Nicotinamide Mononucleotide (NMN) Improves the Senescence of Mouse Vascular Smooth Muscle Cells Induced by Ang II Through Activating p-AMPK/KLF4 Pathway

Abstract

Background: Vascular smooth muscle cells (VSMCs) senescence exacerbates vascular diseases like atherosclerosis and hypertension. Angiotensin II (Ang II) is a strong inducer of VSMCs senescence, causing vascular damage, though its exact mechanism is unclear. Nicotinamide mononucleotide (NMN), a NAD⁺ precursor, has gained attention for its anti-senescence potential, yet its role in inhibiting VSMCs senescence is not fully understood. Methods: This study assessed senescence markers, including β-galactosidase activity (SA-β-gal) and the senescence-associated secretory phenotype (SASP), in mouse VSMCs treated with Ang II alone or with NMN and relevant activators/inhibitors. Results: Compared to controls, SA-β-gal levels and SASP secretion significantly increased in Ang II-exposed cells. In contrast, NMN reduced the expression of both markers. NMN also reversed Ang II-induced VSMCs senescence by downregulating KLF4 and p16 through AMPK activation, which Ang II inhibited, while decreasing mRNA levels of key SASP components. The effects of the AMPK activator AICAR were similar to those of NMN, whereas the AMPK inhibitor Compound C negated NMN's effects. Conclusions: In summary, NMN mitigates Ang II-induced mouse VSMCs senescence via the AMPK/KLF4/p16 pathway. This study underscores the anti-senescence effects of NMN on mouse VSMCs, supporting further exploration of its potential as a food supplement for preventing and treating vascular senescence.

Keywords: AMPK/KLF4/p16 pathway; Angiotensin II; nicotinamide mononucleotide; senescence; vascular smooth muscle cells.

Figure 1

NMN improved the degree of Ang II-induced mouse VSMCs cell senescence and reduced the expression of key SASP factor transcription levels. (A,B) Representative images of SA-β-gal staining and quantitative analyses of SA-β-gal-positive cells (scale, 50 μm). (C–J) qRT-PCR analysis of IL-18, IL-1β, TNF-α, TNF-β, MCP-1, MMP9, MMP12, and MMP13 in mouse VSMCs. Data are expressed as the mean ± SD (n = 3, representing three independent experiments). * p < 0.05, ** p < 0.01 vs. the con group; ^# p < 0.05, ^## p < 0.01 vs. the Ang II group.

Figure 2

NMN regulates the AMPK/KLF4/p16 pathway associated with senescence in mouse VSMCs. (A) Representative Western blotting of AMPK, p-AMPK, KLF4, p16, IL-1β, IL-18, and GAPDH in mouse VSMCs. (B) p-AMPK/AMPK ratio. (C–F) Quantitative density analysis of KLF4, p16, IL-1β, and IL-18 normalized to GAPDH. Data are expressed as mean ± SD (n = 3, representing three independent experiments). * p < 0.05, ** p < 0.01 vs. the con group; ^# p < 0.05, ^## p < 0.01 vs. the Ang II group.

Figure 3

AICAR can improve the senescence of mouse VSMCs induced by Ang II and reduce the expression of key SASP factor transcription levels. (A) The effect of AICAR on the cell viability of mouse VSMCs. Data are expressed as mean ± SD (n = 3). (B,C) Representative images of SA-β-gal staining and quantitative analyses of SA-β-gal-positive cells (scale, 50 μm). (D–K) qRT-PCR analysis of IL-18, IL-1β, TNF-α, TNF-β, MCP-1, MMP9, MMP12, and MMP13 in mouse VSMCs. Data are expressed as the mean ± SD (n = 3, representing three independent experiments). * p < 0.05, ** p < 0.01 vs. the con group; ^# p < 0.05, ^## p < 0.01 vs. the Ang II group.

Figure 4

The activated AMPK/KLF4/p16 pathway is involved in Ang II-induced mouse VSMCs cell senescence. (A) Representative Western blotting of AMPK, p-AMPK, KLF4, p16, IL-1β, IL-18, and GAPDH in mouse VSMCs. (B) p-AMPK/AMPK ratio. (C–F) Quantitative density analysis of KLF4, p16, IL-1β, and IL-18 normalized to GAPDH. Data are expressed as mean ± SD (n = 3, representing three independent experiments). * p < 0.05, ** p < 0.01 vs. the con group; ^## p < 0.01 vs. the Ang II group.

Figure 5

Compound C could mask the effect of NMN on the senescence of VSMCs induced by Ang II. (A) The effect of Compound C on the cell viability of mouse VSMCs. Data are expressed as mean ± SD (n = 3). (B,C) Representative images of SA-β-gal staining and quantitative analyses of SA-β-gal-positive cells (scale, 50 μm). (D–K) qRT-PCR analysis of IL-18, IL-1β, TNF-α, TNF-β, MCP-1, MMP9, MMP12, and MMP13 in mouse VSMCs. Data are expressed as the mean ± SD (n = 3, representing three independent experiments). * p < 0.05, ** p < 0.01 vs. the con group; ^# p < 0.05, ^## p < 0.01 vs. the Ang II group, ^& p < 0.05, ^&& p < 0.01 vs. the Ang II + NMN group.

Figure 6

The AMPK inhibitor reversed the effects of NMN on Ang II-induced markers of senescence. (A) Representative Western blotting of AMPK, p-AMPK, KLF4, p16, IL-1β, IL-18, and GAPDH in mouse VSMCs. (B) p-AMPK/AMPK ratio. (C–F) Quantitative density analysis of KLF4, p16, IL-1β, and IL-18 normalized to GAPDH. Data are expressed as mean ± SD (n = 3, representing three independent experiments). ** p < 0.01 vs. the con group, ^# p < 0.05, ^## p < 0.01 vs. the Ang II group; ^&& p < 0.01 vs. the Ang II + NMN group.

Figure 7

Mechanism of Ang II-induced senescence in mouse VSMCs Model. The red color represents the effect of NMN, while the black color indicates the effect of Ang II. Arrows signify activation, and horizontal lines represent inhibition.