A Study On The Effects Of Linarin On Osteoclasts And The Molecular Mechanisms

BackgroundOsteoporosis is a metabolic skeletal disease characterized by declined bone strength and increased bone fragility,which leads to a consequent increased risk of fracture.Severe osteoporosis is often accompanied by an increased fracture incidence,disability and mortality.It not only brings great pain in physical and mental to patients,but also makes burden and stress to families and society.Osteoporosis has become one of the most common global public health problems with the acceleration of the aging population t.It is estimated that by 2020,the number of osteoporosis patients in China will reach 286 million,and by 2050,the number will be more than 500 million,while Asia will have more than half of all osteoporotic fractures.As there is no way to fully supplement the decline in bone mineral density caused by the disease,osteoporosis is currently incurable.It is particularly important and urgent to take effective early intervention measures.As a highly dynamic tissue,bone forms a homeostasis under the regulation of various factors by continuously remodeling between osteoblast bone formation and osteoclast bone absorption.Once the number of osteoclasts or bone resorption function is increased,the homeostasis is broken,causing excessive loss of bone mass,which could result in many diseases,such as osteoporosis,paget disease,osteoarthritis,rheumatoid arthritis,malignant tumor bone metastasis and so on.Therefore,osteoclasts have become the main targets to treat lesions of bone excessive absorption.Osteoclasts,the multinuclear giant cells derived from the monocyte-macrophage precursor of bone marrow hematopoietic stem cells,were formed by gradually proliferation,differentiation and fusion under the effect of some regulatory factors such as macrophage colony stimulating factor(M-CSF)and tumor necrosis factor(TNF).In this process,the binding of RANKL to its receptor activation of nuclear factor kappa B(RANK),a member of TNF receptor family,leads to the recruitment and activation of TNF receptor-associated factor 6(TRAF6),which makes IKK2 to phosphorylate and degrade I?B?,and releases RelA/p50 or cRel/p50 heterodimer to translocate into the nucleus and triggers transcription.Nuclear factor of activated T cells cytoplasmic ?(NFATcl)is the main transcription and regulatory factor for the formation and maintenance function of osteoclasts.Therefore,the expression of NF-?B and NFAT(nuclear factor of activated T cells)can reflect the activation of RANKL/RANK signaling pathway.Currently,drugs that can inhibit osteoclasts differentiation and fuction in clinical include bisphosphonates,calcitonin,estrogen,selective estrogen receptor antagonist and so on.Although the agents are effective in the treatment of lesions of bone excessive absorption,most of them have some complications or serious side effects,especially in long-term use.For instance,bisphosphonates can cause osteonecrosis of the mandible,atypical fracture of femur and esophageal carcinoma,the prolonged use of estrogen increases the risk of breast cancer and heart disease,etc.Therefore,developing or discovering new drugs with safety to body and good therapeutic effects on osteolytic diseases has always been a hot topic.Since natural extracts have fewer undesirable side effects and more suitable for long-term use than chemical synthetic drugs,the potential bone active compounds in traditional Chinese medicine have attracted more and more attention.Linarin,a natural flavonoid glycoside,has been reported to possess pharmacological effects of promoting osteoblast differentiation and protecting osteoblasts.However,the effects of linarin on osteoclasts have not been studied.In the present study,we have performed a series of experiments in vitro investigating the effect of linarin on osteoclastogenesis and bone resorption and the involvement of the molecular biological mechanism of its action,and provided a sufficient theoretical basis for clinical application.Objectives1.Establish an osteoclast differentiation model using raw264.7 cells and bone marrow macrophages derived from mouse bone marrow to investigate the inhibitory effect of linarin on osteoclast differentiation and activity in vitro.2.To explore the molecular biological mechanism of linarin inhibiting osteoclast differentiation.Methods1.Bone marrow macrophages were isolated from the long bones of 8-week-old c57b1/6 mice.Osteoclasts were cultured in vitro and induced by RANKL and M-SCF.The cultured cells were washed and fixed at room temperature.Fixed cells were then dyed with TRAP staining kit.Cells containing three or more nuclei with positive staining were identified as osteoclasts.2.The toxicity of linarin to osteoclasts was determined by CCK-8 kit after incubation of BMMs with different concentrations of linarin for 24 hours,48 hours and 5 days.The semi-inhibitory concentration was calculated to select an appropriate experimental concentration of linarin.3.BMMs were seeded in 24-well plate and cultured in the presence of complete?-MEM medium containing M-CSF,RAN KL and different concentrations of linarin(0,0.1,1,and 10 ?g/mL).After 5 days,the effects of linarin on osteoclast formation and differentiation were measured.The next day,the area of the bone resorption region per well was observed under an optical microscope.4.Models of RANKL-induced osteoclast differentiation were treated with different concentrations of linarin and real time-quantitative polymerase chain reaction(RT-qPCR)was used to test the expression of osteoclast associated genes.5.To explore the impact of linarin on NFATc1 protein expression in RANKL-induced osteoclast differentiation,Raw264.7 cells were pretreated with 10 ?g/mL linarin for 4 hours and then co-cultured with 50 ng/mL RANKL,subsequently,Western blot analysis was performed to test the levels of NFATc1 protein at Day 0,1 and 3 respectively.6.Raw264.7 cells were pretreated with different concentrations of linarin(0,0.1,1 and 10 ?g/mL)for 4 hours and then stimulated by RANKL,following western blot was used to analyze the effect of linarin on the NF-?B signaling pathway involved in RANKL-induced osteoclastogenesis.Results1.Compared with the control group which was treated by M-CSF alone,when the concentration of linarin was lower than 15?g/mL,the activity of osteoclasts was not significantly affected(P>0.05),which was detected by CCK-8 testing kit.However,when the concentration of linarin was higher than 20?g/mL,osteoclast differentiation from BMMs decreased obviously(P<0.0001).2.BMMs were cultured with various concentrations of linarin for 5 days and then dyed with TRAP staining kit,the number of TRAP positive multinucleated cells was significantly decreased,and the cells are smaller in sizes and fewer numbers of nuclei.The results showed that linarin could effectively inhibit the differentiation of osteoclasts,and the higher the concentration of linarin was,the stronger the inhibitory effect was(F=300.4,P<0.0001).Bone plate absorption test showed that the bone resorption area of osteoclasts decreased obviously even when only 0.1?g/mL of linarin was treated on osteoclasts,indicating that linarin can effectively suppress the activity of osteoclasts(F=277.3,P<0.0001).3.Compared with the positive control group which was treated by RANKL alone,linarin significantly reduced the mRNA expression levels of osteoclast specific markers,which were examined through RT-qPCR assay.When the concentration of linarin was 0.1?g/mL,the expression of NFATcl and TRAP decreased significantly(t=12.19,P=0.0067;t=5.14,P=0.0358),but c-Fos and OSCAR did not change obviously(t=0.11,P=0.9213;t=0.01,P=0.9945).However,the expression of NFATc1?TRAP,c-Fos and OSCAR decreased significantly when the concentration of linarin reached 10?g/mL(t=19.44,P=0.0026;t=9.83,P=0.01;t=14.33,P=0.0007;t=8.01,P=0.004).4.RAW264.7 cells were pretreated with 10 ?g/mL linarin for 4 h and then stimulated with RANKL(50ng/mL),western blot analysis was performed to test the levels of NFATcl protein at Day 0,1 and 3.Compared with the positive control group,the protein levels of NFATc1 at the Day 0 were no obviously change(P>0.05),but significantly decreased at the Day 1 and 3(F=181.2,P<0.0001).The results showed that the protein expression of NFATc1 was not only dose-dependent,but also time-dependent.5.RAW264.7 cells were treated with RANKL in the presence or absence of linarin and NF-?B p65 levels were assessed using Western blot analysis.The results showed that the ratio of p-p65 to p65 increased significantly between 5 and 30 minutes after adding RANKL and reached the peak at 15 minutes(2.134 ± 0.368);however,the ratio of p-p65 to p65 decreased significantly after adding linarin and hit a low point at 15 minutes(0.116 ±0.072).These results indicate that linarin significantly inhibits the RANKL-induced NF-?B signaling pathway.6.Western blot analysis showed that the NF-KB-p65 nuclear translocation in the pretreated RAW264.7 cells with various concentrations of linarin decreased gradually with the increase of linarin concentration(F=42.7,P<0.0001).These results indicated that linarin inhibited the expression of NF-?B p65 in a dose-dependent manner.Conclusion1.In osteoclastogenesis model from BMMs and RAW264.7 cells in vitro,linarin can significantly suppress the formation,differentiation and bone resorption activity of osteoclasts.It can also inhibit the expression of osteoclast specific markers in the process of differentiation.2.The inhibitory effects of linarin on osteoclast happened in the early stage of osteoclast formation and the mechanism is related to the inhibition of the RANKL-induced NF-?B signaling pathway.3.Linarin has potential value in the treatment of osteolytic diseases.