The Relationship Between The Methyl At Ion Of ERα And Kidney Deficiency PMOP And Intervention Of Chinese Herbal Medicine

ObjectivesTo investigate the relationship between Postmenopausal Osteoporosis(PMOP)and DNA methylation in promoter region of ER a,and the effects of Chinese herbal medicine on the Methylation of ERα、proliferation and osteoblastic differentiation.Methods(1)20 cases of Postmenopausal Osteoporosis patients and 20 cases of Postmenopausal women were collected as observation group and normal control group,respectively.The general and physical information of all patients were collected through questionnaire survey and physical examination,the bone mineral density(BMD)of lumbar spine was detected by DEXA,the methylation of ER a gene promoter was detected by methylation PCR(MSP).The difference of the methylation of ER a gene promoter and BMD value in two groups,the relationship between the methylation of ER a gene promoter and BMD value were analysis by SPSS19.0 software.(2)Primary osteoblasts were isolated using double enzyme digestion,and treated with Liuwei Dihuang pill and Jinkui Shenqi pill(the concentration were 10、50、100 ng/μ L).The methylation of ER a gene promoter and expression of ER a in mRNA and protein levels were detected by MSP、RT-PCR and WB,respectively.The proliferation of osteoblasts was detected using CCK8 kit,the viability of ALP was detected by ALP kit,the expression changes of Collagen I and OPG were detected by IHC,the expression changes of AMPK and β-catenin,which are the key molecular in osteoblast proliferation and differentiation,were detected by western blot.Results(1)Compared with normal control group,the BMD value decreased and the methylation level of ERα gene promoter increased in observation group,and the results of Statistical analysis showed there is a negative correlation between the methylation of ERα gene promoter and BMD value.(2)Chinese herbal medicine can inhibit the methylation of ERα gene promoter and increase the mRNA and protein expression of ERα,and the effects of Jinkui Shenqi pill is more obvious.The results of CCK8 showed:the viability of osteoblasts had no obvious changes after the intervention of Liuwei Dihuang pill;and the viability increased notably after intervened by Jinkui Shenqi pill(the concentration is 50,100ng/μL)for 48 h.Meanwhile,the results of osteoblastic differentiation showed:For the expression of ALP and collagen I in osteoblast early differentiation stage,Liuwei Dihuang pill could increase their expression at the concentration of 100 ng/μ L,and Jinkui Shenqi pill could also increase their expression at the concentration of 50 ng/μL and 100 ng/μL,and the effect of Jinkui Shenqi Pill on early differentiation of osteoblast was more obvious.In addition,the expression of AMPK and β-catenin,which are the key molecular in the processes of proliferation and osteoblastic differentiation.Decreased and increased notably after the intervention of Chinese herbal medicine.ConclusionThere is a negative correlation between BMD and DNA methylation in promoter region of ERα;Jinkui Shenqi pill reduces DNA methylation in promoter region of ER a in osteoblasts and enhances the expressions of ER a in mRNA and protein levels.And above results possible influence its proliferation and osteoblastic differentiation.