Background The clinical features of keloids consist of aberrant proliferation, secretion, differentiation and apoptosis of keloid dermis-derived fibroblasts (KFBs). Notably, the apoptosis rate of KFBs is lower than the proliferation rate. Though the anti-fibrotic effect of adipose-derived stem cells (ADSCs) on keloids has become a hot topic of research, the exact anti-fibrotic mechanism of the paracrine effect remains unclear. This study aimed to find out how the conditioned medium of ADSCs (ADSC-CM) exerts an anti-fibrotic effect in KFBs. Methods KFBs and ADSCs were extracted and cultured. Then, ADSC-CM was prepared. Whether ADSC-CM could inhibit KFB growth and induce apoptosis was verified by the use of a cell counting kit-8, an 5-Ethynyl-2-deoxyuridine (Edu) kit and flow cytometry. The expressions of cyclooxygenase-1 (COX-1), COX-2, caspase 3 and B-cell lymphoma-2 (Bcl-2) in ADSC-CM-cultured KFBs were tested by real-time PCR and western blotting. To clarify the role of COX-2 in ADSC-CM-induced KFB apoptosis, a specific COX-2 inhibitor, celecoxib, was applied to KFBs cultured in ADSC-CM. Moreover, we tested the production of arachidonic acid (AA) and prostaglandin E2 (PGE2) by ELISA. Then, we established a keloid transplantation model in a nude mouse to validate the therapeutic effect in vivo. Results The proliferation ability of KFBs cultured in ADSC-CM was found to be weakened and apoptosis was significantly increased. Caspase 3 expression was significantly upregulated and Bcl-2 was downregulated in ADSC-CM-cultured KFBs. Furthermore, ADSC-CM strikingly elevated COX-2 mRNA and protein expressions, but COX-1 expression was unaltered. COX-2 inhibitors reduced ADSC-CM-induced apoptosis. Additionally, COX-2 inhibition blocked the elevation of caspase 3 and reversed the decrease in Bcl-2 expression. ADSC-CM increased PGE2 levels by 1.5-fold and this effect was restrained by COX-2 inhibition. In the nude mouse model, expressions of AA, COX-2 and PGE2 were higher in the translated keloid tissues after ADSC-CM injection than in the controls. Conclusions We showed activation of the COX-2/PGE2 cascade in KFBs in response to ADSC-CM. By employing a specific COX-2 inhibitor, COX-2/PGE2 cascade activation played a crucial role in mediating the ADSC-CM-induced KFB apoptosis and anti-proliferation effects.

中文翻译：

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脂肪干细胞通过旁分泌的花生四烯酸衍生的环氧合酶-2/前列腺素E2级联抑制真皮成纤维细胞生长并诱导瘢痕疙瘩细胞凋亡

背景 瘢痕疙瘩的临床特征包括瘢痕疙瘩真皮源性成纤维细胞（KFBs）的异常增殖、分泌、分化和凋亡。值得注意的是，KFBs的凋亡率低于增殖率。尽管脂肪干细胞（ADSCs）对瘢痕疙瘩的抗纤维化作用已成为研究热点，但其旁分泌作用的确切抗纤维化机制仍不清楚。本研究旨在了解 ADSCs 条件培养基 (ADSC-CM) 如何在 KFBs 中发挥抗纤维化作用。方法提取KFBs和ADSCs并进行培养。然后，制备ADSC-CM。通过使用细胞计数试剂盒-8、5-乙炔基-2-脱氧尿苷 (Edu) 试剂盒和流式细胞术验证 ADSC-CM 是否可以抑制 KFB 生长和诱导细胞凋亡。环氧合酶-1 (COX-1)、COX-2、通过实时 PCR 和蛋白质印迹检测 ADSC-CM 培养的 KFB 中的半胱天冬酶 3 和 B 细胞淋巴瘤 2 (Bcl-2)。为了阐明 COX-2 在 ADSC-CM 诱导的 KFB 细胞凋亡中的作用，将一种特定的 COX-2 抑制剂塞来昔布应用于在 ADSC-CM 中培养的 KFB。此外，我们通过ELISA测试了花生四烯酸（AA）和前列腺素E2（PGE2）的产生。然后，我们在裸鼠体内建立了瘢痕疙瘩移植模型，以验证体内治疗效果。结果ADSC-CM培养的KFBs增殖能力减弱，细胞凋亡明显增加。在 ADSC-CM 培养的 KFB 中，Caspase 3 表达显着上调，Bcl-2 下调。此外，ADSC-CM 显着提高了 COX-2 mRNA 和蛋白质的表达，但 COX-1 的表达没有改变。COX-2 抑制剂减少了 ADSC-CM 诱导的细胞凋亡。此外，COX-2 抑制阻断了 caspase 3 的升高并逆转了 Bcl-2 表达的下降。ADSC-CM 将 PGE2 水平提高了 1.5 倍，并且这种效应受到 COX-2 抑制的抑制。在裸鼠模型中，ADSC-CM 注射后，翻译的瘢痕疙瘩组织中 AA、COX-2 和 PGE2 的表达高于对照组。结论 我们展示了响应 ADSC-CM 的 KFB 中 COX-2/PGE2 级联反应的激活。通过使用特定的 COX-2 抑制剂，COX-2/PGE2 级联激活在介导 ADSC-CM 诱导的 KFB 细胞凋亡和抗增殖作用中起关键作用。5 倍，这种效果受到 COX-2 抑制的抑制。在裸鼠模型中，ADSC-CM 注射后，翻译的瘢痕疙瘩组织中 AA、COX-2 和 PGE2 的表达高于对照组。结论 我们展示了响应 ADSC-CM 的 KFB 中 COX-2/PGE2 级联反应的激活。通过使用特定的 COX-2 抑制剂，COX-2/PGE2 级联激活在介导 ADSC-CM 诱导的 KFB 细胞凋亡和抗增殖作用中起关键作用。5 倍，这种效果受到 COX-2 抑制的抑制。在裸鼠模型中，ADSC-CM 注射后，翻译的瘢痕疙瘩组织中 AA、COX-2 和 PGE2 的表达高于对照组。结论 我们展示了响应 ADSC-CM 的 KFB 中 COX-2/PGE2 级联反应的激活。通过使用特定的 COX-2 抑制剂，COX-2/PGE2 级联激活在介导 ADSC-CM 诱导的 KFB 细胞凋亡和抗增殖作用中起关键作用。