| The main function of bone is to protect the internal organs and support the body. It is also involved in calcium metabolism. Bone is a dynamic metabolic organ which is essential for maintaining the fundamental life processes, and it can be affected by the internal and external environment changes, genetic, lifestyle and exercise. Mechanical stimulation by any motion can modulate bone growth, such as its shape, size, strength, and the anatomical structure. Exercise training can increase its density, reduce the occurrence of osteoporosis, and affect the growth of bone metabolism. These have been proved to be more effective on young people rather than the old people. Bone mass increases during the growing period, it reaches the peak in the mature stage, and then starts to decrease with the increase of age. The accumulation of bone mass during growth period can reduce fracture probability, and delay the occurrence of osteoporosis. PTH and CT play a major role in bone metabolism, and they have been clinically applied to treat osteoporosis by America food and Drug Administration (FDA).However, it has not been reported whether exercise could increase the endogenous PTH and CT concentration and if it has an impact on bone metabolism.Objective: In order to seek the mode of motion this can influence bone synthesis and metabolism more significant. We compare the downhill running, flat slope treadmill and swimming exercise on bone density, bone mineral content and bone metabolism related cytokine gene and protein expression, and compare proliferation and differentiation of primary MSCs after8weeks exercise in order to find out the effect on bone metabolism of concentration changes of endogenous PTH and CT which induced by different exercise. Furthermore, with the aim of enriching the theoretical knowledge of exercise, we hope to make human bones strong and seek out a best way that is effective on the treatment of metabolic bone disease. We compare the differences between serum PTH and CT concentrations through different exercises, the effect of PTH and CT concentration on the proliferation and differentiation of osteoblasts In vitro.Methods: We divide120C57BL/6mice into4groups and30rats in each group. The4groups consist of downhill treadmill group (group DT), flat slope treadmill group (group T), swimming group (group S), and control group (group C). Training program:DT group training, the running speed is0.8km/h, the slope is-9°; The running speed of group T is same as the speed of group DT, the slope is0°; The training of S group is swimming without weight bearing. Using brush stir the mice when they are floating motionless. The time of immediate exercise group is40minutes and mice were all dead within30minutes after exercise. The concentration of serum PTH and CT is determined by Elisa assay and the gene expression of thyroid PTH, CT and Destrin gene is determined by qRT-PCR method. Long term exercises are set to be5times a week and every times40minutes. The exercitation usually lasts8weeks. Serum PTH and CT concentration is measured by Elisa assay in30minutes after exercise and24hours after training. Bone mineral density of the lateral humerus is measured by dual energy X ray. The bone mineral content of tibia is measured by atomic absorption method and the expression of OCN, Runx2, ALP, RANKL, Destrin, PTH1R, CTR, Fz, DVL, LRP5/LRP6, β-catenin, JNK, ROCK, NLK, NFAT, Bax and Bcl2gene is detected by qRT-PCR. Crystal violet staining and MTS methods are used for detecting the proliferation of MSCs and the ALP staining is used for detecting the ALP activity of osteoblasts. Von Kossa staining was used to detect the osteoblast mineralization ability. The characteristics of proliferation and differentiation of bone marrow-derived MSC is detected in vitro in primary cultures. Furthermore, effects of hormone treatment of PTH and CT on bone metabolism are detected in vitro in primary bone marrow-derived MSC cultures. The protein expression of PTH1R, CTR, RANKL and β-catenin is detected by Western-blotting technology.Results:(1) Running and swimming exercise had no significant effect on changing body weight in growing mice (P>0.05).Swimming and treadmill exercise improved the humerus bone density(P<0.05) and the tibia bone mineral content in growing mice, and then effects of downhill running exercise is more significant (P<0.01). It is more significant to influence of different types of exercise on the11genes expression of Runx2, Destrin, PTH1R, CTR, Fz, DVL, p-catenin, ROCK, JNK, Bcl-2and Bax (P<0.05). It is in consistency that the gene expression changes of Destrin, Fz and DVL affected by treadmill and swimming exercise, and it also has upward trend than control group. The expressions of other8genes are obviously influenced by the different types of exercise. (2) In group DT, the proliferation of primary MSCs is better than that of T group and S group (P<0.01). The activity of ALP in T group and S group is better than that of DT group. It is different in aspect of osteoblast mineralization ability, the result is this:T group> DT group>S group>C group.(3) It is significant difference (P<0.05) of gene expression of Fz, DVL, LRP5/6, ROCK, ALP, OCN and RANKL of osteoblasts which differentiated from primary MSCs after different modes of exercise. It is in the same trend between the protein expression and the gene expression of RANKL and PTH1R in osteoblasts.(4) It has very significant difference (P<0.01) in serum PTH and CT concentration in mice, both detection immediately after one training and detection immediately after8weeks training. However, it has no significant effect of different ways of motion on serum PTH and CT concentration in mice which rested24hours after8weeks training. Only time training of different types can induce the gene expression of mouse thyroid CT and Destrin varying significantly (P<0.05).(5) In group P, the proliferation ability of MSCs is better than that in C group and O group (P<0.05). ALP activity of osteoblasts in P+C group and C is better than P group and O group. The mineralization ability of osteoblasts in C group is the best among4groups.(6) In group P+C and group C, the gene expression of PTH1R is increased more significantly than that in other two groups (P<0.01). Relative to the O group, the gene expression of CTR gene is significantly up-regulated in other groups (P<0.05). The gene expression of Fz in osteoblasts in group P and C is significantly higher than the other two groups (P<0.05). Compared to control group, it is highly increased the gene expression of LRP5in3groups treated by hormone intervention (P<0.05), that in P+C group is more obvious than P group and P group than C group. Expression of DVL gene in osteoblasts was obviously unregulated after hormone intervention (P<0.05). Compared with P group or C group, it has a significant down regulation of gene expression of Wnt signaling pathway effectors protein such as beta-catenin, ROCK, JNK and NFAT. Compared to other groups, ROCK and NFAT gene in C group increased significantly (P<0.05). Due to the increased of PTH and CT concentration, it has significant difference in expression of osteoblast marker gene ALP, OCN and RANKL(P<0.05).Conclusion:(1) It has no effect on mouse’s weight by running and swimming exercise, but it can increase their bone density, but downhill running is the most effective exercise in changing the growth of the mouse’s bone mineral density and mineral content.(2) The results of gene expression of bone tissue show that different mechanism of regulating bone metabolism can be affected by different ways of motion. Treadmill exercise promotes bone anabolism by inhibiting the gene expression of ROCK and JNK, which are effector proteins in the Wnt/PCP pathway. Different types of exercise can induce Destrin gene expression up-regulated, but the extent of the increasing of gene expression is related to the exercise strength and gravitational loading.(3) It has a significant impact of different types of exercise on the growth of mouse serum PTH and CT concentration. The increase of serum PTH and CT concentration can be induced by treadmill exercise but swimming training cannot effectively promote the increase of serum PTH and CT concentration. It is well proved that treadmill exercise can promote and regulate bone anabolism by increasing endogenous PTH and CT concentration.(4) In the in vitro experiment, the increase of PTH and CT concentration can change gene expression of Fz and LRP5, the receptor protein of Wnt signal pathway. The change of receptor proteins and effector proteins of Wnt signal pathway show that PTH and CT not only through the classic Wnt signal pathway regulating bone metabolism, but it is also through the activation of Wnt/PCP signaling pathway and Wnt/Ca2+signaling pathways to regulate bone metabolism. Effect of PTH and CT on the regulation of Wnt signaling pathway is not the same and the mainly difference in the two non canonical Wnt signal pathways.(5) The changes of PTH and CT concentration have different effects on the expression of Marker gene in osteoblasts. The increase of CT concentration can urge the gene expression of ALP, RANKL and Runx2. Elevated PTH concentration promote bone anabolism by inducing gene expression of OCN down regulated significantly... |
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