凌、孙田甜、姜浩参与实验操作及数据分析；陶乐、马文婷、吴柳参与收集数据，修改论文；刘成负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
刘成，liucheng0082010@163.com

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出版历程
- 收稿日期: 2021-11-23
- 录用日期: 2022-01-21
- 出版日期: 2022-07-20

Mechanism of action of Eupolyphaga steleophaga in improving nonalcoholic steatohepatitis by regulating syndecan 3

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 Natural Science Foundation of China （20ZR1450300）

Putuo District of Shanghai Innovation Project （ptkwws201817）

Putuo District of Shanghai Innovation Project （ptkwws201904）

Putuo District of Shanghai Innovation Project （ptkwws202223）

Putuo District of Shanghai Innovation Project （ptkwws202112）

Scientific Research Fund of Shanghai Sixth People's Hospital Medical Group （21-ly-02）

Xinglin Scholar of Chengdu University of Traditional Chinese Medicine （YYZX2020117）

More Information

Corresponding author: LIU Cheng, liucheng0082010@163.com(ORCID: 0000-0002-8741-6169)

摘要

摘要: 目的探讨土鳖虫调控黏结合蛋白聚糖3(SDC3)对胆碱缺乏(CDAA)诱导的非酒精性脂肪性肝炎的作用及其机制。方法 18只雄性C57BL/6小鼠随机分为胆碱充足(CSAA)组、CDAA组和胆碱缺乏加土鳖虫(CDAA+T)组。造模第12周开始，CDAA+T组小鼠给予0.108 g/kg土鳖虫(相当于成人用量10倍量)灌胃，CSAA、CDAA组以等体积生理盐水灌胃，18周末取血清和肝组织，检测小鼠肝功能、TC和TG，观测肝组织病理形态学。实时定量PCR检测转化生长因子(TGF)β1、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Col1α1)、SDC3 mRNA表达；检测SDC3在人和小鼠原代肝细胞、肝星状细胞(HSC)、内皮细胞和Kupffer细胞中的表达，应用Si-RNA沉默SDC3，检测SDC3在HSC活化中的作用。蛋白免疫印迹法检测SDC3蛋白表达。计量资料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CSAA组相比，CDAA组肝功能(ALT、AST)、血清和肝脏TC、TG水平显著升高(P值均＜0.01)，CDAA+T组小鼠血清ALT、AST、TC、TG水平较CDAA组均显著下降(P值均＜0.05)。HE染色显示CDAA组脂肪变明显，炎症细胞浸润增多，CDAA+T组炎症细胞浸润减轻；天狼星红染色显示CDAA组胶原增生显著增加，CDAA+T组胶原增生则减少；油红染色中CDAA组脂肪沉积明显，而CDAA+T组脂肪沉积减少。与CDAA组相比，CDAA+T组肝组织TGFβ、SDC3、α-SMA、COL1α1的mRNA表达和SDC3、α-SMA蛋白表达水平显著降低。免疫组化结果显示：SDC3在CSAA组中表达非常低，CDAA组中SDC3表达显著升高，主要在间质细胞，土鳖虫干预后表达显著降低(P值均＜0.05)。PCR结果显示：人和小鼠肝脏各类型细胞中SDC3在HSC细胞表达最高(P值均＜0.001)。体外培养LX2细胞，土鳖虫处理能显著降低TGFβ诱导的α-SMA、Col1α1上调，基因沉默SDC3后，土鳖虫不能抑制α-SMA、Col1α1升高(P值均＜0.05)。结论土鳖虫显著改善CDAA诱导的非酒精性脂肪性肝炎，其机制可能通过调控HSC中SDC3表达实现的。
- 土鳖虫 /
- 非酒精性脂肪肝炎 /
- 多配体蛋白聚糖3
Abstract: Objective To investigate the effect of Eupolyphaga steleophaga on nonalcoholic steatohepatitis induced by choline-deficient L-amino acid-defined diet (CDAA) and its mechanism by regulating syndecan 3. Methods A total of 18 male C57BL/6 mice were randomly divided into choline-sufficient L-amino acid-defined diet (CSAA) group, CDAA group, and CDAA+Eupolyphaga steleophaga group (CDAA+T group). Since week 12 of modeling, the mice in the CDAA+T group were fed with Eupolyphaga steleophaga 0.108 g/kg (10 times that the dose for adults) by gavage, and those in the CSAA and CDAA groups were given an equal volume of normal saline by gavage. Serum and liver tissue samples were collected at the end of week 18 to measure liver function, total cholesterol (TC), and triglyceride (TG) and observe liver pathology. Quantitative real-time PCR was used to measure the mRNA expression levels of transforming growth factor β (TGFβ), α-smooth muscle actin (α-SMA), collagen type Ⅰ α1 (Col1α1), and SDC3; the mRNA expression of SDC3 was measured in human and mouse primary hepatocytes, hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs), and SDC3 was silenced by si-RNA to investigate the role of SDC3 in HSC activation. Western blotting was used to measure the protein expression of SDC3. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the SNK test or the least significant difference t-test was used for further comparison between two groups. Results Compared with the CSAA group, the CDAA group had significant increases in liver function parameters [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] and the levels of TC and TG in serum and the liver (all P < 0.05), and compared with the CDAA group, the CDAA+T group had significant reductions in the serum levels of ALT, AST, TC, and TG (all P < 0.05). HE staining showed that the CDAA group had marked hepatocyte steatosis and increased inflammatory cell infiltration, and the CDAA+T group had alleviated inflammatory cell infiltration; Sirius Red staining showed a significant increase in collagen hyperplasia in the CDAA group and a significant reduction in collagen hyperplasia in the CDAA+T group; oil red staining showed marked fat deposition in the CDAA group and a reduction in fat deposition in the CDAA+T group. Compared with the CDAA group, the CDAA+T group had significant reductions in the mRNA expression levels of TGFβ, SDC3, α-SMA, and COL1α1 and the protein expression levels of SDC3 and α-SMA. Immunohistochemistry showed a very low expression level of SDC3 in the CSAA group and a significant increase in the expression of SDC3 in the CDAA group, mainly in the interstitial cells, and there was a significant reduction after Eupolyphaga steleophaga intervention (all P < 0.05). PCR results showed the highest expression of SDC3 in HSCs of human and mouse liver (all P < 0.001). LX2 cells were cultured in vitro, and Eupolyphaga steleophaga treatment significantly reduced the upregulation of α-SMA and Col1α1 induced by TGFβ, while after SDC3 gene silencing, Eupolyphaga steleophaga did not inhibit the increases in α-SMA and Col1α1 (all P < 0.05). Conclusion Eupolyphaga steleophaga can significantly improve nonalcoholic steatohepatitis induced by CDAA, possibly by regulating the expression of SDC3 in HSCs.
- Eupolyphaga /
- Non-alcoholic Steatohepatitis /
- Syndecan-3

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图 1 肝组织病理结果

注：a，HE染色(×200)；b，油红染色(×400)；c，天狼星红染色(×100)。

Figure 1. Pathological results of liver tissue

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图 2 肝组织α-SMA免疫组化染色结果

注：红色箭头表示α-SMA阳性(×400)。

Figure 2. α-SMA immunohistochemical staining results of liver tissue

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图 3 α-SMA与SDC3 mRNA表达及其相关性

Figure 3. mRNA expression of α-SMA and SDC3 and its correlation

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图 4 肝组织SDC3免疫组化染色结果

注：a，×200；b, 上图方框内的放大(×400)，红色箭头表示SDC3阳性。

Figure 4. SDC3 immunohistochemical staining results of liver tissue

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图 5 SDC3在CDAA模型中的蛋白表达

Figure 5. Protein expression of SDC3 in CDAA model

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表 1 RT-PCR引物序列

Table 1. RT-PCR primer sequence

基因	上游	下游
18 s rRNA	5′-AGTCCCTGCCCTTTGTACACA-3′	5′-CGATCCGAGGGCCTCACTA-3′
mα-SMA	5′-GTTCAGTGGTGCCTCTGTCA-3′	5′-ACTGGGACGACATGGAAAAG-3′
mCol1α1	5′-TAGGCCATTGTGTATGCAGC-3′	5′-ACATGTTCAGCTTTGTGGACC-3′
hSDC3	5′-GGCGCAGTGAGAACTTCG-3′	5′-CCCCGAGTAGAGGTCATCCAG-3′
mSDC3	5′-GTCCAGCCAGAAAGCTACCAC-3′	5′-CATCCCCTGTATGCCGGTG-3′
mTGFβ	5′-GCAAAGACCATCTGTCTCACA-3′	5′-CTCCTCATCGTGTTGGTGG-3′
注：m，小鼠引物；h，人源引物。

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表 2 各组肝功能、血脂、肝脏脂肪表达及病理学改变

Table 2. Changes of liver function, serum lipids, liver fat expression and pathological in each group

指标	CSAA组	CDAA组	CDAA+T组	F值	P值
ALT(U/L)	11.56±2.93	72.79±7.06¹⁾	26.17±3.76²⁾	253.68	＜0.001
AST(U/L)	18.47±4.85	52.94±7.23¹⁾	26.43±2.73²⁾	70.38	＜0.001
血清TC(mmol/L)	2.95±0.64	7.97±1.22¹⁾	4.24±0.90²⁾	45.21	＜0.001
血清TG(mmol/L)	0.27±0.08	0.63±0.10¹⁾	0.35±0.07²⁾	27.82	＜0.001
肝脏TC(mmol/gprot)	1.57±0.45	2.49±0.67¹⁾	1.67±0.41²⁾	5.70	＜0.05
肝脏TG(mmol/gprot)	8.63±2.53	19.85±1.66¹⁾	16.03±1.49²⁾	51.57	＜0.001
NAS评分(分)	0.67±0.52	6.83±0.75¹⁾	4.50±1.05²⁾	90.26	＜0.001
天狼星红面积(%)	1.01±0.14	6.39±0.65¹⁾	3.17±0.26²⁾	344.94	＜0.001
油红面积(%)	0.83±0.12	7.90±1.44¹⁾	3.34±0.50²⁾	131.45	＜0.001
注：与CSAA组比较，1)P＜0.01；与CDAA组比较，2)P＜0.05。

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表 3 纤维化指标和SDC3在CDAA模型中表达

Table 3. Expression of fibrosis index and SDC3 in CDAA model

指标	CSAA组	CDAA组	CDAA+T组	F值	P值
RT-PCR
TGFβ mRNA	1.00±0.37	3.12±1.70¹⁾	1.50±0.29²⁾	14.25	＜0.001
Col1α1 mRNA	0.98±0.28	4.11±1.19¹⁾	0.81±0.23²⁾	16.71	＜0.001
SDC3 mRNA	1.00±0.57	5.88±1.83¹⁾	2.66±1.03²⁾	46.69	＜0.001
α-SMA mRNA	1.54±1.88	10.10±3.37¹⁾	3.53±0.86²⁾	46.33	＜0.001
免疫印迹
SDC3/β-Actin	1.00±0.19	2.52±0.43¹⁾	1.29±0.08²⁾	15.92	＜0.05
免疫组化
SDC3阳性面积(%)	0.09±0.03	0.40±0.05¹⁾	0.25±0.13²⁾	134.37	＜0.001
α-SMA阳性面积(%)	1.09±0.20	2.97±0.21¹⁾	1.78±0.23²⁾	160.52	＜0.001
注：与CSAA组比较，1)P＜0.01；与CDAA组比较，2)P＜0.01。

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表 4 SDC3在肝脏各类细胞中表达

Table 4. Expression of SDC3 in various liver cells

组别	SDC3 mRNA相对表达量
组别	人	鼠
Hepato	0.92±0.24	1.00±0.31
HSC	32.74±1.65	45.67±2.98
LSEC	21.27±1.75	29.47±3.97
Kupffer细胞	26.67±1.87	30.53±3.82
F值	490.38	282.88
P值	＜0.001	＜0.001

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表 5 土鳖虫提取物抑制TGFβ诱导的LX2纤维化

Table 5. The LX2 fibrosis induced by TGFβ was inhibited by the extract of eupolyphaga

组别	α-SMA mRNA	Col1α1 mRNA
Si-NC-PBS-TGFβ组	1.00±0.11	1.00±0.04
Si-NC-T-TGFβ组	0.53±0.07¹⁾	0.34±0.02¹⁾
Si-SDC3-PBS-TGFβ组	2.57±0.12¹⁾	1.79±0.36¹⁾
Si-SDC3-T-TGFβ组	0.96±0.10	0.89±0.11
F值	157.98	19.69
P值	＜0.001	＜0.01
注：与Si-NC-PBS-TGFβ组比较，1)P＜0.001。

下载: 导出CSV

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