Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.

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 (4­PBA). We found that 4­PBA 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 TM­induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X­box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP­homologous protein (Chop), caspase­9, 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 TM­stimulated chondrocytes not treated with BZD. Additionally, all our findings demonstrated that there was no significant difference between the TM­stimulated chondrocytes treated with BZD and those treated with 4­PBA. Taken together, our results indicate that BZD inhibits TM­induced chondrocyte apoptosis mediated by ER stress. Thus, BZD may be a potential therapeutic agent for use in the treatment of OA.

Duhuo Jisheng decoction inhibits endoplasmic reticulum stress in chondrocytes induced by tunicamycin through the downregulation of miR-34a.

Our previous study showed that Duhuo Jisheng decoction (DHJSD) inhibited chondrocyte apoptosis by the mitochondria-dependent signaling pathway. Endoplasmic reticulum (ER) stress is upstream of the mitochondria-dependent signaling pathway and has been shown to promote chondrocyte apoptosis that occurs in osteoarthritis (OA). The present study aimed to evaluate whether DHJSD inhibits the chondrocyte apoptosis by regulating ER stress. DHJSD enhanced the viability of tunicamycin (TM)­exposed chondrocytes, a model of ER stress-induced apoptosis, in a dose­ and time­dependent manner, as shown by MTT assay. The present results showed that DHJSD and sodium 4-phenylbutyrate (PBA), an ER stress inhibitor, reduced TM­induced chondrocyte apoptosis by 4',6-diamidino­2-phenylindole staining. To gain insight into the mechanisms of DHJSD that are responsible for enhancing the viability and inhibiting TM­induced chondrocyte apoptosis, the associated mRNA expressions and protein levels were detected by reverse transcription­polymerase chain reaction (RT­PCR) and western blot analysis, respectively. The results showed that the expression levels of Xbp1, Xbp1s and Bcl­2 were increased, and the expression levels of Bip, Atf4, Chop, Bax, caspase­9 and ­3 were decreased in the TM­exposed chondrocytes treated with DHJSD or PBA compared with that in the TM­exposed chondrocytes. To identify the possible mechanisms, the expression of miR­34a was examined by the TaqMan microRNA assay, and was downregulated in the TM­exposed chondrocytes treated with DHJSD or PBA compared with that in the TM-exposed chondrocytes. DHJSD inhibits ER stress in chondrocytes induced by exposure to TM by downregulating miR­34a, suggesting that DHJSD may be a potential therapeutic agent for OA.

Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice.

The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild­type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression­associated congenital dysplasia of the TMJ in mice.

Bushen Zhuangjin Decoction promotes chondrocyte proliferation by stimulating cell cycle progression.

Bushen Zhuangjin Decoction (BZD), a well-known formulation in Traditional Chinese Medicine, has been widely used for the treatment of osteoarthritis (OA). Due to the poor intrinsic repair capacity of chondrocytes, promoting the proliferation of chondrocytes is an efficient treatment to delay the progression of cartilage degradation. The present study, therefore, focused on the effect of BZD on chondrocyte proliferation, exploring the mechanism of BZD on the inhibition of cartilage degradation. Chondrocytes isolated from the knee articular cartilage of Sprague Dawley rats were cultured and identified by type II collagen immunohistochemistry. It was found that BZD promoted chondrocyte viability in a dose- and time-dependent manner. To investigate if BZD promoted the chondrocyte viability by stimulating the cell cycle progression a flow cytometer was used, and the results showed that the percentage proportion of G0/G1 cells was significantly lower, and the percentage proportion of S cells was significantly higher, in treated cells compared with that in untreated cells. To gain insight into the mechanism underlying the effect of BZD on the cell cycle progression, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and p21 was measured by reverse transcription-polymerase chain reaction and western blotting, respectively. The mRNA and protein expression of cyclin D1, CDK4 and CDK6 in the BZD-treated chondrocytes was significantly upregulated, while the mRNA and protein expression of p21 was significantly downregulated, compared with that in the untreated chondrocytes. These results suggested that BZD promoted chondrocyte proliferation by accelerating G1/S transition, indicating that BZD is a potential therapeutic agent for the treatment of OA.

Tougu Xiaotong formula induces chondrogenic differentiation in association with transforming growth factor-ß1 and promotes proliferation in bone marrow stromal cells.

Indian hedgehog (Ihh), one of the hedgehog gene families, is indicated in the regulation of chondrocyte differentiation. Tougu Xiaotong formula (TXF), a traditional Chinese medicinal compound, has been used for the treatment of bone and joint disease. However, the underlying molecular mechanisms of TXF on the function of bone marrow stromal cells (BMSCs) remain unclear. In the present study, the affect of TXF on proliferation and chondrogenic differentiation was investigated in primary BMSCs from four­week­old Sprague Dawley rats. The cell viability in BMSCs treated with TXF was higher compared to the untreated cells. Additionally, the percentage of G(0)/G(1) phase cells was significantly decreased, whereas that of the S phase cells was significantly increased. Furthermore, following TXF treatment, cyclin D1, cyclin­dependent kinase 4 (CDK4) and CDK6 expression in BMSCs was significantly enhanced. The results showed that TXF had no cytotoxicity to BMSCs. To explore the effect of TXF on the differentiation in BMSCs, whether TXF induced chondrogenic differentiation of BMSCs by the regulation of Ihh signaling pathway was investigated. The protein expression of Ihh, Patched and Smoothened in the induction group were significantly increased when compared to those in the control group, and the highest protein level of Ihh was in the induction group that was treated with the combination of TXF and transforming growth factor­ß1 (TGF­ß1). In addition, TXF combined with TGF­ß1 significantly induced the protein expression of cartilage oligomeric matrix protein and collagen II compared to the TGF­ß1 group. Taken together, these results indicate that TXF promotes the proliferation via accelerating the G(1)/S transition, and induces chondrogenic differentiation in BMSCs by activation of the Ihh signaling pathway in association with TGF­ß1.

Duhuo Jisheng decoction treatment inhibits the sodium nitroprussiate­induced apoptosis of chondrocytes through the mitochondrial­dependent signaling pathway.

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 transcription­polymerase 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.

Achyranthes bidentata polysaccharides activate the Wnt/ß-catenin signaling pathway to promote chondrocyte proliferation.

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/ß­catenin 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, ß-catenin and cyclin D1, and downregulated the expression of glycogen synthase kinase 3ß (GSK-3ß), as shown by reverse transcription PCR (RT-PCR) and western blot analysis. Using immunofluorescence, we also found that ABPS induced ß-catenin nuclear translocation. Importantly, the expression of ß-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/ß-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/ß-catenin signaling pathway.

Overexpression of Shox2 leads to congenital dysplasia of the temporomandibular joint in mice.

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 upregulate Wnt/ß-catenin signaling in chondrocytes.

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/ß-catenin signaling pathway plays an important role in the proliferation of chondrocytes. In the present study, the effects of BCBPs on Wnt/ß-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 Sprague­Dawley 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, ß-catenin, Frizzled-2, glycogen synthase kinase (GSK)-3ß, 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, ß-catenin, Frizzled-2 and cyclin D1 in the BCBP-treated groups markedly increased, whereas the mRNA and protein expression of GSK-3ß 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/ß-catenin signaling, thus promoting chondrocyte proliferation.

[Effect of water extracts from duhuo jisheng decoction on expression of chondrocyte G1 phase regulator mRNA].

OBJECTIVE: To investigate the effect of water extracts from Duhuo Jisheng decoction on chondrocyte G1 phase. METHODS: Chondrocytes were collected from four-week-old SD rats to establish the chondrocyte in vitro culture system. The third generation of chondrocytes was intervened. MTT method was used to measure the effect of water extracts from different concentrations of Duhuo Jisheng decoction on chondrocyte activity. The expressions of Chondrocyte Cyclin D1, CDK4, CDK6 and P21 mRNA in the blank group and low, middle and high-dose groups (100, 200, 400 mg x L(-1)) were detected by RT-PCR method. RESULT: The MTT assay showed that the chondrocyte activity significantly increased within specific drug concentrations (50-800 mg x L(-1)) (P < 0.01); After the intervention for 24 h, the expressions of CyclinD1, CDK4 and CDK6 mRNA in all dose groups notably increased (P < 0.05), with the maximum expressions at the concentration of 200 mg x L(-1); The expression of P21 mRNA decreased, particularly at the concentration of 200 mg x L(-1) (P < 0.01). CONCLUSION: Water extracts from Duhuo Jisheng decoction can promote chondrocyte proliferation by effecting the expression of chondrocyte G1 phase regulator mRNA.