| Background:As senior people become more and more,Chinese orthopedical sugeons face many magnifying problems,such as osteoporosis,post-arthroplasty periprosthetic osteolysi,tumor bone metastasis,etc.Osteoporosis is one of the main factors that endanger the health of the elderly,and has become the fourth major factor in the death of the population.China had 210 million people whose bone mass is lower than normal,and the percentage of osteoporotic patients is up to 15.7%among population over 50.Osteoporosis is a systemic disease,with manifestations including reduced bone mass,degenation of microstructure of bone tissue and increased fragility.Osteoporosis will compromise patient with joint pain,thoracolumbar pain and even fracture,and increase mobidity and motality.It has rapidly developed into a major public health problem in our country.Another age-related problem is osteoarthritis,where the number of prosthetic joint replacements has exploded over the past 20 years.This problem is called"granulopathy".Granulopathy is a kind of disease which is caused by wear of joint prosthesis to stimulate bone tissue,which trigers a series of pathophysiological changes lead to bone tissue degradation,and then lead to prosthesis loosening and corresponding symptoms.Bone metastasis is a disease with bone damage and pain caused by some malignant tumors outside of bone tissue.With the increase of tumor patients,bone metastasis increases correspondingly.In addition to limb pain,spinal metastases can also cause spinal cord compression and paralyze patients.Breast cancer is the most likely cancer to have bone metastases.A study shows that 70%of patients who die from breast or prostate cancer have bone metastases.One point that these problems have in common is the activation of osteoclasts.Osteoclasts and osteoblasts are the two most important types of cells that maintain the balance of bone remolding and ensure the health of bone tissue.Osteoclasts are the only cells in the body that absorb bone.When they are activated,increased in number or function,they can lead to osteoporosis,periprosthetic osteolysis,tumor bone metastasis,osteoarthritis,Paget disease and so on.Therefore the study of osteoclast related diseases has high basic research value,clinical diagnosis and treatment value,and social/economic value.Although the current clinical use of drugs such as calcitonin,diphosphate,estrogen and so on showed effect in the treatment of osteoclast-related diseases,but there still were reports of fracture,osteonecrosis,tumor and other complications.Novel drugs,with high specificity and less side effects to inhibit osteoclast bone resorption,have been a hot research field in recent years.At present,More and more attention has been paid to researches adopting natural herbal drug extracts to inhibit osteoclast differentiation and function and to prevent and treat osteolysis related diseases mediated by osteoclasts.In the previous drug screening,we found that the Chinese herbal monomer Raddeanin A can effectively inhibit osteoclasts' migration,differentiation and bone resorption.In this study,we will further explore the molecular mechanism of Raddeanin A's inhibition of osteoclasts,and confirm its role in the prevention and treatment of two major diseases related to osteoclasts through animal experiments,and then to provide a theoretical basis for the clinical application of Raddeanin A.Objective:To study the molecular mechanism of Raddeanin A's inhibition of osteoclast,and to study the effect and mechanism of Raddeanin A on osteolysis induced by titanium particles and bone metastasis in mice models.Method:Osteoclasts and osteoblasts were cultured in vitro from cells in 6-weeks-old C57BL/6 female mice's bone marrow.Osteoclasts were identified as cells with three positive TRAP staining nuclears.ALP staining and alizarin red stainiing were used to evaluate the differentiation and function of osteoblasts.CCK-8 kit was used to determine the cytotoxicity of Raddeanin A and IC50 was calculated.qPCR was used to determine the expression of specific genes in osteoclast and osteoblast differentiation.The bone resorption region was qualitatively and quantitatively analyzed by scanning the surface of bovine bone slice by electron microscope.Western blot was used to determine the signaling pathway of the Raddeanin A's inhibition of osteoclast differentiation and bone resorption.A mice model of osteolysis of calvarium induced by titanium granules in was established,which get local injection of Raddeanin A solution or PBS once a day for 14 days.Finally,all the calvarial specimens of mice were collected and fixed with 4%paraformaldehyde.Then histological examination and micro-CT scanning were performed to study the effect of Raddeanin A on osteoclastic osteolysis induced by titanium granules in vivo.In order to test the effect of Raddeanin A on tumor bone metastasis in vivo,an animal model of breast cancer bone metastasis was established,which get intraperitoneal injection of Raddeanin A solution or PBS once every two day for 28 days.The tibial specimens of all the mice were collected and fixed with 4%paraformaldehyde.Histological examination and micro-CT scanning were performed respectively.Results:The toxicity test confirmed that the survival of BMMs was not compromised under the condition of less than 0.781?M in 48 hours and less than 0.391?M in 72 hours and less than 0.098 ?M in 96 hours.At the same time,we calculated that the half inhibitory concentration of IC50 on BMMs was 3.73 p.M at 48 hours,2.91?M at 72 hours and 1.94 ?M at 96 hours.The inhibitory effects of differentiation were in a concentration-gradient dependent manner.We cultured BMMs under the stimulation of MSC and RANKL with different concentrations of Raddeanin A(0.2 ?M,0.4 ?M and 0.8 ?M)or with 0.4 ?M Raddeanin A for 3,5 and 7 days.The results showed that the number and the area of osteoclasts in the drug groups were decreased in comparison with the control group.The inhibition of osteoclast differentiation by Raddeanin A showed dose-and time-dependent characteristics.We further exzamined the expression of osteoclast specific genes in osteoclasts stimulated by RANKL.The expression of osteoclast specific genes in the control group was significantly up-regulated,while in drug groups,the expression of these genes was down-regulated in varying degrees.The expression of osteoclast specific gene was inhibited by Raddeanin A in a dose-and time-dependent manner.We added different concentrations of Raddeanin A into osteoblast culture system.The results of ALP staining on the 7th day showed that the osteoblasts were not inhibited at 0.2?m 0.4 ?M and 0.8 ?M,compared with the control group.On the 21st day,alizarin red staining showed that the osteoblast mineralization in 0.2 ?M group was more than that in the control group,but there was no significant difference between theother concentration groups with control group.On the 14th day,the expression of SPARC in the 0.4 ?M drug group was significantly increased.These results indicated that the differentiation and function of osteoblasts were at least not inhibited by Raddeanin A in vitro.BMMs were cultured on bovine bone slices(aseptic)and different concentrationsof Raddeanin A were added.The results showed that the bone resorption area of bovine bone slices in control group was about 43%;while in 0.2 ?M group,the area of bone resorption was reduced to about 18%;in 0.4 ?M group,the area was reduced to about 7%;in 0.8 ?M group,bone resorption was almost invisible.Therefore,it is clear that Raddeanin A has the function of inhibiting osteoclast differentiation and bone resorption in vitro.We also investigated the effects of Raddeanin A on osteoclast differentiation related signaling pathways including MAPK,NF-?B and SRC/AKT signaling pathways.AKT phosphorylated 10 minutes after RANKL stimulation.On the other hand,the phosphorylation of AKT was significantly inhibited at 10 min and 30 min after RANKL stimulation.The expression of SRC was upregulated 3 days after RANKL stimulation,while Raddeanin A reversed it.However,there was no significant difference in the expression of JNK,P38,ERK and I?B? between the control group and the drug group.We added AKT agonist SC79,then we observed that SRC in the control group increased significantly after three days of stimulation by RANKL,and that SRC was significantly inhibited in the group treated with Raddeanin A.Meanwhile,the inhibitory trend of SRC by Raddeanin A was saved by SC79.These results suggested that the differentiation and function of osteoclasts could be inhibited by the Raddeanin A's inhibition of SRC/AKT signaling pathway.A mouse osteolysis model induced by titanium granules was established to study the effect of Raddeanin A on osteoclast associated osteolysis in vivo.We injected saline(control group)or different concentrations of Raddeanin A,and we also set up a blank control group(no titanium granules,only saline every day).4 weeks later,micro-CT showed that compared with the blank control group,the osteolysis of the mice in the control group was obvious.However,the osteolysis of the low concentration and high concentration drug groups was alleviated in varying degrees.Furthermore,the porosity of bone mass to tissue mass ratio(BV/TV)and porosity in reaction area(ROI)were measured on micro-CT 3D reconstruction images.Compared with those in the control group,BV/TV increased in different degrees and porosity decreased in varying degrees in both low and high concentration groups.Meanwhile,TRAP staining showed that there were a large number of multinucleated osteoclasts.on the surface of bone in the osteolytic region of mice in the control group.However,osteoclasts in low concentration and high concentration groups decreased in varying degrees.These data suggest that the osteoclast differentiation and bone resorption function can be inhibited by the growth of Raddeanin A in vivo,and so was titanium particles inducing osteolysis.The cytotoxicity test of MDA-MB-231 breast cancer cell line at 48 and 96 hours showed that the growth of MDA-MB-231 cells was significantly inhibited by Raddeanin A over 6.25?M.The IC50 of MDA-MB-231 cell was 18.01?M and 15.77?M,respectively.Compared with the osteoclasts,MDA-MB-231 cells were more tolerant to Raddeanin A.The proliferation of MDA-MB-231 cell line was significantly inhibited 24 hours after treatment with different concentrations of Raddeanin A.Cell flow analysis showed that the percentage of apoptotic cells was significantly increased by Raddeanin A.The cell invasion assay showed that the proliferation of MDA-MB-231 cells was significantly decreased by Raddeanin A.The inhibition of the invasion behavior of MDA-MB-231 cells was concentration-dependent.We then used another breast cancer cell line BCAP37,and similar results were obtained.We also constructed a nude mouse model of breast cancer bone metastasis and injected MDA-MB-231 breast cancer cells into the tibial plateau of nude mice.The results of micro-CT and histology examination 28 days after intraperitoneal injection of PBS or Raddeanin A(100 ?g/kg)showed that the bone trabecular bone loss in the control group was significantly higher than that in the drug group.The BV/TV was higher and the bone trabecular space was narrower in the drug group than in the control group,while the bone cortex in the drug group was still intact and the bone trabecular absorption was serious and the bone cortex was separated in the control group.TUNEL analysis showed that the apoptotic degree of breast cancer cells in the drug group was significantly increased.These results suggested that Raddeanin A could inhibit osteolysis induced by breast cancer cells in vivo.Finally,we found that the phosphorylation of AKT and the expression of mTOR were down-regulated in MDA-MB-231 cells treated by Raddeanin A at the concentration of 3?M at different time.The results suggest that Raddeanin A's inhibition of growth and invasion of MDA-MB-231 cells may play via AKT/mTOR signaling pathway.Conclusion:Raddeanin A could inhibit differentiation and bone resorption function of osteoclasts,but not inhibit osteoblast differentiation and function in vitro.Raddeanin A could also inhibit the growth and invasion of MDA-MB-231 and BCAP37 breast cancer cells in vitrao.The osteolysis induced by titanium granules and MDA-MB-231 breast cancer cells can be inhibited by Raddeanin A in vivo.Therefore,we speculate that Raddeanin A has potential value in the treatment of osteoclast-related diseases and bone metastasis of breast cancer. |
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