Objective

To investigate the effects of everolimus (EVE) on postoperative fibrosis in the knee joint and the potentially relevant signaling pathways.

Methods

CCK-8 and flow cytometry assays were used to detect the effect of EVE on human fibroblast viability and apoptosis induction. IF and TEM were used to assess fibroblast autophagy. 3-methyladenine (3-MA) was applied to inhibit autophagy to clarify the relationship between autophagy and apoptosis. WB was used to measure the expression of proteins related to apoptosis, autophagy and the mTOR signaling pathway. A rabbit model of knee joint fibrosis was established and topically treated with various concentrations of EVE. IF-P was applied to identify that the main components cells of the fibrotic tissue and histomorphological staining was used to detect the degree of fibrosis and the content of collagen.

Results

Histomorphological staining demonstrated that EVE could reduce the degree of postoperative fibrosis and collagen deposition in the knee joint. The results of IF, TEM, flow cytometry assays and WB detection showed that EVE could activate autophagy and induce fibroblasts apoptosis. Meanwhile, the expression levels of p-PI3K, p-Akt, p-mTOR were downregulated with EVE treatment. After the inhibition of autophagy by 3-MA treatment, the increased fibroblasts apoptosis by EVE treatment was partially decreased.

Conclusion

Everolimus can reduce surgery-induced knee fibrosis by inducing autophagy-mediated fibroblast apoptosis, which may be involved with the regulation of the PI3K/Akt/mTOR signaling pathway.

中文翻译：

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依维莫司通过PI3K / Akt / mTOR信号转导途径诱导自噬介导的成纤维细胞凋亡，从而减轻了兔术后关节纤维化。

目的

调查依维莫司（EVE）对膝关节术后纤维化及潜在相关信号通路的影响。

方法

使用CCK-8和流式细胞仪检测EVE对人成纤维细胞活力和凋亡诱导的影响。IF和TEM用于评估成纤维细胞自噬。应用3-甲基腺嘌呤（3-MA）抑制自噬，以阐明自噬与细胞凋亡之间的关系。WB用于测量与细胞凋亡，自噬和mTOR信号通路相关的蛋白质的表达。建立了兔膝关节纤维化模型，并用各种浓度的EVE局部治疗。IF-P用于鉴定纤维化组织的主要成分细胞和组织形态学染色以检测纤维化程度和胶原蛋白含量。

结果

组织形态学染色表明，EVE可以减少术后纤维化程度和膝关节胶原沉积。IF，TEM，流式细胞术和WB检测的结果表明，EVE可以激活自噬并诱导成纤维细胞凋亡。同时，EVE处理下调了p-PI3K，p-Akt，p-mTOR的表达水平。3-MA处理抑制自噬后，EVE处理增加的成纤维细胞凋亡被部分减少。

结论

依维莫司可以通过诱导自噬介导的成纤维细胞凋亡减少手术引起的膝部纤维化，这可能与PI3K / Akt / mTOR信号通路的调节有关。