Polyphyllin I alleviates osteoblasts injuries induced by TCP wear particles in vitro via inhibition of autophagy / 中草药

Objective: To observe the effect of polyphyllin I (PPI) on osteoblasts injuries induced by tricalcium phosphate (TCP) wear particles in vitro, and explain its regulation mechanisms. Methods: Primary osteoblasts obtained from the calvaria of neonatal SD rat by the series of digestion were identified with ALP staining. The osteoblasts were treated with TCP wear particles (TCP, 0.1 mg/mL) for 48 h to establish an in vitro injuries model of the calvarial osteoblasts. The experiment was randomly divided into control group, model (TCP, 0.1 mg/mL) group, PPI (30 μg/mL) group and PPI (100 μg/mL) group. CCK-8 and chemical colorimetry were used to examine cell viability and lactic dehydrogenase (LDH) content in culture media; Real-time PCR was performed to detect mRNA levels of ALP, Collagen I and RUNX2 in osteoblasts; The flow cytometry was used to examine apoptosis of osteoblasts using Annexin V/PI double staining; When the osteoblasts were treated for 14 d, mineral nodules formation was observed with alizarin S staining; Western blot was applied to examine proteins expression of Bax, Bcl-2, cleaved Caspase-3, Atg5, p62, and microtubule associated protein 1 light chain3 (LC-3). Results: Compared with control group, model group showed that the cell viability of osteoblasts, mRNA levels of ALP, Collagen I and RUNX2, and mineral nodules formation were significantly decreased; LDH content, percentage of apoptosis and proteins expression of Bax, cleaved Caspase-3, Atg5, LC-3 and the ratio of LC-3II/LC-3I were obviously increased in calvarial osteoblasts, whereas proteins expression of Bcl-2 and p62 was remarkably decreased. Compared with model group, PPI groups indicated that cell viability of osteoblasts, mRNA levels of ALP, Collagen I and RUNX2, and mineral nodules formation were dramatically increased; LDH content, percentage of apoptosis, protein expressions of Bax, cleaved Caspase-3, Atg5, and LC-3 and the ratio of LC-3II/LC-3I were obviously decreased, but Bcl-2 and p62 expression were obviously increased. Conclusion: PPI alleviates osteoblasts injuries induced by TCP wear particles via inhibition of autophagy.

Effect of psoralen on rat osteoblasts injuries induced by TCP wear particles in vitro and its mechanism / 中国应用生理学杂志

To investigate the effect and mechanism of psoralen on calvarial osteoblasts injuries caused by tricalcium phosphate (TCP) wear particles in vitro. Primary osteoblasts were obtained from the calvaria of neonatal SD rat by the series of digestion and were identified with ALP staining. Calvarial osteoblasts were treated with TCP wear particles for 48 h to establish the in vitro model of osteoblasts injuries. The rat osteoblasts were randomly divided into control group, TCP wear particles (0.1 mg/ml) group, psoralen treated (at the concentrations of 10, 10, 10 mol/L) groups. WST assay and the flow cytometry were used to detect the cell viability of osteoblasts and apoptosis, respectively. Chemical colorimetry was performed to examine ALP activity of osteobalsts. When the osteoblasts were treated for 14 day, mineral nodules formation was observed with alizarin red S staining. Western blot was applied to examine protein expressions of glucose regulated protein78/94(GRP78/94), inositol dependent enzyme 1 alpha (IREα), spliced X-box binding protein 1 (XBP1s) and phosphorylated c-Jun N-terminal kinase (p-JNK) in calvarial osteoblasts. Compared with control group, the cell viability of osteoblasts, ALP activity and mineral nodules formation in TCP group were decreased significantly (P＜0.05), while the percentage of apoptosis and protein expressions of GRP78/94, IRE1α, XBP1 and p-JNK were obviously increased in calvarial osteoblasts (P＜0.05). Compared with TCP group, the injuries of calvarial osteoblasts and cell apoptosis in psoralen treated groups were obviously decreased (P＜0.05), and the expression levels of GRP78/94, IRE1α, XBP1 and p-JNK were down-regulated remarkably (P＜0.05). Psoralen prevents osteoblasts injuries caused by TCP wear particles through IRE1α-XBP1s-JNK signaling pathway activation.

TCP wear particles causes injury of periprosthetic osteocytes in the mouse calvaria / 中国应用生理学杂志

OBJECTIVE@#To study whether tricalcium phosphate(TCP) wear particles cause injuries of periprosthetic osteocytes in the mouse calvaria, and to explain its molecular mechanism.@*METHODS@#Thirty six-week(ICR)male mice were randomly divided into sham group, model (TCP) group and 3-methyladenine (3-MA) group. A murine calvarial model of osteolysis was established by 30 mg of TCP wear particles implantation over the periosteum around the middle suture of calvaria in mice. On the second postoperative day, the autophagy specific inhibitor 3-MA (1.0 mg/kg) was subcutaneously injected to the calvaria in the 3-MA-treated mice every other day. After 2 weeks, blood and the calvaria were obtained. Micro-CT was used to detect bone mineral density(BMD), bone volume fraction (BVF) and porosity number. HE staining and flow cytometry were performed to analyze the viability and apoptosis of periprosthetic osteocytes. The serum levels of dentin matrix protein 1(DMP-1) and sclerostin (SOST) were determined by ELISA. The proteins expressions of DMP-1, SOST, Beclin-1 and microtuble-associated protein 1 light chain 3 (LC-3) were detected by Western blot in the calvaria osteocytes.@*RESULTS@#Compared with the sham group, the mice in the TCP group showed that a significant decrease in the viability of periprosthetic osteocytes, but obvious increases in number of osteocytes death and osteocytes apoptosis (<0.05), and in serum level and protein expression of SOST; significant decreases in serum level and protein expression of DMP-1 (<0.05), and remarkable up-regulation of autophagy-related factors beclin-1 and the conversion of LC3-Ⅱ from LC3-I in the calvaria osteocytes. Compared with TCP group, the mice in the 3-MA group showed that injuries of calvaria osteocytes were obviously aggravated, and osteocytes apoptosis was significantly increased (<0.05).@*CONCLUSIONS@#TCP wear particles can cause injuries of periprosthetic osteocytes via activation of apoptosis and autophagy, which promotes osteolysis around the prosthesis osteolysis and joint aseptic loosening.

Effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To explore the effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism.@*METHODS@#Thirty-six male ICR mice were randomly divided into three groups (=12):sham group, TCP wear particles (TCP) group and N-acetyl-L-cysteine (NAC) group. Aperiprosthetic osteolysis model in mouse was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the 2nd day post-operation, NAC (1.0 mg/kg) was locally injected to the calvarium under the periosteum every other day for 2 weeks. Then, all the mice were sacrificed to obtain blood and the calvaria. Periprosthetic osteolysis in the mouse calvaria was observed by tartrate resistant acid phosphatase (TRAP) staining; serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6); total anti-oxidation capacity (T-AOC) and superoxide dismutase (SOD) activity were examined by ELISA and chemical colorimetry, respectively; protein levels of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α) and phospho-eIF2α (p-eIF2α) in periprosthetic bone tissue were detected by Western blot.@*RESULTS@#Compared with sham group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were increased obviously in TCP group (<0.05), and serum level of T-AOC and SOD activity were decreased significantly in TCP group (<0.05), GRP78 expression, the ratio of p-PERK and PERK, p-eIF2α and eIF2α in the mouse calvaria of TCP group were up-regulated markedly. Compared with TCP group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were decreased markedly in NAC group (<0.05), serum level of T-AOC and SOD activity were increased obviously in NAC group (<0.05), and GRP78 expression, the ratio of p-PERK/PERK and p-eIF2α/eIF2α were obviously down-regulated.@*CONCLUSIONS@#Inhibition of oxidative stress can prevent periprosthetic osteolysis induced by TCP wear particles, which may be mediated by inactivation of PERK/eIF2α signaling pathway.

Role of PI3K/Akt Signaling Pathway in the TCP Wear Particles-induced Calvarial Osteolysis in Mice Model / 中国运动医学杂志

Objective To explore the role of the PI3K/Akt signaling pathway in the calvarial osteolysis induced by TCP wear particles in mice model.Methods Thirty-six male ICR mice were randomly divided into a sham group (n=12),TCP group (n=12)and a LY294002-treated group (n=12).A murine calvarial model of osteolysis was established through implanting 30 mg of TCP particles onto the surface of bilateral parietal bones following the removal of the periosteum.On the second postoperative day,LY294002 (5 mg·kg-1)was locally injected to the calvarium under the periosteum three times a week;mice in the sham group received local injection of normal saline (N.S.)in the calvarium,and the injection time was consistent with that of LY294002.Two weeks later,the calvaria and periostea were obtained after the mice were executed.The calvarial osteolysis,bone mineral density (BMD)and bone mineral content(BMC)were analyzed using Micro-CT,Hematoxylin-Eosin (HE)staining was conducted to observe the inflammatrory response and formation of osteoclasts.Real-time PCR was applied to detect the mRNA level of tartrate-resistant acid phosphatase (TRAP),the marker of osteoclasts formation,cathepsin K (CstK),receptor activator for nuclear factor-κB kigand (RANKL)and c-Fos.The release of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6)and IL-1β were measured using enzyme-linked immumsorbent assay (ELISA).Results Micro-CT and histological analysis indicated that LY294002,the specific inhibitor of PI3K,significantly prevented TCP wear particles-induced osteolysis and osteoclastogenesis,and increased BMD and BMC in the calvaria of mice.Real-time PCR data revealed LY294002 significantly suppressed the increase in mRNA level of osteoclastogenic genes such as TRAP,CstK,RANKL and c-Fos in the calvaria of TCP wear particles-implanted group.ELISA assay showed that TCP wear particles-induced release of TNF-α,IL-1β and IL-6 was significantly inhibited by LY294002 treatment.Furthermore,LY294002 significantly attenuated TCP wear particles-triggered activation of Akt,and down-regulated the level of p-AktSer473 and p-AktThr308.Conclusion PI3K/Akt signaling pathway contributes to TCP wear particle-induced osteolysis,and can be developed as a new therapeutic target for the prevention and treatment of bone destruction diseases caused by wear debris.