Ke-Ming Chen

Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling

Increasingly natural products particularly flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. This study has reviewed previous studies on the two better known flavonoids, genistein and icariin, their structures, functions, action mechanisms, relative potency, and potential application in regulating bone remodeling and preventing bone loss. Genistein, an isoflavone abundant in soy, has dual functions on bone cells, able to inhibit bone resorption activity of osteoclasts and stimulate osteogenic differentiation and maturation of bone marrow stromal progenitor cells (BMSCs) and osteoblasts. Genistein is an estrogen receptor (ER)‐selective binding phytoestrogen, with a greater affinity to ERβ. Genistein inhibits tyrosine kinases and inhibits DNA topoisomerases I and II, and may act as an antioxidant. Genistein enhances osteoblastic differentiation and maturation by activation of ER, p38MAPK‐Runx2, and NO/cGMP pathways, and it inhibits osteoclast formation and bone resorption through inducing osteoclastogenic inhibitor osteoprotegerin (OPG) and blocking NF‐κB signaling. Icariin, a prenylated flavonol glycoside isolated from Epimedium herb, stimulates osteogenic differentiation of BMSCs and inhibits bone resorption activity of osteoclasts. Icariin, whose metabolites include icariside I, icariside II, icaritin, and desmethylicaritin, has no estrogenic activity. However, icariin is more potent than genistein in promoting osteogenic differentiation and maturation of osteoblasts. The existence of a prenyl group on C‐8 of icariin molecular structure has been suggested to be the reason why icariin is more potent than genistein in osteogenic activity. Thus, the prenylflavonoids may represent a class of flavonoids with a higher osteogenic activity. J. Cell. Physiol. 228: 513–521, 2013.

Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro.

There has been a strong interest in searching for natural therapies for osteoporosis. Genistein, an isoflavone abundant in soy, and icariin, a prenylated flavonol glycoside isolated from Epimedium Herb, have both been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the relative potency in osteogenesis between the individual phytoestrogen flavonoids remains unknown. The present study compared ability of genistein and icariin in enhancing differentiation and mineralization of cultured rat calvarial osteoblasts in vitro. Dose‐dependent studies in osteoblast differentiation measuring alkaline phosphatase (ALP) activity revealed optimal concentrations of genistein and icarrin for stimulating osteogenesis to be both at 10−5 M. Time course studies comparing the two compounds both at 10−5 M demonstrated that icariin treatment always produced higher ALP activity, more and larger areas of CFU‐FALP colonies and mineralized nodules, more osteocalcin secretion, and calcium deposition, and a higher level of mRNA expression of osteogenesis‐related genes COL1α2, BMP‐2, OSX, and RUNX‐2. However, they inhibited the proliferation of osteoblasts to a similar degree. In conclusion, although future in vivo studies are required to investigate whether icariin is more efficient in improving bone mass and/or preventing bone loss, our in vitro studies have demonstrated that icariin has a stronger osteogenic activity than genistein. In addition, while the prenyl group on C‐8 of icariin could be the active group that takes part in osteoblastic differentiation and explains its greater potency in osteogenesis, mechanisms of action, and reasons for the relative potency of icariin versus genistein need to be further studied. J. Cell. Biochem. 112: 916–923, 2011.

Icariin stimulates the osteogenic differentiation of rat bone marrow stromal cells via activating the PI3K–AKT–eNOS–NO–cGMP–PKG

Icariin, a prenylated flavonol glycoside isolated from Epimedii herba, has been found to be a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of icariin is related to the nitric oxide (NO) signal pathway and PI3K/AKT pathway in its upstream. Rat bone marrow stromal cells (rBMSCs) were cultured in osteogenic medium and treated with icariin or together with L-NAME, ODQ, PDE5, and/or LY294002 (the inhibitor of NOS, sGC, cGMP, and PI3K respectively), and effects were examined on the expression of signal messengers (NOS, NO, sGC, cGMP, PKG and PI3K) and the levels of osteogenic markers (alkaline phosphatase or ALP, osteocalcin and calcified nodules). It was found that icariin dose-dependently increased ALP activity, and treatment at the optimal concentration (10(-5)M) increased NOS activity, iNOS and eNOS expression, NO production, sGC and cGMP contents and PKG expression besides the phosphorylation of AKT. The addition of L-NAME, ODQ and PDE5 significantly inhibited the icariin effects on above markers respectively. The addition of LY294002 decreased the p-AKT level, NOS activity, eNOS expression and NO production significantly, but had no significant effect on iNOS expression. The addition of any of the four inhibitors also abolished the osteogenic effect of icariin on rBMSCs as indicated by ALP activity, osteocalcin synthesis, calcium deposition and the number and areas of calcified nodules. These results suggest that the osteogenic effect of icariin involves the PI3K-AKT-eNOS-NO-cGMP-PKG signal pathway. Furthermore, dosage response studies showed that icariin at 10(-6)M (a physiologically achievable concentration in vivo) also activated this signal pathway.

Osthol, a coumarin isolated from common cnidium fruit, enhances the differentiation and maturation of osteoblasts in vitro.

The effect of osthol on osteoblasts was investigated in primary osteoblastic cells isolated from newborn Wistar rats. Osthol was supplemented into cultured medium at 10–7, 10–6, 10–5 and 10–4 mol/l, respectively. No stimulating effect was found on cell proliferation, but 10–5 mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity. Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol. The results showed that the ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 1.52, 2.74 and 2.0 times higher, respectively, than in the control cells. Results of ALP histochemical staining and mineralized bone nodule assays both showed that the number and area achieved in osthol-treated cells were 1.53-fold higher than in control cells. The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, bone morphogenetic protein 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with bone development, were also investigated. The increase in mRNA expression was 1.94, 1.74, 1.68, 1.83 and 2.31 times, respectively, higher compared to the control. Furthermore, osthol increased the protein expression of p38 mitogen-activated protein kinase (MAPK) and type I collagen. p38MAPK protein and collagen in osthol-treated cells were 1.42 and 1.58 times higher in osthol-treated cells compared to the control. The results of these studies support the conclusion that osthol significantly enhances the osteogenic differentiation of cultured osteoblasts. The results also indicated that osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway and that osthol may be used as an important compound in the development of new antiosteoporosis drugs.

The Prenyl Group Contributes to Activities of Phytoestrogen 8-Prenynaringenin in Enhancing Bone Formation and Inhibiting Bone Resorption In Vitro

Previous studies have found that 8-prenylflavonoids have a higher osteogenic activity than do flavonoids, which suggested that the 8-prenyl group may play an active role in bone-protective properties. To address this hypothesis, activities of 8-prenylnaringenin (PNG) and naringenin (NG) in osteoblast and osteoclast differentiation and function were compared in vitro. PNG was found to have a stronger ability than NG to improve osteoblast differentiation and osteogenic function in cultured rat calvarial osteoblasts, as demonstrated by levels of alkaline phosphatase activity, osteocalcin, calcium deposition, and the number and area of mineralized bone nodules, as well as mRNA expression of osteogenesis-related genes Bmp-2, OSX, and Runx-2. In addition, although expression of osteoclastogenic inducer receptor activator of nuclear factor kappa-B ligand (RANKL) was not affected, that of osteoclastogenesis inhibitor osteoprotegerin (OPG) and consequently the OPG/RANKL ratio were increased, more potently by PNG than NG. PNG was also found to have a higher potency than NG in inhibiting the osteoclast formation in rabbit bone marrow cells and their resorptive activity, as revealed by lower numbers of osteoclasts formed, lower numbers and areas of bone resorption pits, and lower mRNA expression levels of tartrate-resistant acid phosphatase and cathepsin K. Furthermore, PNG induced apoptosis of mature osteoclasts at a higher degree and at an earlier time than did NG. These results indicate that the 8-prenyl group plays an important role and contributes to the higher bone-protective activity of PNG in comparison with NG.

Anti-hypoxic activity at simulated high altitude was isolated in petroleum ether extract of Saussurea involucrata.

ETHNOPHARMACOLOGICAL RELEVANCE Rhodiola algida, Saussurea involucrata, and other herbs grown in Qinghai-Tibetan plateau have long been used to prevent and treat acute mountain sickness. AIM OF THE STUDY To screen and identify the anti-hypoxic constituents in the herbs grown in Qinghai-Tibetan plateau of Northwestern China. MATERIALS AND METHODS The anti-hypoxic activities of 20 selected plateau herbs were examined against two positive controls, Rhodiola algida and acetazolamide, using the normobaric hypoxia model of mice. The herb with the highest activity was successively extracted with 70% ethanol, petroleum ether, chloroform, ethyl acetate and n-butanol. The extract with the highest activity was identified by comparing the survival time of mice under normobaric hypoxia condition after being subjected to different extracts. The identified extract was further tested by simulating high altitudes through an acute decompression model and a chronic decompression model for mice. RESULTS The herb found to have the highest anti-hypoxic activity was Saussurea involucrate (Kar. et Kir.) Sch.-Bip, and the most effective fraction was in the petroleum ether extract. Administration of petroleum ether extract of Saussurea involucrata (PESI) to mice at 50mg/kg significantly decreased the mortality of animals under acute decompression conditions. Changes in biochemical indicators for glycometabolism and energy metabolism, including adenosine triphosphate (ATP) content and adenosine triphosphatase (ATPase) activity in brain and cardiac muscle, lactic acid (LAC) and lactate dehydrogenase (LDH) in blood and cardiac muscles, blood sugar, and glycogen content in liver and skeletal muscle were reversed under chronic decompression conditions. CONCLUSIONS Saussurea involucrata (Kar. et Kir.) Sch.-Bip exhibits high anti-hypoxic activity that may be effective in preventing acute mountain sickness, and the active constituents are mainly in the petroleum ether extract.

Icariin attenuates hypoxia-induced oxidative stress and apoptosis in osteoblasts and preserves their osteogenic differentiation potential in vitro

Icariin, a prenylated flavonol glycoside isolated from traditional Chinese medicinal herb of the genus Epimedium, has been demonstrated to be a potential alternative therapy for osteoporosis, and its action mechanism so far has been mainly attributed to its phytoestrogenic property. As blood supply to bone is considerably reduced with ageing and by the menopause, we hypothesized that icariin treatment would reduce bone loss by preventing ischaemia‐induced hypoxic damages to bone.

Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia

Although pulsed electromagnetic fields (PEMFs) have been approved as a therapy for osteoporosis, action mechanisms and optimal parameters are elusive. To determine the optimal intensity, exposure effects of 50 Hz PEMFs of 0.6-3.6 mT (0.6 interval at 90 min/day) were investigated on proliferation and osteogenic differentiation of cultured calvarial osteoblasts. All intensity groups stimulated proliferation significantly with the highest effect at 0.6 mT. The 0.6 mT group also obtained the optimal osteogenic effect as demonstrated by the highest ALP activity, ALP(+) CFU-f colony formation, nodule mineralization, and expression of COL-1 and BMP-2. To verify our hypothesis that the primary cilia are the cellular sensors for PEMFs, osteoblasts were also transfected with IFT88 siRNA or scrambled control, and osteogenesis-promoting effects of 0.6 mT PEMFs were found abrogated when primary cilia were inhibited by IFT88 siRNA. Thus primary cilia of osteoblasts play an indispensable role in mediating PEMF osteogenic effect in vitro.

Different electromagnetic field waveforms have different effects on proliferation, differentiation and mineralization of osteoblasts in vitro

Noninvasive electromagnetic fields (EMFs) have been known to be able to improve bone health; however, their optimal application parameters and action mechanisms remain unclear. This study compared the effects of different forms of EMFs (sinusoidal, triangular, square, and serrated, all set at 50 Hz frequency and 1.8 mT intensity) on proliferation, differentiation and mineralization of rat calvarial osteoblasts. Square EMFs stimulated osteoblast proliferation but sinusoidal EMFs inhibited it. Sinusoidal and triangular EMFs produced significantly greater alkaline phosphatase (ALP) activity, ALP staining areas, calcium deposition, mineralized nodule areas, and mRNA expression of Runx-2, osteoprotegerin and insulin-like growth factor-I than square and serrated EMFs (P < 0.01). Triangular EMFs had a greater effect than sinusoidal EMFs on every indices except for Runx-2 mRNA expression (P < 0.05). These results indicated that while square EMFs promoted proliferation and had no effect on the differentiation of osteoblasts, sinusoidal EMFs inhibited proliferation but enhanced osteogenic differentiation. Triangular EMFs did not affect cell proliferation but induced the strongest osteogenic activity among the four waveforms of EMFs. Thus, the effects of EMFs on proliferation and differentiation of osteoblasts in vitro were dependent on their waveforms.

Icariin induces osteoblast differentiation and mineralization without dexamethasone in vitro.

An effective method for preventing bone loss is by promoting osteoblast differentiation and bone formation. While dexamethasone has been routinely used as a classical inducer for osteoblast differentiation, limitations have been observed with its usage, including its varied effects on expression of osteoblast genes in different species and its potentials in suppressing osteoblastic differentiation and mineralization. In this study, we assessed the ability of flavonoid icariin in enhancing differentiation and mineralization of cultured rat primary osteoblasts in the absence of dexamethasone. It was found that, compared to the non-stimulated control, icariin at 10(-5) M produced a higher alkaline phosphatase activity, more and larger areas of alkaline phosphatase-positive colonies (CFU-FALP) and mineralized nodules, more osteocalcin secretion and calcium deposition, higher levels of mRNA expression of alkaline phosphatase, osteoblastic transcription factors osterix and runt-related transcription factor 2, and collagen 1α, higher levels of protein expression of collagen 1α, alkaline phosphatese, osterix, and runt-related transcription factor 2. In addition, icariin at 10(-5) M was always more potent than dexamethasone at its optimal concentration of 10(-8) M on the above osteoblast differentiation and mineralization markers. Taken together, our studies demonstrated that icariin has a pronounced ability in promoting osteoblast differentiation in vitro in the absence of dexamethasone.