| ObjectiveTo explore the changed proteins which is related with Zuoguiwan as a regulatory role by the methods of proteomics in the bone marrow of the model rats which have osteoporosis caused by bilateral ovariectomy.And then reveal the mechanism of Zuoguiwan during the treatment of the rats’osteoporosis induced by bilateral ovariectomy at the protein level which will also to provide further experimental evidence for exploring the scientific connotation of the mechanism of "The kidney dominating bone"Methods1Rats grouping and drawnFirstly,one hundred female Wistar rats were randomly divided into three groups:twenty in normal control group,twenty in the pseudo-operation group,twenty in model group,twenty in stilbestrol group, twenty in Zuoguiwan group.Both ovaries of all rats in the operation group were extirpated,while only a little fatty tissue nearby the ovaries in the pseudo-operation group were extirpated.Distilled water was administrated orally to normal control group,pseudo-operation group and model group at the same time. The rats in Stilbestrol group and Zuoguiwan group were treated with stilbestrol tablets and decoction of Zuoguiwan.The Stilbestrol tablets was crushed,used distilled water as the the suspension concentration0.0046mg/ml,and the dose was0.046g/kg weight.The concentration of Zuoguiwan was0.968g/ml, and the dose was9.68/kg weight. The drugs were given once a day and last six days. There is one day paused every six days. The whole period last twelve weeks.At the third day and the fifteenth day before the day all the rats were killed,hydrochloric acheomycin were respectively injected into the abdomen of the rats according to30mg/kg to mark the bone with fluorescence.After being killed the right tibia of the rats were retained for bone tissue section. At the same time the left tibia of the rats were used for Frozen tissue section after being decalcified to observe the protein expression by means of immunohistochemistry comfirm the result of mass spectrographic analysis, and the bilateral femur for2-DE and Western-blot. 2Bone histomorphometry indicators analysisDetected the rats’tibia TBV%,TRS%,TFS%,MAR.OSW and mAR by Qwin V3image analysis system.3Bone marrow protein analysis and identificationGet the femur bone marrow from the rats of normal group.model group and Zuoguiwan group, and extract the proteins. With2-D electrophoresis technology to analyze and compare the expressing proteins in the marrow of the three groups, and find thedeferent proteins adjusted by Zuoguiwan which are considered to be the related protein with postmenopausal osteoporosis.Then these diferent proteins were identified by mass spectrographic analysis and get their relateded informations.Chose protein ADLOA to be verified by Western-blot and protein CA1and PRDX6to be verified by immunohistochemical methods.Results1Effects of Zuoguiwan on the bone histomorphometry indicators in ovariectomized rats suffered osteoporosisAfter the bilateral ovaries of the rats were extiroated,TBV%of tibia significantly decreased, while TRS%、TFS%、MAR、OSW and mAR significantly increased,which indicated that the model established by extirpating bilateral ovaries is a kind of high transformed osteoporosis model which has higher resorption than formation in bone.Compared with model group, TBV%significantly increased while TRS%、 TFS%、MAR、OSW and mAR significantly decreased after being treated by zuoguiwan. So Zuoguiwan can treat osteoporosis by reducing the high transformation of the bone.2Result of2-DEThrough Image Master2D Platinum5.0analysis,the numbers of protein expression in the femur bone marrow showed two-dimensional gel electrophoresis profiles is(826±35).Three-fold expression is used as the standard, and twenty-three differentially expressed protein spots were detected which were regulated and corrected by zuoguiwan in the model group.3Results of Mass spectrometryEleven differentially expressed protein spots were detected by MALDI-TOP masss pectra,and11proteins spots obtain correct identification results. Among these proteins, the proteins which were highly expressed in model group and corrected by Zuoguiwan are carbonic anhydrase1, calreticulin, fructose-bisphosphate, aldolaseA, peroxiredoxin-6, enolase3, albumin, disulfide-isomerase A3, enolasel; the proteins which were lowly expressed in model group and corrected by Zuoguiwan are carbonic anhydrase1and protein S100-A8.4Results of verifications4.1Results of western-blotAldolase A was verified by western-blot.The map of western-blot shows: Aldolase A is expressed in the3groups.The level of expression is highest in the model group. The level of expression in zuoguiwan group is significantly decreased and is similar to that in stilbestrol group This result is in accorded with gel analysis and mass identification.4.2Results of immunohistochemistryCA1and PRDX6were verified by immunohistochemical methods.The results show:The expression of CA1is lowest in the model group and significantly increased in zuoguiwan group.The expression of PRDX6is highest in the model group and significantly decreased in zuoguiwan group.Conclusion1. After bilateral ovariectomy, Rats’bone resorption and bone formation were significantly enhanced, but bone resorption is more than bone formation, which resulted in a significant reduction in bone mass. This is a kind of high conversion of osteoporosis. Zuoguiwan can improve the abnormal changes in bone resorption and bone formation, reduce bone loss,and prevent the occurrence of ovariectomized induced osteoporosis disease.2. Eleven differentially expressed proteins in the rats’ femur bone marrow are screened and identified by proteomic methods. Among these proteins, the proteins which were highly expressed in model group but reduced by Zuoguiwan include carbonic anhydrase1, calreticulin, fructose-bisphosphate, aldolaseA, peroxiredoxin6, enolase3, albumin, disulfide-isomerase A3, alpha-enolase. The proteins which were lowly expressed in model group and increased by Zuoguiwan are carbonic anhydrase1and protein S100-A8. The functions of these proteins are related to energy metabolism, antioxidant, signal transduction, cell differentiation, proliferation and apoptosis, etc. Then we conclude that Zuoguiwan’ treatment effect on the osteoporosis of the ovariectomized rat is achieved by regulating cell proliferation, differentiation, metabolism, apoptosis and oxidative reactions in the bone marrow. These proteins are also closely related with Postmenopausal osteoporosis and "The kidney dominating bone". |
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