Effect Of Zoledronic Acid On MMPs/TIMPs System In Prevention Of Steroid-induced Femoral Head Necrosis In Rabbits

Background and Objective MMPs and their specific inhibition factors, the tissueinhibitor of TIMPs are the family numbers of matrix proteases, which are involved inthe degradation of various tissues including bones in the whole body. It has beenconfirmed that MMPs/TIMPs system connects closely with the incidence ofsteroid-induced osteonecrosis. Recently, researches have shown that Zoledronic acid,the commonly used the third-generation bisphosphonates for the treatment ofosteoporosis, can suppress the mRNA expression of MMP-2and MMP-9, reduce theratio of MMPs/TIMPs and inhibit the bone matrix degradation. Therefore, it is worth todo a further research to find out whether zoledronic acid can be the role of theMMP/TIMP system to antagonize steroid-induced osteonecrosis of the femoral head. Inour research, we have prophylactically given the New Zealand white rabbits withzoledronic acid which have been given a large number of glucocorticoidaforehand.Then the protein expression of MMP-2, MMP-9, TIMP-1, TIMP-2of rabbitfemoral head bone tissue have been tested by Western blotting (Western blot), aimed toevaluate whether zoledronic acid could prevent the steroid-induced osteonecrosisaccording to the MMPs/TIMPs system. Finally, we hope to provide an experimentalbasis for the clinical application of zoledronic acid to prevent steroid-inducedosteonecrosis. The objective of this study is to establish an experimental rabbit model ofsteroid-induced avascular necrosis of femoral head (SANFH). Zoledronate acid wasused to intervene and we evaluated the efficacy of this drug in preventing SANFH anddisclose the mechanism of its action. Materials and Methods Seventy healthy28-week-old New Zealand white rabbitswere selected,3.0±0.3kg in weight, and they were randomly divided into3groups:Model group(Group A), Zoledronic acid group(Group B),30/group,and Controlgroup(Group C). All the animals were on a standard feed and forage experimentalfeeding one week. Then Group A and Group B were treated by ear intravenous injectionof lipopolysaccharide,10μg/kg, one time. After24hours all the animals were treated bygluteal muscle injection of methylprednisone,20mg/kg, totally three times with intervalof24hours. Rabbits in the zoledronic acid group were treated with zoledronic acid byear intravenous injection,100μg/kg, at the same time, total two time with interval offour weeks. Group C in the same way to give the same volume of saline at the samepoint. Animal spirits, appetite, weight changes and capture reaction were observed afterinjection. Six weeks after MPS injection, all animals underwent X-ray examination andMRI of bilateral hips to observe changes of femoral head. Then we killed all animals byair embolism. The bilateral femoral heads were harvested and split along the sagittalplane into halves. One half was detected by HE staining and calculated emptyosteocytes rates. Then we evaluated avascular necrosis of the femoral head. In anotherhalf, the total protein of femoral head bone tissue was extracted and the expressionlevels of MMP-2、MMP-9、TIMP-1and TIMP-2were examined by Western Blotting.Results At the end of sixth week, the empty osteocytes rate of model group was27.131.17, the empty osteocytes rate of zoledronate acid group was18.361.22,Zoledronate acid group was lower than model group, the difference was statisticallysignificant (P<0.05). The avascular necrosis rate group A was78.57%（22/28） and theavascular necrosis rate B was14.81%（4/27）, Zoledronic acid group of femoral headnecrosis rate is much lower than model group, the difference was statisticallysignificant (P<0.05). The MMP-2, MMP-9, TIMP-1, TIMP-2protein expression levelswas detected by Western Blot analysis. Comparition between model group and control group: the expression of MMP-2、MMP-9protein in model group increased, theexpression of TIMP-1、TIMP-2protein in model group decreased, the difference wasstatistically significant (P<0.05). Comparition between Zoledronic acid group andmodel group: the expression of MMP-2、MMP-9protein in Zoledronic acid groupdecreased, the expression of TIMP-1、TIMP-2protein in Zoledronic acid groupincreased, the difference was statistically significant (P<0.05).Conclusion1. Zoledronic acid can be effective in preventing the occurrence ofsteroid-induced of femoral head necrosis in rabbits by antagonizing up regulation of theexpression of protein in MMP-2, MMP-9and down regulation of the expression ofmRNA and protein in TIMP-1,TIMP-2,which are regulated by Glucocorticoid,as aresult, the ratio of MMPs/TIMPs decreases,which is the mechanism of preventing theoccurrence of steroid-induced of femoral head necrosis;2. In bone tissue of femoralhead, MPS raised expression of MMP-2, MMP-9protein, lowered expression ofTIMP-1, TIMP-2protein, increasing the ratio of MMPs/TIMPs.3. Early SANFHmodel was successfully manufactured by Single low-dose LPS combined withhigh-dose MPS three times.