背景:瘢痕疙瘩,一种纤维增生性疾病,显著影响患者的生活质量,然而,有效的治疗方法仍然难以捉摸。本研究探讨沉默信息调节因子6(SIRT6)在调节细胞增殖中的作用,入侵,和瘢痕疙瘩成纤维细胞的胶原合成。
方法:收集瘢痕疙瘩和正常皮肤标本,从瘢痕疙瘩组织中分离成纤维细胞。构建SIRT6重组腺病毒(Ad)感染瘢痕疙瘩成纤维细胞以过表达SIRT6。本研究分为三组:对照组,腺病毒阴性对照(Ad-NC)组,和Ad-SIRT6组。通过蛋白质印迹和定量逆转录聚合酶链反应(qRT-PCR)测量SIRT6蛋白和mRNA水平,分别。使用5-乙炔基-2'-脱氧尿苷(EdU)测定法测定细胞活力。利用流式细胞术测量细胞凋亡。为了研究细胞迁移,采用伤口愈合试验和Transwell试验。蛋白质印迹也用于研究凋亡蛋白的表达水平,胶原沉积相关蛋白,和丝裂原激活蛋白激酶(MAPK)/细胞外调节蛋白激酶(ERK)途径相关蛋白。
结果:与对照组和Ad-NC组相比,Ad-SIRT6组SIRT6水平显著升高;细胞增殖减少,迁移和侵袭;α-平滑肌肌动蛋白(α-SMA)的蛋白质水平降低,胶原蛋白I,胶原蛋白III,磷酸SMAD家族成员3(p-Smad3),转化生长因子-β1(TGF-β1),和MAPK/ERK通路蛋白(磷酸化细胞外信号调节蛋白激酶1/2(p-ERK1/2),磷酸MAP激酶-ERK激酶(p-MEK)和磷酸-c-Raf(p-c-Raf)。用表皮生长因子(EGF)治疗,MAPK/ERK通路激动剂,SIRT6逆转了瘢痕疙瘩成纤维细胞对细胞活性的抑制作用,抑制了细胞凋亡。
结论:SIRT6在瘢痕疙瘩成纤维细胞中的过表达减弱增殖,入侵,和胶原蛋白合成,在促进细胞凋亡的同时,可能通过抑制MAPK/ERK通路活性。这表明瘢痕疙瘩治疗的潜在治疗靶标。
BACKGROUND: Keloid, a fibroproliferative disorder, significantly impacts patients\' quality of life, yet effective therapies remain elusive. This study explored the role of silent information regulator 6 (SIRT6) in modulating the proliferation, invasion, and collagen synthesis of keloid fibroblasts.
METHODS: Keloid and normal skin specimens were collected, and fibroblasts were isolated from the keloid tissue. SIRT6 recombinant adenovirus (Ad) was constructed to infect keloid fibroblasts to overexpress SIRT6. This study entails three groups: Control group, adenovirus-Negative Control (Ad-NC) group, and Ad-SIRT6 group. SIRT6 protein and mRNA levels were measured via Western blotting and Quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Cell viability was determined using 5-ethynyl-2\'-deoxyuridine (EdU) assay. Flow cytometry was exploited to measure cell apoptosis. To investigate cell migration, wound healing assay and Transwell assay were employed. Western blotting was also utilized to study the expression levels of apoptotic proteins, collagen deposition-related proteins, and Mitogen-Activated Protein Kinases (MAPK)/extracellular regulated protein kinases (ERK) pathway-related proteins.
RESULTS: Compared to the control and Ad-NC groups, the Ad-SIRT6 group exhibited significantly elevated SIRT6 level; diminished cell proliferation, migration and invasion; reduced protein levels of α-smooth muscle actin (α-SMA), collagen I, collagen III, phospho SMAD Family Member 3 (p-Smad3), transforming growth factor-β 1 (TGF-β1), and MAPK/ERK pathway proteins (phospho extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phospho MAP kinase-ERK kinase (p-MEK) and phospho-c-Raf (p-c-Raf)). Treatment with epidermal growth factor (EGF), an MAPK/ERK pathway agonists, reversed the inhibitory effect of SIRT6 on cell activity and inhibited apoptosis in keloid fibroblasts.
CONCLUSIONS: SIRT6 overexpression in keloid fibroblasts attenuates proliferation, invasion, and collagen synthesis, while fostering apoptosis, likely through the suppression of MAPK/ERK pathway activity. This suggests a potential therapeutic target for keloid treatment.