| Background:Osteoporosis is the most common systemic metabolic disease related to aging,especially in postmenopausal women.In addition to the fragile fracture of femur and hip bone,it also reduces the density of the jaw and the height of the alveolar bone,which accelerate tooth losing and affect the oral treatment such as denture repair,implant implantation and maxillofacial plastic surgery,seriously affects the patients’health and quality of life.Some studies have shown that the drugs for osteoporosis can effectively improve the loss of alveolar bone and promote bone integration around the implant.However,bisphosphonates,estrogen replacement therapy,selective estrogen receptor modulators and other commonly used clinically can improve osteoporosis to a certain extent,long-term use has serious side effects.How to prevent the occurrence and development of osteoporosis has always been a research hotspot.Traditional Chinese medicine monomer has been used in the prevention and treatment of osteoporosis for a long time.Compared with chemical synthetic drugs,it has less adverse reactions and is more suitable for long-term use.Ginsenoside is the main active component of ginseng,which is similar to endogenous estrogen and can be used as the selective ligand of estrogen receptor.Estrogen receptor（ER）is widely present in osteoblasts and osteoclasts of bone tissue.it plays an important role in coordinating the balance of bone metabolism and preventing bone loss in postmenopausal women.Estrogen receptorβ（ERβ）is the main expression form of bone tissue,which is always at a high expression level during the differentiation process of osteoblast.This indicates that ERβplays a key role in the process of osteogenesis and is a potential target for the treatment of osteoporosis.Objective:In this study,we screened ginsenoside monomers that target to up-regulate the transcription and expression of ERβ,and explored its effect on the proliferation and differentiation of MC3T3-E1 cells.PHTPP,a selective ERβantagonist,was further used to explore its effect on the osteogenesis of ginsenoside Rh1.Methods:1.To Screen ginsenoside monomers that can target up-regulation of ERβtranscription and expression:the experiment is divided into blank control group,ginsenoside Rb1 group,Rb2 group,Rd group,Rg1 group,Rg2 group and Rh1 group(1×10-5mmol·L-1),estradiol group(1×10-6mmol·L-1),293T cells were co-transfected with PGL2-ERβand the internal reference Renilla luciferase plasmid Prep7-Rluc,and the dual luciferase activity in each group were detected by dual luciferase reporter gene experiment.The experimental groups were the same as above.The drugs of each group were applied to MC3T3-E1 cells,and the expression level of ERβprotein in each group was detected by western blot.The MC3T3-E1 cells were further divided into blank control group,estradiol(1×10-6mmol·L-1)group,ginsenoside Rh1 group(1×10-6,1×10-5 and 1×10-4mmol·L-1),Western blot was used to detect the expression level of ERβprotein in each group.Finally,the Auto Dock molecular docking experiment was used to simulate the binding of ginsenoside Rh1 and ERβprotein molecules.2.To study the effect of ginsenoside Rh1 on the proliferation and differentiation of osteoblasts:MC3T3-E1 cells were divided into blank control group and ginsenoside Rh1 group(5×10-6,1×10-5,5×10-5,1×10-4,1×10-3 and 5×10-3mmol·L-1)and estradiol group(1×10-6mmol·L-1)and after 24,48 and 72 h,CCK-8 method was used to detect cell proliferation rate in each group.Different concentrations of ginsenoside Rh1(0,1×10-5 and 1×10-4mmol·L-1)were applied to MC3T3-E1 cells,and the expression of osteogenic proteins COL-I,BMP2,OCN,OPG and Runx2 in MC3T3-E1 cells were detected by Western blot.MC3T3-E1 cells were divided into blank control group,estradiol group(1×10-6mmol·L-1)and ginsenoside Rh1 group(1×10-5mmol·L-1 and1×10-4 mmol·L-1),MC3T3-E1 cells were induced for 7,14 and 21 d respectively,ALP staining and alizarin red staining were used to detect the staining area of ALP and calcified nodules,and the effect of ginsenoside Rh1 on the osteogenic differentiation of MC3T3-E1 cells was observed.3.To study the inhibitory effect of PHTPP on the osteogenesis of ginsenoside Rh1:MC3T3-E1 cells were divided into blank control group,Rh1 group(1×10-4 mmol·L-1)and Rh1+PHTPP group.The expression of osteogenic genes BMP2,COL-I,Runx2were detected by real-time PCR;the expression of osteogenic proteins COL-I,BMP2,OCN,OPG and Runx2 were detected by Western blot,The ALP activity was detected by ALP activity assay.Results:1.In the dual luciferase reporter gene experiment,the luciferase activity of ginsenoside Rh1 group(1×10-5mmol·L-1)was significantly higher than that in blank control group（P<0.05）after transfection of ERβ-PGL2 plasmid into 293T cells.Compared with blank control group,the expression level of ERβprotein in ginsenoside Rh1 group was significantly increased,and the expression level of ERβprotein reached the highest at the concentration of 1×10-4 mmol·L-1,even higher than that in estradiol group(1×10-6 mmol·L-1).Auto dock analysis showed that ginsenoside Rh1 could bind in the ligand binding pocket of ERβprotein.2.In CCK-8 experiment,compared with blank control group,the proliferation rate of MC3T3-E1 cells in different concentrations of ginsenoside Rh1 group was significantly increased（P<0.01）,and the highest proliferation rate was reached at 72h.Compared with blank control group,the expression levels of bone related proteins COL-I,BMP2,OPG and Runx2 in ginsenoside Rh1 group were significantly increased by Western blot.After osteogenic differentiation,compared with blank control group,the staining area of ALP in ginsenoside Rh1 group was significantly increased.When the concentration of ginsenoside was 1×10-4 mmol·L-1,the staining area of ALP was the largest,and the staining area of ALP at 14 d was significantly increased compared with that at 7 d,which was time and concentration dependent;compared with the blank control group,the staining area of alizarin red in mineralized nodules in ginsenoside Rh1 group was significantly increased.3.After PHTPP was applied to cells,compared with blank control group,the expression levels of osteogenic genes BMP2,COL-I and Runx2 were significantly decreased in the real-time PCR experiment,and the expression levels of osteogenic proteins COL-I,BMP2,OPG and Runx2 were significantly decreased in the Western blot experiment;the ALP activity was significantly decreased in the ALP activity test at 7 and 14 d（P<0.01）.Conclusion:Ginsenoside Rh1 can bind to ERβin cells,which can up regulate the transcription and expression of ERβin cells,and promote the proliferation and differentiation of osteoblasts,the optimal concentration is 1×10-4 mmol·L-1.The results of this study provide a basis for ginsenoside Rh1 in the prevention and treatment of osteoporosis. |
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