Study On The Role And Mechanism Of NPNT In Promoting Bone Microvessel Formation In Osteoporosis Mice By Exercise

BackgroundOsteoporosis is a bone metabolic disease characterized by decreased bone density and destruction of the bone tissue microstructure.With the accelerated progress of social aging,the incidence rate of osteoporosis is increasing,and the burden of osteoporotic fractures is gradually increasing,which has gradually become a global public health problem that needs to be solved.Exercise can prevent and treat osteoporosis to a certain extent,but the underlying mechanism has not been fully elucidated.Our previous research found that exercise can reduce bone loss in an elderly osteoporosis mouse model and effectively increase the peak bone mass in growing mice.The mechanism may be that mechanical stress stimulates bone formation,exercise changes hormone and cytokine levels to promote bone formation,and exercise stimulates the regulation of related signaling pathways in cells and non-coding RNA.Recent research has shown that the decline in angiogenesis in the bone microenvironment may be one of the key factors inducing osteoporosis.The regulation of osteogenesis by promoting angiogenesis in the bone microenvironment and preventing and treating osteoporosis has become a hot research topic in recent years.Further research has found that osteoblasts secrete a variety of cytokines and regulate angiogenesis in the bone microenvironment through signal transmission with endothelial cells,thus affecting bone formation and bone remodeling,which has an important regulatory role in osteoporosis.Among these factors,nephronectin(NPNT)is expressed and secreted by osteoblasts as an extracellular matrix.Its expression levels decrease in the bones of both mice and patients with osteoporosis.In addition,NPNT can promote endothelial cell migration,tubular structure formation,and fetal rat metatarsal bone angiogenesis in vitro.In the preliminary research of the research group,m RNA chip scanning was used to find that a 9-week treadmill exercise program can increase the m RNA expression of NPNT in the tibia of ovariectomized mice.This finding suggests that exercise may prevent osteoporosis by stimulating angiogenesis in the bone microenvironment.Exercise may promote the expression of osteoblasts and secrete the angiogenesis-promoting cytokine NPNT.However,this hypothesis has not yet been confirmed.ObjectiveThis study aimed to conduct incremental load treadmill exercise intervention on ovariectomized osteoporosis model mice and explore the effects of exercise on bone density,bone biomechanical indicators,and bone microenvironment angiogenesis in mice.Simultaneously,in cell experiments,we further verified whether mechanical stress stimulation increases bone angiogenesis by promoting the expression and secretion of NPNT by osteoblasts.To deepen our understanding of bone angiogenesis and bone remodeling,it will help to elucidate the molecular mechanisms of exercise prevention and treatment of osteoporosis and provide a new target for the treatment of bone-related diseases such as osteoporosis.Methods and Materials Part 1: Effects of exercise on bone mineral density,bone strength,bone blood vessels,and NPNT expression in osteoporosis mice1.44 C57BL/6 female mice were selected for ovariectomy at 12 weeks of age and randomly divided into 4 groups: sham surgery group(S),sham surgery exercise group(SE),ovariectomy group(O),and ovariectomy exercise group(OE),with 11 mice in each group.Starting from 4 weeks after surgery,that is,16 weeks old,a 9-week incremental load treadmill exercise intervention was conducted: the first week was the pre-adaptation week,with intervention intensity of 6m/min,30 min/d,and a slope of0 °;the second week was the 9th week,with intervention intensity of 8-15m/min,60min/day,and a slope of 25 °.The intervention lasted for five days per week.2.After the treadmill intervention,the bilateral femurs and tibias of mice were extracted,and micro CT was used to detect the relevant indicators of the bone mineral density of mice femurs(bone volume percentage,number of trabeculae,the thickness of trabeculae,separation degree of trabeculae,the bone mineral density of trabeculae,the thickness of cortical bone,etc.),as well as the generation of blood vessels in the microenvironment of mouse femurs;Use a universal testing machine to detect biomechanical indicators of mouse femoral bones(maximum load,fracture load,bending strength,deflection,failure strain,elastic modulus,etc.)HE staining was used to detect changes in bone morphology,immunohistochemical detection of the distribution changes of NPNT;Immunofluorescence detection of CD31 and EMCN,and RT-PCR and western blot techniques were used to detect changes in bone formation-related factors Runx2,OSX,ALP,and NPNT in bone tissue.Part 2: The effect of the mechanical distraction of NPNT knockdown MC3T3-E1 osteoblasts on vascular endothelial cells1.NPNT-knockout MC3T3-E1 osteoblasts were constructed using gene knockout strains by electroporation.They were divided into four groups: control(C),knockout(KO),stretch(C+S),and knockout stretch(KO+S).The stretch group and knockout stretch group were subjected to 4% stretch intensity,Sin1/2 waveform,frequency 0.5Hz,and mechanical stretch stress stimulation for 4 hours per day for 7 consecutive days using the Flexcell-5000 cell stretch system.The control group and knockout group cells were cultured in a static state,the culture medium was replaced every 48 h for subsequent experiments,and the cells were collected after the intervention.2.After the mechanical stretching intervention,the expression of NPNT protein was detected by immunofluorescence;RT-PCR technology was used to detect changes in bone formation-related factors such as ALP,OSX,Runx2,ATF4,and angiogenesisrelated factors such as NPNT,Noggin,CXCL9,CXCL10,Nrp1,and Nrp2;Western blot technology was used to detect changes in related proteins such as NPNT,ALP,Runx2,Nrp2,etc;Detect the changes in NPNT protein in the collected culture medium through TCA protein precipitation;Use the collected culture medium to culture vascular endothelial cell lines(SVEC cells),and observe the effects of NPNT on SVEC cell proliferation,migration,and tubular formation ability through scratch wound healing and tubular formation tests.Part 3: The effect of mechanically distracted NPNT overexpression of MC3T3-E1 osteoblasts on vascular endothelial cells1.Construction of NPNT-overexpressing MC3T3-E1 osteoblasts through virusmediated stable transfection was divided into four groups: control group(E1),overexpression group(NPNT),stretch group(E1+S),and overexpression stretch group(NPNT+S).The stretch group and overexpression stretch group were subjected to 4%stretch intensity,Sin1/2 waveform,frequency 0.5 Hz,and mechanical stretch stress stimulation for 4 hours per day for 7 consecutive days using the Flexcell-5000 cell stretch system.The control and overexpression groups were cultured in a static state,the culture medium was replaced every 48 h for subsequent experiments,and the cells were collected after the intervention.2.After the mechanical stretching intervention,the expression of NPNT protein was detected by immunofluorescence;RT-PCR technology was used to detect changes in bone formation-related factors such as ALP,OSX,Runx2,ATF4,and angiogenesisrelated factors such as NPNT,Noggin,CXCL9,CXCL10,Nrp1,and Nrp2;Western blot technology was used to detect changes in related proteins such as NPNT,ALP,Runx2,Nrp2;Detect the changes in NPNT protein in the collected culture medium through TCA protein precipitation;Intervention was conducted on SVEC cells using the collected culture medium,and the effects of NPNT on SVEC cell proliferation,migration,and tubular formation were observed using scratch wound healing and tubular formation tests;Simultaneously observe the activation of signaling pathways in SVEC cells by the collected culture medium.Results Part 11.The Micro CT detection results showed that compared with the sham surgery group,the ovariectomized group had a significantly reduced number of femoral trabeculae,bone trabecular volume percentage,bone trabecular thickness,bone trabecular density,and significantly increased bone trabecular separation.Compared to the ovariectomized group,the ovariectomized group had a significantly increased number of femoral trabeculae,bone trabecular volume percentage,bone trabecular thickness,and bone trabecular density in mice,and significantly reduced bone trabecular separation,indicating a decrease in bone density after ovariectomy.Increasing treadmill exercise load can slow down the decrease in bone density caused by ovariectomy in mice.2.The bone biomechanical indicators showed that compared to the sham surgery group,the maximum load,fracture load,bending strength,deflection,and destructive strain of the femur in the ovariectomized group mice were significantly reduced.Compared with the ovariectomized group mice,the above indicators in the ovariectomized exercise group mice were significantly increased,indicating a decrease in bone biomechanical performance after ovariectomy,Incremental load treadmill which can improve the decrease in bone biomechanical properties caused by ovariectomy.3.The HE staining results showed that compared with the sham-operated group,the ovariectomized group had a decrease in the number of bone trabeculae and an increase in adipocytes.Compared with the corresponding sham surgery group,the sham surgery exercise group and ovariectomized exercise group showed an increase in bone trabeculae and a significant decrease in adipocytes.4.Micro-CT scanning of bone vessels showed that compared to the sham-operated group,the ovariectomized group showed a significant decrease in femoral vessel density and volume,while the sham-operated exercise group mice showed a significant increase in femoral vessel density and volume,while the ovariectomized group showed a significant increase in femoral vessel density and volume.5.The immunofluorescence test results showed that compared to the sham operation group,the fluorescence intensity and protein expression of CD31 and EMCN proteins in the femur of ovariectomized mice decreased,while the fluorescence intensity and protein expression of CD31 and EMCN proteins in the femur of sham operation exercise group mice increased;Compared with the ovariectomized group mice,the ovariectomized exercise group mice showed increased fluorescence intensity and protein expression levels of CD31 and EMCN proteins.Immunohistochemical tests showed that compared with the shamoperated group,the ovariectomized group had a decrease in the NPNT positive area of the femur,while the sham-operated exercise group had an increase in the NPNT-positive area;Compared with the ovariectomized group mice,the ovariectomized exercise group mice showed an increase in NPNTpositive area.6.RT-PCR results showed that compared with the sham operation group,the m RNA expression of Runx2,OSX,ALP,and NPNT in the bone tissue of the sham operation exercise group was significantly upregulated,while the above indicators in the bone tissue of the ovariectomized group mice were significantly reduced.Compared with the ovariectomized mice,the above-mentioned indicators in the bone tissue of the ovariectomized mice were significantly upregulated.The Western Blot test results showed that compared with the sham operation group,the expression of ALP and NPNT proteins in the bone tissue of the sham operation exercise group mice was upregulated,while the above indicators in the bone tissue of the ovariectomized group mice were significantly reduced;Compared with the ovariectomized group mice,the abovementioned indicators in the bone tissue of the ovariectomized exercise group mice were all upregulated.Part 21.The RT-PCR detection results showed that compared with the control group,the m RNA expression of genes such as ALP,Runx2,ATF4,OSX,NPNT,Noggin,Nrp1,Nrp2 in the knockout group cells was significantly downregulated,while the above indicators in the stretch group cells were significantly upregulated;Compared with the traction group,the above indicators in the knockout traction group were significantly downregulated.Compared to the control group,the m RNA expression of CXCL9 and CXCL10 in the knockout group was significantly upregulated.Compared with the knockout group,the above indicators in the knockout stretch group were significantly reduced.2.Western Blot analysis showed that compared with the control group,the expression levels of NPNT,ALP,Runx2,and Nrp2 proteins in the knockout group cells were significantly reduced,while the above indicators were upregulated in the stretch group cells.3.Immunofluorescence detection showed that compared with the control group,the knockout group showed a decrease in the fluorescence intensity of the NPNT protein and a significant decrease in the expression of the NPNT protein.The fluorescence intensity of the NPNT protein in the stretching group was enhanced,and the expression level of the NPNT protein was significantly increased compared to that in the stretching group.The knockout stretching group showed a decrease in the fluorescence intensity of the NPNT protein and a significant decrease in its expression of NPNT protein.4.TCA protein precipitation detection showed that compared with the control group,the expression level of NPNT protein in the cell supernatant of the knockout group was significantly reduced;The content of NPNT protein in the supernatant of the stretching group cells significantly increased,and compared to the stretching group,the content of NPNT protein in the supernatant of the knockout stretching group cells significantly decreased.5.The scratch wound healing test showed that compared with the control group,the knockout group had a decrease in the growth area of SVEC cells,while the stretching group had a significant increase in the growth area of SVEC cells;Compared to the knockout group,the knockout stretch group significantly increased the growth area of SVEC cells.6.The tube formation test showed that compared with the control group,the knockout group had a significant decrease in the ability of SVEC cells to generate tubules,while the stretching group had a significant increase in the ability of SVEC cells to generate tubules;Compared with the stretching group,the tubule production ability of SVEC cells in the knockout stretching group was significantly reduced.Part 31.The m RNA results of RT-PCR detection of related factors showed that compared with the control group,the m RNA expression of genes such as ALP,Runx2,ATF4,OSX,NPNT,Noggin,Nrp1,Nrp2 in the overexpression group cells significantly increased,while the above indicators in the stretch group cells were significantly upregulated;Compared with the traction group,the overexpression of the above indicators in the traction group was significantly upregulated.Compared to the control group,the m RNA expression of CXCL9 and CXCL10 in the overexpression group was significantly downregulated.Compared with the overexpression group,the above indicators in the overexpression stretch group were significantly upregulated.2.The Western Blot detection results showed that compared with the control group,the expression levels of NPNT,ALP,Runx2,and Nrp2 proteins in the overexpression group cells were significantly increased,and the above indicators in the stretching group cells also showed an upward trend.3.Immunofluorescence analysis showed that compared with the control group,the overexpression group had an increased fluorescence intensity of NPNT protein and a significant increase in NPNT protein expression,whereas the overexpression stretch group showed an increased fluorescence intensity of NPNT protein and a significant increase in NPNT protein expression compared to the stretch group;the overexpression stretch group showed an increased fluorescence intensity of NPNT protein and a significant increase in NPNT protein expression.4.TCA protein precipitation detection showed that compared with the control group,the expression level of NPNT protein in the cell supernatant of the overexpression group was significantly increased;The content of NPNT protein in the supernatant of cells in the stretching group also significantly increased;Compared with the overexpression group,the overexpression stretch group showed an increase in the content of NPNT protein in the cell supernatant.5.The scratch wound healing test showed that compared with the control group,the overexpression group had a significant increase in the growth area of SVEC cells,while the stretch group had an increase in the growth area of SVEC cells;Compared to the overexpression group,the overexpression stretching group showed an increase in the growth area of SVEC cells;Compared with the stretching group,the overexpression of stretching SVEC significantly increased the cell growth area.6.The tube formation test showed that compared with the control group,the overexpression group significantly improved the ability of SVEC cells to generate tubules,and the stretching group also significantly improved the ability of SVEC cells to generate tubules;Compared with the stretch group,the tubular production ability of SVEC cells overexpressed in the stretch group was also significantly upregulated.7.The detection of activation signaling pathways in SVEC cells showed that compared to the control group,the knockout group had a significant decrease in P38 phosphorylation levels,while the overexpression group had a significant increase in P38 phosphorylation levels compared to the control group;Compared with the corresponding control group,there was no significant change in the phosphorylation level of Akt protein in the knockout group and overexpression group.Conclusion1.Incremental load treadmill exercise can alleviate bone loss in ovariectomized mice,partially reverse the decrease in bone density and strength,and thus,prevent and treat osteoporosis.2.Incremental load treadmill exercise can promote the expression of NPNT and the formation of blood vessels in the bone microenvironment of mice and improve the decline of blood vessel volume in the bone microenvironment of ovariectomized mice.3.Mechanical stress stimulation can promote the expression and secretion of NPNT by MC3T3-E1 osteoblasts,thereby promoting the proliferation,migration,and tubulogenesis of SVEC.The P38 signaling pathway may be involved in its regulatory role.