Jiang Hesong, Gao Qingqiang, Che Xiaoyan, Zhu Leilei, Zhang Zheng, Chen Yun, Dai Yutian
Department of Andrology, Drum Tower Hospital, Affiliated to School of Medicine, Nanjing University, Nanjing, Jiangsu 210008, P.R. China.
Exp Ther Med. 2017 Nov;14(5):5149-5156. doi: 10.3892/etm.2017.5179. Epub 2017 Sep 21.
The activation of tunica albuginea myofibroblasts (MFs) serves an essential role in Peyronie's disease (PD). Increasing evidence has reported that adipose tissue-derived stem cells (ADSCs) have been demonstrated to attenuate the symptoms of PD in animal models. However, the mechanisms of the antifibrotic effects of ADSCs in PD remain to be fully elucidated. In the present study, the inhibitory effects and possible mechanism of ADSCs on the activation of MFs derived from rat penile tunica albuginea were investigated. ADSCs were obtained from the paratesticular fat of Sprague Dawley rats. MFs were transformed from rat penile tunica albuginea fibroblasts through stimulation with 5 ng/ml tumor growth factor-β1. Transwell cell cultures were adopted for co-culture of ADSCs and MFs. Western blot analysis was used to assess changes in the expression levels of α smooth muscle actin (αSMA), collagen I, phosphorylated (p)-SMAD family member 2 (Smad2), Smad2, ras homolog family member A (RhoA), Rho associated coiled-coil containing protein kinase (ROCK)1 and ROCK2, caspase3, caspase9, and matrix metalloproteinases (MMPs). Collagen gel assays were used to assess cell contractility. Additionally, the concentration of hydroxyproline in the culture medium was detected using commercially available kits. It was demonstrated that ADSCs reduced the expression of αSMA and collagen I of MFs. Furthermore, p-Smad2, RhoA, ROCK1 and ROCK2 expression was significantly reduced in the MFs+ADSCs group compared with that in the MFs-only culture, while the expression of MMPs (MMP2, MMP3, MMP9 and MMP13) and caspases (caspase3 and caspase9) was upregulated. In addition, ADSCs were able to downregulate the concentration of hydroxyproline in the culture medium of MFs and reverse the contraction of MFs. Collectively, these results suggested that ADSCs inhibited the activation of MFs, decreased collagen production, and suppressed the contraction of myofibroblasts, via Smad and RhoA/ROCK signaling pathways. Furthermore, ADSCs reduced the deposition of collagen and promoted the apoptosis of MFs via MMPs, and caspases. Accordingly, the application of ADSCs may provide a novel therapeutic strategy for PD.
白膜肌成纤维细胞（MFs）的激活在佩罗尼氏病（PD）中起着至关重要的作用。越来越多的证据表明，脂肪组织来源的干细胞（ADSCs）已被证明可减轻动物模型中PD的症状。然而，ADSCs在PD中的抗纤维化作用机制仍有待充分阐明。在本研究中，研究了ADSCs对大鼠阴茎白膜来源的MFs激活的抑制作用及其可能的机制。ADSCs取自Sprague Dawley大鼠的睾丸旁脂肪。MFs通过用5 ng/ml肿瘤生长因子-β1刺激从大鼠阴茎白膜成纤维细胞转化而来。采用Transwell细胞培养法对ADSCs和MFs进行共培养。蛋白质免疫印迹分析用于评估α平滑肌肌动蛋白（αSMA）、I型胶原、磷酸化（p）-SMAD家族成员2（Smad2）、Smad2、Ras同源家族成员A（RhoA）、Rho相关卷曲螺旋蛋白激酶（ROCK）1和ROCK2、半胱天冬酶3、半胱天冬酶9和基质金属蛋白酶（MMPs）表达水平的变化。胶原凝胶试验用于评估细胞收缩性。此外，使用市售试剂盒检测培养基中羟脯氨酸的浓度。结果表明，ADSCs降低了MFs中αSMA和I型胶原的表达。此外，与仅MFs培养组相比，MFs + ADSCs组中p-Smad2、RhoA、ROCK1和ROCK2的表达显著降低，而MMPs（MMP2、MMP3、MMP9和MMP13）和半胱天冬酶（半胱天冬酶3和半胱天冬酶9）的表达上调。此外，ADSCs能够下调MFs培养基中羟脯氨酸的浓度并逆转MFs的收缩。总的来说，这些结果表明，ADSCs通过Smad和RhoA/ROCK信号通路抑制MFs的激活，减少胶原蛋白的产生，并抑制肌成纤维细胞的收缩。此外，ADSCs通过MMPs和半胱天冬酶减少胶原蛋白的沉积并促进MFs的凋亡。因此，ADSCs的应用可能为PD提供一种新的治疗策略。
