School of Basic Medicine, Guangxi Medical University, Nanning, Guangxi 530021, China.
Key Laboratory of Longevity and Aging-Related Diseases of Chinese Ministry of Education, Nanning, Guangxi 530021, China.
J Immunol Res. 2022 May 18;2022:3371982. doi: 10.1155/2022/3371982. eCollection 2022.
Skin aging could be regulated by the aberrant expression of microRNAs. In this manuscript, we explain that endothelial cell-derived extracellular vesicles could act as supporters to deliver exogenous miR-326-3p to accelerate skin fibroblasts senescence.
-galactosidase senescence staining assay, Hoechst 33258 apoptosis staining assay, and Ki67 staining assay were used to evaluate the biological function of mouse skin fibroblasts. Real-time PCR was applied to assay miRNAs and mRNAs expressions. Western blot was used to detect TLR4 protein expression. The target gene of miRNA were identified using a double luciferase reporter assay. miR-326-3p mimic/inhibitor and siRNA-TLR4 can demonstrate a nonnegligible link between miR-326-3p-TLR4 and skin aging.
In coculture experiment, senescence endothelial cells could promote the skin fibroblasts senescence and apoptosis via extracellular vesicles pathway. In contrast, miR-326-3p mimics accelerated senescence and apoptosis of skin fibroblasts, while miR-326-3p inhibitor could dramatically delay skin fibroblasts senescence and apoptosis. TLR4 was proved to be a miR-326-3p directly target gene via double luciferase assay. After skin fibroblasts transfected with siRNA-TLR4, cellular senescence and apoptosis were significantly increased. Furthermore, the skin tissues of aging mice were shown with overexpression of miR-326-3p and decrease of TLR4 gene and protein expression levels.
Endothelial cell-derived extracellular vesicles delivery of miR-326-3p was found to have a function in skin fibroblasts via target TLR4. Therefore, endothelial cell-derived extracellular vesicles in antiaging therapies might be a new treatment way for delaying skin aging.
皮肤衰老可能是由 microRNAs 的异常表达调控的。在本研究中，我们解释了内皮细胞衍生的细胞外囊泡可以作为载体，将外源性 miR-326-3p 递送到皮肤成纤维细胞中，从而加速其衰老。
β-半乳糖苷酶衰老染色实验、Hoechst 33258 凋亡染色实验和 Ki67 染色实验用于评估小鼠皮肤成纤维细胞的生物学功能。实时 PCR 用于检测 miRNA 和 mRNA 的表达。Western blot 用于检测 TLR4 蛋白的表达。采用双荧光素酶报告基因实验鉴定 miRNA 的靶基因。miR-326-3p 模拟物/抑制剂和 siRNA-TLR4 可证明 miR-326-3p-TLR4 与皮肤衰老之间存在不可忽视的联系。
在共培养实验中，衰老的内皮细胞可通过细胞外囊泡途径促进皮肤成纤维细胞衰老和凋亡。相反，miR-326-3p 模拟物加速了皮肤成纤维细胞的衰老和凋亡，而 miR-326-3p 抑制剂则显著延缓了皮肤成纤维细胞的衰老和凋亡。双荧光素酶报告基因实验证实 TLR4 是 miR-326-3p 的直接靶基因。用 siRNA-TLR4 转染皮肤成纤维细胞后，细胞衰老和凋亡明显增加。此外，衰老小鼠皮肤组织中 miR-326-3p 表达上调，TLR4 基因和蛋白表达水平下调。
内皮细胞衍生的细胞外囊泡携带的 miR-326-3p 通过靶向 TLR4 发挥作用。因此，内皮细胞衍生的细胞外囊泡在抗衰老治疗中可能成为延缓皮肤衰老的一种新的治疗方法。
