Effect Of Eucommia Ulmoides On MC3T3-E1 Cells Before And After Being Processed With Salt

Eucommia ulmoides possesses the function of strengthening bone,tonifying liver and kidney,and the effect promoted after processed with salt.Lignans in Eucommia ulmoides was one of the active ingredients of anti-osteoporosis,such as Pinoresinol di-O-p-D-glucopyranoside(PDG).But Pinoresinol had no report on anti-osteoporosis.The aim of this article is to investigate osteogenic effect of water extract of Eucommia ulmoides and Eucommia ulmoides processed with salt,and its 30% part of water extract,PDG and Pinoresinol have and how these effects works on MC3T3-E1 cells by assaying the cell proliferation,differ-e ntiation and effects on OPG/RANKl/RANK system.Methods:Osteogenic like cell line MC3T3-E1 was used as cell model,and different concentrations of water extract of Eucommia ulmoides and Eucommia ulmoides processed with salt,and its 30% part of water extract,PDG and Pinoresinol were added in MC3T3-E1 cells for 48 h and 72 h,and then the cell viability,ALP activity,protein expression of OPG and RANKL were tested at different time point.MTT was used for cell viability,ELISA for leskage of OPG and RANKL,and Western blot assays for cell protein expression.Results:1.After treatment with different concentrations of water extract of Eucommia ulmoides and Eucommia ulmoides processed with salt for 48 h and 72 h,the cell viability was significant increased.The crude showed better effects in medium and low concentration,and after processed with salt showed better effects than the crude in all concentrations.After treatment with of the 30% part of water extract of Eucommia ulmoides for 48 h,the medium and low concentration promoted cell viability,and the high concentration effect at 72 h.All concentrations of water extract of Eucommia ulmoides processed with salt promoted cell viability at 48 h and 72 h.PDG had no significant effect at 48 h,but showed better effects in high and medium concentration at 72 h.Pinoresinol promoted cell viability only in 10-3ug/L and 10-2ug/L at 48 h,and in 10-1ug/L at 72 h.PDG showed better effects than Pinoresinol.2.After treatment with different concentrations of water extract of Eucommia ulmoides for 48 h,all concentrations promote ALP secretion,while high and low concentrations promote ALP secretion for 72 h.All concentrations of water extract from Eucommia ulmoides processed with salt promote ALP secretion for 48 h and 72 h,especially better than the crude in long-term.All concentrations of the 30% of water extract of Eucommia ulmoides and Eucommia ulmoides processed with salt promote ALP secretion for 48 h and 72 h.The 30% of water extract of Eucommia ulmoides perform better than processed with salt in medium and high concentration,while less in low concentration.PDG promote ALP secretion in all concentrations for 48 h,and in high and medium concentration for 72 h.All concentrations of Pinoresinol promote ALP secretion for 48h and 72h.3.After treatment with different concentrations of water extract of Eucommia ulmoides and processed with salt for 48 h,all concentrations inhibit RANKL secretion and high concentration promote OPG secretion.After treatment with different concentrations of water extract of Eucommia ulmoides processed with salt for 72 h,all concentrations promote OPG and inhibit RANKL secretion.Water extract of Eucommia ulmoides could promote OPG secretion in 10-4ug/L,10-3ug/L,10-1ug/L,10ug/L,while all concentrations inhibit RANKL secretion.Water extract of Eucommia ulmoides and processed with salt could up-regulate OPG/RANKL for 48 h and 72 h,and processed with salt showed better than the crude for 72 h.All concentrations of the 30% of water extract of Eucommia ulmoides promote OPG secretion for 48 h,especially in 10-2ug/L and 10-1ug/L,and inhibit RANKL secretion for 48 h and 72 h.All concentrations of the 30% of water extract of processed with salt promote OPG secretion for 48 h,and inhibit RANKL secretion for 48 h and 72 h.The 30% of water extract of Eucommia ulmoides and processed with salt could up-regulate OPG/RANKL for 48 h.The 30% of water extract of processed with salt could up-regulate OPG/RANKL only in 10-5ug/L for 72 h.PDG could promote OPG secretion in 10-5ug/L,10-3ug/L,10-1ug/L for 48 h and 72 h.Pinoresinol could promote OPG secretion in 10-5ug/L,10-3ug/L,10-1ug/L,10ug/L for 48 h and in 10-1ug/L,10ug/L for 72 h,while inhibit RANKL secretion in 10-5ug/L,10-3ug/L for 48h and 72h.4.Water extract of Eucommia ulmoides could inhibit RANKL protein expression in 1ug/L,10-2ug/L,10-4ug/L to up-regulate OPG/RANKL.Water extract from processed with salt could promote OPG protein expression and inhibit RANKL protein expression in 10-2ug/L,1ug/L,102ug/L to up-regulate OPG/RANKL.The 30% part of water extract of Eucommia ulmoides could promote OPG protein expression in 10-1ug/L,10-5ug/L to up-regulate OPG/RANKL.The 30% part of water extract of processed with salt mainly inhibit RANKL protein expression in 10-3ug/L,10-5ug/L to up-regulate OPG/RANKL.PDG could up-regulate OPG and RANKL protein expression in 10ug/L,10-1ug/L 10-3ug/L,10-5ug/L.Pinoresinol mainly promote OPG protein expression inhibit RANKL protein expression in 10-3ug/L,10-5ug/L,10ug/L to up-regulate OPG/RANKL.5.After treated with certain concentrations of water extract of Eucommia ulmoides and Eucommia ulmoides processed with salt,and the 30% part of water extract,PDG and Pinoresinol,inhibitor(10-5mol/L),and mixture of drugs with inhibitors for 72 h,LY and SB showed significant inhibition on MC3T3-E1 cell viability,and LY,SB,PDTC and PD showed significant effects on cell proliferation induced by drugs.Conclusions:Water extract of Eucommia ulmoides and processed with salt,and its 30% part of water extract,PDG and Pinoresinol could promote proliferation and differentiation of MC3T3-E1 cells,promote protein secretion and expression of OPG and inhibit RANKL protein on MC3T3-E1 cells,which Eucommia ulmoides processed with salt showed better effects than the crude,Pinoresinol better than PDG.The effects of water extract of Eucommia ulmoides and processed with salt,and the 30% part of water extract,PDG and Pinoresinol on cell proliferation signaling pathways related to ERK,p38 MAPK,NF-κB,AKT.