Ronghua Zhang

Puerarin stimulates osteoblasts differentiation and bone formation through estrogen receptor, p38 MAPK, and Wnt/β-catenin pathways.

Puerarin is an isoflavone extracted from Radix Puerariae, a traditional Chinese herb used to treat many diseases such as osteoporosis. In this study, puerarin was shown to stimulate alkaline phosphatase (ALP) activity, type I collagen (Col I) secretion, and mineralized nodules formation of primary osteoblasts. Whereas the estrogen receptor (ER) antagonist ICI 182780 was able to reduce the increase in ALP activity and Col I secretion induced by puerarin. Furthermore, puerarin was shown to elevate levels of phospho-p38 mitogen-activated protein kinase (MAPK) and β-catenin proteins in a time-dependent manner. Pretreatment of osteoblasts with ICI 182780 can reduce this elevation, whereas pretreatment with p38 MAPK inhibitor SB 203580 did not affect the increase of β-catenin protein. Meanwhile, intragastric administration of puerarin protected against reduction in bone mineral density and bone mineral content in ovariectomized rats, and improved femur trabecular bone structure. Taken together, ER, p38 MAPK, and Wnt/β-catenin pathways were involved in puerarin-stimulated osteoblasts differentiation and bone formation.

Antiosteoporotic effect of icariin in ovariectomized rats is mediated via the Wnt/β-catenin pathway

Icariin (ICA), the main active flavonoid glucoside isolated from Herba Epimedii, has been shown to prevent postmenopausal bone loss in vitro. However, the mechanisms by which ICA prevents bone loss in vivo remain poorly understood. In the present study, the effect of ICA in an ovariectomized (OVX) rat model of osteoporosis was evaluated. Sprague-Dawley rats were divided into sham-operated and OVX groups. The OVX rats were randomly divided into five groups: OVX group (water only), Fosamax (positive) group (5.04 mg/kg, weekly, administered orally), and OVX-ICA groups (125, 250 or 500 mg/kg, daily, administered orally) and treated for 12 weeks. The 125, 250 and 500 mg/kg doses of ICA were designated as low (L-ICA), medium (M-ICA) and high (H-ICA), respectively. Compared with the sham-operated group, the OVX rats had significantly decreased bone mineral density (BMD), reduced serum osteoprotegerin (OPG) and increased serum bone gla protein (BGP) concentrations. ICA significantly increased BMD, biomechanical strength, trabecular bone number and trabecular bone thickness, and reduced lumbar trabecular bone separation. Treatment with ICA also completely normalized the expression of osteoblast markers by increasing serum concentrations of OPG and BGP. Enhanced mineralization was demonstrated by increased expression of differentiation markers. Although further in vivo studies are required to investigate the efficacy of ICA in improving bone mass, this study demonstrates that ICA has strong osteogenic activity, inducing osteogenic differentiation and inhibiting resorption by osteoclasts. It also demonstrates an antiosteoporotic effect for ICA on the basis of BMD, biochemical markers, biomechanical tests and histopathological parameters. Compared with L-ICA and H-ICA, M-ICA was more effective and caused no liver or kidney damage.

Icariin improves osteoporosis, inhibits the expression of PPARγ, C/EBPα, FABP4 mRNA, N1ICD and jagged1 proteins, and increases Notch2 mRNA in ovariectomized rats

Icariin (ICA) is a pharmacologically active flavonoid glycoside that shows promise in the treatment and prevention of osteoporosis (OP). However, the mechanisms underlying the anti-osteoporotic effects of ICA remain largely unclear. The present study used quantitative polymerase chain reaction, western blot and immunohistochemical analysis to examine the effects of ICA on several key targets in the Notch signaling pathway in bone tissue in ovariectomized rats. It was observed that ICA has a pronounced beneficial effect on OP rats and inhibits the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα) and fatty acid-binding protein 4 (FABP4) mRNA. In addition, it was identified that ICA downregulates the expression of notch1 intracellular domain (N1ICD) and Jagged1 proteins in bone tissue, and suppresses the effect of N1ICD on Notch2 mRNA expression. It is proposed that ICA inhibits the differentiation of mesenchymal stem cells into adipocytes by inhibiting the expression of PPARγ, C/EBPα and FABP4 mRNA via the Notch signaling pathway. In addition, it is proposed that ICA inhibits the expression of Notch2 mRNA by suppressing the effect of N1ICD. In conclusion, the results provide further mechanistic evidence for the clinical efficacy of ICA in the treatment of OP.

Icaritin induces MC3T3-E1 subclone14 cell differentiation through estrogen receptor-mediated ERK1/2 and p38 signaling activation

Icaritin (ICT), a hydrolytic product of icariin from the genus Epimedium, has many indicated pharmacological and biological activities. Several studies have shown that ICT has potential osteoprotective effects, including stimulation of osteoblast differentiation and inhibition of osteoclast differentiation. However, the molecular mechanism for this anabolic action of ICT remains largely unknown. Here, we found that ICT could enhance MC3T3-E1 subclone 14 preosteoblastic cell differentiation associated with increased mRNA levels and protein expression of the differentiation markers alkaline phosphatase (ALP), type 1 collagen (COL1), osteocalcin (OC), osteoponin (OPN) and runt-related transcription factor 2 (RUNX2), and improved mineralization, confirmed by bone nodule formation and collagen synthesis. To characterize the underlying mechanisms, we examined the effect of ICT on estrogen receptor (ER) and mitogen-activated protein kinase (MAPK) signaling. ICT treatment induced p38 kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) activation, but it demonstrated at the same time point no effect on activation of c-Jun N-terminal kinase (JNK). ER antagonist ICI182780, p38 antagonist SB203580 and ERK1/2 antagonist PD98059 markedly inhibited the ICT-induced the mRNA expression of ALP, COL1, OC and OPN. ICI182780 attenuated the ICT-induced phosphorylation of p38 and ERK1/2. These observations indicate a potential mechanism of osteogenic effects of ICT involving the ERK1/2 and p38 pathway activation through the ER.

Effects of water extract from epimedium on neuropeptide signaling in an ovariectomized osteoporosis rat model

ETHNOPHARMACOLOGICAL RELEVANCE For the past millennium, water extract from Epimedium (dried leaves of Epimedium brevicornu Maxim.) has been widely used for bone disease therapy in traditional Chinese medicine and has been reported to exhibit salutary effects on osteoporosis in clinical trials. The therapeutic effect of Epimedium is associated with the function of the brain in traditional Chinese medicine theory. STUDY AIM To determine the potential relationship between treating osteoporosis with Epimedium and neuropeptide regulation. MATERIALS AND METHODS Water extract from Epimedium was qualitatively and quantitatively analyzed with HPLC-TOF-MS. Ovariectomized rats were used as an osteoporosis model and were treated orally with water extract from Epimedium 16 weeks after surgery to mimic clinical therapy. After treatment, gene expression and protein levels of four neuropeptides, as well as their main receptors or receptor precursors including; neuropeptide Y (NPY) and its receptors NPY 1 (NPYR1) and 2; calcitonin gene-related peptide and its receptor precursor calcitonin receptor-like receptor (CRLR); vasoactive intestinal peptide (VIP) and its receptor VIP 1 (VIP1R) and 2; and substance P (SP) and its receptor neurokinin 1 receptor (NK1R) were detected in samples taken from bone, brain and spinal cord. RESULTS Treatment with water extract from Epimedium prevented bone mineral loss and reduced femoral bone strength decline associated with osteoporosis. Detection of neuropeptides showed that treatment also affected neuropeptide in the brain/spinal cord/bone axis; specifically, treatment increased brain NPY, bone NPY1R, bone CRLR, bone and spinal cord VIP and VIP2R, bone SP, and brain and spinal cord NK1R. CONCLUSION The effects of osteoporosis can largely be reduced by treatment with Epimedium most likely through a mechanism associated with several neuropeptides involved in regulation of the brain/spinal cord/bone axis. These novel results contribute to existing literature regarding the possible mechanisms of habitual use of Epimedium in the treatment of osteoporosis.

Postmenopausal osteoporosis is associated with the regulation of SP, CGRP, VIP, and NPY

Estrogen deficiency is the main factor underlying postmenopausal osteoporosis. A large number of neuropeptides, which regulate skeletal metabolism, potentially represent a regulatory pathway for the pathogenesis of osteoporosis. The aim of this study was to explore factors involved in the regulation of bone-related neuropeptides and their association with estrogen deficiency and bone metabolism. Thirty adult female Sprague-Dawley (SD) rats were randomly divided into a control group with sham surgery (n = 15) and an ovariectomy group with bilateral oophorectomy (n = 15). After 16 weeks, serum estrogen was reduced,CTX-1 was increased and P1NP was not significantly affected in the ovariectomy group and a model of osteoporosis was established. We then investigate the gene expression and protein levels of a range of neuropeptides and their receptors, including substance P (SP) and tachykinin receptor 1 (TACR1), calcitonin gene-related peptide (CGRP) and calcitonin receptor-like (CALCRL), vasoactive intestinal polypeptide (VIP) and receptor 1 and 2 (VPAC1, 2), neuropeptide Y (NPY) and receptor Y1 and Y2, in the brain and femora. Ovariectomy reduced TACR1, CGRP, CALCRL, NPY, NPY Y2 in the brain, but increased TACR1 and decreased SP, CALCRL, VIP, VPAC2 in the bone. Collectively, our data revealed that the pathogenesis of postmenopausal osteoporosis is associated with the regulation of SP, CGRP, VIP, and NPY. These novel results are of significant importance in the development of neuropeptides as therapeutic targets.