枸杞多糖联合有氧运动对非酒精性脂肪肝炎大鼠模型的改善作用及其机制研究

马佳敏; 高璐璐; 张梦伟; 高清菡; 陶秀娟; 范彦娜; 杨建军

doi:10.3969/j.issn.1001-5256.2021.06.026

枸杞多糖联合有氧运动对非酒精性脂肪肝炎大鼠模型的改善作用及其机制研究

DOI: 10.3969/j.issn.1001-5256.2021.06.026

宁夏医科大学公共卫生与管理学院，银川 750004

基金项目:

国家自然科学基金 (81660537);

宁夏自然科学基金 (2020AAC03181)

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：马佳敏、高璐璐、杨建军负责课题设计，资料分析，撰写论文；张梦伟参与收集数据；高清菡、陶秀娟、范彦娜负责拟定写作思路; 马佳敏、杨建军负责指导撰写文章并最后定稿。

详细信息

作者简介:
马佳敏(1994—)，女，主要从事营养与慢性病研究

通信作者:
杨建军，yangjianjun_1970@163.com

中图分类号: R575.5
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出版历程
- 收稿日期: 2020-12-06
- 录用日期: 2021-01-05
- 出版日期: 2021-06-20

Role and mechanism of Lycium barbarum polysaccharide combined with aerobic exercise in improving nonalcoholic steatohepatitis in rats

School of Public Health and Management, Ningxia Medical University, Yinchuan 750004, China

摘要

摘要: 目的基于p38 MAPK/NF-κB途径探讨枸杞多糖联合有氧运动对高脂饮食诱导的非酒精性脂肪肝炎(NASH)大鼠肝脏的保护作用。方法 8周龄SD大鼠共45只，适应性饲养1周后随机选取10只作为对照组给予普通饲料，剩余大鼠为高脂组(35只)，食用高脂饲料。28周时将高脂组大鼠随机分为4组进行干预(每组8只)，分别为模型组、枸杞多糖组、有氧运动组、联合组(枸杞多糖+有氧运动)，干预周期为10周。实验结束时，测定所有大鼠空腹血糖，收集血清样本，取肝组织和内脏脂肪。生化试剂盒检测血清TG、TC、ALT、AST水平。ELISA试剂盒测定大鼠血清胰岛素(FINS)、TNFα、IL-6和单核细胞趋化蛋白-1(MCP-1)水平。实时定量PCR和Western Blot测定肝组织Toll样受体4(TLR4)、p38 MAPK和NF-κB的表达水平。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LSD-t检验。结果与对照组相比，模型组大鼠TG、TC、AST、ALT、FINS和胰岛素抵抗指数(HOMA-IR)水平均显著升高(P值均＜0.05)，血清炎症因子MCP-1、TNFα和IL-6水平均呈现增高趋势(P值均＜0.05)，肝组织TLR4、p38 MAPK和NF-κB mRNA和蛋白表达水平均显著升高(P值均＜0.05)。与模型组相比，各干预组大鼠TC、AST、ALT、FINS和HOMA-IR水平均显著降低(P值均＜0.05)，血清炎症因子MCP-1、TNFα和IL-6水平均呈现降低趋势(P值均＜0.05)；肝组织TLR4、p38 MAPK、NF-κB相关mRMA和蛋白水平均显著降低(P值均＜0.05)。与枸杞多糖组相比, 联合组TG、ALT、FINS、HOMA-IR水平均显著降低(P值均＜0.05)，血清MCP-1水平呈现降低趋势(P＜0.05)，TLR4 mRNA相对表达水平显著降低(P＜0.05)，p38 MAPK、NF-κB蛋白水平均显著降低(P值均＜0.05)。与有氧运动组相比，联合组FINS、HOMA-IR水平均显著降低(P值均＜0.05)，IL-6、MCP-1水平均显著降低(P值均＜0.05)，TLR4 mRNA相对表达水平显著降低(P值均＜0.05)。结论枸杞多糖联合有氧运动可能通过调节p38 MAPK/NF-κB途径改善NASH大鼠炎症水平。
- 非酒精性脂肪肝炎 /
- 枸杞多糖 /
- 运动 /
- 大鼠，Sprague-Dawley
Abstract: Objective To investigate the protective effect of Lycium barbarum polysaccharide (LBP) combined with aerobic exercise (AE) on the liver of rats with nonalcoholic steatohepatitis (NASH) induced by high-fat diet based on the p38 mitogen-activated protein kinase (p38 MAPK)-nuclear factor-κB (NF-κB) pathway. Methods After 1 week of adaptive feeding, 45 Sprague-Dawley rats, aged 8 weeks, were randomly divided into control group (10 rats fed with normal diet) and high-fat group (35 rats fed with high-fat diet). At the end of week 28, the high-fat group was randomly divided into model group, LBP group, AE group, and LBP+AE group, with 8 rats in each group, and intervention was performed for 10 weeks. At the end of the experiment, fasting blood glucose was measure for all rats, and serum samples, liver tissue, and visceral fat were collected. Biochemical kits were used to measure the serum levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST); ELISA kits were used to measure the serum levels of fasting insulin (FINS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1); quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of Toll-like receptor 4 (TLR4), p38 MAPK, and NF-κB in liver tissue. 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 further comparison between two groups. Results Compared with the control group, the model group had significant increases in TG, TC, AST, ALT, FINS, and homeostasis model assessment of insulin resistance (HOMA-IR) (all P < 0.05), a tendency of increases in the serum levels of the inflammatory factors MCP-1, TNF-α, and IL-6 (all P < 0.05), and significant increases in the mRNA and protein expression levels of TLR4, p38 MAPK, and NF-κB in liver tissue (all P < 0.05). Compared with the model group, each intervention group had significant reductions in TG, TC, AST, ALT, FINS, and HOMA-IR (all P < 0.05), a tendency of reductions in the serum levels of the inflammatory factors MCP-1, TNF-α, and IL-6 (all P < 0.05), and significant reductions in the mRNA and protein expression levels of TLR4, p38 MAPK, and NF-κB (all P < 0.05). Compared with LBP group, the LBP+AE group had significant reductions in TG, ALT, FINS, HOMA-IR, MCP-1, the mRNA expression level of TLR4, protein expression levels of p38 MAPK and NF-κB(all P < 0.05). Compared with Ae group, the LBP+ AE group had significant reductions in FINS, HOMA-IR, IL-6, MCP-1, the mRNA expression level of TLR4 (all P < 0.05). Conclusion LBP combined with AE may improve inflammation in NASH rats by regulating the p38 MAPK/NF-κB pathway.
- Non-Alcoholic Steatohepatitis /
- Lycium Barbarum Polysaccharides /
- Exercise /
- Rats, Sprague-Dawley

HTML全文

图 1 各组大鼠肝组织(HE染色，×200)

注：a，对照组；b，模型组；c，枸杞多糖组；d，有氧运动组；e，联合组。

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图 2 各组大鼠肝组织蛋白表达电泳图

下载: 全尺寸图片幻灯片

表 1 反转录聚合酶链反应引物序列

基因	引物序列(5′→3′)
β-actin	上游：TGTCACCAACTGGGACGATA
	下游：GGGGTGTTGAAGGTCTCAAA
TLR4	上游：AAGTTATTGTGGTGGTGTCTAG
	下游：GAGGTAGGTGTTTCTGCTAAG
p38 MAPK	上游：AGAGTCTCTGTCGACCTGCT
	下游：CATCAGGGTCGTGGTACTGAG
NF-κB	上游：TGCATTCTGACCTTGCCTAT
	下游：TCCAGTCTCCGAGTGAAGC

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表 2 各组NASH大鼠体质量、肝湿重、内脏脂肪质量的比较

指标	对照组(n=8)	模型组(n=8)	枸杞多糖组(n=8)	有氧运动组(n=8)	联合组(n=8)	F值	P值
体质量(g)	625.6±36.0	832.4±57.8¹⁾	809.35±63.83	731.96±67.16²⁾	719.64±58.50²⁾³⁾	17.17	＜0.05
肝湿质量(g)	15.18±1.62	16.62±1.30¹⁾	17.38±1.30	15.32±2.36²⁾	14.55±1.06²⁾³⁾	6.12	＜0.05
内脏脂肪质量(g)	21.66±9.94	84.63±19.15¹⁾	80.02±11.70	58.38±21.10²⁾	56.89±15.30²⁾³⁾	20.45	＜0.05
注: 与对照组相比，1) P＜0.05；与模型组相比，2) P＜0.05；与枸杞多糖组相比，3) P＜0.05。

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表 3 各组NASH大鼠NAS积分的比较

组别	动物数(只)	NAS积分
对照组	8	0.67±0.58
模型组	8	6.67±1.53¹⁾
枸杞多糖组	8	3.67±0.58²⁾
有氧运动组	8	3.33±0.58²⁾
联合组	8	2.33±0.58²⁾
F值		19.77
P值		＜0.05
注：与对照组相比，1) P＜0.05；与模型组相比，2) P＜0.05。

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表 4 各组大鼠血脂、肝功能指标、FINS和HOMA-IR的比较

指标	对照组(n=8)	模型组(n=8)	枸杞多糖组(n=8)	有氧运动组(n=8)	联合组(n=8)	F值	P值
TG(mmol/L)	1.17±0.25	1.55±0.50¹⁾	1.37±0.18	0.94±0.26²⁾	0.88±0.15²⁾³⁾	6.70	＜0.05
TC(mmol/L)	3.84±0.50	4.93±0.98¹⁾	3.52±0.80²⁾	3.52±0.47²⁾	3.29±0.75²⁾	5.05	＜0.05
AST(U/L)	28.51±2.78	35.07±2.57¹⁾	28.23±3.23²⁾	27.33±5.35²⁾	24.78±4.63²⁾	7.99	＜0.05
ALT(U/L)	32.48±6.70	55.29±16.59¹⁾	32.94±8.59²⁾	23.13±9.66²⁾	20.74±7.96²⁾³⁾	6.68	＜0.05
FINS(mg/dl)	1.15±0.53	3.85±0.55¹⁾	2.21±0.82²⁾	2.29±0.75²⁾	1.40±0.82²⁾³⁾⁴⁾	22.58	＜0.05
HOMA-IR	5.80±2.87	24.65±10.59¹⁾	13.78±4.66²⁾	16.06±5.47²⁾	8.68±3.42²⁾³⁾⁴⁾	11.33	＜0.05
注：与对照组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与枸杞多糖组相比，3)P＜0.05；与有氧运动组相比，4)P＜0.05。

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表 5 各组大鼠血清TNFα、IL-6和MCP-1水平的比较

指标	对照组(n=8)	模型组(n=8)	枸杞多糖组(n=8)	有氧运动组(n=8)	联合组(n=8)	F值	P值
TNFα(pg/ml)	54.14±12.48	234.57±55.01¹⁾	157.05±24.16²⁾	166.96±33.02²⁾	115.33±52.17²⁾	18.28	＜0.05
IL-6(pg/ml)	86.84±12.37	315.71±42.00¹⁾	133.63±10.13²⁾	159.75±20.64²⁾	129.27±24.01²⁾⁴⁾	77.23	＜0.05
MCP-1(pg/ml)	4.63±0.37	6.01±1.02¹⁾	3.91±1.37²⁾	3.58±1.03²⁾	2.37±0.40²⁾³⁾⁴⁾	15.13	＜0.05
注：与对照组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与枸杞多糖组相比，3)P＜0.05；与有氧运动组相比，4)P＜0.05。

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表 6 各组大鼠肝组织TLR4、p38 MAPK和NF-κB mRMA表达水平的比较

指标	对照组(n=8)	模型组(n=8)	枸杞多糖组(n=8)	有氧运动组(n=8)	联合组(n=8)	F值	P值
TLR4mRNA	1.10±0.24	2.52±0.44¹⁾	1.85±0.64²⁾	1.41±0.57²⁾	0.60±0.14²⁾³⁾⁴⁾	20.69	＜0.05
p38MAPKmRNA	1.06±0.06	2.47±0.40¹⁾	2.00±0.49²⁾	1.87±0.43²⁾	1.81±0.13²⁾	10.07	＜0.05
NF-κBmRNA	1.22±0.22	1.58±0.19¹⁾	1.33±0.20²⁾	1.31±0.11²⁾	1.29±0.11²⁾	3.27	0.03
注：与对照组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与枸杞多糖组相比，3)P＜0.05；与有氧运动组相比，4)P＜0.05。

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表 7 组大鼠肝组织TLR4、p38 MAPK和NF-κB蛋白表达水平的比较

指标	对照组(n=8)	模型组(n=8)	枸杞多糖组(n=8)	有氧运动组(n=8)	联合组(n=8)	F值	P值
TLR4	0.53±0.06	0.86±0.09¹⁾	0.64±0.16²⁾	0.60±0.13²⁾	0.49±0.08²⁾	5.25	0.02
p38MAPK	0.81±0.06	1.18±0.05¹⁾	0.90±0.06²⁾	0.86±0.04²⁾	0.78±0.05²⁾³⁾	35.42	＜0.05
NF-κB	0.75±0.05	1.12±0.10¹⁾	0.90±0.05²⁾	0.80±0.16²⁾	0.71±0.08²⁾³⁾	8.43	＜0.05
注：与对照组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与枸杞多糖组相比，3)P＜0.05。

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