| A large number of the osteoclast differentiation and activation is the root cause of postmenopausal osteoporosis. The rapid declining of estrogen leading to the bone osteoclast differentiation and activation in postmenopausal women. The rapid increase in bone resorption caused by the osteoclast generation and activation lead to the bone resorption rate faster than bone formation rate. The dynamic equilibrium of bone resorption and bone formation is destroyed, resulting in high conversion of osteoporosis. Studies have shown that exercise can regulate bone metabolism in many ways. Bone can adjust its structure and characteristics according to its mechanical environment. As the astronauts in a long-term microgravity environment, the rate of their bone loss is amazing. The bone loss rate of load-bearing bone is about0.5-2%per month, and appropriate exercise can improve bone density and prevent bone loss. A large number of experiments have reported the osteogenic effect of swimming, jumping, climbing, and resistance training intervention. However, the cell and molecular mechanisms of the exercise regulation to postmenopausal bone reconstruction is still lacking, especially the effects of exercise to osteoclast differentiation of postmenopausal has not yet been reported.Objective:In this study, though imitating the characteristics of bone remodeling in postmenopausal women by the ovariectomized mice modeling, we compared to the effects for the prevention of postmenopausal osteoporosis of uphill running and downhill treadmill running, and focus on the different effects on the osteoclast differentiation and activation of the two different exercise pattern. The purpose of this study is to reveal the mechanism of the different sports impacting bone remodeling in postmenopausal in the cellular and molecular level.Methods:There were648weeks old C57BL/6female mice, they were divided into4groups randomly:sham surgery quiet group (S, n=164), ovariectomized quiet group (O, n=16), ovariectomized uphill running group (U, n=16) and ovariectomized downhill running group (D, n=16),16mice in each group. The mice of group S were implemented of ovariectomized sham surgery, and the remaining three groups of mice were ovariectomized. Training programs:the mice of group U were implemented uphill running training, and the mice of group D were implemented downhill running training, at the speed of0.8km/h, the slope is9°and-9°respectively, each training last40minutes, five trainings a week, the total training period is eight weeks. After eight weeks, we test femur and tibia bone mineral density by a Kodak X-ray absorptiometry; We test tibial bone tissue morphometry index though resin-embedded sections and histomorphometry surgery; We test femur of OPG, RANKL, RANK, M-CSF and TNF-α, IL-6and TRAP gene expression levels though fluorescent quantitative PCR; Bone TRAP staining to detect the protein level of the femur TRAP; Osteoclast differentiation detected by bone marrow osteoclasts in primary culture; Western blot test osteoclast differentiation p65,IκBα, p-p65and p-IκBα protein levels in NF-κB signaling pathway.Results:(1) Compared to the mice in group S, the speed of ovariectomized mice weight gain increased significantly, treadmill exercise can effectively inhibit the weight excessive growth of mice caused by ovariectomized surgery.(2) Compared to the mice in group S, the femoral and tibial bone mineral density of ovariectomized mice decreased significantly, treadmill exercise can effectively prevent bone density decline after the mice were ovariectomized, and the downhill running is better than uphill running.(3) Compared to the mice in group S, the ovariectomized mice tibial trabecular number and volume density reduced significantly, the distance between the trabecular bone increased, the thickness of the trabecular bone thinning and inter-arranged messy, the normal three-dimensional network structure were damaged. Treadmill exercise can effectively prevent the osteoporotic changes of the ovariectomized mice, and downhill running better than uphill running.(4) Compared to the mice in group S, ovariectomized mice bone osteoclast-specific enzyme TRAP gene and protein expression levels were significantly increased, indicating that ovarian surgery enhanced the ability of mouse osteoclast differentiation and activation. Treadmill exercise can effectively inhibit the ovariectomized-induced osteoclast differentiation and activation of the mice, and the downhill running is better than uphill running.(5) Compared to the mice in group S, ovariectomized mice bone RANKL M-CSF and RANK gene expression was significantly increased, and the gene expression of OPG in ovariectomized mice decreased significantly, this shows that the changes of cell factors in OPG-RANKL-RANK system of ovariectomized mice are conducive to osteoclast differentiation and bone resorption. Treadmill exercise can effectively reduce the RANKL and M-CSF gene expression and increase OPG gene expression of ovariectomized mice, and the downhill running is better than uphill running, and the effect of treadmill exercise on bone RANK gene expression is not obvious.(6) Compared to the mice in group S, the bone IL-6and TNF-α gene expression in ovariectomized mice was significantly elevated, while IL-6and TNF-α can promote osteoclast differentiation and bone resorption through OPG-RANKL-RANK system-dependent and-independent pathway. Treadmill exercise can effectively reduce the bone IL-6and TNF-α gene expression in ovariectomized mice, and the downhill running is better than uphill running.(7) Compared to the mice in group S, Ovariectomized mice bone marrow hematopoietic stem cells showed a higher ability to differentiate to osteoclast precursors and osteoclast differentiation capacity. Treadmill running can effectively inhibit the differentiation process of bone marrow hematopoietic stem cells to the osteoclast precursors and the differentiation process of osteoclast precursors to the osteoclast, and the downhill running is better than uphill running.(8)The ovariectomized surgery can lead to a significant increase in mouse osteoclast precursors p-p65and p-of IκBα protein expression, and the p65and IκBα protein expression was not affected by the ovariectomized surgery, this shows that the ovariectomized surgery can activate NF-κB signaling pathway for osteoclast differentiation through the promotion of p65and IκBα protein phosphorylation. Treadmill exercise can effectively inhibit the activation of NF-κB signaling pathway in osteoblast differentiation caused by ovariectomized surgery, and the downhill running is better than uphill running.Conclusion:(1)Treadmill exercise can be effective in preventing the decline in bone mineral density and the osteoporotic changes of bone structure of the ovariectomized mice, and the downhill running is better than uphill running.(2) By adjusting the expression of factors of OPG-RANKL-RANK system and the expression of IL-6, TNF-α and other cytokines, treadmill running can inhibit osteoclast differentiation and bone resorption of the mice after ovariectomy, and prevent the occurrence of osteoporosis.(3) Treadmill running have a significant inhibitory effect on the differentiation process of the bone marrow hematopoietic stem cells to the osteoclast precursors and the differentiation process of the osteoclast precursors to the osteoclast, hereby reducing osteoclastogenesis of the ovariectomized mice.(4) Treadmill running can inhibit the activation of NF-κB signaling pathway of osteoclast differentiation through inhibiting osteoclast precursors p65and IκBα protein phosphorylation in ovariectomized mice, thereby reducing osteoclastogenesis.(5) The effects of downhill running to the ovariectomized mice bone remodeling regulation is better than uphill running, mainly due to the different stress on bone tissue in the exercise.
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