| Periodontitis, one of the oldest oral infectious diseases, has a high prevalence around the whole world. Periodontitis which harms periodontal and oral health, is a main reason for adults’ tooth corruption. With the coming of the aging society in china, Periodontitis is becoming a more prominent oral health problem. The key to cure periodontal disease is to prevent alveolar absorption, meanwhile, stabilize and rebuild the alveolar. Traditional Chinese medicine attaches more importance on holism and dialectical therapy. Recently Stomatological Department of Hebei Chinese Medicine Hospital has achieved the exciting clinical effects by tonifying kidney, activating blood and strengthening teeth to cure periodontitis. However, the relevant detailed mechanism is unclear until now. As we know,osteoblast, the important cells in periodontium tissue, acts as the core role in anabolism metabolism of bore cells, and also have an important function in bone growth and restoration. Bone morphogenetic protein-2, can the generation of new bore, meanwhile, Collagen Type Ⅰ(COL I), the special collagen osteoblast synthesized by osteoblast, is the maturation sign of matrix differentiated from osteoblast. In this study, we researched on the mechanism involving in the effect of this prescription on MC3T3-E1 cells, by observing the effect of "Bushen Huoxue Gu Chi Fang" on the expression of BMP-2 and synthesis of CLL I in MC3T3-E1 cells, providing the theoretical basis for treatment of Periodontitis using this prescription.Objective: This study is to explore mechanism of periodontitis treatment using this prescription by performing a series of experiments: feeding the mouse with “Bushen Huoxue Gu Chi Fang” for a week, the serum containing this prescription obtained from the mouse was added to cultivate MC3T3-EⅠcells in vitro. The expression of BMP-2 in the supernatant was tested by ELLISA, and with the Immunohistochemical method and QRT-PCR, the effect on the COLⅠsynthesis by "Bushen Huoxue Gu Chi Fang" was observed, thus completing exploration of action mechanism for the periodontitisMethods:1 Culture MC3T3-E1 cell line in vitro.2 Contained-herb serum was prepared from rats by gavage with “Bushen Huoxue Gu Chi Fang” of body surface area converted equivalent dose; obtaining blank-contained serum as control group by rate gavage with normal saline and 10% FBS as blank control group.3 MC3T3-EⅠcells were cultured by three different serum, ELLISA assay was employed to detect the expression of BMP-2 in the clear supernatant liquid at 24 hs, 48 hs and 72 hs.4 MC3T3-EⅠcells were cultured by three different serum,the expression of COLⅠm RNA was measured by fluorescence quantitative PCR at 24 hs, 48 hs and 72 hs.5 MC3T3-E Ⅰ cells were cultured by blank-contained serum, contained-herb serum and blank control group Immunohistochemical assay was employed to test the protein level of COLⅠat 72 hs.Results:1 Concentration and content of BMP-2 in the clear supernatant liquid at 24 hs, 48 hs and 72 hs after "Bushen Huoxue Gu Chi Fang" acting on MC3T3-E1 cells;In accordance with statistics, the optical density value of medicated serum group for liquid supernatant were obvious high than that of not medicated serum and fetal calf serum group. their disparity was P<0.05, and this indicates that "Bushen Huoxue Gu Chi Fang" was capability of facilitating the concentration and content of bone inducing factor for BMP2 as well as participating in action on inducing bone growth.2 Effects on expression of COLⅠm RNA caused by action of medicated serum containing of components of "Bushen Huoxue Gu Chi Fang " acting on MC3T3-E1 for 24 hours, 48 hours and 72 hours:In vitro, along with the extension of time, the concentration and content of COL I m RNA of the three groups cells in vitro increased gradually. There was, in accordance with statistics, no obvious disparity(P>0.05), in the not medicated serum group and fetal calf serum group of COL Ⅰ m RNA concentration and content, while there was significantly upregulated for the concentration and content of COLⅠm RNA indicated in statistics(P<0.05).3 Effect on COL I synthesis caused by action of medicated serum containing components of “Bushen Huoxue Gu Chi Fang” acting on MC3T3-E1 at 72 hours :Under the microscope, cytoplasm of cells in the medicated serum group was dark brown staining while the not medicated serum and cell cytoplasm of fetal calf serum group was light brown staining. There was, in accordance with statistics, no obvious disparity between FBS and Blank-contained serum from rat(P>0.05).Difference had statistical significance between Contained-herb from rat and FBS, between Contained-herb from rat and Blank-contained serum from rat with χ2 test(P<0.05).Conclusion: "Bushen Huoxue Gu Chi Fang" is capability of promoting the concentration and content of BMP2 for gown-up bone inducing factor, thus inducing cells of MC3T3-E1 to osteoblast differentiation, thus promoting synthesis and secretion of COLⅠin MC3T3-E 1, promoting osteoblast differentiation and maturation and promoting bone formation. Besides, this may provide new insights into the treatment for periodontitis by promoting regeneration of the alveolar bone. |
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