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