Platelet-rich plasma pretreatment protects anterior cruciate ligament fibroblasts correlated with PI3K-Akt-mTOR pathway under hypoxia condition

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.