Platelet-rich plasma pretreatment protects anterior cruciate ligament fibroblasts correlated with PI3K-Akt-mTOR pathway under hypoxia condition,Journal of Orthopaedic Translation

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Platelet-rich plasma pretreatment protects anterior cruciate ligament fibroblasts correlated with PI3K-Akt-mTOR pathway under hypoxia condition
Journal of Orthopaedic Translation ( IF 5.9 ) Pub Date : 2022-03-10 , DOI: 10.1016/j.jot.2022.02.002
Yanwei Cao _{1,

2} , Yue Li _{1,

2} , Sai Chuen Fu _{3,

4} , Jiewei Shen _{1,

2} , Hui Zhang _{1,

2} , Chunyan Jiang _{1,

2} , Patrick Shu-Hang Yung _{3,

4}

Affiliation

Background

/Objective: Biological factors such as platelet-rich plasma (PRP) combined with anterior cruciate ligament (ACL) primary repair technology are used to treat ACL injury. However, the protective mechanism of PRP for ACL fibroblasts under hypoxia condition is still unknown. The aim of this study was to investigate the protective effect of PRP on ACL fibroblasts under hypoxia condition and illustrate the mechanism of PRP regulating the ACL fibroblasts under hypoxia condition.

Methods

The cells were divided into three groups: control group, hypoxia group and PRP pretreatment group. Lethal dose (LD) 50 for hypoxia induction time and the maximum efficacy of PRP concentration were confirmed by CCK-8 assay. The ability of cell apoptosis, cell proliferation, and cell migration were tested by flow cytometry, scratch assay and transwell assay, respectively. Extracellular matrix (ECM) synthesis and hypoxia-inducible factor 1α (HIF-1α) were identified by immunofluorescence staining, Masson's staining and transmission electron microscope analysis. Inflammatory cell infiltration was assessed by hematoxylin and eosin staining as well as immunofluorescence staining. Western blot analysis and real-time PCR were performed to assess the associated gene and protein expression, respectively. The ratio of phosphorylated/total PI3K, Akt and mTOR were also assessed by western blot analysis.

Results

① LD 50 of hypoxia was 48 h and the maximum efficacy of PRP concentration was 600 × 10⁹/L. ② ANNEXIN V-FITC/PI flow cytometry showed that the hypoxia condition significantly increased the apoptosis of cells (P < 0.001) whereas PRP pretreatment significantly decreased the apoptosis of cells under hypoxia (P < 0.001). The expressions of gene and protein of Bax, Bcl-2, cleaved-caspase 3 were consistent with the results of flow cytometric analysis. ③ Cell cycle analysis for flow cytometry showed the inhibitory effect of hypoxia and promotive effect of PRP pretreatment. ④ Immunofluorescence staining (HIF-1α, collagen I and III) showed the positive effect of hypoxia and negative effect of PRP on these parameters. Real-time PCR showed that type I and III collagen were 2.1 folds and 2.5 folds higher after 48 h hypoxia induction compared to the control group. PRP pretreatment significantly reduced the type I and III collagen mRNA expression of the hypoxia induced ACL fibroblasts to 78.5% and 77.7% at 48 h compared to hypoxia group (P < 0.001), respectively.⑤ Cell migration assay showed that hypoxia condition significantly restrained cell migration compared with the control group. PRP could alleviate the inhibitory effect of hypoxia on fibroblasts. ⑥ Western blot analysis showed the ratio of phosphorylated/total PI3K, Akt and mTOR in hypoxia group increased to 31%, 20% and 44/% compared to control group, respectively. ⑦ The results of in vivo analysis was in accordance with the results of in vitro analysis.

Conclusion

PRP can protect ACL fibroblasts via decreasing apoptosis and increasing cell viability, cell migration and cell proliferation under hypoxia condition. And such PRP protective effect was correlated with PI3K/Akt/mTOR pathway.

The translational potential of this article

PRP can be used to treat patients with ACL tear by injection under arthroscopy or ultrasound guiding.

中文翻译：

富血小板血浆预处理保护缺氧条件下与PI3K-Akt-mTOR通路相关的前交叉韧带成纤维细胞

背景

/目的：利用富血小板血浆（PRP）等生物因子联合前交叉韧带（ACL）一级修复技术治疗ACL损伤。然而，PRP在缺氧条件下对ACL成纤维细胞的保护机制尚不清楚。本研究旨在探讨PRP在缺氧条件下对ACL成纤维细胞的保护作用，并阐明PRP在缺氧条件下调节ACL成纤维细胞的机制。

方法

将细胞分为三组：对照组、缺氧组和PRP预处理组。CCK-8测定证实了缺氧诱导时间的致死剂量(LD) 50 和PRP浓度的最大功效。分别采用流式细胞仪、划痕法和transwell法检测细胞凋亡、细胞增殖和细胞迁移能力。通过免疫荧光染色、Masson染色和透射电镜分析鉴定细胞外基质（ECM）合成和缺氧诱导因子1α（HIF-1α）。通过苏木精和伊红染色以及免疫荧光染色评估炎症细胞浸润。分别进行蛋白质印迹分析和实时 PCR 以评估相关基因和蛋白质的表达。磷酸化/总 PI3K 的比例，

结果

①缺氧LD 50 为48小时，PRP浓度最大功效为600×10 ⁹/L。② ANNEXIN V-FITC/PI流式细胞仪显示缺氧条件下细胞凋亡显着增加（P<0.001），而PRP预处理显着降低缺氧条件下细胞凋亡（P<0.001）。Bax、Bcl-2、cleaved-caspase 3基因和蛋白表达与流式细胞仪分析结果一致。③流式细胞仪的细胞周期分析显示PRP预处理有缺氧抑制作用和促进作用。④免疫荧光染色（HIF-1α、胶原蛋白I和III）显示缺氧对这些参数有积极影响，而PRP对这些参数有消极影响。实时荧光定量 PCR 显示，与对照组相比，低氧诱导 48 小时后，I 型和 III 型胶原蛋白分别高出 2.1 倍和 2.5 倍。与缺氧组相比，PRP预处理后48 h缺氧诱导的ACL成纤维细胞I型和III型胶原mRNA表达量分别显着降低至78.5%和77.7%（P<<0.001）。⑤细胞迁移实验显示缺氧条件下与对照组相比，细胞迁移明显受到抑制。PRP可减轻缺氧对成纤维细胞的抑制作用。⑥Western blot分析显示，缺氧组磷酸化/总PI3K、Akt、mTOR比值较对照组分别升高31%、20%和44/%。⑦体内分析结果与体外分析结果一致。⑤细胞迁移实验表明，与对照组相比，缺氧条件显着抑制了细胞迁移。PRP可减轻缺氧对成纤维细胞的抑制作用。⑥Western blot分析显示，缺氧组磷酸化/总PI3K、Akt、mTOR比值较对照组分别升高31%、20%和44/%。⑦体内分析结果与体外分析结果一致。⑤细胞迁移实验表明，与对照组相比，缺氧条件显着抑制了细胞迁移。PRP可减轻缺氧对成纤维细胞的抑制作用。⑥Western blot分析显示，缺氧组磷酸化/总PI3K、Akt、mTOR比值较对照组分别升高31%、20%和44/%。⑦体内分析结果与体外分析结果一致。