Study Of The Intervention Effects Of Zuoguiwan,YouguiWan Containing Serum With Different Osteoporosis Syndromes On Osteoblast Through ERK1/2 And Wnt/β-catenin Signal Pathway

Objective:To clarify the effects of Zuoguiwan YouguiWan containing serum of different osteoporosis(OP)syndromes on osteoblast proliferation and alkaline phosphatase(ALP)expression and the effects of Zuoguiwan YouguiWan containing serum of different osteoporosis(OP)syndromes on the expression of β-catenin,ERK1,ERK2 mRNA and protein of osteoblast through ERK1/2,Wnt/β-catenin signaling pathway.Method:Establish rats osteoporosis(OP)model by surgery for the preparation of drug-contained serum and osteoblast cultureunder OP model;and 10 weeks after surgery,inject hydrocortisone and take thyroxine to establish OP-kidney-yang-deficiency rat model,and OP-kidney-yin-deficiency rat model,for osteoblast culture anddrug-contained serum preparation.Respectively use femur of OP,OP-kidney-yang-deficiency,OP-kidney-yin-deficiency,two weeks old rats and skull of neonatal 24h rat for in vitro osteoblast culture,and to select the best osteoblasts by cell status and activity to meet the test requirements.Osteoblast get from 24h newborn rat skull were identified by alkaline phosphatase and alizarin red staining,and measured growth curve,investigated the amount of osteoblast in 96-seeded plate,to find out the best condition ot the test.After the confirmation of time and concentration,use the zuoguiwan,youguiwan containing serum of OP,OP-kidney-yang-deficiency,OP-kidney-yin-deficiency,normal rats and blank serum to influence osteoblast,and to investigate the OB proliferation by MTS,the expression of ERK1,ERK2,β-catenin mRNA and protein by RT-PCR and Western Blot.Results:10 weeks after surgery,histopathologic findings showed that the osteoporosis model was established;intragastric injection of hydrocortisone and mouth-taken thyroxine for 10 days,histopathology,the appearance of signs and detection of cAMP/cGMP showed OP-kidney-yang-deficiency,OP-kidney-yin-deficiency model were established.Osteoblast culture of OP,OP-kidney-yang-deficiency,OP-kidney-yin-deficiency,two weeks old rats femur and 24h newborn rats skull were successful,but there are significant differences of cells in status and activity.Cells get from the 24h newborn rats skull has the best activity and is time-saving,while the process of the osteoblast culture of OP,OP-kidney-yang-deficiency,OP-kidney-yin-deficiency are long and can be easily polluted.Therefore,we choose the 24h neonatal rat skull to isolate osteoblasts in vitro.Alkaline phosphatase staining and alizarin red staining confirmed osteoblasts,which has better activity.3000/well were seeded in 96-well plate according to the results;MTS results showed that:20%Zuoguiwan containing serum and 20%serum containing YouguiWan After the effect of 72h,have the most obvious aproliferation effect on bone cells.Four different states(OP,OP-kidney-yang-deficiency,OP-kidney-yin-deficiency,normal state)left YouguiWan containing serum and blank serum intervention osteoblasts showed:left four states YouguiWan containing serum can promote the proliferation and ALP expression in bone cells,and there are differences.For Zuoguiwan concerned,OP and kidney state Zuoguiwan promoted containing serum on osteoblast proliferation and ALP strongest expression,with the normal group,OP group,OP and deficiency Zuoguiwan medicated serum compared with a significant difference(p<0.05).For the purposes of YouguiWan YouguiWan and deficiency state under OP-containing serum on a role in promoting bone cell proliferation and ALP expressed the strongest with the normal group,OP group,OP and kidney group Zuoguiwan containing serum,compared with a significant difference(p<0.05).Four different state(OP,OP-kidney-yang-deficiency,OP-kidney-yin-deficiency,normal state)left YouguiWan containing serum and blank serum intervention osteoblasts ERK1/2,Wnt/β-catenin signal mRNA and protein expression related factor results show four different states left path,YouguiWan containing serum in the regulation of osteoblast β-catenin,there are differences ERK1 ERK2 mRNA and protein expression.Regulating the expression of specific mRNA expression;Compared with the blank serum each state,OP,OP kidney,OP deficiency,left in a normal state YouguiWan containing serum could increase β-catenin mRNA expression,except OP deficiency Zuoguiwan containing serum,but left the remaining state YouguiWan containing serum can promote ERK2 mRNA expression,in addition to the normal group YouguiWan containing serum,the rest of the state Left YouguiWan containing serum can promote the expression of ERK1 mRNA,wherein the OP,OP and kidney group Zuoguiwan containing serum,OP,OP and deficiency group YouguiWan containing serum β upregulated-catenin,ERK1 mRNA expression of respect,and the corresponding state control serum,compared with a significant difference(p<0.05);OP and kidney,OP and deficiency group Zuoguiwan containing serum,OP,OP and YouguiWan deficiency group containing serum raised ERK2 mRNA expression in context,with the blank serum corresponding state,compared with a significant difference(p<0.05);normal,OP and kidney YouguiWan containing serum and blank serum in the role of the three aspects of mRNA expression was no significant difference;OP and kidney Zuoguiwan containing serum containing serum compared with OP and Zuoguiwan deficiency,can significantly increase β-catenin,ERK1,ERK2 mRNA expression(p<0.05);and the control group,compared to the OP group Zuoguiwan containing serum,can significantly upregulated ERK1,ERK2 mRNA expression(p<0.05),but the role of β-catenin for free significant differences;compared to OP and deficiency YouguiWan containing serum,and OP,OP and kidney YouguiWan Under normal conditions containing serum,can significantly increase β-catenin,ERK1,ERK2 mRNA expression(p<0.05).Regulating the expression of specific performance is:OP,OP and kidney,OP and deficiency,left in a normal state YouguiWan containing serum could increase β-catenin expression,ERK1,ERK2 protein,wherein OP,OP and left kidney,YouguiWan containing serum,normal seruβ containing Zuoguiwan,OP and deficiency YouguiWan containing serum,the regulated expression ofβ-catenin protein aspect,and the corresponding state blank serum,compared with a significant difference(p<0.05);OP and deficiency Zuoguiwan containing serum,OP,OP and kidney,OP and deficiency YouguiWan containing serum upregulated ERK2 terms of protein,compared with the corresponding state serum,a significant difference(p<0.05);OP YouguiWan containing serum upregulated ERK1 protein expression,compared with the corresponding serum blank state,with significant difference(p<0.05),Zuoguiwan each group containing serum proteins in the regulation of the expression of ERK1,compared with the corresponding serum blank state,no significant difference;and OP and deficiency Zuoguiwan containing serum phase ratio,OP and kidney Zuoguiwan containing serum could significantly increase the expression of β-catenin protein(p<0.05),it can also increase the expression of ERK1,ERK2 protein,but no significant difference;and OP and kidney Compared YouguiWan containing serum,OP and deficiency YouguiWan containing serum could significantly increase β-catenin,ERK2 protein expression(p<0.05)Conclusion:This is the first study to investigate the effects of Zuoguiwan YouguiWan containing serum of osteoporosis(OP)with different syndromes on bone cell proliferation and differentiation and the expression of mRNA and protein of ERK,Wnt signaling pathway related factors.We find out that there is difference betweern drug contained serum and provide experimental evidence for the treatment of osteoporosis with TCM-youguiwan and zuoguiwan.Also,This is the first study to show that YouguiWan contained serum under kidney yang-deficiency state has the best effects on osteoblast proliferation,ALP expression,β-catenin,ERK1,ERK2 mRNA and protein expression;while Zuoguiwan ontained serum under kidney yin-deficiency state has the best effects on osteoblast proliferation,ALP expression,β-catenin,ERK1,ERK2 mRNA and protein expression,which is consistent with the TCM theory tha t"Youguiwan is for kidney yang difficiency,Zuoguiwan is for Kidney Yin difficiency",and provide scientific basis for clinical dialectical treatment of osteoporosis Zuoguiwan and YouguiWan regulate the proliferation and diferentiation of bone cells by ERK1/2 and Wnt/β-catenin signaling pathway,which may be the mechanisms of zuoguiwan and youguiwan for the prevention of osteoporosis.