ABSTRACT
Objectives To observe the effect of low intensity pulsed ultrasound (LIPUS) on osteocyte injuries induced by the tricalcium phosphate(TCP) wear particles in the calvaria of mice.Methods Thirty ICR male mice of 6 to 8 weeks were randomly divided into a normal control group(n=10),a model group (n=10) and a LIPUS-treated group(n=10).A murine calvarial model of osteolysis was established in the model and LIPUS-treated groups through injecting TCP particles onto the surface of bilateral parietal bones at week 1,3,5,7 and 11.Mice in the normal group received negative ultrasound probe pressing,while those in the LIPUS-treated received LIPUS radiation.Three months later,the calvarias were obtained.The micro-CT,HE staining,flow cytometry and Western blotting were performed to estimate the calvarial osteolysis,osteocyte death,apoptosis and proteins expression of the dentin matrix protein 1 (DMP-1),sclerosis protein (SOST),glucose-regulated protein78 (GRP78),inositol-requiring enzyme(IRE 1 α),spliced X-box binding protein 1 (XBP1 s),c-Jun N-terminal kinase (JNK) and phosphorylated c-Jun N-terminal kinase(p-JNK) respectively.Results Compared with the normal control group,in the model group the viability of prosthetic osteocytes decreased significantly,and cell apoptosis was more obvious(P＜0.05);the osteocytic marker protein DMP-1 down-regulated significantly,but another marker protein SOST up-regulated significantly,which caused the decline in DMP-1/SOST(P＜0.05).Moreover,the expression levels of GRP78,IRE1,XBPls and p-JNK of the model group increased significantly(P＜0.05) in the calvaria osteocytes compared to the control group.However,in the LIPUS treatment group,osteocyte injuries and endoplasmic reticulum(ER) stress both decreased significantly,shown by a significant increase in the number and activity of osteocytes,DMP-1/SOST,and significant inhibition of the IRE1α-XBP1-JNK activation(P＜0.05).Conclusion LIPUS prevents osteocyte injuries induced by TCP wear particles in the calvaria of mice,which may be due to the inhibition of IRE1α-XBP1-JNK pathway activation through ER stress reaction.
ABSTRACT
AIM: To observe the effect of osthole on tricalcium phosphate (TCP) particles-induced calvarial osteolysis in vivo.METHODS:Male ICR mice were randomly divided into sham group , TCP group and osthole group .A mouse calvarial model of osteolysis was established by TCP particles .On the second postoperative day , osthole (20 mg/kg) was locally injected into the calvarium under the periosteum 3 times a week.Two weeks after osthole treatment , blood and calvaria were collected to determine the level of bone turnover markers such as alkaline phosphatase ( ALP) , osteocalcin and tartrate-resistant acid phosphatase ( TRACP) .The periosteum was performed to examine the release of tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6) and IL-1βby ELISA.The calvaria was obtained for histological and molecular analyses.RESULTS:Data from HE and TRACP staining revealed that osthole prevented TCP particles-induced obvious increase in osteoclastogenesis and resorption area in the metaphysis of mouse calvaria .Osthole treatment increased ALP ac-tivity and osteocalcin level , and dncreased the activity of TRACP in the mouse serum compared with TCP group .Further-more, TCP particles-induced the releases of TNF-α, IL-6 and IL-1βwere significantly suppressed by osthole treatment .In addition, Western blot demonstrated that endoplasmic reticulum ( ER) stress markers such as glucose-regulated protein 78 (GRP78) and CAAT/enhancer binding protein homologous protein (CHOP) were significantly up-regulated in TCP parti-cles-implanted calvarial mice , indicating that TCP particles triggered an ER stress response in the mouse calvarial osteolysis model , which obviously attenuated by osthole .CONCLUSION:Osthole inhibits TCP particles-induced calvarial osteolysis in mice, which is mediated by inhibition of ER stress signaling pathway .