3.1. NUDFS4 was identified as a potential target for UVB-induced photoaging through RNA sequencing
Figure 1. RNA-seq was employed to identify potential targets in UV-induced photoaging
(a) Volcano plots highlighted differentially expressed RNAs in BJ cells between the UVB-treated group and the control, using a threshold of fold change ≥ 1.0 and a P-value < 0.05. (b) Cluster analysis was conducted, with samples represented on the x-axis and genes on the y-axis. Various colors represented different levels of gene expression. (c) Gene Ontology (GO) enrichment analysis was performed on all differentially expressed genes (DEGs). (d) The top 30 pathways were identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs. (e) Quantitative real-time PCR (qRT-PCR) confirmed the expression levels of 20 differentially expressed RNAs in BJ cells.
To investigate the potential mechanisms of UVB irradiation-induced skin photoaging, we conducted RNA sequencing (RNA-seq) on BJ cells. RNA-seq identified regulatory differences in numerous genes following UV irradiation compared to normal controls. Differentially expressed genes were determined based on fold-change (FC) filtering (|log2FC|>1) and a P-value <0.05. The comparison between control and UVB-treated groups using volcano plots and heat maps of RNA-seq data (Fig. 1a, b) revealed that 4331 genes were up-regulated and 4390 genes down-regulated post-UVB treatment. Gene Ontology (GO) biological process (BP) analysis highlighted enrichment in cellular respiration and mitochondrial function (Fig. 1c). Given the link between mitochondrial dysfunction and skin aging, we focused on the regulation of mitochondrial function by ultraviolet radiation[25]. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis indicated that the differentially expressed genes post-treatment were involved in various pathways, including oxidative phosphorylation, cell cycle, apoptosis, DNA replication, and cellular senescence (Fig. 1d). Oxidative phosphorylation-related pathways, in particular, were noted for their relevance in UV-mediated photoaging[26]. Consequently, genes involved in oxidative phosphorylation were cross-referenced with the up-regulated genes, and the top 20 differentially expressed genes were selected for further validation. Subsequently, we assessed their expression differences using quantitative real-time PCR (qRT-PCR) in UVB-irradiated and non-irradiated cells. The results indicated significant differences in all RNAs, with NDUFS4 exhibiting the lowest P-value and a relatively high expression difference (Fig. 1e).
3.2 NDUFS4 mediates UV-induced photoaging
Figure 2. NDUFS4 expression is regulated by the ultraviolet ray in the cell damage induced by ultraviolet light
(a) Genes related to UVB-induced photoaging were identified through quantitative PCR (qPCR). (b) Dual-Luciferase Assay of Ndufs-4: This assay evaluated UVB-induced changes in NDUFS4 expression in BJ cells. (c, d) Western Blot and PCR Analysis: These methods confirmed the effect of NDUFS knockdown on its expression and the subsequent alterations following UVB exposure. (e) Flow Cytometry: Employed to determine the impact of NDUFS knockdown on cell apoptosis. (f) MTT assays in BJ cells. All data are averages from three independent experiments ± SD, *p ≤ 0.05, **p ≤ 0.01, *** p ≤0.005.
Drawing from previous literature and our findings, UVB-induced skin damage is associated not only with mitochondrial oxygen free radical production but also with a reduction in antioxidant enzyme activities. NDUFS4 was chosen for further investigation due to its crucial role in maintaining the stability and function of mitochondrial complex I[27]. To explore this, we constructed a luciferase reporter recombinant plasmid using the NDUFS4 gene and a dual luciferase reporter gene. We compared the luciferase activity between untreated and UVB-treated groups to assess the impact of UVB on NDUFS4 transcription. Our results indicated a significant increase in NDUFS4 expression in BJ cells following UVB irradiation (Fig. 2a). To corroborate these findings, we conducted additional independent experiments, which revealed that NDUFS4 expression in NDUFS4-KD cells was upregulated after UVB exposure, as demonstrated by both qPCR and WB analyses (Fig. 2b,c). These findings suggest that UVB induces NDUFS4 expression in cells. Using the MTT method and flow cytometry, we examined the effects of NDUFS4 on cell proliferation and apoptosis induced by UVB (Fig. 2d,e). Our observations showed that UVB-treated BJ cells exhibited reduced proliferation and enhanced apoptosis , which were mitigated upon knockdown of NDUFS4. Consequently, NDUFS4 is implicated in cellular damage induced by UVB light.
3.3.UVB regulates Ferroptosis by inducing NDUFS4
Figure 3. NDUFS4 plays a regulatory role in ferroptosis
(a) Representative images of intracellular ROS immunofluorescence staining in BJ cells. Scale bars: 100 microns (100x), 25 microns (640x). Each group had N=3 samples. (b) Transmission electron microscopy was used to observe mitochondrial morphology in different treatment groups. (c, d) Western blot analysis of H2AX, γ-H2AX, SLC7A11, FTH1, GPX4, EPR, p-EPR, p38 and p-p38 , GAPDH served as a loading control.
An important characteristic of ferroptosis, a new type of cell death, is the alteration in mitochondrial microstructure. In the NC+UVB group, compared to the NDUFS4-KD+UVB group, significant morphological changes in the mitochondria were observed, including reduced mitochondrial size, intensified membrane staining, and marked fragmentation of the inner mitochondrial cristae (Fig. 3a). Another hallmark of ferroptosis is the accumulation of lethal lipid peroxidation products, resulting in cytotoxicity[28]. Consequently, we investigated the effect of reduced NDUFS4 expression in cells on the expression of ROS, a marker of ferroptosis, to elucidate the role of NDUFS4 in the progression of ferroptosis. Our findings revealed that regardless of NDUFS4 knockdown, cells exposed to UV light exhibited significantly higher ROS levels compared to those not irradiated. These results suggest that UV irradiation alters mitochondrial microstructure and increases ROS expression in cells, potentially inducing ferroptosis (Fig. 3b). Moreover, reduced ROS expression following NDUFS4 knockdown indicates that UV-induced ferroptosis occurs via the NDUFS4 pathway. To corroborate these observations, we investigated whether knocking down NDUFS4 affected the expression of ferroptosis-associated proteins. NDUFS4 knockdown notably increased the γ-H2AX, while decreasing downregulated the expression of thesolute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and GPX4 levels (Fig. 3c). The elevation of DNA damage marker γ-H2AX, ACSL4, coupled with the reduction of the anti-ferroptosis proteins FTH1, SLC7A11 and the ferroptosis inhibitor GPX4, suggests that ferroptosis progression is hindered after NDUFS4 knockdown. This indicates that NDUFS4 plays a regulatory role in ferroptosis. Additionally, NDUFS4 knockdown enhanced ERK and p38 phosphorylation, aligning with previous studies that identified the MAPK signaling pathway's involvement in ferroptosis (Fig. 3d). Thus, we hypothesize that the MAPK pathway contributes to NDUFS4's influence on ferroptosis in BJ cells, playing a regulatory role.
3.4. Overexpression of GPX4 reversed NDUFS4-induced ferroptosis
Figure 4. Overexpression of GPX4 reversed NDUFS4-mediated ferroptosis
(a) Western Blot analysis confirmed the efficiency of NDUFS4 knockdown and GPX4 overexpression. (b) Flow cytometry for cell apoptosis detection: Assessment revealed that GPX4 overexpression mitigates cell apoptosis.(c) Immunofluorescence staining of ROS in BJ cells: This technique visualized ROS, with scale bars representing 100 microns (100x magnification) and 25 microns (640x magnification). Each group had N = 3 samples. (d, f) Western Blot analysis assessed the expression of proteins H2AX, γ-H2AX, SLC7A11, FTH1, GPX4, EPR, p-EPR, p38 and p-p38, with GAPDH as the loading control.
To further elucidate the role of NDUFS4 in ferroptosis, we genetically modified BJ cells to stably express GPX4 and monitored changes in ferroptosis markers. Ferroptosis initiation typically involves GPX4 inactivation, a lipid peroxidation (LPO) scavenger, due to glutathione depletion[29]. Our protein imprinting results confirmed successful low expression of NDUFS4 and GPX4 expression (Fig. 4a). GPX4, as an essential antioxidant enzyme, plays a critical role in REDOX balance and is significant in regulating ferroptosis. Previous studies have shown that GPX4 inhibits ferroptosis primarily by reducing elevated lipid peroxidation levels[30]. Notably, cells expressing GPX4 could interrupt the ferroptosis induced by low NDUFS4 expression, further decreasing ROS levels and limiting ferroptosis progression (Fig. 4b). The increased expression of FTH1 in WB results, also corroborate this finding. However, GPX4 overexpression significantly reduced cell apoptosis, as evidenced by flow cytometry (Fig. 4c), and downregulated the expression of the DNA damage marker γ-H2AX and upregulated the expression of the anti-ferroptosis protein SLC7A11, as shown by WB experiments (Fig. 4d). These findings indicate that GPX4 overexpression can mitigate cell damage caused by ferroptosis. Furthermore, GPX4 overexpression, inhibiting ferroptosis, coincided with decreased phosphorylation of proteins in the MAPK pathway (Fig. 4e). The MAPK signaling pathway, particularly its subtypes p38 and ERK, plays a crucial role in cell proliferation, differentiation, apoptosis, and stress responses[31]. Our study focused on these MAPK subtypes in inflammation induced by ultraviolet light. We observed that UV exposure inhibited the phosphorylation of ERK and p38 in BJ cells, which could be reversed by GPX4 overexpression or NDUFS4 knockdown. These observations suggest that the MAPK pathway is involved in the progression of UVB-induced ferroptosis.
3.5. Protective effect of ferroptosis inhibitor on UVB induced photoaging in mice
Figure 5. In vivo animal experiments were performed to verify the mechanism of NDUFS4 regulating ferroptosis
a. Photographs of SKH1 hairless mice skins treated in different ways. (b) Hematoxylin-eosin (HE) staining of tissue samples (n=4), magnification×100, ×200. (c, d) Immunofluorescence confocal microscopy for γ-H2AX and NDUFS4 visualization in skin tissues of treated mice. (e, f) Western Blot analysis of protein expression: NDUFS4, H2AX, γH2AX, SLC7A11, FTH1, GPX4, EPR, p-EPR, p38 and p-p38, with GAPDH as the loading control. (g) EPR and p-EPR immunohistochemistry
Further in vivo functional experiments are necessary to elucidate the mechanism through which ultraviolet light induces ferroptosis by upregulating NDUFS4 expression and modulating the MAPK signaling pathway. To investigate the effects of ferroptosis inhibition in an animal model, we utilized the common ferroptosis inhibitor Fer-1 in mice. Fer-1 impedes ferroptosis by lowering free iron levels and inhibiting lipid peroxidation[32,33]. UVB-induced skin photoaging is typified by symptoms such as erythema, laxity, dryness, and wrinkling. Our experiments revealed that UVB irradiation resulted in pronounced erythema and skin folding, indicative of photoaging, on the dorsal skin of mice. However, Fer-1 treatment alleviated UVB-induced skin lesions, including erythema and wrinkles (Fig. 5a). Post UVB exposure and Fer-1 application, we noted increased epidermal thickness, normalized subepidermal keratinocyte arrangement, and reduced inflammatory cell presence in skin tissues (Fig. 5b). Immunofluorescence staining demonstrated that Fer-1 reduced γ-H2AX levels post-UVB exposure, indicating diminished DNA damage (Fig. 5c). Notably, in line with in vitro cellular findings, UVB light induced NDUFS4 expression, which was reversed by Fer-1(Fig. 5d). Inhibition of ferroptosis progression led to the downregulation of γ-H2AX, and the upregulation of FTH1, SLC7A11 and GPX4 expression in subsequent experiments (Fig. 5e). Additionally, it resulted in reduced phosphorylation of ERK and p38, proteins associated with the MAPK pathway (Fig. 5f). Similar results were obtained in another immunohistochemical study (Fig. 4g). In summary, these results indicate that UVB radiation induces ferroptosis through NDUFS4 activation. Fer-1 suppresses ferroptosis by reducing NDUFS4 expression, which leads to decreased intracellular lipid ROS and diminished phosphorylation of proteins in the MAPK signaling pathway.