Effect Of Bushen Jianpi Huoxue Recipe On Osteogenic Differentiation And Apoptosis Of MC3T3-E1 Cells In High Glucose Environment

Purpose:Discussion on the Bushen Jianpi Huoxue Formula sugar environment MC3T3-E1 cell proliferation,the influence of the osteogenetic differentiation and apoptosis.Material and method:Wistar rats,18,male and female half,six weeks of age,weight 200 ± 20 g,were randomly divided into three groups: blank control group,Bushen Jianpi Huoxue Formula group,metformin group,each group of six.According to clinical dose converse gastric drug delivery: blank control group took distilled water irrigation,Bushen Jianpi Huoxue Formula take Bushen Jianpi Huoxue Formula hydrochloride suspension liquid;metformin group take metformin hydrochloride suspension liquid,lavage 1 times a day.After 7 d,intraperitoneal injection of anesthesia,abdominal aorta in blood,let stand for 2 h after centrifugal(2500 rpm,4 ?,20 min),collect serum,group blending,56 ? water bath inactivated 30 min,after the membrane filtration repackaging,preserved-86 ?.Experiment is divided into blank control group,the high sugar group,low dose group,high dose group in traditional Chinese medicine(TCM)dose group,the metformin group.By using the method of CCK 8 screening of glucose to the cells of MC3T3-E1 intervention the optimal dose and testing groups osteoblast proliferation rate.With p-nitrophenyl phosphate method(PNPP)groups of alkaline phosphatase(ALP)activity.Using the ELISA method to detect the contents of osteocalcin(OC).Flow cytometry Anexin ?/PI with double staining detecting osteoblast apoptosis rate.Immune protein imprinting technology(Western blot)detection osteogenetic differentiation related protein expression of BMP2 and Runx2 and apoptosis related proteins Bax and the expression of Bcl-2.Real-time fluorescent quantitative PCR(RT-q PCR)test BMP2,Runx2,Bax and Bcl-2 m RNA expression.Results:1.Different concentrations of glucose on MC3T3-E1 the influence of cell proliferation Compared with blank control group,cell culture in 24 h and 48 h glucose concentration of 25 mmol/L and 33 mmol/L tendency for cell inhibition rate were significantly increased,with statistical significance(p < 0.01),cell culture,in 72 hours,glucose concentration tendency for 16.7 mmol/L,25 mmol/L and 33 mmol/L tendency for cell inhibition rate of L group were increased(p < 0.01 or p < 0.05).2.Invigorating Bushen Jianpi Huoxue Formula on sugar environment under the influence of MC3T3-E1 cell proliferation ability Compared with blank control group,cell culture in 24 h,48 h and 72 h,high sugar group were significantly lower(p<0.01).Compared with the high sugar group,cell culture in 24 h,48 h,cell proliferation ability to rise in the lower dosage treatment groups in TCM(p < 0.05),cell culture for 24 h,,48 h and 72 h,high-dose group in TCM,and metformin group significantly increased cell proliferation(p < 0.01).Compared with metformin group,cell culture in 24 h,48 h and 72 h,cell proliferation ability of low and high dose group in TCM significantly reduced(p<0.01).Compared with low dose group in TCM,cell culture in 24 h,48 h and 72 h,cell proliferation ability of middle dose group in TCM was significantly increased(p<0.01).3.Bushen Jianpi Huoxue Formula on sugar environment under the influence of MC3T3-E1 cell proliferation ability.3.1 Influence of alkaline phosphatase(ALP)Compared with blank control group,cell culture in 1 d,3 d,5 d,7 d,high sugar group were significantly lower(p<0.01).Compared with the high sugar group,cell culture in 3 d,5d,7 d,cell proliferation ability to rise in the lower dosage treatment groups in TCM(p < 0.05 or p < 0.01),cell culture in 1 d,3 d,5 d,7 d,low and high dose group in TCM and metformin group was significantly increased(p < 0.05 or p < 0.01).Compared with metformin group,cell culture in 1 d,3 d,5 d,7 d,low dosage treatment groups in TCM(p <0.05 or p < 0.01),and other groups have no significant difference(p > 0.05).Compared with low dose group in TCM,cell culture in 3 d,5 d,7 d,middle dose group in TCM was significantly increased(p<0.01).3.2 Impact on osteocalcin(OCN)Compared with blank control group,cell culture in 3 d,5 d,7 d and 14 d,high sugar group were significantly lower(p<0.01).Compared with the high sugar group,cell culture in3 d,5 d,7 d and 14 d,high-dose group and metformin group in TCM were significantly higher(p<0.01).Compared with metformin group,cell culture in 3 d,5 d,7 d and 14 d,low dose group in TCM osteocalcin production capacity significantly reduced(p < 0.01),cell culture in 5 d,7 d and 14 d,high dose group in TCM osteocalcin secretion capacity reduction(p < 0.05 or p < 0.01);Compared with low dose group in TCM,cell culture in 3 d,5 d,7 d and14 d,the high dose group in TCM osteocalcin secretion capacity significantly higher(p<0.01).3.3 Bushen Jianpi Huoxue Formula on sugar environment of osteoblast BMP2,Runx2 protein expression After 48 h,compared with blank control group,high sugar BMP2,Runx2 protein expression were significantly reduced(p<0.01).Compared with the high sugar group,BMP2,Runx2 protein expression quantity of low,medium and high dose group in TCM and metformin group significantly higher(p < 0.05 or p < 0.01).3.4 Bushen Jianpi Huoxue Formula on high sugar environment of osteoblast BMP2,Runx2 m RNA expression After 48 h,drug intervention group compared with blank control group,high sugar BMP2,Runx2 gene expression were significantly reduced(p<0.01).Compared with the high sugar group,high,medium and low dose group in TCM and metformin group significantly higher(p < 0.05 or p < 0.01).Compared with metformin group,low,medium and high dose group in TCM of BMP2 gene expression were significantly reduced(p<0.01),low,middle dose group in TCM were reduced(p < 0.05 or p < 0.01).Compared with low dose group in TCM,high dose group in TCM of Runx2 gene expression increased significantly(p<0.01).4.Bushen Jianpi Huoxue Formula on high sugar environment of osteoblast apoptosis4.1Flow cytometry detection MC3T3-E1 cell apoptosis Compared with the blank control group,high sugar group apoptosis rate was significantly higher(p<0.01).Compared with the high sugar group,high,medium and low dose group in TCM,the metformin group of cell apoptosis rate were significantly decreased(p<0.01).Compared with the high sugar group,high,medium and low dose group in TCM,the metformin group of cell apoptosis rate were significantly decreased(p<0.01).Compared with metformin group,low,high dose group in TCM,apoptosis rate increased(p < 0.05 or p< 0.01).Compared with low dose group in TCM,medium and high dose group in TCM cells in early apoptosis rate was significantly reduced(p<0.01).4.2 Bushen Jianpi Huoxue Formula on sugar environment of osteoblast the influence of Bax,Bcl-2 protein expression.Compared with blank control group,high sugar group of the Bcl-2/Bax ratio significantly reduced(p<0.01).Compared with the high sugar group,high,medium and low dose group in TCM,the metformin group increased(p < 0.05 or p < 0.01).Compared with metformin group,high dose group in TCM increased significantly(p< 0.01),low,middle dose group in TCM were significantly reduced(p<0.01).Compared with low dose group in TCM,high dose group in TCM increased significantly(p<0.01).4.3 Bushen Jianpi Huoxue Formula on sugar environment of osteoblast the influence of Bax,Bcl-2 m RNA expression.After 48 h,compared with blank control group,high sugar group of the Bcl-2/Bax ratio significantly reduced(p<0.01).Compared with the high sugar group,high,medium and low dose group in TCM,the metformin group increased significantly(p<0.01).Compared with metformin group,high dose group increased(p < 0.05),low,middle dose group in TCM were significantly reduced(p<0.01).Compared with low dose group in TCM,high dose group in TCM the Bcl-2/Bax ratio increased significantly(p<0.01).Conclusion:1.Bushen Jianpi Huoxue Formula can promote high sugar induced MC3T3-E1 cell proliferation and differentiation.2.Bushen Jianpi Huoxue Formula can prevent high sugar induced MC3T3-E1 cell apoptosis.