Danshen attenuates cartilage injuries in osteoarthritis in vivo and in vitro by activating JAK2/STAT3 and AKT pathways

Abstract

Articular cartilage degradation is a main feature of osteoarthritis (OA). The effects of Danshen, a traditional Chinese herb, in mitigating cartilage damage have been reported before. This study was conducted to investigate the effects of Danshen on cartilage injuries in OA. Rabbit OA models were established by surgical destabilization of the medial meniscus and the anterior and posterior cruciate ligaments in the left knee joint. Injection of Danshen into the articular cavity attenuated OA cartilage destruction in vivo. The levels of phosphorylated Janus kinase 2 (JAK2) and phosphorylated signal transducer and activator of transcription 3 (STAT3) were decreased in osteoarthritic cartilage, while they were rescued upon Danshen treatment. Furthermore, chondrocytes isolated from normal rabbit cartilage were exposed to 2 mM sodium nitroprusside (SNP) to establish an OA model in vitro. We found that the oxidative stress and chondrocyte apoptosis induced by SNP were suppressed by Danshen. The phosphorylation levels of JAK2 and STAT3 were decreased in response to SNP treatment, whereas they were rescued by Danshen. Additionally, AG490, a specific JAK2 inhibitor, counteracted the anti-apoptotic effect of Danshen. The phosphorylation level of protein kinase B (AKT) was also altered in response to SNP and reversed by Danshen. The anti-apoptotic effect of Danshen was counteracted by AKT pathway inhibitor LY194002. Taken together, Danshen attenuates OA cartilage destruction by regulating the JAK2/STAT3 and AKT signaling pathways.

Keywords: AKT; Danshen; JAK2/STAT3; apoptosis; articular cartilage degradation.

Figures

Fig. 1.

Danshen attenuated articular cartilage degradation in vivo and activated the JAK2/STAT3 pathway. (A) Histological examination (H&E staining) of articular cartilage in Danshen- or SH-treated OA models. Scale bar=100µm. (B) Western blot was performed to assess the levels of phosphorylated and total JAK2 and STAT3 in articular cartilage.

Fig. 2.

Danshen rescued the antioxidant system decreased by SNP. The chondrocytes were randomized into five groups: the control group, Danshen group, SNP group, SH+SNP group, and Danshen+SNP group. The levels of GSH, SOD, and CAT in each group were measured with corresponding kits. Error bars indicate the standard deviation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Fig. 3.

Danshen ameliorated SNP-induced chondrocyte apoptosis and enhanced SNP-reduced proliferation of chondrocytes. (A) Chondrocyte apoptosis was determined by flow cytometry assay. FL1 corresponds to Annexin-FITC and FL2 corresponds to PI. (B) Proliferation of chondrocytes was detected by MTT assay. Error bars indicate the standard deviation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Fig. 4.

Danshen activated the JAK2/ STAT3 pathway. JAK and p-JAK2 (A) and STAT3 and p-STAT3 (B) were detected by western blot. The levels of p-JAK and p-STAT3 were upregulated by Danshen in SNP-treated chondrocytes.

Fig. 5.

Danshen attenuated SNP-induced chondrocyte apoptosis via the JAK2/STAT3 signaling pathway. (A) Flow cytometry showed that Danshen’s inhibitory effects on SNP-induced apoptosis of chondrocytes were abrogated by AG490 treatment. FL1 corresponds to Annexin-FITC and FL2 corresponds to PI. (B) An MTT assay showed that Danshen’s effects on proliferation of chondrocytes were inhibited by AG490 treatment. Error bars indicate the standard deviation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Fig. 6.

Danshen ameliorated SNP-induced chondrocyte apoptosis via the AKT signaling pathway. (A) Western blot was performed to assess AKT and p-AKT levels. The level of p-AKT was increased by Danshen in SNP-treated chondrocytes. (B) Flow cytometry showed that Danshen’s inhibitory effects on SNP-induced apoptosis of chondrocytes were abrogated by LY294002. FL1 corresponds to Annexin-FITC and FL2 corresponds to PI. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.