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

Abstract

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.

Figure 1

Morphology of chondrocytes (magnification, ×200). (A) Newly isolated chondrocytes. (B) Primary cells cultured for 3 days. (C) Primary cells cultured for 6 days. (D) First-passage cells cultured for 3 days. (E) Second-passage cells cultured for 3 days.

Figure 2

Chondrocyte identification by immunohistochemical stainaing for type II collagen (magnification, ×200). (A) Positive P2 chondrocytes stained brown in the cytoplasm. (B) P2 chondrocytes negative for type II collagen did not stain.

Figure 3

Bushen Zhuangjin decoction (BZD) enhances the viability of tunicamycin (TM)-stimulated chondrocytes. (A) Chondrocytes treated with TM and BZD at various concentrations for 24 h. Values are means ± SD (vertical bars); ^▲▲P<0.01 compared to untreated cells (not treated with either TM or BZD); ^**P<0.01, ^*P<0.05 compared to TM-stimulated chondrocytes. (B) Viability of TM-stimulated chondrocytes treated with 400 µg/ml BZD for various periods of time. Values are the means ± SD (vertical bars), ^**P<0.01 and ^*P<0.05, compared to 24 h.

Figure 4

Bushen Zhuangjin decoction (BZD) prevents morphological changes of tunicamycin (TM)-stimulated chondrocytes (magnification, ×200). (A) Untreated cells, the control group. (B) TM-stimulated chondrocytes, the model group. (C) The viability of TM-stimulated chondrocytes treated with 400 µg/ml of BZD for 48 h, the dosing group. (D) The viability of TM-stimulated chondrocytes treated with 5 mM 4-phenylbutyric acid (4-PBA) for 20 h, the positive control group.

Figure 5

Bushen Zhuangjin decoction (BZD) inhibits the apoptosis and decrease in mitochondrial membrane potential (ΔΨm) in tunicamycin (TM)-stimulated chondrocytes. (A) Morphological changes in chondrocytes associated with apoptosis were examined by 4′,6-diamidino-2-phenylindole (DAPI) staining. (B) Chondrocyte apoptotic rate was assessed by Annexin V/propidium iodide (PI) staining. (C) The changes in ΔΨm in chondrocytes treated with TM and BZD at various concentrations and chondrocytes left untreated were revealed by JC-1 staining. (D) Percentage of apoptotic cells in chondrocytes treated with TM and BZD at various concentrations and untreated chondrocytes. (E) Percentage of live cells (P2) in chondrocytes treated with TM and BZD at various concentrations and untreated chondrocytes. ^▲▲P<0.01, compared to untreated cells; ^**P<0.01, compared to TM-stimulated chondrocytes. 4-PBA, 4-phenylbutyric acid.

Figure 6

Bushen Zhuangjin decoction (BZD) prevents tunicamycin (TM)-induced chondrocyte apoptosis. (A–C) mRNA expression levels of apoptosis-related factros in chondrocytes left untreated and in those treated with TM and BZD or 4-phenylbutyric acid (4-PBA) in the presence of TM for 24 h were measured by reverse transcription-polymerase chain reaction (RT-PCR). We measured the mRNA expression levels of binding immunoglobin protein (Bip), X-box binding protein 1 (Xbp1), activation transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), (B) Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), caspase-3 and caspase-9. β-actin was used as the internal control for normalization. Values are means ± SD (vertical bars). ^▲▲P<0.01 compared to untreated cells; ^**P<0.01, ^*P<0.05 compared to TM-stimulated chondrocytes.

Figure 7

Bushen Zhuangjin decoction (BZD) inhibits the apoptosis of tunicamycin (TM)-stimulated chondrocytes mediated by endoplasmic reticulum (ER) stress. (A–C) Protein expression levels in chondrocytes left untreated and treated with TM and BZD or 4-phenylbutyric acid (4-PBA) in the presence of TM for 48 h were measured by western blot analysis. The protein expression levels of (C) binding immunoglobin protein (BiP), X-box binding protein 1 (Xbp1), activation transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), (B) Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), caspase-3 and caspase-9 were measured. β-actin was used as the internal control for normalization. Values are means ± SD (vertical bars). ^▲▲P<0.01 compared to untreated cells; ^**P<0.01, compared to TM-stimulated chondrocytes.