Achyranthes bidentata polysaccharides (ABPS) are the active components of Radix Achyranthis Bidentatae (AB), which has been extensively used in Traditional Chinese medicine (TCM) in the treatment of osteoarthritis (OA). Our previous study provided evidence that ABPS regulated the G1/S transition to promote chondrocyte proliferation. However, the precise mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of ABPS on the Wnt/β‑catenin signaling pathway in chondrocytes. Chondrocytes, obtained from the knee cartilage of Sprague-Dawley rats, were identified by type II collagen immunohistochemistry. ABPS upregulated the expression of Wnt-4, Frizzled-2, β-catenin and cyclin D1, and downregulated the expression of glycogen synthase kinase 3β (GSK-3β), as shown by reverse transcription PCR (RT-PCR) and western blot analysis. Using immunofluorescence, we also found that ABPS induced β-catenin nuclear translocation. Importantly, the expression of β-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/β-catenin signaling pathway. In addition, we found that ABPS increased the expression of type II collagen in chondrocytes. These results suggest that ABPS promote chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway.

Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3 and Bax were significantly decreased, whereas the mRNA and protein expression levels of Xbp1 and Bcl-2 were significantly increased compared with the TM-stimulated chondrocytes not treated with BZD. Additionally, all our findings demonstrated that there was no significant difference between the TM-stimulated chondrocytes treated with BZD and those treated with 4-PBA. Taken together, our results indicate that BZD inhibits TM-induced chondrocyte apoptosis mediated by ER stress. Thus, BZD may be a potential therapeutic agent for use in the treatment of OA.

Our previous study reported that inactivation of Shox2 led to dysplasia and ankylosis of the temporomandibular joint (TMJ), and that replacing Shox2 with human Shox partially rescued the phenotype with a prematurely worn out articular disc. However, the mechanisms of Shox2 activity in TMJ development remain to be elucidated. In this study, we investigated the molecular and cellular basis for the congenital dysplasia of TMJ in Wnt1-Cre; pMes-stop Shox2 mice. We found that condyle and glenoid fossa dysplasia occurs primarily in the second week after the birth. The dysplastic TMJ of Wnt1-Cre; pMes-stop Shox2 mice exhibits a loss of Collagen type I, Collagen type II, Ihh and Gli2. In situ zymography and immunohistochemistry further demonstrate an up-regulation of matrix metalloproteinases (MMPs), MMP9 and MMP13, accompanied by a significantly increased cell apoptosis. In addition, the cell proliferation and expressions of Sox9, Runx2 and Ihh are no different in the embryonic TMJ between the wild type and mutant mice. Our results show that overexpression of Shox2 leads to the loss of extracellular matrix and the increase of cell apoptosis in TMJ dysplasia by up-regulating MMPs and down-regulating the Ihh signaling pathway.

Bauhinia championi (Benth.) Benth. polysaccharides (BCBPs), extracted from Bauhinia championi (Benth.) Benth., which has been used in traditional Chinese medicine (TCM) for the treatment of osteoarthritis (OA), are the bioactive constituents of Bauhinia championi (Benth.) rattan. However, the molecular mechanisms responsible for their effects on OA are poorly understood. The Wnt/β-catenin signaling pathway plays an important role in the proliferation of chondrocytes. In the present study, the effects of BCBPs on Wnt/β-catenin signaling in chondrocytes were investigated. BCBPs were obtained by hot-water extraction and identified by the modified high performance liquid chromatography (HPLC) method. Chondrocytes were isolated from the knees of Sprague‑Dawley rats and identified by type II collagen immunohistochemistry. The chondrocytes were treated with or without BCBPs for 48 h. Cell viability was evaluated by MTT assay. The mRNA and protein levels of Wnt-4, β-catenin, Frizzled-2, glycogen synthase kinase (GSK)-3β, cyclin D1 and collagen II were detected by western blot analysis and reverse transcription PCR (RT-PCR), respectively. We found that the BCBPs contained at least seven monosaccharides, including D-mannose, rhamnose, D-(+) glucuronic acid, D-(+) galacturonic acid, D-glucose, galactose and arabinose. The cell viability of the chondrocytes treated with 50, 100 and 200 µg/ml BCBPs was significantly higher than that of the chondroctyes in the control group (treated with 0 µg/ml BCBPs). Furthermore, compared with the control group, the mRNA and protein expression of Wnt-4, β-catenin, Frizzled-2 and cyclin D1 in the BCBP-treated groups markedly increased, whereas the mRNA and protein expression of GSK-3β significantly decreased. Of note, the dose of 100 µg/ml BCBPs was more effective than the dose of 50 µg/ml BCBPs and 200 µg/ml BCBPs. In addition, we found that treatment with BCBPs upregulated the protein levels of collagen II in the chondrocytes. These results indicate that BCBPs upregulate Wnt/β-catenin signaling, thus promoting chondrocyte proliferation.

Chondrocyte apoptosis activated by the mitochondrial-dependent signaling pathway plays a crucial role in the cartilage degeneration of osteoarthritis. Duhuo Jisheng decoction (DHJSD), a herbal formula from traditional Chinese medicine, has been widely used for treating osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effect of DHJSD remain to be elucidated. In the present study, the effects of DHJSD on the mitochondrial-dependent signaling pathway in sodium nitroprussiate (SNP)-induced chondrocyte apoptosis were investigated. Chondrocytes, from the knee articular cartilage of Sprague Dawley rats, were identified by type II collagen immunohistochemistry. The chondrocytes, stimulated with or without SNP to induce apoptosis, were treated by DHJSD for various concentrations and times. The viability of SNP-induced chondrocytes treated with DHJSD was enhanced compared to SNP-induced chondrocytes in a dose- and time-dependent manner, as assessed by the MTT assay. The apoptosis of SNP-induced chondrocytes treated by DHJSD was significantly decreased compared to SNP-induced chondrocyte, as shown by 4',6-diamidino-2-phenylindole and Annexin V/propidium iodide staining. The mitochondrial membrane potential (∆Ψm) of SNP-induced chondrocytes treated by DHJSD was significantly decreased compared to SNP-induced chondrocyte, as shown by JC-1 staining. To understand the mechanism, the mRNA and protein levels of Bax, B-cell lymphoma 2 (Bcl-2), caspase-9 and caspase-3 were detected by reverse transcription‑polymerase chain reaction and western blot analysis, respectively. In SNP-induced chondrocyte treated by DHJSD, the Bcl-2 expression was increased, whereas the expression of Bax, caspase-9 and caspase-3 was decreased compared to SNP-induced chondrocyte. Taken together, these results indicated that DHJSD inhibits the apoptosis of SNP-induced chondrocyte by the mitochondrial-dependent apoptotic pathway, and this may partly explain its therapeutic efficacy for OA.