Effects of naringin on proliferation, differentiation and maturation of rat calvarial osteoblasts in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial osteoblasts (ROB).</p><p><b>METHOD</b>Segregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differentiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERbeta was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERbeta and collagen I was examined by Western blot. After the addition of ICI 182.780, an inhibitor of the estrogen signal pathway, these index also was examined and the changes were compared.</p><p><b>RESULT</b>The ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 x 10(-5) mol x L(-1) is the best concentration. Naringin improved the secretion of osteocalcin, osteopontin, bone morphogenetic protein-2 and collagen I significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERalpha and ERbeta. While all these effects can be restrained by ICI 182.780.</p><p><b>CONCLUSION</b>The naringin with final concentration of 1 x 10(-5) mol x L(-1) enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182.780. These results suggesting that naringin is one of the phytoestrogens and have the activity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.</p>

Comparative study on the differentiation and maturation of rat calvarial osteoblasts by naringin and Na-ringenin in vitro / 中国药学杂志

OBJECTIVE: To investigate the effects on the differentiation and maturation of rat osteoblast (ROB) by naringin and it's metabolite-naringenin. METHODS: Primary ROB were obtained from new born SD rat skull after dissected and digested many times by type II collagenase during sterile condition. Serial subcultivation were proceeded when cells covered 80% culture dish. Cell proliferation was detected by MTT, while the alkaline phosphatase (ALP) activity was adopted as osteogenic differentiation marker to screen the best concentration of naringin and naringenin. The secretion of osteocalcin, bone morphogenetic protein-2 (BMP-2), oes-teopontin(OPN) and collagen I, the bone mineralized nodulus, even the gene expression of bFGF, IGF-1, Runx-2, Osterix, OPG and RANKL all were compared among the naringin-supplemented group, naringenin-supplemented group and the control. RESULTS: Both naringin and naringenin can significantly improved ALP activity, the secretion of osteocalcin, BMP-2, OPN and collagen I, the bone mineralized nodulus also were raised. Besides, these two drugs also stimulated the expression of genes which related to the osteogenesis of ROB. However, naringenin is stronger than naringin in above markers significantly. CONCLUSION: The osteoprotective effects of naringenin is stronger than naringin at enhancing the osteogenic differentiation of ROB, suggesting that naringin can be administered via oral and its metabolites developed higher activity to prevent osteoporosis. These results may provide a guide for the new drug develop and dosage forms design during osteoporosis therapy.

The changes of iNOS and NO in the osteogenic differentiation process of rat bone marrow stromal cells promoted by icariside II / 药学学报

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.

Effect of Osthol on the proliferation and differentiate of osteoblasts in vitro / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the effects of Osthol on the proliferation and differentiation of osteoblasts of rats (rat calvarial osteoblasts, ROB) cultured in vitro.</p><p><b>METHODS</b>The neonatal SD rat skull was segregated, and enzyme digestion was used to obtain bone cells which were cultured in MEM containing 10% FBS. The medium was changed every three days, and serial subcultivation was performed when cells covered with 90% of the culture dish. The Osthol was added to 96-well plates with final concentration of 1 x 10(-4) mol/L, 1 x 10(-5) mol/L, 1 x l0(-6) mol/L and 1 x10(-7) mol/L, and MTT method was used to evaluate the proliferation. Differentiation analysis: the alkaline phosphatase (ALP) activity was determined at the 3rd, 6th, 9th, 12th and 15th days separately after osteogenic induction culture. The synthesis of type I collagen was observed using immunohistochemical method at the 8th day. The ALP stain was performed at the 12th day. The alizarin red staining was done and calcified nodules was counted at the 14th day.</p><p><b>RESULTS</b>The Osthol with final concentration of 1 x 10(-4) mo/L inhibit the proliferation of ROB. The Osthol with final concentration of 1 x 10(-5) mol/L had no obvious influence on the proliferation of ROB, but it significantly promoted the activity of ALP, enhanced the synthesis of collagen type I and increased the number of calcified nodules.</p><p><b>CONCLUSION</b>The Osthol with final concentration of 1 x 10(-5) mol/L can promote differentiation and maturation of ROB, which may be active ingredients of Chinese drugs for the osteoporosis prophylaxis.</p>