补阳还五汤通过调控外泌体miR-590-5p介导的巨噬细胞极化延缓大鼠血管衰老

doi:10.12122/j.issn.1673-4254.2025.06.14

摘要/Abstract

摘要：

目的探讨补阳还五汤对血管衰老的延缓作用机制是否与外泌体miR-590-5p介导的巨噬细胞极化有关。方法将18只雄性SD大鼠随机分为年轻组、衰老组和补阳还五汤组，6只/组。通过腹腔注射D-半乳糖300 mg·kg^-1·d^-1建立衰老模型，补阳还五汤组灌胃补阳还五汤4.4 g/kg，年轻组、衰老组给予等量生理盐水。Vevo 3100高分辨率超声成像系统检测大鼠腹主动脉脉搏波传导速度（PWV）。分离胸主动脉，ELISA法测定血管组织衰老相关β-半乳糖苷酶（SA-β-gal）活性；免疫组织化学法检测p16、p21和SA-β-gal表达。提取各组大鼠血清外泌体并通过纳米颗粒跟踪分析及透射电镜观察外泌体的大小及形态；Western blotting检测血清外泌体表面标记物（CD63和CD81）、血管内皮细胞和血管平滑肌细胞标记蛋白（CD31和SM-22）；qRT-PCR检测血清外泌体中miR-590-5p表达；免疫荧光染色和qRT-PCR检测巨噬细胞M1/M2比例及炎性细胞因子表达；生物信息学分析预测miR-590-5p靶基因；双荧光素酶报告基因实验验证miR-590-5p与SLC8A3的靶向关系；Western blotting检测血管组织中SLC8A3表达。结果与年轻组相比，衰老组大鼠腹主动脉PWV升高（P<0.001），血管中p16、p21和SA-β-gal表达增加（P<0.05）；与衰老组相比，补阳还五汤组能够逆转衰老引起的上述变化（P<0.05）。提取的血清外泌体大小为50~150 nm，呈现经典的双凹状囊性小泡结构。分离的血清外泌体不仅表达外泌体表面标记CD63和CD81，还可表达血管内皮细胞及血管平滑肌细胞标记蛋白CD31和SM-22。与年轻组相比，衰老组大鼠血清外泌体中miR-590-5p表达下调（P<0.001），血管中M1和M2巨噬细胞比例（P<0.01）以及M1巨噬细胞特异性细胞因子白细胞介素6和白细胞介素1β升高（P<0.01），M2巨噬细胞特异性细胞因子白细胞介素4和白细胞介素10降低（P<0.01）。与衰老组相比，补阳还五汤组上调衰老大鼠血清外泌体中miR-590-5p表达（P<0.001），抑制衰老血管巨噬细胞M1极化（P<0.05）。Pearson相关性分析显示，血清外泌体miR-590-5p表达升高与M1/M2巨噬细胞比例呈负相关（P<0.05）。生物信息学分析显示，miR-590-5p与SLC8A3基因序列存在特异性互补结合位点，双荧光素酶报告实验证实miR-590-5p靶向SLC8A3基因并负调控SLC8A3表达（P<0.05）。Western blotting表明，与年轻组相比，衰老组血管组织中SLC8A3表达升高（P<0.05）；与衰老组相比，补阳还五汤组中SLC8A3表达下调（P<0.05）。结论补阳还五汤通过增加外泌体转运miR-590-5p靶向抑制SLC8A3基因表达，改善血管巨噬细胞M1极化及炎性细胞激活，进而延缓血管衰老。

关键词: 补阳还五汤, miR-590-5p, 外泌体, 巨噬细胞极化, 血管衰老

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

涂舒谕, 陈祥宇, 李程辉, 黄丹萍, 张莉. 补阳还五汤通过调控外泌体miR-590-5p介导的巨噬细胞极化延缓大鼠血管衰老[J]. 南方医科大学学报, 2025, 45(6): 1251-1259.

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.

图/表 5

表1 引物序列

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

图1 补阳还五汤延缓血管衰老

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.

图2 补阳还五汤上调血清外泌体miR-590-5p表达

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.

图3 血清外泌体miR-590-5p与血管巨噬细胞激活相关

图4 miR-590-5p靶向并负调控SLC8A3基因表达

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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