Icariin Exerts Estrogen-Like Actions on Proliferation of Osteoblasts in Vitro via Membrane Estrogen Receptors-Mediated Non-nuclear Effects.

Background: According to reports, icariin (ICA) is a bone anabolic agent able to prevent osteoporosis in both ovariectomized rats and postmenopausal women. However, its effect on osteoblast proliferation remains to be determined, and the underlying mechanism remains to be elucidated. Methods: Icariin-bovine serum albumin (BSA) conjugates were purified by Sephadex G-25 gel chromatography technology. Primary osteoblasts from neonatal rats were used to evaluate the effects of ICA, ICA-BSA, ICA-BSA + ICI182780, and ICA-BSA + PD98059. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI)-staining assays were used to detect the proliferation of osteoblasts after drug exposure. The intracellular calcium ions were detected using a confocal microscope with Fluo-3/AM as the fluorescent indicator. Western blot was capitalized on to measure the relative content of phospho-extracellular signal-regulated kinase (p-ERK). Results: Primary osteoblasts in culture were detected by histochemical staining of alkaline phosphatase, and calcified nodules were obtained by sequential digestion. Icariin and bovine serum albumin could form conjugate, which could be purified by Sephadex G-25 gel chromatography technology. MTT and flow cytometry results show that ICA-BSA conjugate significantly facilitated the proliferation of osteoblasts (P < 0.05). The intracellular calcium ions also ascended vastly in the cells treated with ICA-BSA conjugate (P < 0.01). Icariin-bovine serum albumin exposure rapidly activated the extracellular signal-regulated kinase (ERK) signaling. Furthermore, ICA- and ICA-BSA-mediated actions on osteoblasts were signally alleviated after dealing with ERK inhibitor PD98059 or estrogen receptor (ER) antagonist ICI182780, which might have a relation to the repression of ERK phosphorylation. Conclusions: Icariin could serve as estrogen in osteoblast cells by the rapid nongenomic ER signaling pathway independent of ligand and estrogen response element (ERE) and mediated by mitogen-activated protein kinase (MAPK).

Secretome analysis of rat osteoblasts during icariin treatment induced osteogenesis.

Osteoporosis is a serious public health problem and icariin (ICA) is the active component of the Epimedium sagittatum, a traditional Chinese medicinal herb. The present study aimed to investigate the effects and underlying mechanisms of ICA as a potential therapy for osteoporosis. Calvaria osteoblasts were isolated from newborn rats and treated with ICA. Cell viability, apoptosis, alkaline phosphatase activity and calcium deposition were analyzed. Bioinformatics analyses were performed to identify differentially expressed proteins (DEPs) in response to ICA treatment. Western blot analysis was performed to validate the expression of DEPs. ICA administration promoted osteoblast viability, alkaline phosphatase activity, calcium deposition and inhibited osteoblast apoptosis. Secretome analysis of ICA­treated cells was performed using two­dimensional gel electrophoresis and matrix­assisted laser desorption/ionization time­of­flight mass spectrometry. A total of 56 DEPs were identified, including serpin family F member 1 (PEDF), protein disulfide isomerase family A, member 3 (PDIA3), nuclear protein, co­activator of histone transcription (NPAT), c­Myc and heat shock protein 70 (HSP70). These proteins were associated with signaling pathways, including Fas and p53. Bioinformatics and western blot analyses confirmed that the expression levels of the six DEPs were upregulated following ICA treatment. These genes may be directly or indirectly involved in ICA­mediated osteogenic differentiation and osteogenesis. It was demonstrated that ICA treatment promoted osteogenesis by modulating the expression of PEDF, PDIA3, NPAT and HSP70 through signaling pathways, including Fas and p53.

Neuroprotective effect of combining tanshinone IIA with low-dose methylprednisolone following acute spinal cord injury in rats.

The present study compared the potential neuroprotective effect of tanshinone IIA (TIIA) monotherapy, methylprednisolone (MP) monotherapy and combined treatment in an adult acute spinal cord injury (ASCI) rat model. The current study used the weight-drop method (Allen's Impactor) in the rat model and the mechanical scratch method in primary spinal cord neuron culture to determine whether the combined treatment was able to reduce the required dosage of MP in the treatment of ASCI to produce a similar or improved therapeutic effect. In vivo male Sprague Dawley rats (n=60) were randomly divided into 5 groups, of which 12 rats were selected for the sham group and T9-T11 laminectomies, leading to ASCI, were performed on 48 of the 60 rats using a 10 g ×25 mm weight-drop at the level of T10 spinal cord. Therefore, the ASCI group (n=12) included the 'laminectomy and weight-drop'. The remaining 36 ASCI model animals were subdivided into 3 groups (n=12 each group): TIIA group (30 mg/kg/day), MP group (30 mg/kg) and combined treatment group (TIIA 30 mg/kg/day + MP 20 mg/kg). Neuronal function following ASCI was evaluated using the Basso Beattie Bresnahan (BBB) locomotor rating scale. Levels of the anti-apoptotic factor B-cell lymphoma-2 (Bcl-2), the pro-apoptotic factors Bcl-2 associated protein X (Bax) and caspase-3, and the inflammatory associated factor nuclear factor-κB, were analyzed by western blot analysis. Immunohistochemistry was used to detect caspase-3. To investigate the underlying mechanism, the anti-oxidative effect of combination TIIA and MP treatment was assessed by measuring the activity of malondialdehyde (MDA) and superoxide dismutase (SOD) in ASCI. In agreement with the experiment in vivo, primary neurons were prepared from the spinal cord of one-day-old Sprague-Dawley rats' and co-cultured with astrocytes from the brain cortex. The injury of neurons was induced by mechanical scratch and levels of apoptosis factors were analyzed by western blot analysis. The results of the current study indicated that injured animals in the combined treatment group exhibited a significant increase in BBB scores (P<0.05). TIIA + MP combined treatment and MP treatment was observed to reduce the expression of pro-apoptotic factors and promote neuron survival in vivo and in vitro. Combined treatment may promote neuroprotection through reduced apoptosis and inflammation caused by ASCI, similar to MP alone. Combined treatment reversed the decrease of SOD and the increase of MDA level caused by ASCI. In addition, combined treatment decreased the expression of caspase-3 in the neurons following ASCI in rats, as indicated by immunofluorescence double labeling. Overall, the present study indicates that the combined treatment of TIIA and MP may protect the neurons by stimulating the rapid initiation of neuroprotection following ASCI and reduce the dosage of MP in the treatment of ASCI required to produce the same or improved neuroprotective effects in vivo and in vitro.