Effects Of MAPK On Expression Of TRPV6 In Osteoblast Of Rat

Mitogen activated protein kinase (MAPK) signal path is an improtant path in which extra cell signal inducing nucleolus reaction, Composed by a group of step actived MAPK that steped actived by amino-acid residue through phosphorylation during signal transduction. In these years it is founded that MAPK has intimate relationship with the proliferation, differentiation and apoptosis of osteoblast. In the resent research it has suggested that as transent receptor potential pathway, TRPV5 and TRPV 6 were a part of Transent receptor potential pathway (TRP) familiy specified for epithelial calcium ion channel, they play an important role in the transduction of crossing cell Ca2+. Thoughly study the founction of TRPV5 and TRPV6 in the bone metabolism, we could point out the moleculor therapy to the disease of bone metabolism. In this study, we have investigated the relationship between the MAPK and the TRPV6 in osteoblast, to further provide the clue and evidence of mechanism of bone metabolism disease such as osteoporosis.Material and method: We used the suckling Wistar rat (after borned 1-2 days), extracted osteoblast by means of continue digestive enzyme and observed and made the photo of the primary cell by inverted microscope everyday. The proliferation and morphology of osteoblast was observed. We identified alkaline phosphatase staining of osteoblast and alizarin red staining of mineralized node through the method of modified Kaplow technology. Group: 1. control group;2. PD98059 5uml/L ; 3. PD98059 10uml/L;4.PD98059 20uml/L ;5 .SB203580 5uml/L;6.SB203580 10uml/L;7.SB203580 20uml/L;8.PD98059 5uml/L+SB203580 5uml/L;9.PD98059 10uml/L+SB203580 10uml/L;10.PD98059 20uml/L+SB203580 20uml/L。The TRPV6 mRNA of osteoblast was examed by RT-PCR. Statistics result: x±S, t-test. Result:1. Osteoblast culture: Inverted microscope observed that osteoblast with irregularly shape (triangle or fusiform) had 1~3 nuclear, possesed abundoned and clear cytoplasm, while the mononuclear osteoblast is orbicular-ovate. The ALP of osteoblast became purple-black microgranule after Calcium-Coalt staining. There were lilac or purple-black microgranule or a mass of black deposit in most of cytoplasm. The positive rate of ALP staining was above 90%. The opacity zone could present sporadic red mineralization nodes after alizarin red staining. We use MTT to investigated the cell activity suggested that the osteoblast entered logarithmic growth phase after inoculation 4~5 days, reached the peak of proliferation in the 10th day, and went into the phase of decline.2. The influnce of SB203580 blocking p38MAPK pathway to osteoblast: The result suggested that the cell proliferation activity has the decline trend with the rise of SB203580 concentration, but no obviously difference with the control group, p>0.05. Bacteria count examed the influnce of SB203580 to the porliferation of osteoblast, revealed that there were no evidence change of cell number in the group of SB203580 (5μmol/L), while the other medicine groups cell number obviously dose-depended delined. With the rise of the drug concentration, ALP positive granule reduced, apperanced as lilac. Alizarin red staining result demonstrated that: Compared with normal control, the number of calcium nodes has reduced, and small in size; and in the group of 40μmol/L, the reduction of cell number was much more obviouse, the cell extention narrow, no formation of calcium. SB203580 suppressed the expression of TRPV6 mRNA, which reduced with the rise of the concentration of SB203580.3.The influnce of PD98059 blocking p44/42 MAPK pathway to osteoblast: The result suggested that the cell proliferation activity has the decline trend with the rise of PD98059 concentration, but no obviously difference with the control group, p>0.05. Bacteria count examed the influnce of PD98059 to the porliferation of osteoblast, revealed that there were no evidence change of cell number in the group of 5μmol/L PD98059, while the cell number has obviously reduced in group of 10μmol/L PD98059, p<0.05, in the group of 80μmol/L PD98059 the cell has evidencely decreased, p<0.01. ALP staining: Compared with the control group, with the increasing of medicine concentration there was obviously reduction of cell number, the cell extention became much smaller, nuclear size evidencely shrinked and no multinuclear cell. ALP positive granule reduced, apperanced as lilac. Alizarin red staining result demonstrated that: Compared with normal control, the number of calcium nodes has reduced, and small in size; and in the group of 30μmol/L and 60μmol/L, the reduction of cell number was much more obviouse, the cell extention narrow, no formation of calcium. PD98059 suppressed the expression of TRPV6 mRNA, which reduced with the rise of the concentration of PD98059.4.The influnce of PD98059+SB203580 blocking p44/42 and p38 MAPK pathway to osteoblast: The result suggested that the cell proliferation activity has the decline trend with the rise of PD98059 and SB203580 concentration, and the cell possessed lower activity while combination of PD98059 and SB203580 compared with seperated. Bacteria count demonstrated that cell number obviously lower than control when combination of PD98059 and SB203580, p<0.01; and evidencely lower than the seperated group. The result revealed that, in control group, cell concentrated as fusiform, polygon, and cell extention thicking. ALP staining showed that cell number obviously lower than the seperated group when combination of PD98059 and SB203580, the size of cell was small and cell extention narrow, the ALP staining positive granule reduced. The variation became much more obviously with the increase of medicine concentration. Alizarin red staining showed that: compared with control, the number of calcium node has reduced, and small in size; In the group of conbination (10μmol/L and 20μmol/L), the number of cell has reduced more evidencely, no calcium nodes. The combination of PD98059 and SB203580 obviously suppressed the expression of TRPV6 mRNA, while the 5μmol/L group could evidencely suppressed, and with the increased of the concentration, the expression of TRPV6 mRNA obviously reduced.Conclusion:1. Wistar rat calvaria bone can be used for enzymatic digestion to obtain a large number of osteoblasts, through the primary and passaged cell morphology, ALP staining and alizarin red staining to prove the high purity of osteoblasts, the growth of good, the ability of form the ore nodules.2. The PD98059 that is inhibitor of the p44/42 MAPK pathway, can suppress osteoblast proliferation, obviously reduced osteoblast TRPV6 mRNA,and droped the formation of calcium node. 3. The SB203580 that is inhibitor of the p38 MAPK pathway, can suppress osteoblast proliferation, obviously reduced osteoblast TRPV6 mRNA, droped the formation of calcium node.4.The PD98059 and SB203580 exist synergy in the suppression of osteoblast proliferation, reduced osteoblast TRPV6 mRNA level ,droped the formation of calcium node.