植物乳杆菌ZDY04促进胆固醇逆向转运减轻TMAO诱导

汤景辉; 秦嫚嫚; 唐乐; 魏华; 裘梁; 余军

doi:10.13381/j.cnki.cjm.00000-00

植物乳杆菌ZDY04促进胆固醇逆向转运减轻TMAO诱导

doi: 10.13381/j.cnki.cjm.00000-00

汤景辉^{1, 2,},
秦嫚嫚^{1, 2},
唐乐^{1, 2},
魏华³,
裘梁^{1, 2,*, ,},
余军⁴

1.
江西中医药大学医学转化中心, 江西南昌 330006
2.
江西中医药大学江西省中药防治血管重塑相关疾病重点实验室
3.
南昌大学食品与科学技术国家重点实验室
4.
Department of Physiology and Center for Metabolic Disease Research, Lewis Katz School of Medicine, Temple University, Philadelphia, PA19140, USA

基金项目: 国家自然科学基金（81860090）；江西省教育厅一般项目（GJJ190677）；江西中医药大学博士启动基金（2019WBZR009）；江西省自然科学基金（20202BABL206007）；江西省重点实验室建设项目（20202BCD42014）；南昌市科技局项目（2018-NCZDSY-005）

详细信息

作者简介:
汤景辉（1992-），男，学士，从事益生菌与心血管保护作用研究，E-mail:xxxxxxxx@qq.com

通讯作者:
裘梁（1981-），男，副教授，博士，E-mail：xxxxxxx.edu.cn

中图分类号: Q93
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出版历程
- 收稿日期: 2020-12-22
- 修回日期: 2021-03-24
- 网络出版日期: 2022-09-19
- 刊出日期: 2023-05-31

Lactobacillus plantarum ZDY04 reduces Trimethylamine N-Oxide-induced atherosclerosis by promoting reverse cholesterol transport in ApoE^-/- mice

TANG Jing-hui^{1, 2
,},
QIN Man-man^{1, 2},
TANG Le^{1, 2},
WEI Hua³,
QIU Liang^{1, 2,*
, ,},
YU Jun⁴

1.
Translational Medicine Center, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330006, China
2.
Jiangxi Key Laboratory of Traditional Chinese Medicine for Prevention and Treatment of Vascular Remodeling Related Diseases, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330006, China

More Information

Corresponding author: QIU Liang, E-mail: liangqiu@jxutcm.edu.cn

摘要

摘要:
目的探讨植物乳杆菌ZDY04能否改善氧化三甲胺（TMAO）诱导的小鼠动脉粥样硬化及其机制研究。
方法 6~8周龄雌性载脂蛋白E基因敲除（apolipoprotein E-deficient mice，ApoE^-/-）小鼠被随机分成3组：正常饮食组（Chow组）、胆碱+溶剂组（Choline+PBS组）和Choline+ZDY04组，通过自由饮用1.3%高胆碱饮食构建小鼠动脉粥样硬化模型，灌胃含有15%甘油的无菌PBS或菌株，持续16周，检测血脂水平，uHPLC-MS/MS测定血浆TMAO和粪便三甲胺（TMA）的含量，油红“O”染色检测全长主动脉粥样硬化病变程度，苏木素-伊红、油红“O”以及Masson染色分别检测主动脉根部斑块面积、脂质占比以及胶原纤维含量，免疫荧光检测主动脉根部斑块中巨噬细胞以及平滑肌细胞数量，RT-qPCR和Western blotting检测小鼠肝脏胆固醇逆向转运相关基因及Fmo3基因的表达水平。
结果与Choline+PBS组小鼠相比，Choline+ZDY04组小鼠主动脉脂质沉积区域显著减少，主动脉根部动脉粥样硬化斑块面积，脂滴及巨噬细胞阳性区域明显减少，胶原纤维和平滑肌细胞在斑块中占比没有明显变化。粪便TMA及血清TMAO含量显著降低。血清总胆固醇、低密度脂蛋白胆固醇含量具有下降趋势但差异无统计学意义，高密度脂蛋白胆固醇含量显著上升。胆固醇逆向转运相关基因Sr-b1、Abcg5和Cyp7a1等显著升高，Fmo3基因表达水平没有变化。
结论植物乳杆菌ZDY04通过促进胆固醇的逆向转运和分解，抑制ApoE^-/-小鼠体内的动脉粥样硬化。
- 动脉粥样硬化 /
- 植物乳杆菌ZDY04 /
- 氧化三甲胺 /
- 胆固醇逆向转运
Abstract:
Objective To observe the effect of Lactobacillus plantarum ZDY04 in Trimethylamine N-Oxide (TMAO)-induced atherosclerosis in mice and its underlying mechanisms.
Methods Thirty female (six to eight-weeks old) apolipoprotein E-deficient (ApoE^-/-) mice were randomly assigned into three groups as follows: Chow group, Choline+PBS group and Choline+ZDY04 group, and fed with 1.3% high-choline diet to establish atherosclerosis models, then treated with sterile PBS or ZDY04 containing 15% glycerol for 16 weeks. After treatment, serum lipids were measured using cholesterol kit. The serum TMAO and fecal TMA content were determined by using liquid chromatography-tandem mass spectrometry LC-MS. Atherosclerotic lesion formation in whole aorta was detected with Oil red O staining. Hematoxylin-eosin, Oil red O and Masson staining were used to detect the atherosclerotic area, lipid deposition and collagen content in the aortic roots. Immunofluorescent staining of macrophage specific antigen CD68 and smooth muscle a-actin (SMA) were used to examine the lesion composition in the aortic root. The expression of genes related to reverse cholesterol transport and Fmo3 in liver was detected with RT-qPCR and Western blot.
Results Compared with Choline+PBS group, the atherosclerotic lesions in the whole aortas significantly reduced in Choline+ZDY04 group. ZDY04 treatment significantly decreased atherosclerotic lesion size, lipid deposition and macrophage content, but not the contents of lesion smooth muscle cells or collagen, when compared to Choline+PBS fed mice. The content of cecum TMA and serum TMAO decreased significantly in Choline+ZDY04 group; serum high density lipoprotein-cholesterol significantly increased, but total cholesterol and low density lipoprotein-cholesterol only marginally decreased. ZDY04 significantly increased the mRNA expression of reverse cholesterol transport genes Sr-b1, Abcg5, Cyp7a1 but not Fmo3. Liver CYP7A1 protein level was also increased by ZDY04 compared to controls.
Conclusion Lactobacillus plantarum ZDY04 can inhibit TMAO induced thermogenesis by promoting reverse transport and decomposition of cholesterol in ApoE^-/- mice.
- Atherosclerosis /
- Lactobacillus plantarum ZDY04 /
- Trimethylamine N-Oxide /
- Reverse cholesterol transport

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图 1 植物乳杆菌ZDY04抑制ApoE^-/-小鼠全长主动脉中动脉粥样硬化病变的形成

注：A为全长主动脉油红“O”染色；B~E为全长主动脉、主动脉弓、胸主动脉和腹主动脉斑块病变区域统计结果。与Choline+PBS组相比，^*P<0.050，^**P<0.010，^***P<0.001。

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图 2 植物乳杆菌ZDY04抑制ApoE^-/-小鼠主动脉根部中动脉粥样硬化病变的形成

注：A分别为主动脉根部H＆E、油红“O”和Masson染色；B~D分别为斑块面积、斑块内脂滴占比和胶原纤维占比统计结果。与Choline+PBS组相比，^*P<0.05，^**P<0.01。

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图 3 植物乳杆菌ZDY04减少主动脉根部中巨噬细胞的阳性区域

注：A为小鼠主动脉根部AS斑块CD68和α-SMA免疫荧光染色；B~C分别为CD68及α-SMA阳性区域统计结果，与Choline+PBS组相比，^*P<0.05，^**P<0.01。

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图 4 植物乳杆菌ZDY04减少ApoE^-/-小鼠中血清TMAO和粪便TMA的水平

注：A、B分别为盲肠内TMA和血清内TMAO含量；C为肝脏Fmo3基因表达；D为肝脏FMO3蛋白表达及统计结果。与Choline+PBS 组相比，^*P<0.05，^**P<0.01。

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图 5 植物乳杆菌ZDY04对小鼠血清胆固醇的影响

注：A~C分别为血清总胆固醇、低密度脂蛋白胆固醇及高密度脂蛋白胆固醇统计结果。与Choline+PBS组相比，^*P<0.05，^**P<0.01。

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图 6 植物乳杆菌ZDY04升高小鼠肝脏中胆固醇逆转运相关基因的表达

注：A~E分别为肝脏Sr-b1、Abcg5、Abcg8、Abcg1和Cyp7a1基因mRNA表达；F为肝脏CYP7A1蛋白表达。与Choline+PBS组相比，^*P<0.05，^**P<0.01，^***P<0.001，^****P<0.000 1。

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表 1 引物序列

基因		序列（5′→3′）	序列长度（bp）
Fmo3	F	GTGGTTCTTGGGTGATGAGTC	109
Fmo3	R	GAGATGGCGGTGGGTAAGTT
Scarb1	F	TTCTCGCCCTTCAGGATCT	147
Scarb1	R	GCTCATCAAGCAGCAGGTC
Cyp7a1	F	AACAACCTGCCAGTACTAGATAGC	99
Cyp7a1	R	GTGTAGAGTGAAGTCCTCCTTAGC
Abcg1	F	ATAATGGCCACCAACTCACC	112
Abcg1	R	GGGACCTTTCCTATTCGGTT
Abcg5	F	CGCGAGAGGTTGCGATACA	128
Abcg5	R	CTGCCAATCATTTGGTCCGC
Abcg8	F	TGGGCATCCGAAATCTAAG	222
Abcg8	R	TTGGGCAGCAGTTGGTCAT
β-actin	F	TGTTACCAACTGGGACGACA	165
β-actin	R	GGGGTGTTGAAGGTCTCAAA

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