不同运动对2型糖尿病小鼠骨中cAMP/CREB/Atf4途径及骨形成的影响

Effects of Different Exercises on cAMP/CREB/ATF4 Pathway and Bone Formation in Bone of T2DM Mice

目的:探讨2型糖尿病(T2DM)小鼠骨形成变化及不同运动对T2DM小鼠骨中环磷酸腺苷(c AMP)/环磷腺苷效应元件结合蛋白(CREB)/激活转录因子4(ATF4)途径和骨形成的影响。方法:40只4周龄C57BL/6雄性小鼠,适应性喂养1周后随机分为正常对照组(ZC,10只)和T2DM造模组(30只)。T2DM造模组小鼠造模成功后随机分为T2DM对照组(TC组,9只)、T2DM游泳组(TS组,9只)和T2DM下坡跑组(TD组,9只)。利用游泳和下坡跑对TS组和TD组小鼠进行8周训练。结束后,取小鼠血清并利用ELISA法检测c AMP浓度;取右侧股骨并利用RT-PCR法检测CREB、ATF4、骨钙素(OC)、骨钙蛋白(OCN)和骨涎蛋白(BSP)的m RNA表达;取右侧胫骨并利用West-blotting法检测CREB蛋白表达;取小鼠骨髓间充质干细胞(BMSCs)并诱导其向骨细胞(OB)分化,利用碱性磷酸酶(ALP)染液对OB染色;取左侧股骨并用Micro-CT对股骨远端BMD进行扫描。结果:与ZC组相比,TC组c AMP浓度下降,CREB、ATF4、OC、OCN和BSP m RNA及CREB蛋白表达均显著下降(P<0.05或P<0.01),OB成骨能力和BMD显著下降(P<0.01)。与TC组相比,TS组OC和OCN m RNA表达上调(P<0.05或P<0.01),OB骨形成能力增强;TD组c AMP浓度下降,CREB、ATF4、OC和OCN m RNA及CREB蛋白表达均显著上调(P<0.05或P<0.01),OB骨形成能力和BMD显著增加(P<0.01)。与TS组相比,TD组c AMP浓度升高,CREB、ATF4和OC的m RNA表达显著上调(P<0.05或P<0.01),OB骨形成能力增强。结论:2型糖尿病小鼠骨形成代谢被抑制;下坡跑通过激活2型糖尿病小鼠骨中c AMP/CREB/ATF4途径,促进成骨细胞分化及骨形成能力,提高骨密度,且其作用效果优于游泳。

Objective To observe the changes of bone formation in type 2 diabetes mellitus （T2DM） mice and the effect of different exercises on their cAMP/CREB/Atf4 pathway and bone formation. Meth- ods Forty four-week-old C57BL/6 male mice were randomly divided into a normal control group （ZC） of 10 and a T2DM group of 30. T2DM was induced using the high-fat diet and injection of strepto- zotocin. Then,the T2DM mice were randomly divided into a T2DM control group （TC）,a T2DM swim- ming group （TS） and a T2DM downhill running group （TD）,eaeh of 10. The TS and TD groups were trained for eight weeks as their group names indicated. Then the concentration of cAMP in serum was tested using the enzyme-linked immunosorbent assay （ELISA）. The reverse transcription-polymerase chain reaction （RT-PCR） was used to test the mRNA expression of the cAMP-response element bind- ing protein （CREB）,activate transcription factor 4 （ATF4） ,osteocalcin （OC） ,bone gla-protein （OCN） and bone sialoprotein （BSP） in the left tibia,and Western blotting was employed to test the protein ex- pression of CREB in the right femur. The bone marrow mesenehymal stem cells （BMSCs） were taken and induced to differentiate into osteoblasts （OBs） and dyed using the alkaline phosphatase （ALP） so- lution. The left side hindlimb bone was taken and scanned the bone mineral density （BMD） of the dis- tal end using the Skyscan Micro-CT. Results Compared with group ZC,the concentration of cAMP de- clined in group TC. Moreover,the mRNA expression of CREB,ATF4,OC,OCN and BSP as well as the protein expression of CREB of group TC were significantly down-regulated（P〈0.01 or P〈0.05）,together with the OB osteogenic capacity and BMD （P〈0.01） compared to group ZC. Compared with group TC, significant increase was observed in the mRNA expression of OC and OCN （P〈0.01 or P〈0.05）,as well as the OB osteogenic capacity of group TS. The concentration of cAMP of group TD decreased, the mRNA expression of CREB,ATF4, OC and OCN, as well as the protein expression of CREB were all significantly up-regulated （P〈0.01 or P〈0.05） compared with group TD. The OB osteogenic capaci- ty and BMD（P〈0.05） of group TD also increased significantly. Compared with group TS,the concentra- tion of cAMP（P〈0.05） and the OB osteogenic capacity increased,and the mRNA expression of CREB, ATF4 and OC of group TD increased significantly（P〈0.01 or P〈0.05）. Conclusion The bone forma- tion metabolism of type 2 diabetic mice is inhibited. The downhill running is superior to swimming in promoting the osteoblast differentiation and bone formation, as well as the bone mineral density through activating the cAMP/CREB/Atf4 pathway in the bone of T2DM mice.