| Background:The increase of inflammatory factors in vivo is an important pathogenesis of postmenopausal osteoporosis.With the increase of age,the level of estrogen,which maintains bone mass balance,decreases,which leads to the increase of inflammatory factors,destruction of bone microenvironment,bone loss and osteoporosis.Tumor necrosis factor-α(TNF-α)was the most obvious inflammatory factor.Previous studies have shown that TNF-α inhibits the differentiation of bone marrow mesenchymal stem cells into osteoblasts and reduces bone formation by increasing the P2Y2 receptor of bone marrow mesenchymal stem cells.However,the formation of osteoporosis is affected by osteoblasts on the one hand and osteoclasts on the other.Therefore,we hypothesized that the increase of TNF-α can also affect osteoclast formation by affecting the P2 receptor of bone marrow hematopoietic stem cell(Bone Marrow Hematopoietic Stem cells,BMHCs),and there are no clear conclusions and mechanisms reported.Therefore,we designed this experiment to explore the ways in which TNF-α affects the formation of osteoclasts.Objective:1.To make it clear that TNF-α can promote the differentiation of BMHCs into osteoclasts.2.To investigate the effect that TNF-α can increase the expression of specific P2 receptor in BMHCs.3.To determine that the increased expression of specific receptors can promote the differentiation of BMHCs into osteoclasts.4.To explore the signal pathway that TNF-α promotes the expression of BMHCs specific receptors.Methods:1.Animal experimentTo establish a classic postmenopausal osteoporosis model-ovariectomized(ovariectomy,OVX)mice: 30 8-week-old female mice were randomly divided into three groups.The first group underwent sham operation and received caudal vein injection of normal saline(SHAM).The second group underwent ovariectomy and caudal vein injection of normal saline(OVX).The third group underwent ovariectomy and caudal vein injection of anti-TNF-α(OVX+anti-TNF-α).After feeding under the same conditions for 12 weeks,the samples were sampled without difference.The eyeball serum of mice was taken and the level of TNF-α was determined by ELISA method to evaluate the model.The ultrastructural changes of bone(femur and tibia)were evaluated by Micro-CT scanning,and the gross structural changes of bone trabeculae and osteoclasts were evaluated by HE staining,TRAP staining and immunohistochemical staining.2.Cell experiment2.1.Bone marrow hematopoietic stem cells were collected and cultured,and differentiated with different concentrations of TNF-α(10,20,30ng/ml)and different time(1,3,5 days).Finally,TRAP staining was performed and counted,and compared to explore the appropriate concentration and time of TNF-α to promote bone marrow hematopoietic stem cells to differentiate into osteoclasts.2.2 After bone marrow hematopoietic stem cells were cultured with different stimuli(TNF-α concentration 0,20ng/ml),P2 receptors at different time points were detected,repeated many times,different methods(fluorescence quantitative PCR detection,Western blot),confirmed high expression of specific receptors;2.3 Transfection of knockout plasmid / activation plasmid,inhibition / overexpression of specific receptors,detection of plasmid efficiency,TRAP staining,immunostaining and specific gene Western blot assay were used to detect the differentiation of bone marrow hematopoietic stem cells into osteoclasts.;2.4 By co-culture of calf bone slices and biomimetic bone smears,the bone resorption capacity of osteoclasts was detected after the differentiation was stimulated by TNF-α(concentration of 0,20,20ng/ml).Results:1.The results of in vivo experiments showed that the increase of TNF-α could promote the formation of postmenopausal osteoporosis,increase the production of osteoclasts and up-regulate the expression of BMHCs P2X7 receptor,while anti-TNF-α had a significant reversal effect.Through the ELISA method,we can detect the value of TNF-α factor in the serum of mice after modeling,so as to verify the rationality and reliability of the mouse model and grouping used in this experiment.Three-dimensional reconstruction was performed after Micro-CT scanning.The results showed that compared with the sham-operated control group(SHAM),the femur BMD and BV/TV of(OVX)mice decreased and Tb.N increased.However,the trabecular structure of OVX mice treated with anti-TNF-α was significantly improved compared with(OVX)of ovariectomized mice.The results of HE staining(bone trabecular structure)were similar.The results of TRAP staining showed that anti-TNF-α treatment could significantly inhibit the formation of osteoclasts in ovariectomized mice.Immunofluorescence results showed that there were many osteoclasts positive for P2X7 in ovariectomized mice.The results showed that the level of serum TNF-α in ovariectomized mice was significantly higher than that in the other two groups,while the level of serum TNF-α in ovariectomized + anti-tumor necrosis factor-α mice was significantly lower than that in ovariectomized mice.The protein and RNA,of osteoclast progenitor cells were extracted from the three groups of bone marrow hematopoietic stem cells.The results showed that the expression of P2X7 protein and RNA in the cell protein of OVX group was significantly higher than that of the other two groups.2.The results of in vitro experiments showed that TNF-α of 20ng/ml could significantly induce bone marrow hematopoietic stem cells to differentiate into osteoclasts and significantly up-regulate the expression of P2X7.In vitro,bone marrow hematopoietic stem cells were stimulated with different concentrations of TNF-α(0,10,20 and 30ng/ml),and different numbers of osteoclasts positive for TRAP staining could be obtained by differentiation culture.The staining results showed that bone marrow hematopoietic stem cells could differentiate into a large number of osteoclasts under the stimulation of 20 ng/ml.Further experiments showed that the differentiation of osteoclasts was related to time,and a large number of osteoclasts were produced after 5 days.The results of quantitative reverse transcriptase polymerase chain reaction showed that 20 ng/ml TNF-α could significantly up-regulate P2X7 receptor.Western blot also confirmed this.3.In vitro experiments showed that knockout of P2X7 RNA could significantly inhibit the effect of TNF-α on the differentiation of BMHCs into osteoclasts and the bone resorption of osteoclasts.Therefore,P2X7 participates in the process of osteoclast differentiation promoted by TNF-α.In order to verify the role of P2X7 receptor in the process of TNF-α factor promoting the differentiation of BMHCs into osteoclasts,we first transfected the target cells with sh RNA-P2X7 plasmid and silenced the gene of the receptor(overexpression of the receptor did not increase the number of cell differentiation).Western blot analysis showed that the protein of receptor in target cells decreased at different time points(2 days and 5 days)after sh RNA-P2X7 transfection.Bone marrow hematopoietic stem cells with P2X7 receptor silencing were cultured in osteoclast differentiation medium for 5 days,and then different groups of RNA and proteins were extracted.The expression of osteoclast-specific genes,including MMP-9,TRAP,cathepsin K(cathepsin K,CTSK)and C-src,were detected by RT-PCR and Western blot.The results of RT-PCR and Western blot showed that the expression levels of MMP-9,TRAP,CTSK and C-src were significantly decreased in BMHCs transfected with sh RNA-P2X7.In addition,it is speculated that sh RNA-P2X7 can inhibit the potential of BMHCs to differentiate into osteoclasts,as shown by TRAP staining.In addition,specific immunohistochemical staining showed that TNF-α could significantly increase the number of P2X7 receptors in osteoclasts and promote osteoclast differentiation.Bone resorption indentation test showed that TNF-α factor could promote the differentiation of bone marrow stem cells into osteoclasts and improve the ability of osteoclasts,while the inhibition of P2X7 receptor could significantly reduce the effect of TNF-α factor on bone marrow mesenchymal stem cells.In summary,these results suggest that P2X7 receptors are involved in the process of TNF-α promoting the differentiation of BMHSs into osteoclasts.4.TNF-α upregulates the expression of P2X7 through PI3K/Akt signal pathway,thus promoting osteoclast differentiation induced by RANKL.In order to study the signal pathways through which TNF--α up-regulates the expression of P2X7 receptor,we first observed the effects of tumor necrosis factor-α on PI3K/Akt,NF-k B and mitogen-activated protein kinase signal pathways during bone marrow hematopoietic stem cell osteotomy.The results suggest that PI3K/Akt signaling pathway is significantly activated by TNF-α factor in the process of bone marrow hematopoietic stem cells differentiating into osteoclasts,and they are activated by TNF-α factor in a time-dependent manner.When PI3 K was inhibited by inhibitor,the effect of tumor necrosis factor-α on the differentiation of bone marrow hematopoietic stem cells into osteoclasts was significantly reduced.In addition,when the PI3K/Akt signal pathway was activated by agonists,the results showed that P2X7 receptors increased accordingly.CCK-8 results showed that there was no statistical difference in the effect of different reagents on the number of osteoclasts.Conclusion: The results of in vivo experiments showed that TNF-α could form postmenopausal osteoporosis,increase the production of osteoclasts and up-regulate the expression of BMHCs P2X7 receptor,while anti-TNF-α had a significant reversal effect.The results of in vitro experiments showed that TNF-α of 20ng/ml could significantly induce bone marrow hematopoietic stem cells to differentiate into osteoclasts and significantly up-regulate the expression of P2X7.In vitro experiments showed that knockout of P2X7 RNA could significantly inhibit the effect of TNF-α on osteoclast differentiation and osteoclast bone resorption induced by BMHCs.TNF-α upregulates the expression of P2X7 through PI3K/Akt signal pathway,thus promoting osteoclast differentiation induced by RANKL. |
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