不同方式运动对生长期小鼠骨合成代谢和Wnt信号通路的影响
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摘要
骨的主要功能是保护内部器官和支撑身体,另外,还参与钙的代谢。骨组织在整个生命过程中都是动态变化的,受内、外环境变化的影响,其中包括遗传、生活方式和运动锻炼。运动造成的机械刺激能够调节骨的发育,影响骨生长的速率和骨的形状、大小、强度以及解剖学结构。运动锻炼能够增加骨密度,降低骨质疏松症的发生,并且对生长期骨合成代谢的影响要比老年更有效。骨量在生长期不断提高,在成熟期达到峰值,以后会随年龄增长开始降低,生长期骨量积累减少骨折发生,也能延缓骨质疏松症的发生。PTH和CT在骨代谢中起到主要作用,这两种激素已被美国食品与药品管理局(FDA)批准在临床上用来治疗骨质疏松。运动对骨合成代谢的影响是否通过诱导内源性PTH和CT浓度升高进行调控；Wnt信号通路是调节骨代谢的主要信号通路,PTH和CT是否通过Wnt信号通路调节骨形成,这些机制尚未得到证实。
目的：观察下坡跑台运动、平坡跑台运动和游泳运动对骨密度、骨矿含量和骨组织骨合成代谢相关细胞因子基因及蛋白表达的影响,检测8周运动后原代MSCs增殖和分化差别,寻求对骨合成代谢影响更为显著的运动方式。对比不同方式运动对小鼠血清PTH和CT浓度影响,体外PTH和CT浓度变化对成骨细胞增殖和分化的影响,研究不同方式运动诱导内源性PTH和CT浓度变化对骨合成代谢产生影响,探讨不同方式运动健骨机制,丰富运动健骨的理论知识,为骨代谢疾病的治疗寻找合适的靶点。
方法：C57BL/6小鼠120只,分4组,下坡跑台组(DT组)、平坡跑台组(T组)、游泳组(S组)、安静对照组(C组),每组30只。训练方案为：DT组训练,跑速均为0.8km/h,坡度分别为-9°；T组跑速与DT组相同,坡度为0°；S组游泳训练,不负重,漂浮不动时用毛刷驱赶。即刻运动组在一次运动后处死,运动时间40min,Elisa法测血清PTH和CT浓度,qRT-PCR法测甲状腺PTH、CT和Destrin基因表达。长期运动组每次训练40分钟,每周训练5次,共训练8周,用Elisa法测运动后即刻和运动后休息24小时血清PTH和CT浓度,用双能X射线测肱骨远端骨密度,原子吸收法测胫骨骨矿含量,qRT-PCR检测胫骨ALP、OCN、Runx2、RANKL、 Destrin、PTH1R、 CTR、Fz、DVL、LRP5/LRP6、β-catenin、JNK、ROCK、NLK、NFAT、Bax和Bc12基因表达。8周训练后,4组小鼠分别取原代MSCs体外培养,结晶紫染色检测MSCs增殖；诱导分化后ALP染色法检测成骨细胞ALP活性,Von Kossa染色检测成骨细胞矿化能力,qRT-PCR测相关基因表达,Western Blot法测相关蛋白表达,以检测运动对原代MSCs细胞影响的效果在体外培养过程中是否能够持续存在。在MSCs细胞培养过程中添加PTH和CT,分为4组,P+C组两种激素同时添加,P组只添加PTH, C组只添加CT,O组为对照组,不添加激素,MTS法检测MSCs细胞增殖情况,诱导分化后ALP染色法检测成骨细胞ALP活性,Von Kossa染色检测成骨细胞矿化能力,qRT-PCR测相关基因表达,Western Blot法测相关蛋白表达,以检测PTH和CT对MSCs增殖和分化的影响。
结果：(1)跑台运动和游泳运动对生长期小鼠体重没有显著影响(P>0.05)；游泳运动和跑台运动都提高了生长期小鼠肱骨骨密度(P<0.05),提高了胫骨骨矿含量,并且下坡跑台运动效果更显著(P<0.01)。不同方式运动对胫骨Runx2、Destr1R、 CTR、Fz、DVL、β-catenin、ROCK、JNK、Bcl-2和Bax这11种基因的表达产生显著影响(P<0.05)；跑台运动和游泳运动运动后胫骨Destrin、Fz和DVL基因表达有一致性变化,相对于对照组有上调趋势,另外8种基因表达受运动方式影响较显著。
(2)DT组原代MSCs增殖优于T组和S组(P<0.01)；T组和S组成骨细胞ALP活性要优于DT组；成骨细胞骨矿化能力不同,T组>DT组>S组>C组。
(3)不同方式运动后,3个运动组原代MSCs诱导分化的成骨细胞Fz、DVL和RANKL基因表达显著上调(P<0.05),OCN基因表达显著下调(P<0.05)。另外,DT组原代MSCs诱导分化的成骨细胞CTR、LRP5、ROCK和Destrin基因表达显著上调(P<0.05),ALP基因表达显著下调(P<0.05)；S组原代MSCs诱导分化的成骨细胞CTR和ROCK基因表达显著上调(P<0.05)。PTHIR、CTR和RANKL蛋白表达跟基因表达趋势相同,而β-catenin蛋白表达与基因表达不同。
(4)一次训练后即刻和8周训练后即刻,不同方式运动导致小鼠血清PTH和CT浓度呈现非常显著性差异(P<0.01),在8周训练休息24小时后,不同方式运动对小鼠血清PTH和CT浓度变化没有显著影响。一次训练后即刻,DT组小鼠血清PTH和CT浓度显著升高(P<0.05)；8周训练后即刻,S组小鼠血清PTH浓度显著降低(P<0.01),DT组和T组小鼠血清CT浓度都显著升高(P<0.01),其中DT组小鼠血清浓度升高最为显著。一次不同方式运动对小鼠甲状腺CT和Destrin基因表达产生显著影响(P<0.05)。
(5)P组MSCs增殖能力强于C组和O组(P<0.05),P+C组和C组成骨细胞ALP活性优于P组和O组,C组成骨细胞矿化能力强于其他3组。
(6)P+C组和C组PTH1R基因表达相对于另外两组显著上调(P<0.01)；相对O组,P+C组、P组和C组CTR基因表达都显著上调(P<0.05)。P组和C组成骨细胞相对于其他两组Fz基因表达显著上调(P<0.05)；激素干预的3组LRP5基因表达都显著上调(P<0.05),P+C组>P组>C组。激素干预后成骨细胞表达DVL基因也显著上调(P<0.05)；P+C组成骨细胞Wnt信号通路效应蛋白β-catenin、ROCK、JNK和NFAT基因表达相对于P组或者C组都有显著下调趋势； C组的ROCK和NFAT基因相对于其他几组显著上调(P<0.05)。PTH和CT浓度升高后,成骨细胞Marker基因ALP、OCN和RANKL表达出现显著差异(P<0.05),P+C组和C组ALP和RANKL基因表达显著上调(P<0.01),P+C组和P组OCN基因表达显著下调(P<0.01),C组Runx2基因表达显著上调(P<0.05)。
结论：(1)跑台运动和游泳运动对生长期小鼠体重没有影响,但是能够显著增强生长期小鼠骨密度,并且下坡跑台运动对生长期小鼠骨密度和骨矿含量的提高更为有效。
(2)骨组织基因表达结果显示,跑台运动主要通过抑制Wnt/PCP信号通路的效应蛋白ROCK和JNK基因表达,促进骨合成代谢。不同方式运动能够诱导Destrin基因表达上调,并且上调幅度跟重力负荷相关,表明Destrin基因对力学刺激敏感。
(3)不同方式运动会对生长期小鼠血清PTH和CT浓度产生显著影响,下坡跑台运动能够显著提高血清PTH和CT浓度,平坡跑台运动也能提高血清PTH和CT浓度,效果没有下坡跑台显著,游泳运动不能有效促进血清PTH和CT浓度的提高。表明跑台运动能够通过提高内源性PTH和CT浓度进而促进骨合成代谢。
(4)在体外细胞培养实验中,单独添加PTH促进MSCs细胞增殖效果最明显,单独添加CT有效促进成骨细胞活性和骨矿沉积,同时添加PTH和CT能促进受体蛋白PTHIR和CTR表达,促进经典Wnt信号通路受体蛋白LRP5的基因表达和经典Wnt信号通路效应蛋白β-catenin蛋白表达,增加骨矿沉积。另外,单独添加CT促进Wnt/Ca2+信号通路NFAT基因表达。
(5)PTH和CT浓度变化对成骨细胞Marker基因表达产生不同影响。CT浓度升高促进ALP、RANKL和Runx2基因表达上调,提高成骨细胞活性。PTH浓度升高诱导成骨细胞OCN基因表达显著下调,促进骨合成。
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
目的：观察下坡跑台运动、平坡跑台运动和游泳运动对骨密度、骨矿含量和骨组织骨合成代谢相关细胞因子基因及蛋白表达的影响,检测8周运动后原代MSCs增殖和分化差别,寻求对骨合成代谢影响更为显著的运动方式。对比不同方式运动对小鼠血清PTH和CT浓度影响,体外PTH和CT浓度变化对成骨细胞增殖和分化的影响,研究不同方式运动诱导内源性PTH和CT浓度变化对骨合成代谢产生影响,探讨不同方式运动健骨机制,丰富运动健骨的理论知识,为骨代谢疾病的治疗寻找合适的靶点。
方法：C57BL/6小鼠120只,分4组,下坡跑台组(DT组)、平坡跑台组(T组)、游泳组(S组)、安静对照组(C组),每组30只。训练方案为：DT组训练,跑速均为0.8km/h,坡度分别为-9°；T组跑速与DT组相同,坡度为0°；S组游泳训练,不负重,漂浮不动时用毛刷驱赶。即刻运动组在一次运动后处死,运动时间40min,Elisa法测血清PTH和CT浓度,qRT-PCR法测甲状腺PTH、CT和Destrin基因表达。长期运动组每次训练40分钟,每周训练5次,共训练8周,用Elisa法测运动后即刻和运动后休息24小时血清PTH和CT浓度,用双能X射线测肱骨远端骨密度,原子吸收法测胫骨骨矿含量,qRT-PCR检测胫骨ALP、OCN、Runx2、RANKL、 Destrin、PTH1R、 CTR、Fz、DVL、LRP5/LRP6、β-catenin、JNK、ROCK、NLK、NFAT、Bax和Bc12基因表达。8周训练后,4组小鼠分别取原代MSCs体外培养,结晶紫染色检测MSCs增殖；诱导分化后ALP染色法检测成骨细胞ALP活性,Von Kossa染色检测成骨细胞矿化能力,qRT-PCR测相关基因表达,Western Blot法测相关蛋白表达,以检测运动对原代MSCs细胞影响的效果在体外培养过程中是否能够持续存在。在MSCs细胞培养过程中添加PTH和CT,分为4组,P+C组两种激素同时添加,P组只添加PTH, C组只添加CT,O组为对照组,不添加激素,MTS法检测MSCs细胞增殖情况,诱导分化后ALP染色法检测成骨细胞ALP活性,Von Kossa染色检测成骨细胞矿化能力,qRT-PCR测相关基因表达,Western Blot法测相关蛋白表达,以检测PTH和CT对MSCs增殖和分化的影响。
结果：(1)跑台运动和游泳运动对生长期小鼠体重没有显著影响(P>0.05)；游泳运动和跑台运动都提高了生长期小鼠肱骨骨密度(P<0.05),提高了胫骨骨矿含量,并且下坡跑台运动效果更显著(P<0.01)。不同方式运动对胫骨Runx2、Destr1R、 CTR、Fz、DVL、β-catenin、ROCK、JNK、Bcl-2和Bax这11种基因的表达产生显著影响(P<0.05)；跑台运动和游泳运动运动后胫骨Destrin、Fz和DVL基因表达有一致性变化,相对于对照组有上调趋势,另外8种基因表达受运动方式影响较显著。
(2)DT组原代MSCs增殖优于T组和S组(P<0.01)；T组和S组成骨细胞ALP活性要优于DT组；成骨细胞骨矿化能力不同,T组>DT组>S组>C组。
(3)不同方式运动后,3个运动组原代MSCs诱导分化的成骨细胞Fz、DVL和RANKL基因表达显著上调(P<0.05),OCN基因表达显著下调(P<0.05)。另外,DT组原代MSCs诱导分化的成骨细胞CTR、LRP5、ROCK和Destrin基因表达显著上调(P<0.05),ALP基因表达显著下调(P<0.05)；S组原代MSCs诱导分化的成骨细胞CTR和ROCK基因表达显著上调(P<0.05)。PTHIR、CTR和RANKL蛋白表达跟基因表达趋势相同,而β-catenin蛋白表达与基因表达不同。
(4)一次训练后即刻和8周训练后即刻,不同方式运动导致小鼠血清PTH和CT浓度呈现非常显著性差异(P<0.01),在8周训练休息24小时后,不同方式运动对小鼠血清PTH和CT浓度变化没有显著影响。一次训练后即刻,DT组小鼠血清PTH和CT浓度显著升高(P<0.05)；8周训练后即刻,S组小鼠血清PTH浓度显著降低(P<0.01),DT组和T组小鼠血清CT浓度都显著升高(P<0.01),其中DT组小鼠血清浓度升高最为显著。一次不同方式运动对小鼠甲状腺CT和Destrin基因表达产生显著影响(P<0.05)。
(5)P组MSCs增殖能力强于C组和O组(P<0.05),P+C组和C组成骨细胞ALP活性优于P组和O组,C组成骨细胞矿化能力强于其他3组。
(6)P+C组和C组PTH1R基因表达相对于另外两组显著上调(P<0.01)；相对O组,P+C组、P组和C组CTR基因表达都显著上调(P<0.05)。P组和C组成骨细胞相对于其他两组Fz基因表达显著上调(P<0.05)；激素干预的3组LRP5基因表达都显著上调(P<0.05),P+C组>P组>C组。激素干预后成骨细胞表达DVL基因也显著上调(P<0.05)；P+C组成骨细胞Wnt信号通路效应蛋白β-catenin、ROCK、JNK和NFAT基因表达相对于P组或者C组都有显著下调趋势； C组的ROCK和NFAT基因相对于其他几组显著上调(P<0.05)。PTH和CT浓度升高后,成骨细胞Marker基因ALP、OCN和RANKL表达出现显著差异(P<0.05),P+C组和C组ALP和RANKL基因表达显著上调(P<0.01),P+C组和P组OCN基因表达显著下调(P<0.01),C组Runx2基因表达显著上调(P<0.05)。
结论：(1)跑台运动和游泳运动对生长期小鼠体重没有影响,但是能够显著增强生长期小鼠骨密度,并且下坡跑台运动对生长期小鼠骨密度和骨矿含量的提高更为有效。
(2)骨组织基因表达结果显示,跑台运动主要通过抑制Wnt/PCP信号通路的效应蛋白ROCK和JNK基因表达,促进骨合成代谢。不同方式运动能够诱导Destrin基因表达上调,并且上调幅度跟重力负荷相关,表明Destrin基因对力学刺激敏感。
(3)不同方式运动会对生长期小鼠血清PTH和CT浓度产生显著影响,下坡跑台运动能够显著提高血清PTH和CT浓度,平坡跑台运动也能提高血清PTH和CT浓度,效果没有下坡跑台显著,游泳运动不能有效促进血清PTH和CT浓度的提高。表明跑台运动能够通过提高内源性PTH和CT浓度进而促进骨合成代谢。
(4)在体外细胞培养实验中,单独添加PTH促进MSCs细胞增殖效果最明显,单独添加CT有效促进成骨细胞活性和骨矿沉积,同时添加PTH和CT能促进受体蛋白PTHIR和CTR表达,促进经典Wnt信号通路受体蛋白LRP5的基因表达和经典Wnt信号通路效应蛋白β-catenin蛋白表达,增加骨矿沉积。另外,单独添加CT促进Wnt/Ca2+信号通路NFAT基因表达。
(5)PTH和CT浓度变化对成骨细胞Marker基因表达产生不同影响。CT浓度升高促进ALP、RANKL和Runx2基因表达上调,提高成骨细胞活性。PTH浓度升高诱导成骨细胞OCN基因表达显著下调,促进骨合成。
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
引文
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