Effect And Mechanism Research Of Fructus Ligustri Lucidiand Its Constituents On MC3T3-E1Proliferation And OPG Expression

Fructus Ligustri Lucidiis commonly used on nourishing liver and kidney yin and strengthening bones. Some active constituents have been found in Fructus Ligustri lucidi, like Oleanolic acid and Ursolic acid, and Ursolic acid was proved to have osteogenic effects. The aim of this article is to investigate osteogenic effect of Fructus Ligustri Lucidi water extract, Ursolic acid and Oleanolic acid have and how these effects works on MC3T3-E1cells by assaying the cell proliferation, differentiation and effects on OPG/RANKL/RANK system.Methods Osterogenic like cell line MC3T3-E1was used as cell model, different concentrations of Fructus Ligustri Lucidi water extract, Ursolic acid and Oleanolic acid was added in MC3T3-E1cells for48h,72h and96h, then the cell proliferation, cell differentiation, gene or protein expression of OPG and RANKL, and the phosphorylation of proteins were tested at different time point. CCK-8(cell counting kit-8) was used for cell proliferation, ELISA (enzyme-linked immunosorbent assay) for leakage of OPG and RANKL, FQ-PCR (realtime fluorescence quantitative PCR) for OPG and RANKL mRNA expression, and western bolt assays for cell protein expression.Results:After treatment with certain consentrations of Fructus Ligustri Lucidi for48h,72h,96h, and the cell viability was significant increased, especially in long-term and medium-or low-concentration. After treatment with certain consentrations of Ursolic acid and Oleanolic acid for48h and72h, the cell viability showed significant increased, and Ursolic acid showed better effects than Oleanolic acid. High and low concentrations of Fructus Ligustri Lucidi and Ursolic acid promoted differentiation of cells. Fructus Ligustri Lucidi, Ursolic acid and Olieanolic acid could up-regulate OPG and RANKL protein in supernatant at48h and72h except for highest concentration. Certain contrations of Fructus Ligustri Lucidi and its constituents could promot OPG and RANKL mRNA expression at48h and72h.10-5mg/ml of Fructus Ligustri Lucidi,1×10-4μM of Ursolic acid and1×10-3μM of Oleanolic acid up-regulated OPG protein expression, then Fructus Ligustri Lucidi and Oleanolic acid down-regulated RANKL protein expression. After treated with certain concentrations of Fructus Ligustri Lucidi water extract or its constituents, inhibitors (20μM), and mixture of Fructus Ligustri Lucidi water extract with inhibitors for72h, ICI, RO, SB and LY showed significant inhibition on MC3T3-E1cell viability, all inhibitors showed significant effects on OPG leakage, as well as Fructus Ligustri Lucidi-, Ursolic acid-and Oleanolic acid-induced cell proliferation and the leakage of OPG proteins effects in different patterns. Fructus Ligustri Lucidi and Ursolic acid could up-regulate p-ERK, p-p38, inhibit p-JNK, and Fructus Ligustri Lucidi could up-regulate p-AKT. Oleanolic acid could up-regulate p-AKT, and down-regulated p-ERK, p-p38, p-JNK. Conclusions:Fructus Ligustri Lucidi water extract, Ursolic acid and Oleanolic promoted proliferation and differentiation of MC3T3-E1cells, mRNA and protein expression of OPG and RANKL on MC3T3-E1cells. Fructus Ligustri Lucidi showed long-term effects. Ursolic acid is the main constituent of Fructus Ligustri Lucidi on cell proliferation and OPG expression, The effect of Fructus Ligustri Lucidi-induced cell differentiation and RANKL expression was related to Ursolic acid and oleanolic acid. The effects of Fructus Ligustri Lucidiand its constituents on cell proliferation and leakage of OPG related to ER, PKC, NF-κB, MAPK and AKT signaling pathways in different ways. Fructus Ligustri Lucidi, Ursolic acid and Oleanolic acid have different effects on phosphorylation of MAPK and AKT proteins, ERK and p38MAPK signaling pathways were the key to make Oleanolic acid special.