Introduction
Psoriasis is an autoimmune inflammatory skin disease with scales, erythema, and pruritus as the main clinical symptoms, which has a long course and affects about 2–4% of the world’s population.1 One of the most common histological features of psoriatic lesions is epidermal hyperplasia caused by excessive proliferation and abnormal differentiation of keratinocytes. The epidermal barrier plays an important role in keeping skin homeostasis and preventing the human body from many external stressors, including chemical stress, environmental conditions, and physical stress.2 Epidermal barrier dysfunction was observed in psoriatic plaques when compared with uninvolved skin and healthy controls.3 The potential association between proinflammatory cytokines and barrier integrity has been observed in some inflammatory dermatological disorders.4 Additionally, epidermal barrier function could be destroyed by hyperproliferation and defective keratinocyte differentiation.5 Thus, the study was designed to investigate the potential mechanism involved in proliferation, inflammation, and epidermal barrier dysfunction in psoriasis.
Sirtuins (SIRTs) are a protein family of nicotinamide adenine dinucleotide (NAD+)–dependent histone deacetylases, which are involved in a series of biological processes such as DNA damage repair, aging, oxidative stress, and inflammatory response.6 For example, SIRT5 overexpression can reduce the inflammatory response of AD neurons,7 and SIRT5 can reduce cisplatin-induced acute kidney injury by regulating Nrf2/HO-1 and Bcl-2.8 SIRT5 prevents apoptosis of lung epithelial cells induced by cigarette extract by deacetylation of FOXO3.9 These results suggest that SIRT5 has important anti-inflammatory and anti-injury effects. However, it has not been studied in psoriatic lesions. The study has shown that the expression of SIRT5 is reduced in psoriatic keratinocytes and tissues.10 Therefore, this study aims to explore whether SIRT5 can reduce the inflammatory response of psoriatic epithelial cells and the damage to epidermal barrier function.
Methods and Materials
Cell culture
Normal human epidermal keratinocytes (NHEKs) were purchased from Cascade Biologics/Invitrogen (USA) and cultured in serum-free EpiLife cell culture media (Cascade Biologics/Invitrogen) with 0.06 mM Ca2+ and 1 × EpiLife Defined Growth Supplement (EDGS, Cascade Biologics/Invitrogen), along with the addition of 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B at 37°C per 5% CO2. To induce an inflammatory response, interleukin 17A (IL-17A, Absin, Shanghai, China) at concentrations of 10, 50, and 100 ng/ml was used to treat NHEKs for 24 h. To activate the ERK, activator tBHQivator t-butylhydroquinone (tBHQ, MedChemExpress)11 at a concentration of 50 μM was used to treat NHEKs for 24 h.
Plasmid transfection
To determine the effect of SIRT5, its overexpression was induced by the transfection. LipofectamineTM 3000 mixed with plasmids overexpressing SIRT5 (Ov-SIRT5) and empty plasmids (negative control, Ov-NC) were slowly added into the culture medium, mixed, and stood for 20 min at room temperature. NHEKs were cultured in an incubator at 37°C with 5% CO2 for 6 h, and then the culture medium was replaced with a fresh medium for further culture. After 24 h, the cells were used for further experiments. Following transfected cells were treated with IL-17A and tBHQ, and they were divided into IL-17A + Ov-SIRT5/Ov-NC and IL-17A + Ov-SIRT5 + tBHQ.
RT-qPCR assay
TRIzol kit was used to extract total RNA from cells. Total RNAs were reversely transcribed cDNA using the iScript cDNA Synthesis Kit according to the manufacturer’s protocol (BioRad, USA). The SYBR Green assay kit was used to detect the target mRNA expression level on ABI Prism 7900 real-time quantitative PCR with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference. The relative expression of target mRNA was calculated with the 2-ΔΔCt method.
Western blot
Untransfected and transfected cells treated with IL-17A or tBHQ were lysed with RIPA lysis buffer. Protein concentration was determined by a bicinchoninic acid (BCA) protein detection kit. Total protein was subjected to SDS-PAGE electrophoresis and electrically transferred to the polyvinylidene difluoride (PVDF) membrane. The protein strips were blocked with 5% milk powder at room temperature for 1 h, and the corresponding primary antibody (Abcam, England) was added and incubated overnight at 4°C. An appropriate secondary antibody was added and incubated at room temperature for 1 h. The protein strips were exposed to enhanced chemiluminescence reagent. Grayscale values of protein bands were analyzed by Quantity One software with GAPDH used as an internal reference.
CCK8 assay
Cells at the logarithmic growth stage were selected and seeded into 96-well plates at a density of 2.5 × 103 cells/well (100 μL), with three wells for each group. 10 μL of CCK-8 solution was added to each well. After incubation for 2 h at 37°C with 5% CO2, the absorbance at 450 nm was detected by a microplate reader.
EdU staining
The proliferation of NHEKs was stained with BeyoClick™ EdU-488 following the manufacturer’s protocol (Beyotime, Shanghai, China). Cells were fixed with 4% paraformaldehyde and then treated with PBS containing 0.3% Triton X-100. The cells were observed under a fluorescence microscope.
ELISA assay
Commercial sandwich ELISA (Beyotime, Shanghai, China) was used to determine the expression levels according to the manufacturer’s protocol.
Statistical analysis
Experimental data are expressed as the mean ± standard deviation. The statistical difference among different groups was analyzed by one-way analysis variance (ANOVA), followed by Tukey’s test. GraphPad Prism 8.0 was used to perform the analysis. P ˂ 0.05 was determined as significant.
Result
SIRT5 overexpression decreases cell proliferation, inflammation, and barrier dysfunction
Compared with the Vehicle group, IL-17A induced decreased SIRT5 levels in a dose-dependent manner, indicating that abnormally low expression of SIRT5 might have a role in psoriasis (Figures 1A and 1B). As shown in the results, the overexpression of SIRT5 was induced after the transfection of Ov-SIRT5 plasmids compared with Ov-NC (Figures 2A and 2B). SIRT5 overexpression is also accompanied by decreased cell viability and proliferation in IL-17A-treated keratinocytes when compared with the cotreatment group of IL-17A and Ov-NC (Figures 2C and 2D). To determine whether SIRT5 modulates the expression of inflammatory factors, we first analyzed the levels of IL-8, IL-1β, and IL-6. The results revealed that the levels of these interleukins decreased upon SIRT5 overexpression compared with the Ov-NC group in IL-17A-induced keratinocytes (Figure 3A). In addition, SIRT5 overexpression decreased the p-p65 expression level (Figure 3B), indicating the regulatory role of SIRT5 in inflammation.
Figure 1 IL-17A decreased SIRT5 expression. (A, B) The expression of SIRT5 by RT-qPCR and western blot assay. **P ˂ 0.01, ***P ˂ 0.001.
Figure 2 SIRT5 overexpression decreased proliferation in IL-17A-induced keratinocytes. (A, B) The expression levels of SIRT5 by RT-qPCR and western blot assay. (C) The cell viability levels by CCK8 assay. (D) EdU staining analysis for cell proliferation. **P ˂ 0.01, ***P ˂ 0.001.
Figure 3 SIRT5 overexpression decreased the levels of inflammatory factors in IL-17A-induced keratinocytes. (A) The levels of IL-8, IL-1β, and IL-6 by ELISA assay. (B) Western blot analysis for p-p65 levels. ***P ˂ 0.001.
The expression of ELOVL1 and ELOVL4 substantially increased in IL-17A-induced keratinocytes compared with that in the OV-NC group (Figure 4A), followed by increased expression of proteins including filaggrin, loricrin, and aquaporin-3 involved in epidermal barrier (Figures 4B and 4C). We further observed that keratinocytes exhibited a significant decrease in p-ERK and STAT3 levels upon SIRT5 overexpression compared with the Ov-NC group, while ERK levels had no significant changes in IL-17A-treated keratinocytes (Figure 4D).
Figure 4 SIRT5 overexpression decreased barrier dysfunction. (A) RT-qPCR analysis for ELOVL1 and ELOVL4 expression. (B, C) RT-qPCR and western blotting analysis for the expression of filaggrin, loricrin, and aquaporin-3. (D) Western blotting analysis for the expression of p-ERK, ERK, and STAT3. **P ˂ 0.01, ***P ˂ 0.001.
SIRT5 is associated with the ERK/STAT3 pathway
We next analyzed whether the effects of SIRT5 on IL-17A-induced keratinocytes were associated with ERK/STAT3 pathway. tBHQ was used as an ERK1/2 activator. Keratinocytes exhibited a significant increase in p-ERK and STAT3 levels upon tBHQ addition compared with the cotreatment group of IL-17A and Ov-SIRT5 (Figure 5A). However, keratinocytes showed no significant changes in ERK expression. tBHQ treatment resulted in increased cell viability and destroyed the effects of SIRT5 overexpression in IL-17A-treated keratinocytes (Figure 5B). tBHQ was further observed to reverse the effects of SIRT5 overexpression on cell proliferation by EdU staining compared with the cotreatment group of IL-17A and Ov-SIRT5 (Figure 5C), suggesting that SIRT5 overexpression reduces cell proliferation via ERK/STAT3 signaling. To test whether ERK/STAT3 signaling had a similar regulatory role in inflammation and barrier function in the context of SIRT5 overexpression, we used tBHQ to treat cells following the transfection of SIRT5 overexpression in IL-17A-treated keratinocytes. The decreased levels of IL-8, IL-1β, IL-6 (Figure 6A), and p-p65 (Figure 6B) upon SIRT5 overexpression were significantly increased by tBHQ addition, as well as ELOVL1 and ELOVL4 (Figure 6C), and also filaggrin, loricrin, and aquaporin-3 (Figures 6D and 6E), suggesting that ERK/STAT3 signaling is involved in the effects of SIRT5 against IL-17A-induced inflammation and barrier dysfunction.
Figure 5 SIRT5 against proliferation. (A) Western blotting analysis for the expression of p-ERK, ERK, and STAT3. (B) Keratinocyte viability levels by CCK8 assay. (C) Keratinocyte proliferation levels by EdU staining. *P ˂ 0.05, **P ˂ 0.01, ***P ˂ 0.001.
Figure 6 SIRT5 against inflammation and barrier dysfunction. (A) IL-8, IL-1β and IL-6 levels by ELISA assay. (B) Western blotting analysis for the expression of p-p65 and p65. (C) The expression of ELOVL1 and ELOVL4 by RT-qPCR analysis. (D, E) The expression of filaggrin, loricrin, and aquaporin-3 by RT-qPCR and western blotting analysis. **P ˂ 0.01, ***P ˂ 0.001.
Discussion
Psoriasis is a typical chronic inflammatory skin disease characterized by epidermal hyperplasia and impaired skin barrier function. A large number of studies have confirmed that excessive proliferation and abnormal differentiation of keratinocytes are secondary phenomena caused by immune activation. Keratinocytes overexpress several inflammatory mediators and amplify local immune responses.12 Ceramide (CER) synthesized in epidermal keratinocytes functions as a major component of the extracellular lipid matrix and is involved in maintaining normal barrier function.13,14 ELOVL1 and ELOVL4 are involved in the production of CERs with very long-chain Fas.15,16 Thus, we determined the expression of ELOVL. Our findings indicate that SIRT5 overexpression leads to reductions in proliferation and expression of inflammatory factors, as well as increased expression of ELOVL1 and ELOVL4, filaggrin, loricrin, and aquaporin-3 in IL-17A-induced keratinocytes, indicating a protective role of SIRT5 in psoriasis. Mechanically, this beneficial role is confirmed to be linked to ERK/STAT3. The levels of p-ERK and STAT3 were significantly decreased after SIRT5 overexpression. SIRT5 is found to be able to inhibit the expression of ERK.6 Further, the ERK1/2 activator profoundly reverses the effects of SIRT5 overexpression on proliferation, inflammation, and barrier function damage. STAT3 is widely involved in the role of promoting psoriasis17 and can be activated by IL-17A.18 Effective inhibition of STAT3 is crucial to alleviate psoriasis. Existing studies have shown that the ERK signal can regulate the expression of STAT3.19 Previous studies have suggested that inhibition of ERK signal can reduce the proliferation of keratinocytes.20 In the pathogenesis of psoriasis, excessive proliferation of keratinocytes is the direct cause of the development of psoriasis, and STAT3 can promote the proliferation and differentiation of cells.21,22 Additionally, ERK/STAT3 signal participates in promoting the barrier function damage in keratinocytes.23 This study implied that the decrease in proliferation and reduction in barrier function damage by SIRT5 overexpression were mediated by ERK/STAT3 in keratinocytes in response to IL-17A.
In response to IL-17A, keratinocytes expressed increased IL-8, IL-1β, and IL-6 levels, as well as increased p-p65. It was reported that keratinocytes trigger the NF-κB signaling pathway to upregulate the expression levels of proinflammatory factors.24 The SIRT5 overexpression could blunt the impacts of IL-17A on these inflammatory mediators, while tBHQ addition was able to significantly reverse this effect, suggesting that keratinocytes altered the SIRT5/ERK pathway to regulate the NF-κB signaling pathway in response to IL-17A. Regarding the regulatory role of SIRT5 in NF-κB, a study found that SIRT5 promoted acetylation of p65 to regulate NF-κB pathway and affect the expression of downstream cytokines.25 This study identified a potential regulatory mechanism of SIRT5 involved in modulating NF-κB by regulating ERK/STAT3 to downregulate the phosphorylated levels of p65.
Our data indicate that ERK/STAT3 is required for the role of SIRT5 in IL-17A-stimulated keratinocytes in decreasing proliferation, inflammatory response, and epidermal barrier dysfunction. Future studies will shed light on how SIRT5 modulates ERK/STAT3 to play a role in psoriasis.