| Objective: Osteoporosis is a metabolic bone disease which caused by an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation,and osteoblasts are actually differentiated from Bone Marrow Mesenchymal Stem Cells(BMSCs).With the progressive study of the mechanism,it was found that bone formation and bone angiogenesis are closely related and coupled to each other in the process of bone metabolism.Therefore,reduced bone angiogenesis may also be a cause of osteoporosis.A large number of studies have confirmed that exercise can effectively prevent osteoporosis,and our preliminary study showed that exercise can upregulate the expression of lncRNA HOXA11-AS in the femur and tibia of mice,but whether exercise can mediate lncRNA HOXA11-AS to promote bone formation and improve bone angiogenesis is unclear.In this study,we investigated whether exercise and its resulting mechanical stress can mediate the expression of lncRNA HOXA11-AS in BMSCs by performing treadmill training intervention and mechanical distraction in mice and mouse BMSCs,respectively,and inhibiting the expression of lncRNA HOXA11-AS in BMSCs.Supernatants from BMSCs after distraction and knockdown of lncRNA HOXA11-AS were collected for culturing SVEC to investigate whether exercise and mechanical stress can mediate lncRNA HOXA11-AS to promote bone formation and bone angiogenesis,and thus provide theoretical basis for prevention and treatment for osteoporosis by exercise.Methods: 1 Animal experiment A total of 40 female C57BL/6 mice at 12 weeks of age were selected to construct a devitalized osteoporosis mouse model,and they were randomly divided into shamoperated and ovary-removed groups,then divided into sham-operated and quiet(SHAM)group,sham-operated exercise(SHAM+E)group,ovary-removed and quiet(OVX)group,ovary-removed and exercise(OVX+E)group according to whether treadmill training was performed or not,10 mice in each group.After a 2-week rest after the surgery,the mice in the treadmill training group first underwent 1 week of acclimatization treadmill training(Monday to Friday training,1 time/d,30 min/time,speed 6 m/min,increasing 2 m/min every other day,slope 25°,rest at the weekend);then 8 weeks of formal treadmill training(Training from Monday to Friday,1 time/d,1h/time,including 5min warm-up exercise and 55 minutes of formal exercise,the starting speed is 8m/min,and the weekly increment is 1m/ min,the official movement speed is 15m/min,the slope is 25°,and the weekend rests).Mice in SHAM group and OVX group were housed under normal conditions without any intervention.5 mice were randomly selected from each group,and the right femur was formaldehyde-fixed for micro CT scan reconstruction to analyze bone microstructure,and the right tibia was extracted for Real-time PCR to detect the expression of lncRNA HOXA11-AS,osteogenic factors and angiogenic factors.The other five were injected with angiography,the right tibia was removed overnight,formaldehyde-fixed and decalcified,and then used for micro CT scanning of bone vessels.2 Cellular experiment 2.1 Osteogenic differentiation experiments of primary mouse bone marrow mesenchymal stem cells BMSCs cultured to the second generation were inoculated on six-well cell culture plates and divided into Ctrl and Day7 for 7-day osteogenic differentiation experiments,with differentiation induced in osteogenic induction medium for 7 days in the Day7 group and not induced in the Ctrl group.After induction,ALP staining was performed to detect osteogenic differentiation.Meanwhile,differentiation was induced in osteogenic induction medium for 0,3,7,14 and 21 days in the Ctrl,Day3,Day7,Day14 and Day21 groups,respectively,and alizarin red staining and RNA extraction were performed after induction.2.2 Mechanical stretch protocol for primary mouse bone marrow mesenchymal stem cells BMSCs cultured to the second generation were inoculated in Bio Flex six-well plates and divided into five groups of Ctrl,S1,S3,S5 and S7.After the cells reached 80% confluence,the BMSCs were subjected to mechanical stretch(stretch intensity of 3%,frequency of 0.5 Hz,stretch duration of 4h/d,stretch every other day)for 7 days using the Flexcell FX-5000 cell stress loading system,with no intervention for the Ctrl group.The supernatant was collected at the end of retraction intervention and used to culture SVEC for scratch test,and cellular RNA were extracted.Real-time PCR was performed to detect the m RNA expression of lncRNA HOXA11-AS,osteogenic factor and angiogenic factor.2.3 Mouse primary bone marrow mesenchymal stem cells transfection experiment and post-transfection supernatant intervention in SVEC experiment BMSCs cultured to the second generation were inoculated in cell culture plates and divided into BLANK group,sh-NC group and sh-HOXA11-AS group,and the cells were replaced with α-MEM basic medium without serum,penicillin and streptomycin for 12 h when they were walled and confluent.sh RNA plasmids prepared(sh-HOXA11-AS,sh-NC)-liposome complexes were added to the corresponding wells,gently shaken and mixed and put into the cell culture incubator,BLANK group with liposomes only and without sh RNA.After 6-8h-transfection the cell were replaced with osteogenic induced differentiation medium,followed by changing the culture medium every 2-3 days,and terminated after 7 days,the supernatant was collected,and ALP staining and RNA extraction were performed,and Real-time PCR was performed to detect lncRNA HOXA11-AS expression.In addition,cell proliferation was detected by CCK8 at 8h,24 h and 48 h post-transfection.The supernatant collected after transfection was subsequently cultured in SVEC for scratch assay to observe the proliferation and migration of SVEC cells.Results: 1 Animal experimental part(1)The results of mouse femur microstructure scan by micro CT showed that compared with SHAM group,BV/TV,Ct.Ar/Tt.Ar,Tb.N and Tb.Th were significantly decreased in OVX group(P < 0.01),while Tb.Sp was significantly increased(P < 0.01);compared with SHAM+E group,Tb.N was significantly increased(P < 0.05)and BV/TV,Ct.Ar/Tt.Ar and Tb.Th were increased but not statistically different(P > 0.05),and Tb.Sp was significantly decreased(P < 0.05)in the SHAM+E group compared with the SHAM group;BV/TV,Ct.Ar/Tt.Ar and Tb.Th were significantly increased in the OVX+E group compared with the OVX group(P < 0.05),Tb.N increased significantly(P < 0.01),and Tb.Sp decreased significantly(P < 0.01).(2)The results of mouse tibial bone vessels scan by micro CT showed that the area of bone vessels was significantly decreased in the OVX group compared with the SHAM group(P < 0.05);the area of bone vessels was significantly increased in the OXV+E group compared with the OVX group(P < 0.05),and the area of bone vessels was increased but not statistically different in the SHAM+E group compared with the SHAM group(P > 0.05).(3)The PCR results of mouse tibia showed that the lncRNA HOXA11-AS in the tibias of mice in the OVX group was significantly decreased compared with the SHAM group(P < 0.01);the expression of lncRNA HOXA11-AS in the femur and tibia of mice in the SHAM+E group was upregulated but not statistically different(P > 0.05),while the lncRNA in the tibias of mice in the OVX+E group expression of HOXA11-AS was significantly upregulated in the OVX+E group compared with the OVX group(P < 0.05).In addition,the m RNA expression of osteogenic and angiogenic factors such as Osterix,OPN and FGF2 in the OVX group was significantly decreased compared with the SHAM group(P < 0.05),while the expressions of the above factors were significantly up-regulated in the OVX+E group(P < 0.05).Compared with the SHAM group,the expressions of Osterix,FGF2 and EGFL6 in the SHAM+E group had a trend of increasing but no statistical difference(P>0.05),and the expressions of OPN and FGFR1 were significantly increased(P<0.01,P<0.05).2 Cellular experimental sections(1)The results of osteogenic differentiation experiments of BMSCs showed that ALP staining gradually deepened during osteogenic differentiation at 7 days,alizarin red staining gradually deepened and the area of calcium nodules gradually increases during osteogenic differentiation at 21 days;Real-time PCR results showed that the expression of lncRNA-HOXA11-AS was gradually up-regulated,with a significant upregulation at day 7 of differentiation(P < 0.01)and a peak at day 14,followed by a decrease in expression.The expression of OPN and FGF2 was gradually up-regulated during osteogenic differentiation and was significantly up-regulated at day 7(P < 0.01).(2)The results of 7-day mechanical stretch in BMSCs showed that the expression of lncRNA HOXA11-AS was elevated in all stretched cells compared with the Ctrl group and was significantly upregulated at day 1 of retraction(P < 0.05)and reached a peak at 3 days of stretching(P < 0.01),followed by a decrease in expression;the expression of osteogenic factors Runx2,ATF4 was the same as that of lncRNA HOXA11-AS;the m RNA expression of OPN was up-regulated on days 3 and 7 of stretching(P < 0.01),and the m RNA expression of FGF2 was significantly up-regulated on day 1 of stretching(P < 0.05),and then slightly decreased but still higher than that of the Ctrl group.The results of the mechanical stretch experiment showed that the supernatant of BMSCs after 7 days of mechanical stretch gradually promoted the healing of SVEC scratches,but there was no statistical difference(P > 0.05).(3)The results of BMSCs transfection experiment showed that: the results of CCK8 assay showed that: the proliferation of BMSCs was not significantly inhibited at 8h and 24 h of transfection in sh-HOXA11-AS group compared with sh-NC group(P>0.05), while the proliferation of BMSCs was significantly inhibited at 48h(P<0.01);the results of scratch experiment showed that: the supernatant of BMSCs transfected with the results of scratch assay showed that the scratch healing rate of SVEC cells cultured with the supernatant of BMSCs in the sh-HOXA11-AS group was significantly lower than that in the sh-NC group after 24 h of supernatant culture(P < 0.05).Conclusion: Treadmill training can effectively promote the expression of lncRNA HOXA11-AS in tibias of devitalized osteoporotic mice and promote bone formation and bone angiogenesis;and appropriate mechanical stretch stimulation can promote the expression of lncRNA HOXA11-AS in BMSCs and up-regulate the expression of osteogenic factors and angiogenic factors,while knockdown lncRNA HOXA11-AS in BMSCs resulted in the inhibition of both proliferation of BMSCs and angiogenic ability. |
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