miR-146a Decreases Inflammation and ROS Production in Aged Dermal Fibroblasts

Abstract

Aging is associated with a decline in the functionality of various cell types, including dermal fibroblasts, which play a crucial role in maintaining skin homeostasis and wound healing. Chronic inflammation and increased reactive oxygen species (ROS) production are hallmark features of aging, contributing to impaired wound healing. MicroRNA-146a (miR-146a) has been implicated as a critical regulator of inflammation and oxidative stress in different cell types, yet its role in aged dermal fibroblasts and its potential relevance to wound healing remains poorly understood. We hypothesize that miR-146a is differentially expressed in aged dermal fibroblasts and that overexpression of miR-146a will decrease aging-induced inflammatory responses and ROS production. Primary dermal fibroblasts were isolated from the skin of 17-week-old (young) and 88-week-old (aged) mice. Overexpression of miR-146a was achieved through miR-146a mimic transfection. ROS were detected using a reliable fluorogenic marker, 2,7-dichlorofluorescin diacetate. Real-time PCR was used to quantify relative gene expression. Our investigation revealed a significant reduction in miR-146a expression in aged dermal fibroblasts compared to their younger counterparts. Moreover, aged dermal fibroblasts exhibited heightened levels of inflammatory responses and increased ROS production. Importantly, the overexpression of miR-146a through miR-146a mimic transfection led to a substantial reduction in inflammatory responses through modulation of the NF-kB pathway in aged dermal fibroblasts. Additionally, the overexpression of miR-146a led to a substantial decrease in ROS production, achieved through the downregulation of NOX4 expression in aged dermal fibroblasts. These findings underscore the pivotal role of miR-146a in mitigating both inflammatory responses and ROS production in aged dermal fibroblasts, highlighting its potential as a therapeutic target for addressing age-related skin wound healing.

Keywords: aging wound healing; dermal fibroblasts; miR-146a; reactive oxygen species (ROS).

Figure 1

Delayed wound healing in aged mice with reduced miR-146a and induced NOX4 expression. (A) Representative images of wounds on days 0, 4,8, 12, 14, and 16 after wounding. (B) Wound size change during the healing process of initial 8 mm wound in WT (C57BL/6J or B6) mice at different ages by Image J analysis. Young (17 weeks old mice), blue; aged (88 weeks old mice), orange (n = 5 per group). Error bars indicate mean ± SEM. *, p < 0.05. (C) Real-time qPCR analysis of miR-146a gene expression in young and aged dermal fibroblasts (mean ± SD, n = 5 per group). (D) Real-time qPCR analysis of NOX4 gene expression in young and aged dermal fibroblasts (mean ± SD, n = 5 per group). ** p < 0.01.

Figure 2

MiR-146a and NOX4 expression in aged dermal fibroblasts. (A) Real-time qPCR analysis of miR-146a gene expression in young and aged dermal fibroblasts (mean ± SD, n = 3 per group). (B) Real-time qPCR analysis of NOX4 gene expression in young and aged dermal fibroblasts (mean ± SD, n = 3 per group). ** p < 0.01.

Figure 3

Inflammatory responses and ROS production in aged dermal fibroblasts. Real-time qPCR analysis of IL6 (A), IRAK1 (B), TRAF6 (C), and NF-kB (D) gene expression in young and aged dermal fibroblasts (mean ± SD, n = 3 per group). (E) ROS production between young and aged dermal fibroblasts using the DCFDA/H2DCFDA–cellular ROS assay. (F) Young and aged dermal fibroblast size comparison using the Echo Revolve macroscope’s built-in software (https://discover-echo.com/revolve). * p < 0.05, ** p < 0.01.

Figure 4

Effects of miR-146a overexpression on inflammatory responses and ROS production. Dermal fibroblasts were transfected with a miR-146a mimic (146a mimic) or a negative control mimic (Con-mimic). (A) miR-146a expression was detected by real-time qPCR with U6 as an internal control. The gene expression levels of IL6 (B), IRAK1 (C), TRAF6 (D), and NF-kB (E) were determined by RT-qPCR in miR-146a overexpression fibroblasts or control mimic-transfected fibroblasts. (F) ROS production between young and aged dermal fibroblasts transfected with miR-146a mimics or control mimics using the DCFDA/H2DCFDA–cellular ROS assay.

Figure 5

MiR-146a targets NOX4 in dermal fibroblasts. (A) Bioinformatic analysis of miRNA targets revealed a binding site between miR-146a and the 3′-UTR region of human Nox4 mRNA, suggesting a possible regulatory relationship between miR-146a and Nox4. (B) NOX4 gene expression in miR-146a overexpression young or aged fibroblasts (mean ± SD, n = 3 per group). ** p < 0.01. NS: not significant. (C) Cartoon illustration of possible mechanisms of miR-146a/NOX4 signaling in aged dermal fibroblasts.