The roles of PI3K/Akt signal pathway in carboxymethylated chitosan protecting NO-induced chondro-cytes apoptosis and the mechanisms

Abstract

Objective To investigate the protective effects of carboxymethylated chitosan (CMCS) on nitric oxide (NO) induced apoptosis in rat chondrocytes, and the probable roles and mechanisms of PI3K/Akt signaling pathway in these process. Methods Rat knee articular cartilage was used as the source of chon-drocytes, the cells were identified by immunohistochemical staining against collagen type Ⅱ, odium nitro-prusside (SNP, 3 mmol/L) was used to establish the apoptotic models of chondrocytes. Cells were divided into four groups: the control group, the SNP-induced group, the SNP+CMCS treated group, the SNP+CMCS+PI3K inhibitor Wortmannin treated group. Cell proliferation were assessed by cell proliferation assay kit (CCK-8), the apoptotic rate of chondrocytes was determined by FCM with Annexin V-FITC/PI double staining, the expression levels of MMP-13 and TIMP-1 mRNA were detected by real-time polymerase chain reaction (PCR) analysis, the expression of Akt and p-Akt protein levels was detected by Western blotting analysis. One-way analysis of variance (ANOVA) statistical analysis was used to calculate the data. Results Three mmol/L SNP can inhibit proliferation (0.221±0.023), and the proliferation was reduced by 70% compared with the control group (0.736±0.032, F=8.203, P=0.021); and the induced apoptosis in cultured chondrocytes could be observed. The apoptotic rate was (68.8±5.2)%. Increased MMP-3 and decreased TIMP-1 mRNA expression were observed in SNP-induced cells. After adding CMCS to SNP-induced chondrocytes, the proliferation was increased while apoptotic rate was decreased, the apoptotic rate decreased to (14.7±2.3)%. CMCS could promote the activation of p-Akt in SNP-induced chondrocytes and restore SNP-induced MMP-13 and TIMP-1 mRNA expression. Conclusion CMCS could protect apoptosis in SNP induced chon-drocytes via activation of PI3K/Akt pathway. Key words: Chondrocytes; Nitric oxide; Apoptosis; Carboxymethylated chitosan; PI3K/Akt