活化T淋巴细胞核因子5在高盐诱导小鼠平滑肌细胞衰老中的作用及其机制

doi:10.13481/j.1671-587X.20250302

吉林大学学报(医学版) ›› 2025, Vol. 51 ›› Issue (3): 567-575.doi: 10.13481/j.1671-587X.20250302

• 基础研究 • 上一篇

活化T淋巴细胞核因子5在高盐诱导小鼠平滑肌细胞衰老中的作用及其机制

仲威¹,戴芝银¹,崔星钢¹,李波¹,姜瑜^1,²()

^1.江苏大学附属医院心血管内科，江苏镇江 212001
^2.江苏省常州市妇幼保健院心血管内科，江苏常州 213003

收稿日期:2024-08-29 接受日期:2024-10-20 出版日期:2025-05-28 发布日期:2025-07-18
通讯作者: 姜瑜 E-mail:jy950927@163.com
作者简介:仲威（1989-），男，江苏省镇江市人，主治医师，医学博士，主要从事心血管疾病基础方面的研究。
基金资助:
国家自然科学基金项目(82000261);江苏省卫健委科研项目(H201644);江苏省镇江市社会发展项目(SH2022067);江苏大学医教协同创新基金项目(JDY2022007)

Effect of nuclear factor of activated T lymphocytes 5 on senescence of smooth muscle cells of mice induced by high-salt and its mechanism

Wei ZHONG¹,Zhiyin DAI¹,Xinggang CUI¹,Bo LI¹,Yu JIANG^1,²()

^1.Department of Cardiology，Affiliated Hospital，Jiangsu University，Zhenjiang 212001，China
^2.Department of Cardiology，Maternal and Child Health Care Hospital，Changzhou City，Jiangsu Province，Changzhou 213003，China

Received:2024-08-29 Accepted:2024-10-20 Online:2025-05-28 Published:2025-07-18
Contact: Yu JIANG E-mail:jy950927@163.com

摘要/Abstract

摘要：

目的探讨活化T淋巴细胞核因子5（NFAT5）抑制剂KRN5在高盐诱导小鼠血管平滑肌细胞（VSMCs）衰老中的作用，并阐明其作用机制。方法 30只8周龄雄性ApoE^－/－小鼠分为正常组、衰老组和高盐处理衰老组，每组10只，衰老组和高盐处理衰老组构建小鼠自然衰老模型；分离培养小鼠VSMCs，将VSMCs分为正常组、衰老组、高盐处理衰老组和高盐处理衰老+KRN5组。采用β-半乳糖苷酶（Sa-β-gal）染色法检测各组小鼠主动脉组织和VSMCs衰老情况，免疫荧光法检测各组小鼠主动脉组织和VSMCs中NFAT5和磷酸化的组蛋白H2A变异体X（γ-H2AX）蛋白表达情况，实时荧光定量PCR（RT-qPCR）法检测各组细胞中NFAT5、γ-H2AX、细胞周期依赖性激酶抑制剂2A（P16）和细胞周期依赖性激酶抑制剂1A（P21）mRNA表达水平，Western blotting法检测各组VSMCs中NFAT5、γ-H2AX、P16和P21蛋白表达水平。结果 Sa-β-gal染色法，与正常组比较，衰老组和高盐处理衰老组小鼠主动脉组织衰老阳性面积比例均明显增加（P<0.05），小鼠VSMCs衰老细胞阳性比例均明显增加（P<0.05）。与衰老组比较，高盐处理衰老组小鼠VSMCs衰老细胞阳性比例明显增加（P<0.05）；与高盐处理衰老组比较，高盐处理衰老+KRN5组小鼠VSMCs衰老细胞阳性比例明显减少（P<0.01）。免疫荧光法，与正常组比较，衰老组小鼠VSMCs中γ-H2AX蛋白表达量明显增加（P<0.05）；与衰老组比较，高盐处理衰老组小鼠主动脉组织中SA-β-gal染色和NFAT5蛋白表达量均明显增加（P<0.05）；与正常组比较，衰老组和高盐处理衰老组小鼠VSMCs中NFAT5蛋白表达量明显增加（P<0.05）；与衰老组比较，高盐处理衰老组小鼠VSMCs中NFAT5蛋白表达量明显增加（P<0.05）。RT-qPCR法，与正常组比较，衰老组和高盐处理衰老组小鼠VSMCs中NFAT5、γ-H2AX、P16及P21 mRNA表达水平均明显升高（P<0.05）；与衰老组比较，高盐处理衰老组小鼠VSMCs中NFAT5、γ-H2AX、P16和P21 mRNA表达水平均明显升高（P<0.05）；与衰老组比较，高盐处理衰老组和高盐处理衰老+KRN5组小鼠VSMCs中NFAT5、γ-H2AX、P16及P21 mRNA表达水平均明显升高（P<0.05）；与高盐处理衰老组比较，高盐处理衰老+KRN5组小鼠VSMCs中NFAT5、γ-H2AX、P16和P21 mRNA表达水平均明显降低（P<0.05）。Western blotting法，与正常组比较，衰老组和高盐处理衰老组小鼠VSMCs中NFAT5、γ-H2AX、P16及P21蛋白表达水平均明显升高（P<0.05）；与衰老组比较，高盐处理衰老组小鼠VSMCs中NFAT5、γ-H2AX、P16和P21蛋白表达水平均明显升高（P<0.05）；与衰老组比较，高盐处理衰老组和高盐处理衰老+KRN5组小鼠VSMCs中NFAT5、γ-H2AX、P16及P21蛋白表达水平均明显升高（P<0.05）；与高盐处理衰老组比较，高盐处理衰老+KRN5组小鼠VSMCs中NFAT5、γ-H2AX、P16和P21蛋白表达水平均明显降低（P<0.05）。结论 NFAT5对高盐诱导小鼠VSMCs衰老可能具有一定促进作用。

关键词: 血管衰老, 活化T淋巴细胞核因子5, KRN5, 血管平滑肌细胞, β-半乳糖苷酶

Abstract:

Objective To discuss the role of nuclear factor of activated T-cells 5 （NFAT5） inhibitor KRN5 in high salt-induced senescence of mouse vascular smooth muscle cells （VSMCs）， and to clarify its mechanism. Methods Thirty 8-week-old male ApoE^-/- mice were divided into normal group， senescence group and high-salt treatment senecence group， with 10 mice in each group； the mice in senescence group and high-salt treatment senecence group were used to establish natural senecence mouse models； the mouse VSMCs were isolated and cultured， and divided into normal group， senescence group， high-salt treatment senecence group and high-salt treatment senecence+KRN5 group. β-galactosidase （Sa-β-gal） staining was used to detect the senescence of aortic tissues and VSMCs in various groups； immunofluorescence method was used to detect the expressions of NFAT5 and phosphorylated histone H2A variant X （γ-H2AX） proteins in mouse aortic tissues and VSMCs in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the mRNA expression levels of NFAT5， γ-H2AX， cyclin-dependent kinase inhibitor 2A （P16） and cyclin-dependent kinase inhibitor 1A （P21） in the cells in various groups； Western blotting method was used to detect the protein expression levels of NFAT5， γ-H2AX， P16 and P21 in VSMCs in various groups. Results The Sa-β-gal staining results showed that compared with normal group， the proportions of senescence-positive area in aortic tissues of the mice in senescence group and high-salt treatment senecence group were significantly increased （P<0.05）， and the proportion of senescence-positive cells in the VSMCs of the mice was significantly increased （P<0.05）； compared with senecence group， the proportion of senescence-positive cells in the VSMCs mice in high-salt treatment senecence group was significantly increased （P<0.05）； compared with high-salt treatment senecence group， the proportion of senescence-positive cells in the VSMCs of the mice in high-salt treatment senecence+KRN5 group was significantly decreased （P<0.01）. The immunofluorescence results showed that compared with normal group， the expression level of γ-H2AX protein in mouse VSMCs of the mice in senescence group was significantly increased （P<0.05）； compared with senescence group， the expression levels of SA-β-gal staining and NFAT5 protein in aortic tissue of the mice in high-salt treatment senecence group were significantly increased （P<0.05）； compared with normal group， the expression level of NFAT5 protein in the VSMCs of the mice in senecence group and high-salt treatment senecence group was significantly increased （P<0.05）； compared with senecence group， the expression level of NFAT5 protein in the VSMCs of the mice in high-salt treatment senecence group was significantly increased （P<0.05）. The RT-qPCR results showed that compared with normal group， the expression levels of NFAT5， γ-H2AX， P16， and P21 mRNA in the VSMCs of the mice in senescence group and high-salt treatment senecence group were significantly increased （P<0.05）； compared with senecence group， the mRNA expression levels of NFAT5， γ-H2AX， P16， and P21 mRNA in the VSMCs of the mice in high-salt treatment senecence group were significantly increased （P<0.05）； compared with senecence group， the expression levels of NFAT5， γ-H2AX， P16， and P21 mRNA in the VSMCs of the mice in high-salt treatment senecence group and high-salt treatment senecence+KRN5 group were significantly increased （P<0.05）； compared with high-salt treatment senecence group， the mRNA expression levels of NFAT5， γ-H2AX， P16， and P21 in the VSMCs of the mice in high-salt treatment senecence+KRN5 group were significantly decreased （P<0.05）. The Western blotting results showed that compared with normal group， the expression levels of NFAT5， γ-H2AX， P16， and P21 proteins in the VSMCs of the mice in senescence group and high-salt treatment senecence group were significantly increased （P<0.05）； compared with senescence group， the expression levels of NFAT5， γ-H2AX， P16， and P21 proteins in the VSMCs of the mice in high-salt treatment senecence group were significantly increased （P<0.05）； compared with senecence group， the expression levels of NFAT5， γ-H2AX， P16， and P21 proteins in the VSMCs of the mice in high-salt treatment senecence group and high-salt treatment senecence+KRN5 group were significantly increased （P<0.05）； compared with high-salt treatment senecence group， the expression levels of NFAT5， γ-H2AX， P16， and P21 proteins in the VSMCs of the mice in high-salt treatment senecence+KRN5 group were significantly decreased （P<0.05）. Conclusion NFAT5 may play a promoting role in high salt-induced senescence of the mouse VSMCs.

Key words: Vascular aging, Nuclear factor of activated T-cells 5, KRN5, Vascular smooth muscle cells, β-galactosidase

中图分类号:

R339.38

仲威,戴芝银,崔星钢,李波,姜瑜. 活化T淋巴细胞核因子5在高盐诱导小鼠平滑肌细胞衰老中的作用及其机制[J]. 吉林大学学报(医学版), 2025, 51(3): 567-575.

Wei ZHONG,Zhiyin DAI,Xinggang CUI,Bo LI,Yu JIANG. Effect of nuclear factor of activated T lymphocytes 5 on senescence of smooth muscle cells of mice induced by high-salt and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2025, 51(3): 567-575.

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Group	Proportion of positive area
Normal	1.00±0.00
Senescence	0.22±0.04^*
High-salt treatment senescence	0.33±0.05^*

Group	Proportion of positive area ofsenescence cells
Normal	0.00±0.00
Senescence	0.44±0.04^*
High-salt treatment senescence	0.80±0.02^*

Group	Proportion of positive area
Senescence	0.42±0.02
High-salt treatment senescence	0.78±0.07^*
High-salt treatment senescence+KRN5	0.27±0.03^*△

Group	Expression of NFAT5
Normal	0.00±0.00
Senescence	0.52±0.04^*
High-salt treatment senescence	0.70±0.02^*△

Group	NFAT5	γ-H2AX	P16	P21
Normal	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
Senescence	2.34±0.08^*	2.69±0.22^*	1.91±0.18^*	1.84±0.13^*
High-salt treatment senescence	3.07±0.15^*△	3.42±0.09^*△	2.18±0.08^*△	2.35±0.19^*△

活化T淋巴细胞核因子5在高盐诱导小鼠平滑肌细胞衰老中的作用及其机制

Effect of nuclear factor of activated T lymphocytes 5 on senescence of smooth muscle cells of mice induced by high-salt and its mechanism

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