Hydrogen Sulfide Suppresses Skin Fibroblast Proliferation via Oxidative Stress Alleviation and Necroptosis Inhibition

Keloid is a common dermatofibrotic disease with excessive skin fibroblast proliferation. Hydrogen sulfide (H2S) as the third gasotransmitter improves fibrosis of various organs and tissues. Our study is aimed at investigating the effects and possible mechanisms of H2S on skin fibroblast proliferation. Scar tissues from six patients with keloid and discarded skin tissue from six normal control patients were collected after surgery, respectively. Plasma H2S content and skin H2S production in patients with keloid were measured. Keloid fibroblasts and transforming growth factor-β1- (TGF-β1, 10 ng/mL) stimulated normal skin fibroblasts were pretreated with H2S donor as NaHS (50 μM) for 4 h. Cell migration after scratch was assessed. The expressions of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), collagen I, and collagen III were detected by immunofluorescence, real-time PCR, and/or Western blot. Intracellular superoxide anion and mitochondrial superoxide were evaluated by dihydroethidium (DHE) and MitoSOX staining, respectively. Mitochondrial membrane potential was detected by JC-1 staining. Apoptotic cells were detected by TDT-mediated dUTP nick end labeling (TUNEL). The expressions of receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) were measured by Western blot. We found that H2S production was impaired in both the plasma and skin of patients with keloid. In keloid fibroblasts and TGF-β1-stimulated normal skin fibroblasts, exogenous H2S supplementation suppressed the expressions of α-SMA, PCNA, collagen I, and collagen III, reduced intracellular superoxide anion and mitochondrial superoxide, improved the mitochondrial membrane potential, decreased the positive rate of TUNEL staining, and inhibited RIPK1 and RIPK3 expression as well as MLKL phosphorylation. Overall, H2S suppressed skin fibroblast proliferation via oxidative stress alleviation and necroptosis inhibition.


Introduction
Keloid is a common dermatofibrotic disease with main characteristics of excessive skin fibroblast proliferation and extracellular matrix accumulation [1]. Keloid is usually regarded as a benign skin tumor according to clinical manifestation, gene phenotype, pathological features, and prognosis [2]. Keloid has become a cosmetic problem because most protruding scar tissues are beyond the epidermis with obvious pigmentation. In addition, patients suffering from keloid usually feel pruritus and pain, which even cause psychological problems such as inferiority and anxiety [3].
Keloid can be treated by corticosteroid injection, radiotherapy, cryotherapy, 5-fluorouracil, tamponade, laser, and surgery [4]. However, there is still a high recurrence rate because of the lack of "gold standard" for keloid treatment [5]. Therefore, seeking effective interventions for keloid is a hot topic in clinic.
Transforming growth factor-β 1 (TGF-β 1 ) is recognized as one of the most important factors for fibrosis [6]. Previous studies have confirmed that the content of TGF-β 1 in keloid was significantly increased, while TGF-β 1 inhibition prevented keloid fibroblast proliferation and attenuated extracellular matrix synthesis [7]. However, TGF-β 1 blockage is prone to causing extensive damage by diversified biological effects due to nonspecific inhibition. It is necessary to explore novel strategies to suppress keloid fibroblast proliferation and improve dermatofibrotic diseases.
Hydrogen sulfide (H 2 S) is the third gasotransmitter with multiple functions, which plays a vital role in a variety of physiological and pathological processes [8]. H 2 S is mainly generated by catalysis of cystathionine γ-lyase (CSE) in skin [9,10]. Previous researches showed that H 2 S promoted skin wound healing by inflammation inhibition, oxidative stress suppressing, and vascular endothelial growth factor (VEGF) enhancement [11][12][13][14]. H 2 S supplement significantly alleviated skin pruritus in mice [15]. H 2 S also induced apoptosis and blocked the activation of nuclear factors-κB (NF-κB) to inhibit melanoma cells growth and prevent tumor progression [16,17]. That is to say, H 2 S showed protective effects against various skin diseases. Additionally, previous studies found that H 2 S inhibited the activation of fibrosisrelated cells and cytokine expression, then alleviated renal fibrosis, and improved renal function in the rats with renal sclerosis caused by renal tubular epithelial mesenchymal transition (EMT) [18]. Exogenous H 2 S significantly decreased the level of TGF-β 1 in vivo, increased the expression of aortic elastin, and prevented diabetic nephropathy arteriosclerosis in rats [19]. H 2 S improved myocardial fibrosis via suppressing the TGF-β 1 /Smad signaling pathway [20]. H 2 S inhibited the TGF-β 1 /Smad signaling pathway, improved lung function, and alleviated dermatitis, pulmonary alveoli injury, and chronic obstructive pulmonary fibrosis in cigarette smoke-induced chronic obstructive pulmonary disease of rats [21,22]. The evidence demonstrated that H 2 S had potential effects on fibrotic diseases [23]. However, the possible effect of H 2 S on keloid and other dermatofibrotic disease remains unknown. A recent study showed that the impairment of endogenous H 2 S aggravated mitochondrial damage, increased reactive oxygen species (ROS), and induced necroptosis to aggravate diabetic cardiomyopathy. Exogenous H 2 S improved mitochondrial function, inhibited oxidative stress, and reduced necroptosis to improve diabetic cardiomyopathy [24]. Moreover, H 2 S donors inhibited necroptosis and alleviated hypoxiainduced myocardial fibroblast proliferation depending on sirtuin 3 [25]. However, whether oxidative stress and necroptosis were involved in the possible effect of H 2 S on skin fibroblast proliferation has not been known well.
Therefore, NaHS, a common H 2 S donor, was applied to investigate whether exogenous H 2 S supplementation could reverse excessive proliferation in keloid fibroblasts and in TGF-β 1 -induced normal skin fibroblasts. In addition, the mechanisms of H 2 S on skin fibroblast proliferation were further explored from the perspective of oxidative stress and necroptosis. It is beneficial to provide a novel strategy for the prevention and treatment of dermatofibrotic diseases including keloid, which has a potential clinical transformation prospect.

Materials and Methods
2.1. Collection of Human Skin Samples. Scar tissues from six patients with keloid and discarded skin tissue from six nor-mal control patients without any skin diseases were collected after surgery, respectively. All cases of keloid and normal control were free of infection, tumor, cardiovascular diseases, renal diseases, diabetes mellitus, and other systemic diseases. All patients were not subjected to any local drug injection, radiation, or surgical treatment within the latest 2 years, and any topical drug treatment within the latest 1 month. Plasma was also collected from the above patients. The study strictly followed the requirements of the Declaration of Helsinki. All patients signed a written informed consent. The research was approved by the ethics committee of Affiliated Hospital of Nantong University (No. 2020-L146).

Determination of H 2 S Levels in Plasma and Skin.
After polarization, H 2 S-specific microelectrode was inserted into the plasma. The current change was measured by the connected free radical analyzer (World Precision Instruments Inc., Sarasota, Florida, USA). H 2 S concentration was calculated according to the standard curve drawn by the standard concentration and corresponding currents. The concentration of H 2 S in plasma was calibrated with that in normal control.
The collected skin samples were cut into pieces and homogenized (1/10, w/v) in K 2 PO 4 (100 mM). Then 460 μL homogenization buffer was taken, and 20 μL Lcysteine (10 mM) and 20 μL pyridoxal-5 ′ -phosphate (2 mM) were sequentially added. The H 2 S level was measured as the above methods. H 2 S production in the skin was calculated according to the standard curve and normalized with protein concentration, which were calibrated with that in normal control.

Primary Skin Fibroblasts Culture and Treatment.
After removing epidermis and surrounding fat, skin samples were cut into pieces of 1 mm 3 , cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, New York, USA) with 10% fetal bovine serum (FBS, Gibco, Grand Island, New York, USA) and subcultured for further experiments. Keloid fibroblasts from keloid were pretreated with NaHS (50 μM, Sigma-Aldrich, St. Louis, MO) for 4 h. Skin fibroblasts from normal skin were pretreated with NaHS (50 μM) for 4 h followed with or without TGF-β 1 (10 ng/ml, PeproTech, Suzhou, China) stimulation for 12 h. The same amount of culture medium was given as a control group.

Cell Migration Assay after Scratch.
After the above treatment, skin fibroblasts were scratched with pipette tips of 10 μL, and the shedding cells were washed out with PBS. Cell migration was recorded by photograph after scratch for 0, 4, 8, 12, and 24 h. The migration rate of cells was calculated as the ratio of the migrated area to the original area.    Figure 1: H 2 S production was impaired in patients with keloid. Scar tissues from patients with keloid (Keloid) and discarded skin tissue from normal control patients (Control) were collected after surgery, respectively. Plasma was also collected from above patients. (a) Plasma H 2 S content was detected. (b) Skin H 2 S production was assessed. * * P < 0:01 vs. control, n = 6.       anti-β-tubulin (1 : 3000; CMCTAG, Milwaukee, WI, USA), or anti-β-actin (1 : 5000; ABclonal, Wuhan, China) antibodies at 4°C overnight. Next, the secondary antibody (1 : 5000; Beyotime, Shanghai, china) was incubated for 2 h at room temperature. The protein bands were visualized on the membrane with enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) solution.
2.12. Statistical Analysis. All data were expressed as the mean ± standard error of the mean (SEM) and analyzed by t test or one-way ANOVA followed by the Student-

H 2 S Production Is Impaired in Patients with
Keloid. H 2 S levels were measured in plasma and skin tissues of patients. There were lower plasma H 2 S content and skin H 2 S production in patients with keloid than that in normal control (P < 0:01) (Figure 1), suggesting that H 2 S production was impaired in patients with keloid.

NaHS Inhibits Keloid Fibroblast Migration after Scratch.
However, whether impaired H 2 S production was critical in the pathological process of keloid was unknown. Next, we investigated the influence of restoring H 2 S level on keloid fibroblast proliferation. The scratch migration assay showed that the migration rate of keloid fibroblasts after scratch was faster than that of normal skin fibroblasts (P < 0:01), which was significantly reduced by NaHS pretreatment (P < 0:01) (Figure 2). It suggested that exogenous H 2 S supplementation inhibited keloid fibroblast migration after scratch.

NaHS Suppresses α-SMA Expression in Keloid
Fibroblasts. α-SMA is one of the sensitive markers to indicate cells proliferation [26]. Therefore, the expression of α-SMA was detected in cells with real-time PCR, immunofluorescence, and Western blot. All these results consistently showed that there was more α-SMA expression in keloid fibroblasts than that in normal skin fibroblasts (P < 0:01), which was significantly suppressed by NaHS pretreatment (P < 0:01) (Figure 3). It demonstrated that exogenous H 2 S supplementation inhibited keloid fibroblast proliferation.  Figure 7: NaHS improved mitochondrial membrane potential in keloid fibroblasts. Skin fibroblasts were extracted from skin tissues of normal controls (normal skin) and scar tissue from patients with keloid (keloid), respectively. After pretreatment with NaHS (50 μM) or the same amount of culture medium (control) for 4 h, the fresh culture medium was replaced. After 12 h, mitochondrial membrane potential was detected by JC-1 staining. JC-1 aggregates (red) and JC-1 monomers (green) were observed. The nuclei were stained with DAPI (blue). Bar = 25 μm.

NaHS Inhibits PCNA Expression in Keloid Fibroblasts.
PCNA, another sensitive indicator of skin fibroblast proliferation, plays an important role in DNA replication [27]. Therefore, PCNA was also detected to verify the influence of H 2 S on keloid fibroblast proliferation. Real-time PCR, immunofluorescence, and Western blot demonstrated that PCNA expression was higher in keloid fibroblasts than that in normal skin fibroblasts (P < 0:01), which was significantly suppressed by NaHS pretreatment (P < 0:01) (Figure 4), further indicating that exogenous H 2 S supplementation inhibited keloid fibroblast proliferation.

NaHS Blocks Collagen Synthesis in Keloid Fibroblasts.
Then, the content of collagen I and collagen III was measured to evaluate the influence of H 2 S on collagen synthesis.
Both real-time PCR and immunofluorescence verified that compared with normal skin fibroblasts, the expression of collagen I and collagen III in keloid fibroblasts was significantly higher than that in normal skin fibroblasts (P < 0:01), which was reduced by NaHS pretreatment (P < 0:01) ( Figure 5). It suggested that exogenous H 2 S supplementation blocked collagen synthesis in keloid fibroblasts.

NaHS Attenuates Oxidative Stress in Keloid Fibroblasts.
DHE staining and MitoSOX staining were applied to detect the level of intracellular superoxide anion and mitochondrial superoxide, respectively. Compared with normal skin fibroblasts, red fluorescence by DHE and MitoSOX staining in keloid fibroblasts was stronger, which was weakened by NaHS pretreatment (Figure 6), demonstrating that  Oxidative Medicine and Cellular Longevity intracellular superoxide anion and mitochondrial superoxide were both inhibited by NaHS. It suggested that exogenous H 2 S supplementation attenuated oxidative stress in keloid fibroblasts.

NaHS Improves Mitochondrial Membrane Potential in
Keloid Fibroblasts. Decreased mitochondrial membrane potential (Δψm) might contribute to mitochondrial injury and ROS accumulation [28]. Red fluorescence by JC-1 staining indicated normal mitochondria with higher Δψm, while green indicated impaired mitochondria with lower Δψm. Our experiment demonstrated that compared with normal skin fibroblasts, green fluorescence was increased, but red fluorescence was decreased in keloid fibroblasts, while NaHS pretreatment significantly enhanced red and weakened green fluorescence (Figure 7), suggesting that exogenous H 2 S supplementation improved mitochondrial membrane potential to alleviate mitochondrial damage in keloid fibroblasts.

NaHS Alleviates Necroptosis in Keloid Fibroblasts.
Due to the intercellular genomic DNA breakage and 3 ′ OH exposing at the early stage, apoptotic cells were able to be marked by TUNEL staining, which is also one characteristic of necroptosis [29]. Necroptosis is strictly regulated by multiple molecules. RIPK1 and RIPK3, two key signal molecules to mediate necroptosis, are commonly considered specific indicators of necroptosis. In addition, MLKL is a core substrate of RIPK3 downstream to mediate necroptosis. Therefore, TUNEL staining and the above proteins measurement were applied to evaluate cell necroptosis. Compared with normal skin fibroblasts, there were more TUNEL stainingpositive cells, higher RIPK1 and RIPK3 expressions as well as MLKL phosphorylation in keloid fibroblasts (P < 0:01), which were all reversed by NaHS pretreatment (P < 0:01) ( Figure 8). These data indicated that exogenous H 2 S supplementation alleviated necroptosis in keloid fibroblasts.

NaHS Inhibits TGF-β 1 -Stimulated Skin Fibroblast
Migration after Scratch. The above results demonstrated that exogenous H 2 S supplementation inhibited keloid fibroblast proliferation. However, whether the above effects could be extended to other dermatofibrotic diseases is unknown. Therefore, normal skin fibroblasts were extracted and stimulated with TGF-β 1 . Skin fibroblast migration with TGF-β 1 stimulation after scratch was faster than normal skin fibroblasts (P < 0:01), which was reversed by NaHS pretreatment (P < 0:01) (Figure 9). It suggested that exogenous H 2 S supplementation inhibited TGF-β 1 stimulated skin fibroblast migration after scratch.

NaHS Blocks Collagen Synthesis in TGF
3.13. NaHS Attenuates Oxidative Stress in TGF-β 1 -Stimulated Skin Fibroblasts. Compared with normal skin fibroblasts, TGF-β 1 stimulation enhanced red fluorescence in skin fibroblasts, which was weakened by NaHS pretreatment (Figure 13), and demonstrated that intracellular super-oxide anion and mitochondrial superoxide were inhibited by NaHS. It suggested that exogenous H 2 S supplementation attenuated oxidative stress in TGF-β 1 -stimulated skin fibroblasts.

Oxidative Medicine and Cellular Longevity
These results suggested that exogenous H 2 S supplementation alleviated necroptosis in TGF-β 1 -stimulated skin fibroblasts.

Discussion
As an endogenous gasotransmitter, H 2 S plays an important role in a variety of pathophysiological processes. H 2 S dysfunction is reported to be involved in the pathogenesis of hypertension, diabetes, atherosclerosis, and tumor [30][31][32].
Previous studies showed that excessive H 2 S production induced thyroid malignancy, deteriorated pancreatitis, and lung injury [33,34], while impaired H 2 S production promoted obesity and aggravated insulin resistance [35]. In addition, H 2 S content was significantly decreased in patients with psoriasis, which was negatively correlated with the severity of clinical symptoms [36,37]. H 2 S also facilitated melanoma cell apoptosis and inhibited tumor growth by upregulating Fas-associated protein with a novel death domain (FADD) [38]. Exogenous H 2 S suppressed melanoma cells migration and invasion to delay melanoma progression and metastasis formation [39]. Interestingly, a novel drug carrier nanofibrous membrane, blending the recombinant spider silk protein and NaHS by electrospun, 11 Oxidative Medicine and Cellular Longevity promoted skin wound healing [40]. These studies suggest that H 2 S has a potential effect on skin diseases.
Keloid is a skin benign fibroproliferative disease, and the definite pathogenesis of keloid is not clear. We found that the H 2 S level in the plasma and skin of patients with keloid was notably decreased, suggesting that H 2 S production impairment might be involved in human skin fibroblast proliferation. Previous studies have verified that H 2 S improved multiple fibrotic diseases. Specifically, exogenous H 2 S prevented myocardial fibrosis during myocardial infarction, hypertension, and diabetes [41]. H 2 S also attenuated vascular fibrosis by regulating endothelial mesenchymal transition (EndMT) [42]. H 2 S alleviated hypoxia-induced chronic renal fibrosis via restoring ten-eleven translocations (TET), reversing DNA methylation, and enhancing Klotho gene expression [43]. Our present study found that NaHS pretreatment slowed down keloid fibroblast migration, decreased the expression of α-SMA, PCNA, collagen I, and collagen III. It suggested that exogenous H 2 S supplementation inhibited keloid fibroblast proliferation. Then we further explored the possible effect of H 2 S on some other fibrotic diseases. It is widely known that the excess activation of TGF-β 1 signaling pathway contributed to the occurrence and development of dermatofibrotic diseases including keloid [44]. Therefore, TGF-β 1 stimulation is a common experimental strategy to induce skin fibroblast proliferation and imitate dermatofibrotic diseases. Our results showed that TGF-β 1 stimulation accelerated skin fibroblast migration and upregulated the expression of α-SMA, PCNA, collagen I, and collagen III, suggesting that TGF-β 1 successfully induced critical characteristics of skin fibrosis in vitro. Similar to the preventive effects against keloid fibroblasts, NaHS pretreatment also inhibited cell migration and suppressed the expression of α-SMA, PCNA, collagen I, and collagen III in TGF-β 1 -stimulated skin fibroblasts. Taken together, the above evidence indicates that H 2 S possibly has a powerful therapeutic effect on skin fibrosis, which is conducive to developing novel ideas for the prevention and treatment of dermatofibrotic diseases. Previous studies verified that H 2 S attenuated oxidative stress, regulated glucose and fat metabolism, and improved mitochondrial function to alleviate liver cirrhosis [45]. H 2 S inhibited inflammation and oxidative stress, reduced the expression of Toll like receptor 4 (TLR4), NOD like receptor family pyrin domain containing 3 (NLRP3), and caspase-1, improved the renal function and renal histopathological changes, and alleviated kidney injury in sepsis-associated acute kidney injury (SA-AKI) [46]. H 2 S also inhibited oxidative stress to correct the dysfunction in neurodegenerative diseases [47]. H 2 S attenuated paraquat-induced acute liver injury via enhancing antioxidant capacity, regulating mitochondrial function, and inhibiting NLRP3 inflammasome activation [48]. All these studies indicated that the antioxidant effect of H 2 S might be one of the common mechanisms to improve fibrosis in various organs and tissues. DHE, MitoSOX, and JC-1 staining in our experiment confirmed that NaHS pretreatment decreased intracellular superoxide anion and mitochondrial superoxide but increased mitochondrial membrane potential in keloid fibroblasts and TGF-β 1 -stimulated normal skin fibroblasts, suggesting that exogenous H 2 S supplementation attenuated oxidative stress. These results indicated that the antiproliferation effect of H 2 S on skin fibroblasts might be ascribed to oxidative stress inhibition.
Intracellular ROS accumulation can induce cell necroptosis, which is a new type of programmed cell death with the characteristics of both apoptosis and necrosis. Necroptosis plays an important role in maintaining cell homeostasis, regulating tumor immunity and inflammatory response, and mediating ischemic injury and several other pathophysiological processes [49,50]. As two important mediators of necroptosis, RIPK1 combines with RIPK3 to form necrosomes and phosphorylate MLKL, which then transfers to cell membrane and ruptures it, and exaggerates cell necroptosis finally [51]. Due to the enhanced oxidative stress, necroptosis was consequently increased in mice with chronic hepatitis [52]. Some researchers found that ROS accumulation in melanocytes' mitochondria promoted necrosome formation and aggravated necroptosis in the pathogenesis of vitiligo [53]. Our present experiment showed that NaHS pretreatment downregulated the expression of RIPK1 and RIPK3, inhibited MLKL phosphorylation, and decreased the TUNEL staining-positive rate in keloid fibroblasts and TGF-β 1 -stimulated normal skin fibroblasts, suggesting that exogenous H 2 S supplementation alleviated necroptosis in both keloid fibroblasts and TGF-β 1 -stimulated normal skin fibroblasts. However, besides expression of RIPK1, RIPK3, and MLKL, some other proteins including cleaved caspase  Figure 13: NaHS attenuated oxidative stress in TGF-β 1 -stimulated skin fibroblasts. Skin fibroblasts were extracted from skin tissues of normal controls (normal skin). After pretreatment with NaHS (50 μM) for 4 h, skin fibroblasts were stimulated with TGF-β 1 (10 ng/mL) for 12 h. (a) The intracellular superoxide anion was stained by DHE (red). The nuclei were stained with DAPI (blue). Bar = 50 μm. (b) Mitochondrial superoxide was stained with MitoSOX (red). The mitochondria were stained with MitoTracker (green). The nuclei were stained with DAPI (blue). Bar = 25 μm. 13 Oxidative Medicine and Cellular Longevity 3, necrosomes, and inflammasomes had not been detected in our present study. Actually, more necroptosis proteins will be a great benefit to validate the effect of H 2 S on necroptosis in skin fibroblasts. This is one limitation of our present study. Considering the powerful antioxidant effects, the suppressive ability of necroptosis by H 2 S may be attributed to the inhibition of oxidative stress. Interestingly, inhibition of skin fibroblasts necroptosis by H 2 S seems to contradict with the preventive effects on proliferation. Actually, a large amount of cellular contents will be released after necroptosis, such as inflammatory factors, cell metabolites, and other substances, which all further promote cell proliferation. Recently, some researchers found that H 2 S attenuated necroptosis depending on sirtuin3 enhancement to suppress excessive proliferation in hypoxia-induced cardiac fibroblasts [25]. That is to say that the mutual regulation between cell death and proliferation is very complex. Therefore, the inhibitory mechanism of H 2 S on skin fibroblast proliferation may involve some other reactions, such as extracellular matrix degradation, inflammation aggravation, and cytokine release, which need future research.
In conclusion, H 2 S production was impaired in the plasma and skin of patients with keloid. Exogenous H 2 S supplementation suppressed proliferation in keloid fibroblasts and TGF-β 1 -stimulated normal skin fibroblasts. The suppressive mechanisms may be related to oxidative stress alleviation and necroptosis inhibition. Our present study is of benefit to provide a novel strategy for the prevention and treatment of keloid and some other dermatofibrotic diseases.

Data Availability
The data used to support the finding of this study are available from the corresponding authors upon request.