富血小板血浆外泌体促进软骨细胞增殖并抑制凋亡

Platelet-rich plasma-derived exosomes promote chondrocyte proliferation and inhibit apoptosis

目的探讨富含血小板血浆(PRP)来源外泌体(PRP-Exo)对体外白介素1β(IL-1β)处理的软骨细胞的保护作用和分子机制。方法分离和纯化PRP-Exo,并鉴定和表征。IL-1β处理大鼠软骨细胞模拟体外骨关节炎(OA)中的软骨细胞(IL-1β组),IL-1β联合使用PRP-Exo(IL-1β+PRP-Exo组)或活化模式的PRP(PRP-As)(IL-1β+PRP-As组)分别作用于细胞,CCK-8法测细胞增殖,流式细胞术检测细胞凋亡。通过Western blot和qRT-PCR分析Furin介导ALK1/ALK5信号平衡的机制。结果从PRP成功分离纯化了外泌体。IL-1β+PRP-As组和IL-1β+PRP-Exo组的肿瘤坏死因子-α(TNF-α)水平和细胞凋亡率低于IL-1β组,细胞增殖率和Furin的水平均高于IL-1β组(均P<0.05),且IL-1β+PRP-Exo组促进软骨细胞增殖及抑制凋亡作用都优于IL-1β+PRP-As组(均P<0.05)。软骨细胞中加入Furin可明显降低ALK1/ALK5比率(P<0.05)。IL-1β不仅降低软骨细胞中Furin水平,且提高ALK1/ALK5的比率(均P<0.05),而加入PRP-Exo可通过提高Furin水平从而降低ALK1/ALK5比率(均P<0.05)。结论PRP-Exo通过维持Furin介导的ALK1/ALK5平衡促进软骨细胞增殖并抑制凋亡。

Objective To investigate the protective effect and molecular mechanism of platelet rich plasma(PRP)-derived exosome(PRP-Exo)on interleukin-1β(IL-1β)treated chondrocytes in vitro.Methods PRP-Exo was isolated and purified,and then identified and characterized.Rat chondrocytes were treated with IL-1β to simulate in vitro osteoarthritis(OA)chondrocytes(an IL-1β group).Furthermore,the cells were also exposed to IL-1β and PRP-Exo(an IL-1β+PRP-Exo group)or activated PRP(PRP-As)(an IL-1β+PRP-Exo group).Then,cell proliferation was detected by CCK-8 assay,while apoptosis was detected by flow cytometry.Western blot and qRT-PCR were used to analyze the underlying mechanism of Furin-mediated ALK1/ALK5 signaling balance.Results Exosomes were successfully isolated and purified from PRP.The IL-1β+PRP-As and IL-1β+PRP-Exo groups showed decreases in the level of tumor necrosis factor-α(TNF-α)and cell apoptosis rate,and increases in cell proliferation rate and Furin level,compared with the IL-1β group(P<0.05).Moreover,the IL-1β+ PRPExo group presented better effects than the IL-1β+ PRP-As group(P<0.05).Treatment with Furin in chondrocytes significantly decreased the ratio of ALK1/ALK5(P<0.05).IL-1βnot only decreased the level of Furin in chondrocytes,but also increased the ratio of ALK1/ALK5(P<0.05).In contrast,exposure to PRP-Exo resulted in decreases in the ratio of ALK1/ALK5 through increasing the level of Furin(P<0.05).Conclusions PRP-Exo can promote chondrocyte proliferation and inhibit apoptosis through maintaining Furin-mediated ALK1/ALK5 balance.