Buyang Huanwu Decoction delays vascular aging in rats through exosomal miR-590-5p signal-mediated macrophage polarization

doi:10.12122/j.issn.1673-4254.2025.06.14

Abstract

Abstract:

Objective To investigate the mechanism underlying the inhibitory effect of Buyang Huanwu Decoction (BYHWD) on vascular aging. Methods Eighteen male SD rats were randomized into young group, intraperitoneal D-galactose injection-induced aging group, and BYHWD gavage group. The changes in pulse wave velocity (PWV), vascular SA-β-gal activity, and expressions of p16, p21 and SA‑β‑gal of the rats were examined. Serum exosomes were isolated from the rats, and after characterization using NTA and TEM and for surface markers and vascular cell markers, were examined for miR-590-5p expression using qRT-PCR. The M1/M2 macrophage ratio and cytokine levels were evaluated using immunofluorescence staining and qRT-PCR. Bioinformatics analysis and dual-luciferase reporter assays were carried out to predict the potential target genes of miR-590-5p and validate its targeting relationship with SLC8A3, whose expressions were detected in the vascular tissues of the rats by Western blotting. Results Compared with the young rats, the aging rats exhibited significantly increased PWV in the abdominal aorta with elevated vascular expressions of p16, p21 and SA-β-gal, which were all reversed by BYHWD treatment. The isolated serum exosomes were positive for CD63, CD81, CD31 and SM-22, and the exosomes from aging rats showed significantly downregulated expression of miR-590-5p, which was upregulated after BYHWD treatment. The aging rat vessels showed an increased M1/M2 macrophage ratio with elevated M1-specific cytokines and reduced M2-specific cytokines, and BYHWD treatment effectively inhibited M1 polarization of the macrophages. Pearson analysis revealed a negative correlation between exosomal miR-590-5p upregulation and the M1/M2 ratio. Bioinformatics analysis and dual-luciferase assays confirmed that miR-590-5p targets SLC8A3. Western blotting demonstrated increased SLC8A3 expression in aging rat vessels, which was downregulated after BYHWD treatment. Conclusion BYHWD attenuates vascular aging in rats by modulating macrophage M1 polarization and suppressing vascular inflammation via exosomal miR-590-5p-mediated downregulation of SLC8A3.

Key words: Buyang Huanwu Decoction, miR-590-5p, exosomes, macrophage polarization, vascular aging

Shuyu TU, Xiangyu CHEN, Chenghui LI, Danping HUANG, Li ZHANG. Buyang Huanwu Decoction delays vascular aging in rats through exosomal miR-590-5p signal-mediated macrophage polarization[J]. Journal of Southern Medical University, 2025, 45(6): 1251-1259.

Figures/Tables 5

Tab.1 Primer sequences

Gene	Primer sequence (5'-3')
U6	F: CTCGCTTCGGCAGCACA
U6	R: AACGCTTCACGAATTTGCGT
All	R: GAGCTTATTCATAAAGTGCAG
RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGCCT
miR-590-5p	F: GCGCGCGGAGCACAAA
miR-590-5p	R: AGTGCAGGGTCCGAGGTATT
IL-6	F: CTTCTTGGGACTGATGTTGTTGAC
IL-6	R: TCTGTTGTGGGTGGTATCCTCTGTG
IL-1β	F: CACTGCAGGCTTCGAGATGAACAAC
IL-1β	R: TGTCGTTGCTTGTCTCTCCTTGTAC
IL-4	F: GCTGTCACCCTGTTCTGCTTTCTCA
IL-4	R: CATGGAGTCCCTTTTTCTGTG
IL-10	F: GGTTGCCAAGCCTTGTCAGAAATG
IL-10	R: GCTGTATCCAGAGGGTCTTC

Fig.1 BYHWD delays vascular aging in rats. A: Representative PWV images of the abdominal aorta and quantitative comparison among the groups (n=4). B: Expression of SA‑β‑gal determined by ELISA in each group (n=6). C: Immunohistochemistry and quantitative analysis of p16, p21 and SA-β-gal expressions (Original magnification: ×40; n=3). *P<0.05, **P<0.01, ***P<0.001.

Fig.2 BYHWD upregulates miR-590-5p expression in serum exosomes from rats treated with D-gal. A: Size distribution of the exosomes detected by NTA. B: Representative TEM images showing morphology of the exosomes. C, D: Expressions of CD63, CD81, CD31 and SM-22 in the exosomes determined by Western blotting. E: Expression of miR-590-5p in serum exosomes of each group detected by qRT-PCR (n=3). **P<0.01, ***P<0.001.

Fig.4 miR-590-5p targets and negatively regulates the expression of SLC8A3. A: Prediction of miR-590-5p binding sites within the 3'-UTR of SLC8A3 using the TargetScan database. B: Dual-luciferase reporter assay for validating the targeting relationship between miR-590-5p and SLC8A3. C: Expression level of SLC8A3 determined by Western blotting. *P<0.05.

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