Benzylguanine induces cellular senescence and decreased relaxation of vascular smooth muscle in rat aorta

Cellular senescence is characterized by a state of irreversible growth arrest, accompanied by distinct morphological and physiological alterations. This phenomenon is closely associated with DNA damage. Recently, our department identified that the inhibition of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) by O-6-benzylguanine (BG) induces cellular senescence in vascular smooth muscle cells (VSMCs). Consequently, we have resolved to validate these findings in an animal model. The objective of this study is to examine the impact of BG on cellular senescence in vascular smooth muscle and to assess its influence on the relaxation response. Wistar rats were administered BG by gavage with a dose of 12 mg/kg (2 doses per 24 hours). After 12 weeks, animals were sacrificed, the aorta was removed, cleaned, and samples were prepared for Western blotting, and small aortic rings were prepared for vascular response measurement. Senescent markers p27Kip1, p21 Cipl were significantly upregulated after BG treatment, which confirms that MGMT inhibition leads to cellular senescence. We did not observe any statistically significant changes in the relative expression of protein p53 and ERK1/2. This means that the expression of cyclin-dependent kinase inhibitors is regulated independently of the cell cycle regulator p53 and the ERK1/2 branch of the MAPK signaling pathway. Furthermore, we observed significantly decreased relaxation response of aortic smooth muscle to sodium nitroprusside, which confirms known adverse effects of cellular senescence on the vascular system. To conclude, we found that treatment with BG triggered an increase in the relative expression of p27, p21, and PCNA compared to healthy aortic tissues, acting as mediators and markers of senescence. Our findings have provided new insights into the signaling of cellular senescence in a model of MGMT inhibition.