Li Ling, He Ziying, Zhu Yue, Shen Qiyan, Yang Shengju, Cao Shuanglin
Department of Dermatology, Affiliated Hospital of Nantong University, Nantong 226001, China.
Yancheng No.1 People's Hospital, Yancheng 224001, China.
Oxid Med Cell Longev. 2022 Jun 21;2022:7434733. doi: 10.1155/2022/7434733. eCollection 2022.
Keloid is a common dermatofibrotic disease with excessive skin fibroblast proliferation. Hydrogen sulfide (HS) as the third gasotransmitter improves fibrosis of various organs and tissues. Our study is aimed at investigating the effects and possible mechanisms of HS 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 HS content and skin HS production in patients with keloid were measured. Keloid fibroblasts and transforming growth factor- - (TGF- , 10 ng/mL) stimulated normal skin fibroblasts were pretreated with HS donor as NaHS (50 M) for 4 h. Cell migration after scratch was assessed. The expressions of -smooth muscle actin (-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 HS production was impaired in both the plasma and skin of patients with keloid. In keloid fibroblasts and TGF- -stimulated normal skin fibroblasts, exogenous HS supplementation suppressed the expressions of -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, HS suppressed skin fibroblast proliferation oxidative stress alleviation and necroptosis inhibition.
瘢痕疙瘩是一种常见的皮肤纤维化疾病，伴有皮肤成纤维细胞过度增殖。硫化氢（HS）作为第三种气体信号分子可改善各种器官和组织的纤维化。我们的研究旨在探讨HS对皮肤成纤维细胞增殖的影响及可能机制。分别收集6例瘢痕疙瘩患者术后的瘢痕组织和6例正常对照患者废弃的皮肤组织。检测瘢痕疙瘩患者血浆HS含量和皮肤HS生成量。将瘢痕疙瘩成纤维细胞和转化生长因子-β（TGF-β，10 ng/mL）刺激的正常皮肤成纤维细胞用HS供体硫氢化钠（NaHS，50 μM）预处理4小时。评估划痕后细胞迁移情况。通过免疫荧光、实时PCR和/或蛋白质免疫印迹法检测α-平滑肌肌动蛋白（α-SMA）、增殖细胞核抗原（PCNA）、I型胶原和III型胶原的表达。分别通过二氢乙锭（DHE）和MitoSOX染色评估细胞内超氧阴离子和线粒体超氧。通过JC-1染色检测线粒体膜电位。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测凋亡细胞。通过蛋白质免疫印迹法检测受体相互作用蛋白激酶1（RIPK1）、RIPK3和混合谱系激酶结构域样蛋白（MLKL）的表达。我们发现瘢痕疙瘩患者血浆和皮肤中的HS生成均受损。在瘢痕疙瘩成纤维细胞和TGF-β刺激的正常皮肤成纤维细胞中，外源性补充HS可抑制α-SMA、PCNA、I型胶原和III型胶原的表达，减少细胞内超氧阴离子和线粒体超氧，改善线粒体膜电位，降低TUNEL染色阳性率，并抑制RIPK1和RIPK3表达以及MLKL磷酸化。总体而言，HS通过减轻氧化应激和抑制坏死性凋亡来抑制皮肤成纤维细胞增殖。
