Resveratrol protects MC3T3-E1 cells against cadmium-induced suppression of osteogenic differentiation by modulating the ERK1/2 and JNK pathways.

Resveratrol (RES) is a natural polyphenolic compound with a broad range of physiological and pharmacological properties. Previous studies have shown that RES also plays an important role in protecting and promoting early bone metabolism and differentiation. The accumulation of cadmium (Cd), one of the world's most poisonous substances, can inhibit skeletal growth and bone maturation, thus causing osteoporosis. However, whether RES can prevent the Cd-induced inhibition of osteogenic differentiation remains unknown. In this study, we found that RES promoted the early maturity of osteoblastic MC3T3-E1 cells, as demonstrated by the significantly increased mRNA and protein expression of a range of differentiation markers, including alkaline phosphatase (ALP), collagen 1 (COL1), bone morphogenetic protein-2 (BMP-2), and runt-related transcription factor 2 (RUNX2). In contrast, we found that cadmium chloride (CdCl2) inhibited the viability and osteogenic maturity of MC3T3-E1 cells. We also demonstrated that RES pretreatment for 30 min provided significant protection against Cd-induced apoptosis and attenuated the inhibition of osteogenic differentiation induced by Cd by modulating ERK1/2 and JNK signaling. In conclusion, our results indicate that RES is a potential femoral protectant that not only enhance the viability and early differentiation of osteoblasts, but also protect osteoblasts from cadmium damage.

Effects of cadmium on osteoblast cell line: Exportin 1 accumulation, p-JNK activation, DNA damage and cell apoptosis.

Cadmium is an environmental metal pollutant that has been a focus of research in recent years, which is reported to cause bone disease; however, its skeletal toxicity and the mechanism involved are not yet fully known. Therefore, this study used MC3T3-E1 subclone 14 cells to determine the mechanism of cadmium toxicity on bone. Cadmium chloride (Cd) significantly reduced cell viability in a concentration-dependent manner. Exposure to Cd inhibited osteoblast-related proteins (Runx2, Col-1, STC2) and decreased alkaline phosphatase (ALP) activity. Cd caused Exportin-1 accumulation and induced DNA damage. Cd significantly down-regulated caspase 9 and induced cleaved-PARP, cleaved-caspase 3 protein level. Treatment with JNK inhibitor, SP600125, suppressed cadmium-induced elevation in the ratio of phosphorylation of JNK to JNK. Inhibition of caspase with pan-caspase inhibitor, Z-VAD-FMK, prevented MC3T3-E1 subclone 14 cells from cadmium-induced reduction of Runx2, STC2, caspase 9, and accumulation of cleaved PARP and cleaved caspase 3. Cd-induced cell survival enhanced by SP600125 but rescued by Z-VAD-FMK or KPT-335. These results suggest that cadmium cytotoxicity on bone involved exportin 1 accumulation, phosphorylation of JNK, induction of DNA damage and pro-apoptosis, which was induced by activation of caspase-dependent pathways.

Effectiveness associated with different therapies for senile osteoporosis: a network Meta-analysis.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of Chinese herbal medicines for invigorating the kidney (CHMIK) on senile osteoporosis. METHODS: We searched for studies in English-language databases (PubMed, the Cochrane Library, and Web of Science) and Chinese-language databases (China National Knowledge Infrastructure, Wan Fang Data, VIP Chinese periodical service platform, and China Biology Medicine disc from their inception to September 2017. Randomized controlled trials comparing the effectiveness of Traditional Chinese Medicine therapies (alone or in combination) and conventional clinical medicine therapies among older adult patients with osteoporosis were identified. We conducted a network Meta-analysis with a Bayesian hierarchical random-effects model using RStudio software, Version 3.4.1. RESULTS: Forty-three randomized controlled trials assessing the differences between Traditional Chinese Medicine and conventional clinical medicine were identified, including 15 treatments and involving 3316 patients. The results of the network Meta-analysis indicated that alendronate (odds ratio [OR] = 0.20, 95% confidence interval [CI]: 0.047-0.73) and calcium (OR = 0.18, 95% CI: 0.11-0.30) are significantly more effective if combined with oral CHMIK. CHMIK alone is significantly more effective than both alendronate (OR = 0.34, 95% CI: 0.10-1.0) and calcium (OR = 0.13, 95% CI: 0.056-0.28). Moreover, CHMIK + tuina + calcium is more effective than CHMIK + calcium + vitamin D + alendronate (OR = 18.0, 95% CI: 1.1-2.7e + 02). CONCLUSION: The present network Meta-analysis found that alendronate and calcium are more effective if combined with oral CHMIK and that oral CHMIK alone may be more effective than alendronate or calcium. Tuina may have an advantage over oral medicines. Oral CHMIK and calcitonin show the most potential for treating senile osteoporosis.

Icariin stimulates osteogenic differentiation and suppresses adipogenic differentiation of rBMSCs via estrogen receptor signaling.

Icariin (ICA) is a major active ingredient in Herba epimedii, which is commonly used as a Chinese herbal medicine for the treatment of osteoporosis. Previous studies have revealed that ICA exerted a protective effect against bone loss and increased bone regeneration; however, the association between ICA and estrogen receptor (ER) signaling remains unclear. The aim of the present study was to determine the effect of ICA on rat bone marrow stromal cells (rBMSCs). Cell Counting Kit­8 assays were conducted to measure proliferation, alkaline phosphatase (ALP) activity was evaluated to assess osteoblast differentiation, and reverse transcription­quantitative polymerase chain reaction as well as western blotting were performed to detect the expression of cellular and molecular markers of osteogenic or adipogenic differentiation. The results demonstrated that treatment of rBMSCs with 10­6 M ICA stimulated rBMSC proliferation and ALP activity. Furthermore, ICA treatment increased the expression of the osteogenic markers runt­related transcription factor 2, collagen type 1 and bone morphogenetic Protein 2; however, it also decreased the expression of the adipogenic differentiation markers peroxisome proliferator­activated receptor gamma and CCAAT/enhancer­binding protein α. Treatment of rBMSCs with ICI182780, an ER antagonist, blocked the effects of ICA. Taken together, these findings indicated that ICA may stimulate osteoblast differentiation and inhibit adipogenic differentiation via the activation of the ER signaling pathway. Therefore, ICA has the potential to serve as a therapeutic alternative for the prevention and treatment of osteoporosis.

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.

Changes in related circular RNAs following ERß knockdown and the relationship to rBMSC osteogenesis.

Recently, several studies have indicated that circular RNAs (circRNAs) play significant roles in various disease; however, little is known about the chronology of estrogen receptor beta (ERß) deficiency and altered circRNA expression, or their relationship with osteogenesis. Herein, we show through western-blot and quantitative real-time PCR assays, that when ERß is silenced, the expression of osteogenesis-related proteins and mRNAs were down-regulated. We then performed RNA-Seq to analyze differential circRNA expression between the control and ERß knockdown group. This analysis revealed that, 146 circRNAs were differentially expressed by fold-change≥2.0, p ≤ 0.05, and, among this group, 68 circRNAs were down-regulated, while 78 were up-regulated. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER pathway analyses were performed to predict the function of these differentially expressed circRNAs. Finally, co-expressed targets gene, and circRNA-microRNA network were constructed for predicted miRNA sponges. This research suggested that ERß may through 2:27713879|27755789/2:240822115|240867796-miR-328-5p-mRNA axis to regulate osteogenic differentiation.

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.