地五养肝方对非酒精性脂肪肝小鼠循环外泌体溶血甘油磷脂的调控作用

doi:10.12122/j.issn.1673-4254.2024.07.18

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (7): 1382-1388.doi: 10.12122/j.issn.1673-4254.2024.07.18

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地五养肝方对非酒精性脂肪肝小鼠循环外泌体溶血甘油磷脂的调控作用

陈光亚¹^,²(), 向星亮¹, 曾兆祥¹, 黄荣增¹, 金姝娜³, 肖明中⁴, 宋成武¹^,⁵^,⁶

^1.湖北中医药大学，药学院，湖北武汉 430065
^3.湖北中医药大学，基础医学院，湖北武汉 430065
^6.湖北中医药大学，肝脏疾病中医药现代化研究中心，湖北武汉 430065
^2.鄂州市中心医院药学部，湖北鄂州 436000
^4.湖北省中医院肝病研究所，湖北武汉 430000
^5.湖北时珍实验室，湖北武汉 430065

收稿日期:2024-03-11 出版日期:2024-07-20 发布日期:2024-07-25
通讯作者: 宋成武 E-mail:13403032494@163.com
作者简介:陈光亚，硕士，E-mail: 13403032494@163.com
基金资助:
湖北省中医药管理局中医药青年人才项目(ZY2023Q040)

Regulatory effect of Diwu Yanggan Decoction on lysoglycerophospholipids in circulating exosomes in a mouse model of nonalcoholic fatty liver disease

Guangya CHEN¹^,²(), Xingliang XIANG¹, Zhaoxiang ZENG¹, Rongzeng HUANG¹, Shuna JIN³, Mingzhong XIAO⁴, Chengwu SONG¹^,⁵^,⁶

^1.College of Pharmacy,
^3.School of Basic Medical Sciences,
^6.Center of Traditional Chinese Medicine Modernization for Liver Diseases, Hubei University of Chinese Medicine, Wuhan 430065, China
^2.Ezhou Central Hospital, Department of Pharmacy, Ezhou 436000, China
^4.Hepatic Disease Institute, Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan 430000, China
^5.Hubei Shizhen Laboratory, Wuhan 430065, China

Received:2024-03-11 Online:2024-07-20 Published:2024-07-25
Contact: Chengwu SONG E-mail:13403032494@163.com

摘要/Abstract

摘要：

目的研究地五养肝方对非酒精性脂肪肝（NAFLD）小鼠循环外泌体溶血甘油磷脂（Lyso-GPLs）的调控作用。方法采用尺寸排阻色谱法分离血液中循环外泌体，运用透射电镜观察外泌体的外观、粒径仪检测外泌体直径分布、Western blotting法检测外泌体表面特异性蛋白CD9，CD63及TSG101的表达；将24只雄性KM小鼠随机分为正常组、模型组和复方给药组，8只/组，正常组给予标准饲料，模型组和复方给药组饲喂高脂饲料，喂养1周后，各给药组小鼠按剂量给予地五养肝胶囊内容物，正常组和模型组给予等量生理盐水，灌胃给药4周。末次给药后，各小鼠麻醉后取血分离血清，检测总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST），观察其对NAFLD的改善作用并提取血清中循环外泌体；同时，结合多元统计分析，筛选NAFLD小鼠循环外泌体中Lyso-GPLs类成分生物标志物，观察地五养肝方对标志物的调控作用。结果透射电镜结果显示外泌体大小在100 nm左右，形状为典型的具有清晰双层膜的茶托样结构；粒径结果显示循环外泌体粒径分布均匀，平均粒径为137.5 nm；Western blotting检测结果表明外泌体表面特异性蛋白CD9， CD63及TSG101的存在；模型组小鼠血清中TC、HDL-C、LDL-C和AST水平显著高于正常组（P<0.01），经地五养肝方治疗后，TC、LDL-C、AST、ALT水平显著回调（P<0.01），并筛选出43个经地五养肝方治疗后呈现显著回调趋势（P<0.01）的Lyso-GPLs标志物成分。结论地五养肝方防治NAFLD的作用机制涉及对循坏外泌体溶血甘油磷脂的改善和调控作用，为其防治慢性肝病的作用机制研究提供了新的科学视角。

关键词: 地五养肝方, 循环外泌体, 液质联用, 溶血甘油磷脂, 非酒精性脂肪肝

Abstract:

Objective To evaluate the regulatory effect of Diwu Yanggan (DWYG) Decoction on lysoglycerophospholipids (Lyso-GPLs) in circulating exosomes in a mouse model of nonalcoholic fatty liver disease (NAFLD). Methods Circulating exosomes isolated from mouse serum by size exclusion chromatography were morphologically characterized using transmission electron microscope and examined for surface markers CD9, CD63 and TSG101 using Western blotting. Twenty-four male Kunming mice were randomized into 3 groups for normal feeding (control, n=8) or high-fat diet feeding for 1 week to induce NAFLD, after which the latter mice were given DWYG decoction (treatment group, n=8) or normal saline (model group, n=8) by gavage for 4 weeks. After the last treatment, blood samples were collected from the mice for testing serum TC, HDL-C, LDL-C, ALT and AST levels and isolating circulating exosomes. Using multivariate statistical analysis based on targeted metabolomics strategy, the potential biomarkers for Lyso-GPLs in the exosomes were screened. Results The isolated exosomes about 100 nm in size had a typical saucer-like structure with distinct double-layer membranes and a mean particle size of 137.5 nm and expressed the specific surface marker proteins CD9, CD63 and TSG101. The mouse models of NAFLD had significantly increased serum levels of TC, HDL-C, LDL-C and AST and lowered serum ALT level. A total of 43 Lyso-GPLs with significant reduction after DWYG Decoction treatment were identified in NAFLD mice. Conclusion DWYG Decoction can regulate Lyso-GPLs in circulating exosomes in NAFLD mice, which provides a new clue for studying the therapeutic mechanism of DWYG Decoction for liver disease.

Key words: Diwu Yanggan Decoction, circulating exosomes, UPLC-QTOF-MS/MS, lysoglycerophospholipid, nonalcoholic fatty liver disease

陈光亚, 向星亮, 曾兆祥, 黄荣增, 金姝娜, 肖明中, 宋成武. 地五养肝方对非酒精性脂肪肝小鼠循环外泌体溶血甘油磷脂的调控作用[J]. 南方医科大学学报, 2024, 44(7): 1382-1388.

Guangya CHEN, Xingliang XIANG, Zhaoxiang ZENG, Rongzeng HUANG, Shuna JIN, Mingzhong XIAO, Chengwu SONG. Regulatory effect of Diwu Yanggan Decoction on lysoglycerophospholipids in circulating exosomes in a mouse model of nonalcoholic fatty liver disease[J]. Journal of Southern Medical University, 2024, 44(7): 1382-1388.

图/表 5

图2 102个溶血甘油磷脂提取离子色谱图

Fig.2 Chromatogram of the metabolite profile of 102 lysoglycerophospholipids.

表1 小鼠生理生化参数

Tab.1 Physiological and biochemical parameters of the mice (Mean±SD, n=8)

Parameter	Normal	Model	DWYG
Physiological parameter
Body weight (g)	48.25±4.38	50.13±4.13	46.90±3.97
Liver weight (g)	2.01±0.38	2.37±0.22	1.94±0.30^#
Liver/body index	4.14±0.48	4.74±0.45*	4.13±0.44^#
Biochemical parameters
AST	80.71±10.19	114.07±12.70**	73.55±10.48^##
ALT	39.35±11.69	47.36±11.37	32.16±3.96^##
AST/ALT	2.16±0.47	2.52±0.59	2.30±0.32
HDL-C (mmol/L)	2.30±0.54	4.23±0.96**	4.40±0.66**
LDL-C (mmol/L)	0.31±0.06	0.91±0.20**	0.53±0.06**^##
TC (mmol/L)	3.26±0.65	7.29±1.18**	5.85±0.54**^##
AI	0.44±0.27	0.79±0.41	0.35±0.20^#

图3 DWYG对小鼠循环外泌体Lyso-GPLs含量的影响

Fig.3 Effect of DWYG Decoction on contents of Lyso-GPLs in circulating exosomes in NAFLD mice. A: Relative peak areas of blood circulation exosome lysophospholipids in the normal, model and treatment group. B: PCA score plots of normal, model and DWYG groups.

图4 3组样品的OPLS-DA分析结果

Fig.4 Results of OPLS-DA analysis of the samples in the 3 groups. A: OPLS-DA analysis of normal and model groups. B: 999-time permutation validation results of the normal and model groups. C: OPLS-DA analysis of the treatment and model groups. D: 999-time permutation validation results of the treatment and model group.

图5 43个Lyso-GPLs的相对含量

Fig.5 Relative contents of 43 Lyso-GPLs. A: Fold change. B: Relative content in normal, model and compound groups.

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地五养肝方对非酒精性脂肪肝小鼠循环外泌体溶血甘油磷脂的调控作用

Regulatory effect of Diwu Yanggan Decoction on lysoglycerophospholipids in circulating exosomes in a mouse model of nonalcoholic fatty liver disease

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