Low Dose X-ray Irradiation Induced Morphological Changes And Cytoskeleton Reorganization Of Osteoblasts

As is known to all, low dose X-ray irridiationis widelyapplied in different branchesof medicine as therapeutical,especially in orthopaedics. Patients suffering fromorthopaedic disease often need to accept multiple X-rays for therapy. Therefore,researching therole of low dose X-ray has the vital significance. In our previous research,we reported that low dose X-ray irradiation may promote differentiation of osteoblasts andaccelerateing fracture healing in mice. According to the results of microarray, we foundthat low dose X-ray irradiation induced LIMK2gene expression increased significantly,which was closely related to the regulation of cytoskeleton reorganization.Thus, wespeculate that low dose X-ray irradiation can cause cytoskeleton rearrangement and affectcell function.The aim of the present study was to investigate the effects of X-ray radiation onmorphology and cytoskeleton of MC3T3-E1cells. We will also preliminary investigatedthe roles of RhoA/ROCK pathway involved in thisprocess, for further understanding thepossiblemechanismsinvolvedin biological effectsof low dose of ionizingradiation.Part I Effects of differentdoses of X-ray radiation on the morphologyand actin cytoskeleton of osteoblastsObjective: To observe the effects of different doses of X-ray irradiation on themorphology，microstructurechanges and actincytoskeleton of osteoblasts.The findingsofthis research will provide evidence for further study of low dose X-ray irradiationbiological effects.Methods: MC3T3-E1cells were exposed to irradiation of0Gy,0.5Gy,5Gy. Weinvestigated cellular morphological changes by phase contrast microscope andtransmission electron microscopy. The organization of actin microfilaments was determinedby immunofluorescence.Results: The F–actin of0.5Gy,5Gy group were destroyed after2h exposure toirradiation, and the more serious with the increase of radiation dose, which performancedby a dose dependentmanner. But24hourslater, the fluorescence intensity of F–actin in0.5Gy,5Gy group increased gradually and the fiber stress also increased.The mostsignificant changes appeared in the third day after X-ray irrddiation. However,thesechangesgraduallyreturned to normalinthe fifthday.Conclusion: The cytoskeleton of MC3TE-E cells were destroyed after2hours,exposure to X-ray irradiation.Howerver,0.5Gy and5Gy X-ray irradiation inducedreorganizationof actinfilamentsof MC3T3cells1d later. Part II Roles of RhoA/ROCK pathwayon the actin cytoskeletonreorganization induced by X-ray irridiationObjective:To investigate the roles of RhoA/ROCK pathway on the osteoblast’s actincytoskeleton reorganizationinduced by X-ray irridiation．Methods: MC3T3-E cels were exposed to0.5,5Gy X-ray irradiation.Real-time PCRand Western blot were used to detect the levels of RhoA,ROCK and phospho-cofilin.Wealso pretreated the MC3T3-E1cells by ROCK kinase inhibitor Y27632beforeirridiation,thenwe observed cytoskeletal changes afterX-rayirradiation.Results: The expression levels of RhoA, ROCK,LIMK gene in0.5Gy,5Gy groupbegan to rise after24hours,X-ray iridiation.The most obvious changes appeared in thethird day.However,the levels of these genes associated with cytoskeleton reduced to0Gygroup5days later. Y27632pretreatment blocked the X-ray irradiation inducedcytoskeleton reorganizationof osteoblasts.Conclusion: Low dose X-ray irradiation induced reorganization of actin filaments inMC3T3cells andRhoA/ROCK pathway played a significant role in regulating cytoskeletaldynamics. PartIII CytoskeletonreorganizationInhibitionblockedtheproliferationanddifferentiationofosteoblastsinducedbylow doseX-rayirradiationMethods: MC3T3-E1cells were pretreated with ROCK kinase inhibitor Y27632toinhibitthe cytoskeleton reorganization before0.5Gy X-ray irridiation．CCK-8assay wereused toevaluate cell proliferation ability.Wealso investigated alkalinephosphataseactivityand the expression of Runx2,ALP,COL1,OCN and Oeterix as osteoblaets,differentiationmarker.Results: After pretreatment with ROCK kinase inhibitor Y-27632,the proliferationand differentiation of osteoblasts induced by low dose X-ray irradiation wereabolished.The expression of Runx2,ALP,COL1,OCN were reducedto the0Gy group level.Conclusion: RhoA/ROCK signaling pathway involved in low dose X-ray irradiationinduced osteoblast proliferationanddifferentiation.