OBJECTIVE This study aimed to investigate the effects of Buyang Huanwu Decoction (BYHWD) on delaying vascular aging and explore whether the underlying mechanism is associated with microRNA-665 (miR-665)/DNA damage-regulated autophagy modulator1 (DRAM1)-mediated autophagy.
METHODS Male SD rats with natural aging were randomly divided into the aging group, BYHWD low, medium, high dosage groups (9.25, 18.5, 37.0 g·kg-1) and resveratrol group (80 mg·kg-1), with a young group set as well. The rats in each group were dissected and the blood vessels were collected. ELISA was used to assess senescence associated β-galactosidase (SA-β-Gal) activity and advanced glycation end products (AGEs) level in vascular tissues. HE, Masson, and EVG staining were performed to observe the morphological structure of the vascular tissues. The qPCR was performed to detect the expression of miR-665 in vascular tissues. Bioinformatics analysis and dual-luciferase reporter gene experiments were used to validate the targeting relationship between miR-665 and DRAM1. Transmission electron microscope was used to observe the autophagosome. Western blot was performed to determine the protein expression of p16, DRAM1 and autophagy-related proteins Beclin1, p62 and LC3. Immunohistochemistry was used to detect the protein expression of DRAM1 in vascular tissues.
RESULTS Compared to the young group, the aging group showed increased SA-β-Gal activity, AGEs level and p16 protein expression (P < 0.01), disordered arrangement of vascular tissues, thickened media, increased collagen fibers, fractured and disorganized elastic fibers. The expression of miR-665 was upregulated (P < 0.01). The number of autophagosomes was reduced. The protein expression of Beclin1 and LC3Ⅱ/Ⅰ downregulated (P < 0.01), while the protein expression of p62 was upregulated (P < 0.01). In addition, the protein expression of DRAM1 was downregulated in vascular tissues (P < 0.01). Compared to the aging group, intervention with BYHWD and resveratrol reduced SA-β-Gal activity (P < 0.01), AGEs level and p16 protein expression (P < 0.05, P < 0.01), improved vascular morphology and elastic fiber structure, reduced collagen fibers. High dose BYHWD significantly downregulated miR-665 expression (P < 0.01), increased the number of autophagosomes. Medium and high dose of BYHWD significantly upregulated protein expression of Beclin1 and LC3Ⅱ/Ⅰ (P < 0.01), downregulated protein expression of p62 (P < 0.05, P < 0.01). High dose BYHWD significantly upregulated protein expression of DRAM1 in vascular tissues of aging rats (P < 0.05). Bioinformatics analysis revealed the presence of specific complementary binding sites between the sequences of miR-665 and DRAM1. Dual-luciferase reporter assays confirmed that miR-665 targeted DRAM1 gene and negatively regulated DRAM1 protein expression.
CONCLUSION BYHWD may promote the protein expression of DRAM1 by inhibiting the expression of miR-665, thereby promoting vascular autophagy and delaying vascular aging.
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