The Effect Of Tonifying Kidney, Activating Blood And Strengthening Teeth On The Cellular Ultrastructure And Differentiation

Periodontitis is one of the most common oral diseases, second only to the damage of human’s health caused by cancer and cardiovascular. Other than its effect on the chewing function and maxillofacial appearance, Periodontitis can also bring danger thus triggering diabetes,kidney disease,cardiovascular disease, and premature newborn. The severe absorption of alveolar bone in the late stage of periodontitis often leads to tooth loss, therefore the key to cure periodontal disease is to prevent alveolar absorption, meanwhile, stabilize and rebuild the alveolar. Chinese medicine pays more attention to holism and dialectical therapy. Recently Stomatological Department of Hebei Chinese Medicine Hospital has obtained the satisfied clinical effect by tonifying kidney, activating blood and strengthening teeth to cure periodontitis. However,the relevant detailed mechanism has not been explored.Alkaline Phosphosphatase(ALP),a homologous dimeric glycoprotein, takes part in the process of bone metabolism, and also is regarded as the early sign of mature bone extracellular matrix.Collagen Type Ⅰ(COL I),of which 80%~90% is contained in bore tissue, builds up the protein frame in bone tissue, and plays an important role in maintaining bone biomechanics characteristic and structural integrity, meanwhile,deemed as the maturation sign of matrix differentiated from osteoblast.Mineralized nodules were formed through matrix mineralization after collagen deposition,marks the last stage of osteoblastic differentiation.In this study,we researched on the mechanism involving in the effect of this prescription on accelerating bone formation by observing the influence of BUSHENHUOXUEGUCHI on ultrastructure and differentiation of 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 BSHXGC for a week,the serum containing this prescription obtained from the mouse was added to cultivate MC3T3-E1 cells in vitro. Observing the effect of BUSHENHUOXUEGUCHI on ultrastructure,ALP activity,the expression of COLI and mineralized nodule formation in MC3T3-E1 cells.Thus completing investigate of action mechanism involving in the effect of this prescription on bone formation.Methods:1 Culture MC3T3-E1 cell line in vitro.2 Contained-herb serum was prepared from rats by gavage with BUSHENHUOXUEGUCHI 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 To observe the effects of BUSHENHUOXUEGUCHI on ultramicrostructure of MC3T3-E1 cell.MC3T3-E1 cells were cultured by blank-contained serum, contained-herb serum and blank control group,respectively.The ultrastructure changes in MC3T3-E1 cells were observed by means of electron transmission at 1d、4d、7d and 14 d.4 ALP activity test was carried out to evaluate the effect of BUSHENHUOXUEGUCHI with kites on the ALP expression of MC3T3-E1 cell.MC3T3-E1 cells were seeded on 96-well plates at 1300 cells per well and cultured by blank-contained serum,contained-herb serum and blank control group, respectively. ALP detection kits was employed to detect ALP activity in the clear supernatant liquid at 1d、4d、7d and 14 d.5 MC3T3-E1 cells were cultured by blank-contained serum,contained-herb serum and blank control group,respectively. Immunohistochemical assay was employed to test the protein level of COLⅠat 72 hs.6 The effects of BUSHENHUOXUEGUCHI on the mineralized nodules formation of MC3T3-E1 cells were evaluated by alizarin red staining.MC3T3-E1 cells were cultured by blank-contained serum,contained-herb serum and blank control group, respectively. Alizarin red staining was used to observe and count the formation of mineralized nodules.Results:1 The effects of BUSHENHUOXUEGUCHI on ultramicrostructure of MC3T3-E1 cells.With the culture time going on,the amount of mitochondria,Golgi bodies and endoplasmic reticulum showed a slight increase,but were not well developed in MC3T3-E1 cells cultured with blank-contained serum and blank controlled group, respectively. Compared with the two controlled groups, not only the number of protuberances increased,inducing the osteoblast differentiation of MC3T3-E1 cells, the nucleus was also egg- shaped,in addition, nuclear membrane was tortuous and invaginates, and mitochondria, Golgi bodies and endoplasmic reticulum were abundant meanwhile well developed,besides, intracellular secretory more matrix vesicles were found, and metabolic activity developed in MC3T3-E1 cells cultivated with contained-herb serum.2 The influence of BUSHENHUOXUEGUCHI on the ALP activity in the clear supernatant liquid of MC3T3-E1 cell.The activity of ALP increased gradually with the prolonging cultivation time in all three groups. There was no difference for statistics significance between blank-contained serum and blank controlled groups(P<0.05).While the activity of ALP had stronger activity in MC3T3-E1 cells cultivated by contained-herb serum than that in blank-contained serum and blank controlled groups, and difference had statistical significance(P<0.05).3 The effects of BUSHENHUOXUEGUCHI on the expression of COL I in MC3T3-E1 cell.Checked by microscope after immunohistochemical staining,we discovered that cytoplasm stained with dark brown yellow in MC3T3-E1 cells cultivated with contained-herb serum at 72 hs, while light brown yellow in blank-contained serum group. Difference had statistical significance between two groups analyzed with χ2 test(P<0.05).4 The influence of BUSHENHUOXUEGUCHI on the number of mineralized nodules forming in MC3T3-E1 cells.Mineralized nodules were formed in all three groups after cultivated at 14 d.There was no difference for statistical significance between blank-contained serum and blank control groups(P<0.05).While the average volume of mineralized nodules were enlarged manifestly in MC3T3-E1 cells cultivated in contained-herb serum than that in blank-contained serum and blank controlled groups, and difference had statistical significance(P<0.05).Conclusion: BUSHENHUOXUEGUCHI can accelerate the differentiation of MC3T3-E1 cells into osteoblast in vitro, enhancing the activity of ALP, increasing mineralized nodule formation, and also promoting cells differentiation and maturation of MC3T3-E1, meanwhile bone formation in vitro. This may provide new insights into the treatment of periodontitis by promoting the regeneration of alveolar bone.