[Influence and mechanism of extracellular vesicles derived from human dermal papilla cells on skin fibrosis in mice]

Abstract

Objective: To explore the influence and mechanism of extracellular vesicles (EVs) derived from human dermal papilla cells (hDPCs), i. e. hDPC-EVs on skin fibrosis in mice. Methods: This study was an experimental research. One hundred discarded hair follicle units from 2 male patients aged 25 years and 40 years who underwent hair transplantation surgery at the Second Hospital of Lanzhou University in September 2024 were collected, and primary hDPCs were extracted and successfully identified. After hDPCs of passage 3 to 5 were taken and cultured, the hDPC-EVs were extracted and successfully identified. The expression of microRNA-182-5p (miRNA-182-5p) in hDPCs and hDPC-EVs was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR, n=4). Thirty 6-week-old male C57BL/6J mice were taken and injected intradermal bleomycin for 4 weeks to establish mouse skin fibrosis models. Six mice after modeling were selected according to the random number table method (the same grouping method applied hereafter), and another 6 healthy untreated 6-week-old male C57BL/6J mice were taken. The protein expression of transforming growth factor β₁ (TGF-β₁) in normal skin tissue and fibrotic skin tissue of mice was detected by Western blotting (n=3). The remaining 24 mice after modeling were divided into phosphate buffered solution (PBS)+miRNA mimic control group, EV+miRNA mimic control group, EV+miRNA inhibitor group, and miRNA mimic group (n=6). Two weeks after injection of the reagents corresponding to the group names, the protein expressions of α-smooth muscle actin (α-SMA) and type Ⅰ collagen in fibrotic skin tissue was detected by Western blotting (n=3), and the expression of miRNA-182-5p and the mRNA expression of TGF-β₁ in fibrotic skin tissue was detected by real-time fluorescence quantitative RT-PCR (n=4). Human hypertrophic scar fibroblasts (HSFs) were taken and divided into miRNA-182-5p mimic+wild-type TGF-β₁ group, miRNA-182-5p control+wild-type TGF-β₁ group, miRNA-182-5p mimic+mutant-type TGF-β₁ group, and miRNA-182-5p control+mutant-type TGF-β₁ group. Cells in each group were transfected with the corresponding plasmids and cultured for 36 h. Double luciferase reporter gene assay was performed to detect the interaction between miRNA-182-5p and TGF-β₁ (denoted as relative luciferase activity, n=5). Results: The expression of miRNA-182-5p in hDPC-EVs was significantly higher than that in hDPCs (t=5.48, P<0.05). Compared with that in normal skin tissue of mice, the protein expression of TGF-β₁ was increased in fibrotic skin tissue of mice. After 2 weeks of treatment, compared with those in PBS+miRNA mimic control group, the protein expressions of α-SMA and type Ⅰ collagen in the fibrotic skin tissue of mice in EV+miRNA mimic control group were significantly decreased (P<0.05); compared with those in EV+miRNA mimic control group, the protein expressions of α-SMA and type Ⅰ collagen in the fibrotic skin tissue of mice in EV+miRNA inhibitor group were significantly increased (P<0.05); compared with those in EV+miRNA inhibitor group, the protein expressions of α-SMA and type Ⅰ collagen in the fibrotic skin tissue of mice in miRNA mimic group were significantly decreased (P<0.05). After 2 weeks of treatment, compared with those in EV+miRNA mimic control group, the expression of miRNA-182-5p in the fibrotic skin tissue of mice in PBS+miRNA mimic control group and EV+miRNA inhibitor group was significantly decreased (P<0.05), while the mRNA expression of TGF-β₁ was significantly increased (P<0.05). Compared with those in EV+miRNA inhibitor group, the expression of miRNA-182-5p in fibrotic skin tissue of mice in PBS+miRNA mimic control was significantly increased (P<0.05); the expression of miRNA-182-5p in the fibrotic skin tissue of mice was significantly increased (P<0.05), while the mRNA expression of TGF-β₁ was significantly decreased in miRNA mimic group (P<0.05). After 36 h of culture, the relative luciferase activity of HSFs in miRNA-182-5p mimic+wild-type TGF-β₁ group was 0.594±0.019, which was significantly lower than 1.000±0.153 in miRNA-182-5p control+wild-type TGF-β₁ group (t=5.87, P<0.05); the relative luciferase activity of HSFs in miRNA-182-5p mimic+mutant-type TGF-β₁ group was 0.911±0.085, which has no statistically significant difference with 0.934±0.027 of miRNA-182-5p control+mutant-type TGF-β₁ group (P>0.05), indicating that miRNA-182-5p could exerted targeted regulation of TGF-β₁. Conclusions: hDPC-EVs alleviate bleomycin-induced skin fibrosis in mice by delivering miRNA-182-5p to inhibit the TGF-β₁ signal pathway.