Qin Li1, Ying-sai Fan2, Zong-qin Gao3, Kai Fan4, Zhong-jie Liu5. 1. Department of Veterinary Clinic Medicine, College of Veterinary Medicine, CVM, China Agricultural University, Mingyuanxilu No. 2, Haidian District, 100193 Beijing, China. Electronic address: liqin_00753@sina.com. 2. Department of Veterinary Clinic Medicine, College of Veterinary Medicine, CVM, China Agricultural University, Mingyuanxilu No. 2, Haidian District, 100193 Beijing, China. Electronic address: funinside@sina.com. 3. Department of Veterinary Clinic Medicine, College of Veterinary Medicine, CVM, China Agricultural University, Mingyuanxilu No. 2, Haidian District, 100193 Beijing, China. Electronic address: 13141278488@163.com. 4. Department of Veterinary Clinic Medicine, College of Veterinary Medicine, CVM, China Agricultural University, Mingyuanxilu No. 2, Haidian District, 100193 Beijing, China. Electronic address: dirtyfan@sina.com. 5. Department of Veterinary Clinic Medicine, College of Veterinary Medicine, CVM, China Agricultural University, Mingyuanxilu No. 2, Haidian District, 100193 Beijing, China. Electronic address: liuzhongjiecau@163.com.
Abstract
UNLABELLED: Fructus Ligustri Lucidi, fruits of Ligustrum lucidum Ait. (Oleaceae), has the effects of tonifying the liver and the kidney and strengthening the bones and muscles. In ancient times, Fructus Ligustri Lucidi can be prepared in ethanol or in water. Some active compounds have been found in Fructus Ligustri Lucidi, like Oleanolic acid and Ursolic acid, and Ursolic acid were proved to have osteogenic effects. METHODS AND RESULTS: To prove that Fructus Ligustri Lucidi water extract have osteogenic effects on MC3T3-E1 cells and how these effects work, we used CCK-8 (cell counting kit-8), ELISA (enzyme-linked immunosorbent assay), FQ-PCR (realtime fluorescence quantitative PCR) and western blot assays. After treatment with Fructus Ligustri Lucidi for 48h, 72h, 96h, the cell viability was marked increased, on concentration-dependently and time-dependently pattern. High and low concentrations of Fructus Ligustri Lucidi promoted differentiation of cells. Fructus Ligustri Lucidi could up-regulate OPG and RANKL protein in supernatant at 48h and 72h except for highest concentration (10(-1)mg/ml). Fructus Ligustri Lucidi promote OPG and RANKL mRNA expression at 48h and 72h, while the level of promoting at 72 was higher than 48h. 10(-5)mg/ml of Fructus Ligustri Lucidi up-regulates OPG protein expression and down-regulates RANKL protein expression. After treatment with Fructus Ligustri Lucidi water extract, inhibitors, Fructus Ligustri Lucidi water extract with inhibitors for 72h, inhibitors PD 98059, SB 203580, SP600125 and LY 294002 showed Fructus Ligustri Lucidi-induced cell proliferation and the leakage of OPG proteins effects. Fructus Ligustri Lucidi promoted the protein levels of ERK, p-ERK, p-JNK, p38, pp38, AKT and p-AKT, and inhibited the protein levels of JNK. CONCLUSIONS: Fructus Ligustri Lucidi water extract promoted cell proliferation and differentiation, mRNA and protein expression of OPG and RANKL on MC3T3-E1 cells. The effects of cell proliferation and leakage of OPG related to MAPK and AKT signaling pathways in different ways.
UNLABELLED: Fructus Ligustri Lucidi, fruits of Ligustrum lucidum Ait. (Oleaceae), has the effects of tonifying the liver and the kidney and strengthening the bones and muscles. In ancient times, Fructus Ligustri Lucidi can be prepared in ethanol or in water. Some active compounds have been found in Fructus Ligustri Lucidi, like Oleanolic acid and Ursolic acid, and Ursolic acid were proved to have osteogenic effects. METHODS AND RESULTS: To prove that Fructus Ligustri Lucidi water extract have osteogenic effects on MC3T3-E1 cells and how these effects work, we used CCK-8 (cell counting kit-8), ELISA (enzyme-linked immunosorbent assay), FQ-PCR (realtime fluorescence quantitative PCR) and western blot assays. After treatment with Fructus Ligustri Lucidi for 48h, 72h, 96h, the cell viability was marked increased, on concentration-dependently and time-dependently pattern. High and low concentrations of Fructus Ligustri Lucidi promoted differentiation of cells. Fructus Ligustri Lucidi could up-regulate OPG and RANKL protein in supernatant at 48h and 72h except for highest concentration (10(-1)mg/ml). Fructus Ligustri Lucidi promote OPG and RANKL mRNA expression at 48h and 72h, while the level of promoting at 72 was higher than 48h. 10(-5)mg/ml of Fructus Ligustri Lucidi up-regulates OPG protein expression and down-regulates RANKL protein expression. After treatment with Fructus Ligustri Lucidi water extract, inhibitors, Fructus Ligustri Lucidi water extract with inhibitors for 72h, inhibitors PD 98059, SB 203580, SP600125 and LY 294002 showed Fructus Ligustri Lucidi-induced cell proliferation and the leakage of OPG proteins effects. Fructus Ligustri Lucidi promoted the protein levels of ERK, p-ERK, p-JNK, p38, pp38, AKT and p-AKT, and inhibited the protein levels of JNK. CONCLUSIONS: Fructus Ligustri Lucidi water extract promoted cell proliferation and differentiation, mRNA and protein expression of OPG and RANKL on MC3T3-E1 cells. The effects of cell proliferation and leakage of OPG related to MAPK and AKT signaling pathways in different ways.