| Background Osteoporosis is a common bone metabolism disease character by the loss of bone mass and degradation of bone microstructure,resulted in the increased bone fragility and fracture risk.With the acceleration of the aging process of the population,the incidence of osteoporosis is increasing year by year,and the prevention and treatment of osteoporosis has become a major public health problem that needs to be solved urgently.Conventional drug treatment has certain toxic,side effects and cannot completely cured,which limits its clinical application.A large number of basic and clinical studies have shown that appropriate exercise promoting bone formation,delay bone loss in the elderly,and play a key role in preventing and treating osteoporosis.The WHO also regards exercise as one of the important non-drug treatments for osteoporosis,but the mechanism has not yet been fully clarified.The research on the mechanism of exercise prevention and treatment of osteoporosis is mainly carried out from the aspects of hormones,cytokines,signal pathways and so on.In recent years,the role of micro RNA in exercise prevention and treatment of osteoporosis has been gradually discovered,and miR-214 has been shown to be widely involved in the regulation of bone metabolism.However,there is no in vivo animal study on whether miR-214 mediates exercise to prevent and cure osteoporosis.Therefore,this study will take miR-214 as the entry point and MSC as the research object to explore how miR-214 promotes bone formation and prevents osteoporosis.MethodPart Ⅰ : The effect of exercise on the expression of bone miR-214 and bone in growth period and osteoporotic mice1.Twelve wild-type female 6-week-old C57BL/6 mice were randomly divided into control group(Control,C)and exercise group(Exercise,E),with 6 mice in each group.The control group was routinely raised without any intervention.The exercise group had a 6-week treadmill exercise intervention.The speed was 6m/min in the first week,and then the speed increased by 1m/min every week,and the slope was always 25°.Materials will be collected 24 hours after the last intervention.2.The left femur was fixed with 4% PFA and used for micro CT detection.The right femur is used for bone biomechanics testing.The total RNA extracted from the left tibia was used for RT-PCR detection.The total protein extracted from the right tibia was used for Western-blot detection.3.24 wild-type female 12-week-old C57BL/6 mice were randomly divided into 4groups: ovariectomized group(OVX)and sham operation group(SHAM),ovariectomized + exercise group(OVX+EX),sham operation + Exercise group(SHAM+EX),with 6 mice in each group.The non-exercise group is routinely raised without any intervention.The exercise group undergoes a 9-week treadmill exercise intervention.The first week is exercise preconditioning,and the treadmill speed is 6m/min,the time is 30 min,and the treadmill slope is 25°.The next 2-9 weeks are formal interventions.Treadmill exercises include warm-up exercises and formal interventions.The warm-up exercise time is 5 minutes,the treadmill speed is 6m/min,the slope is 25°,the formal intervention time is 55 minutes,and the second week speed is 8m/min.And the weekly speed increases by 1m/min.4.The left femur is used for BMD and bone biomechanics detection,the right femur is used for miro CT detection,and the total RNA from the left tibia is used for RT-PCR detection.The total protein extracted from the right tibia was used for Western-blot detection.Part Ⅱ: The effect of knocking out miR-214 in MSCs on the bones of mice of different ages1.Prrx1 Cre mice was used to cross-pair with Mir214flox/+ mice to obtain mice that specifically knock out miR-214 in MSC.In this study,heterozygous female knockout mice(Mir214-/+)were selected for the experiment,and their bones were observed in12-week-old(12W),24-week-old(24W),and 36-week-old(36W)mice.2.The left femur was fixed with 4% PFA and used for micro CT detection.The right femur is used for bone biomechanics testing.The total RNA extracted from the left tibia was used for RT-PCR detection.The total protein extracted from the right tibia was used for Western-blot detection.Part III: Effects of exercise and ovariectomy on the expression of bone and related genes in miR-214 gene knockout mice1.Twenty-four 24-week-old female heterozygous knockout mice were selected and randomly divided into ovariectomized group(OVX),sham operation group(SHAM),ovariectomized + exercise group(OVX+EX),sham operation + Exercise group(SHAM+EX),with 6 mice in each group.Twelve wild-type mice were used as negative controls,and they were randomly divided into ovariectomized group(OVX)and sham operation group(SHAM),with 6 mice in each group.The operation of ovariectomy and sham operation is the same as the second part of experiment one.Four weeks after the operation,a one-week exercise preconditioning was carried out.The treadmill speed was 6m/min,the time was 30 min,and the treadmill slope was 25°.Afterwards,a 9-week exercise intervention was carried out.The exercise intervention included warmup exercise and formal exercise.The warm-up exercise was 6m/min,the time was 5min,and the treadmill slope was 25°;the first week of the formal exercise was 8m/min.The treadmill time is 55 min,and the slope is 25°.After that,the speed is increased by1m/min every week.2.The left femur was fixed with 4% PFA and used for micro CT detection.The right femur is used for bone biomechanics testing.The total RNA extracted from the left tibia was used for RT-PCR detection.The total protein extracted from the right tibia was used for Western-blot detection.Research resultPart Ⅰ :1.Micro CT and bone biomechanical indicators show that treadmill exercise significantly increases the bone mass,bone trabecular thickness and reduces the number of bone trabeculae in growing mice.2.RT-PCR and Western-blot indicators showed that treadmill exercise significantly inhibited the expression of bone miR-214 in growing mice,and significantly increased the expression of Osterix,ATF4,β-catenin.3.Treadmill exercise significantly improves the bone mass and BMD of osteoporotic mice,and the bone biomechanical indexes of osteoporotic mice also show a certain upward trend after treadmill exercise training.4.The expression of miR-214 in the bone of mice was significantly increased after the ovary was removed.Treadmill exercise can significantly reduce the expression of miR-214 in the bone of osteoporotic mice.And through RT-PCR and Western-blot detection results,it was found that the expression of ATF4 and β-catenin genes increased significantly after exercise in osteoporotic mice.Part Ⅱ:1.Micro CT detection found that the bone mass of gene knockout mice after miR-214 was knocked out in MSC was significantly increased at 12 W,24W,and 36 W.And the thickness of trabecular bone also showed a significant increase trend.In addition,the maximum load and bending strength of the knockout mice were significantly increased at 24 W and 36 W,and the destruction strain of the knockout mice was significantly increased at 12 W and 36 W.2.RT-PCR detection found that the expression of miR-214 in BMSCs of knockout mice and the expression of miR-214 in the bones of 12 W,24W,and 36 W knockout mice were significantly reduced.3.RT-PCR and Western-blot indicators showed that the expression of Osterix,ATF4,β-catenin,FGFR1 and other genes in the bones of miR-214 gene knockout mice increased significantly at 12 W,24W,and 36 W.Part III: Effects of exercise and ovariectomy on the expression of bone and related genes in miR-214 gene knockout mice1.The body weight of wild-type and gene knockout mice increased significantly after the ovaries were removed,and treadmill exercise can significantly reduce the body weight of the knockout ovarian mice.The results of micro CT and bone biomechanics tests found that the bone mass and bone strength of the knockout mice were significantly higher than that of the wild-type mice.The bone mass and bone strength of the wild-type and knockout mice were significantly reduced after the ovaries were removed.After 9 weeks of treadmill exercise intervention,the gene.The bone mass and bone strength of knockout mice and sham-operated mice increased significantly.2.RT-PCR detection revealed that the expression of bone miR-214 in gene knockout mice was significantly lower than that of wild-type mice.The expression of bone miR-214 in gene knockout mice increased significantly after the removal of the ovary,and9 weeks of treadmill exercise intervention can partially reverse the increase in bone miR-214 expression caused by the removal of the ovary.3.RT-PCR and Western-blot results showed that in knockout mice,ovarian removal reduced the expression of m RNA and protein such as Osterix,ATF4 and β-catenin in their bones.After 9 weeks of exercise intervention,the expression of the above genes was higher expressed in miR-214 knockout ovarian mice.At the same time,the expression of genes such as Osterix,FGFR1,ATF4 and β-catenin in the sham-operated mice of gene knockout mice is higher than that of wild-type mice.Conclusion:1.Exercise reduce the expression of miR-214 in the bones of growing mice and osteoporotic mice,and promote the expression of genes related to bone formation,thereby increasing their bone mass.2.miR-214 is a negative regulator in osteoblast differentiation of MSC.Knockout of miR-214 can increase the expression of related genes and increase the bone mass and bone strength of mice at different ages.3.Exercise can partially reverse the bone loss of miR-214 knockout mice caused by estrogen loss by reducing the expression of miR-214. |
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