| Objective: On the basis of previous experimental study,rat serum containing methotrexate,low-dose group,medium dose group and high-dose group of longzuantongbi formula were used to prepare drug containing serum In order to explore the molecular mechanism of longzhuangtongbi formula on bone erosion,provide a new theoretical basis for the treatment of rheumatoid arthritis with longzhuangtongbi formula,and guide clinical medication.Methods: Forty healthy female SD rats were randomly divided into normal control group,methotrexate group,low-dose group,medium dose group and high-dose group.The normal control group was given the same amount of normal saline by gavage every day.The gavage doses of low-dose group,medium dose group and high-dose group were 1.35,2.7 and 5.4g · kg-1 ·d-1,respectively.The gavage dose of methotrexate group was 0.9g · kg-1 · d-1.The normal control group,the low-dose group,the medium dose group and the high-dose group were gavaged at 9 am every day,once a day,and the methotrexate group was gavaged twice a week for 15 days.After15 days of continuous intragastric administration,the serum containing methotrexate and low,medium and high dose longzuantongbi prescription were prepared.MC3T3-E1 subclone 14 cells were cultured with the prepared serum.MTT assay was used to detect the effect of methotrexate and the serum containing Zhuang medicine longzuantongbi formula on the proliferation of MC3T3-E1 subclone 14 cells;RT qPCR was used to detect the effects of methotrexate and medicated serum of low,medium and high dose groups on the gene expression of key factors BMP2,Smad1,Smad4,Smad5,Smad9,Runx2 and Ki67 in BMP2 Smads signaling pathway;Objective to investigate the effect of methotrexate(MTX)and serum containing Zhuang medicine longzuantongbi formula on the protein expression of BMP2,Smad1,Smad4,Smad5,Smad9 and Runx2 in BMP2-Smads signaling pathway by Western blot.Results: 1.Compared with the normal control group,the serum containing methotrexate and longzuantongbi formula at low,medium and high doses could promote the proliferation of MC3T3-E1 subclone 14 cells,but there was no concentration and time-dependent effect.2.Compared with the normal control group,the medicated serum in the low,medium and high dose groups of longzuantongbi formula could promote the expression levels of BMP2,Smad1,Smad4,Smad5,Smad9 and Runx2 genes in MC3T3-E1 subclone 14 cells in varying degrees.The serum containing methotrexate and the low,medium and high dose groups of longzuantongbi formula could promote the expression of BMP2 gene in MC3T3-E1 subclone 14 cells.The serum containing Zhuang medicine longzuantongbi formula could significantly promote the expression of BMP2 gene in MC3T3-E1 subclone 14 cells(P<0.01),and the serum containing Zhuang medicine longzuantongbi formula could significantly promote the expression of BMP2 gene in MC3T3-E1 subclone 14 cells(P<0.05).The expression of Smad4 and Smad9 genes in MC3T3-E1 subclone 14 cells was significantly increased in MTX group and low-,medium-,and high-dose groups.The expression of Smad4 and Smad9 genes in MC3T3-E1 subclone14 cells was significantly increased in low-dose group(P<0.01),In high dose group,drug containing serum promoted the increase of Smad4 and Smad9 genes in MC3T3-E1 subclone 14 cells,but the difference was not statistically significant(P>0.05).Methotrexate group and low-,medium-,and high-dose groups of longzuantongbi formula could promote the expression of Smad1 and Smad5 related genes in MC3T3-E1 subclone 14 cells,but the difference was not statistically significant(P>0.05).The serum containing methotrexate and the low,middle and high dose groups of longzuantongbi formula could promote the expression of Runx2 gene in MC3T3-E1 subclone 14 cells,and the serum containing longzuantongbi formula could significantly promote the expression of Runx2 gene in MC3T3-E1 subclone 14 cells(P<0.01),The serum containing methotrexate could promote the increase of Runx2 gene expression in MC3T3-E1 subclone 14 cells,but there was no statistical significance(P>0.05).3.Compared with the normal control group,the serum containing methotrexate group and the low,medium and high dose groups of Zhuang medicine longzuantongbi formula could promote the protein expression levels of BMP2,Smad1,Smad4,Smad5,Smad9 and Runx2 in MC3T3-E1 subclone14 in varying degrees.The serum containing methotrexate and the low,middle and high dose groups of longzuantongbi formula could promote the expression of BMP2 protein in MC3T3-E1 subclone 14,and the serum containing methotrexate and the low dose group of longzuantongbi formula could promote the expression of BMP2 protein in MC3T3-E1 subclone 14,and the difference was statistically significant(P<0.05),The expression of BMP2 protein in MC3T3-E1 subclone 14 was significantly increased in medium dose group(P<0.01),while the expression of BMP2 protein in MC3T3-E1 subclone 14 was increased in high dose group(P>0.05).The expression of Smad1 protein in MC3T3-E1 subclone 14 was significantly increased in middle dose group(P<0.05).The expression of Smad1 protein in MC3T3-E1 subclone 14 was increased in methotrexate group and low and high dose groups(P>0.05).Methotrexate group and low-,medium-,and high-dose longzuantongbi formula groups could promote the expression of Smad4 and Smad5 related proteins in MC3T3-E1 subclone 14 cells,but there was no statistical significance(P>0.05).The expression of Runx2 and Smad9 protein in MC3T3-E1 subclone 14 cells was significantly increased in MTX group and low,medium and high dose groups of Zhuang medicine longzuantongbi formula(P<0.01),while the expression of Runx2 and Smad9 protein in MC3T3-E1 subclone 14 cells was significantly increased in MTX group and Zhuang medicine longzuantongbi formula(P<0.01),High dose group promoted the expression of Runx2 and Smad9 in MC3T3-E1 subclone14 cells,but there was no significant difference(P>0.05).Conclusion:The results showed that:1.The serum containing longzuantongbi formula could effectively promote the proliferation of MC3T3-E1 subclone 14 cells,but there was no concentration and time dependence.2.The medicated serum of low,middle and high dose groups of Zhuang medicine longzuantongbi formula can effectively promote the expression of BMP2,Smad4,Smad9,Runx2 mRNA and protein of BMP2,Smad1,Smad9,Runx2 in BMP2 Smads signaling pathway.The results showed that the serum containing longzuantongbi formula could promote the proliferation of osteoblasts through BMP2-Smads signaling pathway.Promoting the proliferation of osteoblasts and improving bone erosion may be one of the mechanisms of longzuantongbi formula in the treatment of rheumatoid arthritis. |
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