Reynoutria japonica consisted of emodin-8-ß-D-glucoside ameliorates Dermatophagoides farinae extract-induced atopic dermatitis-like skin inflammation in mice by inhibiting JAK/STAT signaling.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction and chronic inflammatory responses. Reynoutria japonica, known as Huzhang in traditional Chinese Medicine, can enhance blood circulation to eliminate wind pathogens and terminate coughing. Despite pharmacological evidence supporting the efficacy of R. japonica in suppressing edema-induced skin inflammation or connective tissue diseases, its pharmaceutical potential for treating AD-like skin inflammation remains unexplored. This study investigated the possible effects of R. japonica ethanol extract (RJE) on Dermatophagoides farinae extract (DfE)-induced AD-like skin inflammation in NC/Nga mice. To elucidate the underlying mechanisms by which RJE inhibits skin inflammation, we examined the effect of RJE on IFN-γ/TNF-α-induced signal transducer and activator of transcription (STAT) signaling in human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs). Our findings revealed that RJE mitigates DfE-induced AD-like symptoms and skin barrier disruptions in mouse skin lesions. Moreover, RJE attenuated DfE-induced mast cell infiltration and serum levels of inflammatory cytokines (IL-1α, IL-1ß, IL-6, IL-23, IFN-γ, TNF-α, and GM-CSF). RJE also inhibited IFN-γ/TNF-α-induced chemokine levels and STAT3 phosphorylation in HEKs and HDFs. Virtual binding analysis of the RJE components suggested that emodin-8-ß-D-glucoside binds to Janus kinase (JAK) 1/2, thereby suppressing STAT signaling, which was confirmed by Western blot analysis. In conclusion, our results suggest that RJE may alleviate DfE-induced skin barrier dysfunction by inhibiting JAK/STAT signaling and the proinflammatory immune response through the suppression of inflammatory mediators in AD-like skin disease. These findings suggest that RJE has potential as an effective therapy for AD management.

Veronica persica Ethanol Extract Ameliorates Dinitrochlorobenzene-Induced Atopic Dermatitis-like Skin Inflammation in Mice, Likely by Inducing Nrf2/HO-1 Signaling.

Atopic dermatitis (AD) is chronic allergic contact dermatitis with immune dysregulation. Veronica persica has pharmacological activity that prevents asthmatic inflammation by ameliorating inflammatory cell activation. However, the potential effects of the ethanol extract of V. persica (EEVP) on AD remain elusive. This study evaluated the activity and underlying molecular pathway of EEVP in two AD models: dinitrochlorobenzene (DNCB)-induced mice and interferon (IFN)-γ/tumor necrosis factor (TNF)-α-stimulated human HaCaT keratinocytes. EEVP attenuated the DNCB-induced increase in serum immunoglobulin E and histamine levels, mast cell counts in toluidine-blue-stained dorsal skin, inflammatory cytokine (IFN-γ, interleukin [IL]-4, IL-5, and IL-13) levels in cultured splenocytes, and the mRNA expression of IL6, IL13, IL31 receptor, CCR-3, and TNFα in dorsal tissue. Additionally, EEVP inhibited the IFN-γ/TNF-α-induced mRNA expression of IL6, IL13, and CXCL10 in HaCaT cells. Furthermore, EEVP restored the IFN-γ/TNF-α-induced downregulation of heme oxygenase (HO)-1 in HaCaT cells by inducing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. A molecular docking analysis demonstrated that EEVP components have a strong affinity to the Kelch-like ECH-associated protein 1 Kelch domain. In summary, EEVP inhibits inflammatory AD by attenuating immune cell activation and inducing the Nrf2/HO-1 signaling pathway in skin keratinocytes.

The Safety and Efficacy of 1-Monoeicosapentaenoin Isolated from the Trebouxiophyceae Micractinium on Anti-Wrinkle: A Split-Face Randomized, Double-Blind Placebo-Controlled Clinical Study.

The skin aging process is governed by intrinsic and extrinsic factors causing skin wrinkles, sagging, and loosening. The 1-monoeicosapentaenoin (1-MEST) is a component isolated from Micractinium, a genus of microalgae (Chlorophyta, Trebouxiophyceae). However, the anti-wrinkle effects of 1-MEST are not yet known. This study aimed to evaluate the anti-wrinkle effects of 1-MEST in vitro and in clinical trials. The cytotoxicity of 1-MEST was investigated in vitro using the MTS assay in human epidermal keratinocytes (HEKs). Expression of matrix metalloproteinase (MMP)-1 and MMP-9 was determined by ELISA in HEKs irradiated with UVB after treatment with 1-MEST. A split-face randomized, double-blind, placebo-controlled study was conducted to evaluate the safety and efficacy of 1-MEST. The study evaluated wrinkle parameters and visual assessment, self-efficacy and usability questionnaires, and adverse events. The study showed that the 1-MEST was not cytotoxic in HEKs, suppressed MMP-1 secretion and MMP-9 protein expression in HEKs irradiated with UVB. The wrinkle parameters and mean visual assessment score were significantly decreased in the test group after 12 weeks and differed from the control group. There were no significant differences in efficacy and usability. Adverse effects were also not observed. The 1-MEST showed anti-wrinkle properties to slow down or prevent skin aging.

Ethanol extract of Veronica persica ameliorates house dust mite-induced asthmatic inflammation by inhibiting STAT-3 and STAT-6 activation.

Veronica persica is a flowering plant belonging to the family Scrophulariaceae. Here, we aimed to evaluate the pharmacological activity of the ethanol extract of Veronica persica (EEVP) in an airway inflammation model. We examined airway responsiveness to aerosolized methacholine, serum immunoglobulin (Ig)E levels, and total cell numbers in the lung and bronchoalveolar lavage fluid (BALF). Histological analysis of the lung tissue was performed using hematoxylin-eosin, Masson trichrome, or periodic acid-Schiff staining. Fluorescence-activated cell sorting analysis in the lung and BALF was applied to clarify the changes in immune cell types. Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction were applied to investigate cytokine levels and gene expression related to airway inflammation. STAT-3/6 phosphorylation was examined in primary bronchial/tracheal epithelial cells using western blot analysis. EEVP significantly suppressed total IgE levels and methacholine-induced increase of Penh value in the HDM-challenged mouse model. EEVP also attenuated the severity of airway remodeling in lung tissues and decreased eosinophil and neutrophil infiltration in the lungs and BALF. EEVP significantly reduced the production of cytokines in BAL and splenocyte culture medium, and the expression of mRNAs related to airway inflammation in the lung tissue. EEVP suppressed IL-4/13-induced STAT-3/6 phosphorylation in the epithelial cells. We showed for the first time that EEVP effectively inhibits eosinophilic airway inflammation by suppressing the expression of inflammatory factors for T cell activation and polarization, and inhibits MCP-1 production of bronchial/tracheal epithelial cells by suppressing STAT-3/6 activation. EEVP may be a potential pharmacological agent to prevent inflammatory airway diseases.

GC-MS and LC-TOF-MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark.

Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and ß-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1ß secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and ß-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p < 0.001) TRAP activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited significant (p < 0.05) BMM cell viability. NO production was significantly inhibited (all p < 0.0001) by the sample. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and ß-sitosterol (p < 0.0001) and further, chlorogenic acid and naringenin remarkably inhibited IL-1ß production. The P. africana methanolic extract significantly inhibited RANKL-induced TRAP activity. The phytochemical study of P. africana stem bark revealed a number of chemical compounds with anti-osteoporosis activity. There was no observed hepatocyte apoptosis in the liver of zebrafish larvae. In conclusion, the stem bark of P. africana is non-toxic to the liver and its inhibition of TRAP activity makes it an important source for future anti-osteoporosis drug development.

Root Extract of a Micropropagated Prunus africana Medicinal Plant Induced Apoptosis in Human Prostate Cancer Cells (PC-3) via Caspase-3 Activation.

Prostate cancer is one of the major causes of cancer-related deaths among men globally. Medicinal plants have been explored as alternative treatment options. Herein, we assessed the in vitro cytotoxic effects of 70% ethanolic root extracts of six-month-old micropropagated Prunus africana (PIR) on PC-3 prostate cancer cells as an alternative to the traditionally used P. africana stem-bark extract (PWS) treatment. In vitro assays on PC-3 cells included annexin-V and propidium iodide staining, DAPI staining, and caspase-3 activity analysis through western blotting. PC-3 cells were exposed to PWS and PIR at different concentrations, and dose-dependent antiprostate cancer effects were observed. PC-3 cell viability was determined using CCK-8 assay, which yielded IC50 values of 52.30 and 82.40 µg/mL for PWS and PIR, respectively. Annexin-V and PI staining showed dose-dependent apoptosis of PC-3 cells. Significant (p < 0.001) percent of DAPI-stained apoptotic PC-3 cells were observed in PWS, PIR, and doxorubicin treatment compared with the negative control. PWS treatment substantially elevated cleaved caspase-3 levels in PC-3 cells compared with the PIR treatment. These results provide evidence for the antiprostate cancer potential of PIR and sets a basis for further research to enhance future utilization of roots of young micropropagated P. africana for prostate cancer treatment as an alternative to stem bark. Moreover, micropropagation approach may help provide the required raw materials and hence reduce the demand for P. africana from endangered wild population.

Serum Starvation Sensitizes Anticancer Effect of Anemarrhena asphodeloides via p38/JNK-Induced Cell Cycle Arrest and Apoptosis in Colorectal Cancer Cells.

The molecular mechanism underlying the anticancer effects of Anemarrhena asphodeloides (A. asphodeloides) on colon cancer is unknown. This is the first study evaluating the anticancer effect of A. asphodeloides extract (AA-Ex) in serum-starved colorectal cancer cells. Changes in cell proliferation and morphology in serum-starved MC38 and HCT116 colorectal cancer cells were investigated using MTS assay. Cell cycle and apoptosis were investigated using flow cytometry, and cell cycle regulator expression was determined using qRT-PCR. Apoptosis regulator protein levels and mitogen-activated protein kinase (MAPK) phosphorylation were assessed using western blotting. AA-Ex sensitively suppressed proliferation of serum-starved colorectal cancer cells, with MC38 and HCT116 cells showing greater changes in proliferation after treatment with AA-Ex under serum starvation than HaCaT and RAW 264.7 cells. AA-Ex inhibited cell cycle progression in serum-starved MC38 and HCT116 cells and increased the expression of cell cycle inhibitors (p53, p21, and p27). Furthermore, AA-Ex induced apoptosis in serum-starved MC38 and HCT116 cells. Consistently, AA-Ex suppressed the expression of the anti-apoptotic molecule Bcl-2 and upregulated pro-apoptotic molecules (cytochrome c, cleaved caspase-9, cleaved caspase-3, and cleaved-PARP) in serum-starved cells. AA-Ex treatment under serum starvation decreased AKT and ERK1/2 phosphorylation in the cell survival signaling pathway but increased p38 and JNK phosphorylation. Furthermore, AA-Ex treatment with serum starvation increased the levels of the transcription factors of the p38 and JNK pathway. Serum starvation sensitizes colorectal cancer cells to the anticancer effect of A. asphodeloidesvia p38/JNK-induced cell cycle arrest and apoptosis. Hence, AA-Ex possesses therapeutic potential for colon cancer treatment.

Ethanol Extract of Amomum tsao-ko Ameliorates Ovariectomy-Induced Trabecular Loss and Fat Accumulation.

In Asia, Amomum tsao-ko has long been used as a spice or seasoning in food to stimulate digestion. In the present study, we evaluated the effects of ethanol extract of Amomum tsao-ko (EEAT) on menopausal osteoporosis and obesity. After the administration of EEAT in ovariectomy (OVX) mice models for five weeks, microcomputed tomography and a histological analysis were performed to assess, respectively, the trabecular structure and the fat accumulation in adipose, liver, and bone tissues. We also examined the effects of EEAT on a bone marrow macrophage model of osteoclastogenesis by in vitro stimulation from the receptor activator of nuclear factor-kappa Β ligand (RANKL) through real-time PCR and Western blot analysis. In addition, ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with authentic standards was applied to characterize the phytochemical profiling of EEAT. We found that EEAT significantly decreased OVX-induced body weight gain and fat accumulation, significantly prevented OVX-induced deterioration of bone mineral density and microstructure of trabecular tissues, and significantly inhibited osteoclast differentiation by downregulating NF-κB/Fos/NFATc1 signaling in osteoclasts. Furthermore, UHPLC-MS/MS identified eight beneficial phytochemicals in EEAT. Collectively, these results suggest that EEAT might be an effective nutraceutical candidate to attenuate menopausal osteoporosis by inhibiting osteoclastogenesis and to prevent obesity by suppressing fat accumulation.

Water Extract of Fritillariae thunbergii Bulbus Inhibits RANKL-Mediated Osteoclastogenesis and Ovariectomy-Induced Trabecular Bone Loss.

Fritillariae thunbergii bulbus has been widely used to treat symptoms of coughs and airway congestion in the chest due to pathological colds and damp phlegm in traditional Chinese medicine. Despite its long history of traditional use, its pharmacological activities on osteoclastogenesis and osteoporosis have not been evaluated. This study investigated the effects of the water extract of Fritillariae thunbergii bulbus (WEFT) on osteoclast differentiation in bone marrow-derived macrophage cells and on ovariectomy (OVX)-induced osteoporosis in mice. We found that WEFT significantly inhibited osteoclastogenesis by downregulating the receptor activator of the NF-κB ligand (RANKL) signaling-induced nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expression. In an OVX-induced osteoporosis model, WEFT significantly prevented the OVX-induced trabecular loss of femurs, accompanied by a reduction in fat accumulation in the bone marrow and liver. In addition, WEFT significantly prevented weight gain and gonadal fat gain without recovering uterine atrophy. Using ultrahigh-performance liquid chromatography-tandem mass spectrometry, seven alkaloids (peimisine glucoside, yibeissine, peiminoside, sipeimine-glucoside, peimisine, peimine, and peiminine) were identified in WEFT. The results of this study suggest that WEFT can be a potential pharmacological candidate to reduce menopausal osteoporosis and menopause-related symptoms, such as fat accumulation.

In Vitro Antiosteoporosis Activity and Hepatotoxicity Evaluation in Zebrafish Larvae of Bark Extracts of Prunus jamasakura Medicinal Plant.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 µg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 µM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p < 0.001) at all concentrations of P. jamasakura extracts compared to the control treatment. At 50 µg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p < 0.01) BMM cell viability compared to the control treatment. P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p < 0.01) effects on lipopolysaccharide (LPS)-induced NO production at 200 µg/ml and exhibited significant (p < 0.01) and (p < 0.05) stimulative effects, respectively, on RAW 264.7 cell viability. No overt hepatotoxicity was observed in the liver of zebrafish larvae in any of the treatments. Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Water Extract of Lysimachia christinae Inhibits Trabecular Bone Loss and Fat Accumulation in Ovariectomized Mice.

In Asia, extracts of Lysimachia christinae have been used for liver or urinogenital system-related diseases in traditional medicine. In this study, we investigated the effects of the water extract of L. christinae (WELC) on receptor activator of nuclear factor-kappa Β ligand (RANKL)-induced osteoclastic differentiation of bone marrow macrophages, and on osteoporosis and obesity in ovariectomy mice. RANK signaling pathways related to osteoclast differentiation were examined by real-time polymerase chain reaction (PCR) and western blot analysis. Additionally, we performed micro-computed tomography to assess trabecular bone loss, histological analysis for fat accumulation in adipose, liver, and bone tissues, and phytochemical profiling for WELC characterization. WELC significantly inhibited osteoclast differentiation by downregulating RANKL-induced mitogen-activated protein kinase (MAPK)/c-Fos/nuclear factor of activated T-cells (NFAT) signaling in osteoclast precursors and ovariectomy-induced trabecular loss by suppressing osteolcastic bone resorption. WELC markedly decreased ovariectomy-induced body weight gain and fat accumulation in adipose, liver, and bone tissues. Furthermore, ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) identified 16 phytochemicals in WELC when compared with the mass fragmentation of standard chemicals. Collectively, these results suggest that WELC might possess beneficial effects on postmenopausal osteoporosis by inhibiting osteoclast differentiation and obesity by suppressing fat accumulation.

Beneficial Effects of Insect Extracts on Nonalcoholic Fatty Liver Disease.

It is well known that nonalcoholic fatty liver disease (NAFLD) is a common disease worldwide because of unhealthy changes in dietary habits. In this study, we determined the effects of Tenebrio molitor Linnaeus, 1758 extract (TML) and Allomyrina dichotoma Linnaeus, 1771 larvae extract (ADL) in cellular and animal models. In vitro, TML and ADL treatments did not cause cytotoxicity, but attenuated the accumulation of lipid in HepG2 cells induced by free fatty acids. In vivo, mice were orally treated with TML and ADL for 10 weeks during high-fat diet feeding. TML and ADL administration significantly reduced the weight of body, liver tissue, and adipose tissue. Serum lipid profiles, hepatic functional parameters, and glucose levels were ameliorated by TML and ADL. Moreover, TML and ADL suppressed increased lipogenesis and inflammation-related makers, and improved antioxidant enzyme activity. In liver tissue, the decreased lipid accumulation by administration of TML and ADL was observed using Oil Red O and Hematoxylin and Eosin staining. Therefore, we suggest that TML and ADL may be having a therapeutic potential and is used to develop a therapeutic agent for NAFLD.

Effects of Processed Polygonum multiflorum with KIOM Patent on Bone Remodeling-Related Protein Expression in Human Osteoblast-Like SaOS-2 Cells.

This present study evaluated the effects of processed P. multiflorum on osteogenesis using Sarcoma osteogenic (SaOS-2) cell lines and osteoclastogenesis of bone marrow-derived macrophage cells (BMM) and to elucidate differences in effect on the expression of bone-related proteins between commercially sold P. multiflorum and patented, in vitro-propagated Korea Institute of Oriental Medicine (KIOM) P. multiflorum. Raw P. multiflorum and P. multiflorum that were stir-baked and steamed in black bean juice were compared, and western blotting analysis was performed to investigate the expression of bone remodeling-related proteins in SaOS-2 cells. In the cells treated with P. multiflorum steamed in black bean juice, the expression of RANKL was decreased, whereas that of osteoprotegerin, alkaline phosphatase, Runx2, and osterix was increased. Owing to these results, we conclude that processed P. multiflorum can be used as an alternative treatment for bone diseases such as osteoporosis, osteopenia, periodontitis, and Paget's disease.

Pine needles attenuate receptor activator for nuclear factor-B ligand (RANKL)-induced trabecular bone loss by inhibiting osteoclast differentiation.

BACKGROUND: The leaf of Pinus densiflora known as pine needles has been used to treat vascular disease, gastrointestinal diseases, and urinary diseases in traditional medicine. We evaluated anti-osteoporotic effect of water extract of Pinus densiflora (WEPN) on acute bone loss and osteoclastogenesis induced by receptor activator for nuclear factor-κB ligand (RANKL). METHODS: After oral administration of WEPN (0.25 g/kg) for 5 days, femora were collected, and bone parameter [trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), trabecular number (Tb. N), and bone mineral density (BMD)] were analyzed by micro-CT analysis. Anti-osteoclastic effect of WEPN was examined using tartrate-resistant acid phosphatase activity and activation of RANKL signaling pathway. RESULTS: We found that WEPN significantly attenuated RANKL-induced decrease of BV/TV, Tb.Th., Tb.N, and BMD but increase of Tb. Sp in femora. WEPN dose-dependently decreased osteoclastogenesis accompanied by inhibiting the activation of RANKL signaling components (JNK, p38, and p65) and mRNA expression level of osteoclast specific genes (NFATc1, c-Fos, TRAP, cathepsin K, DC-STAMP, and carbonic anhydrate). CONCLUSION: WEPN inhibition on osteoclastogenesis could contribute to attenuate RANKL-induced trabecular bone loss in vivo. Therefore, it might suggest that WEPN could be prescribed in traditional medicine or used in health functional food to prevent or treat osteoporotic bone diseases.

Water extract of Rumex crispus prevents bone loss by inhibiting osteoclastogenesis and inducing osteoblast mineralization.

BACKGROUND: Rumex crispus root has traditionally been used in Asian medicine for the treatment of hemorrhage and dermatolosis. The aim of this study was to explore the pharmaceutical effects of water extract of Rumex crispus (WERC) on osteoblast and osteoclast differentiation. We also studied the effect of WERC on the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced trabecular bone destruction mice model. METHODS: High performance liquid chromatography analysis was used to identify three compounds (emodin, chrysophanol, and physcion) of WERC. The in vivo effect of WERC was examined using an administration of WERC or vehicle on the ICR mice with bone loss induced by intraperitoneal RANKL injection on day 0 and 1. All mice were sacrificed by cervical dislocation at day 7 and the femurs of mice were isolated for soft X-ray and Micro-CT analysis. The in vitro effect of WERC on osteoblast mineralization or osteoclast differentiation was examined by alizarin red S staining or by tartrate-resistant acid phosphatase staining and assay. To determine the transcription level of osteoblast or osteoclast-specific genes, real-time quantitative polymerase chain reaction was used. Western blot analysis was performed to study the effect of WERC on mitogen-activated protein kinases (MAPK) or nuclear factor-κB (NF-κB) signaling molecules. RESULTS: The presence of three compounds in WERC was determined. WERC significantly suppressed RANKL-induced trabecular bone loss by preventing microstructural deterioration. In vitro, WERC increased osteoblast mineralization by enhancing the transcription of runt-related transcription factor 2 and its transcriptional coactivators, and by stimulating extracellular signal-regulated kinase phosphorylation. Furthermore, WERC significantly inhibited osteoclast differentiation by suppressing the activation of the RANKL signalings (MAPK and NF-κB) and the increasing inhibitory factors of nuclear factor of activated T cells cytoplasmic 1. CONCLUSION: This study showed that WERC could protect against osteoporosis and suggested that the possible mechanism of WERC might be related to increased osteoblast differentiation by activating Runx2 signaling and inhibition of osteoclast differentiation by suppression of RANKL signaling.

Water extract of Uncaria sinensis suppresses RANKL-induced bone loss by attenuating osteoclast differentiation and bone resorption.

BACKGROUND: The hooks and stems of Uncaria sinensis have been used to mitigate cardiovascular and central nervous system disorders in Asia traditional medicine. Regulation of osteoclast differentiation and activity is a major target for preventing and treating pathological bone diseases. METHODS: Tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-stained multinucleated cells were used to examine receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. The activation of RANKL-induced signaling pathways and the expression of transcription factors were investigated by western blot analysis and quantitative real-time polymerase chain reaction. The bone resorption activity of osteoclast was studied using a plate coated with hydroxyl-apatite. Trabecular bone destruction was investigated using a RANKL-induced trabecular bone loss mouse model. RESULTS: We found that water extract of the hooks and stems of U. sinensis (WEUS) inhibits RANKL-induced differentiation of murine bone marrow macrophages and RAW264.7 cells into osteoclasts. WEUS inhibited the activation of NF-κB and the expression of nuclear factor of activated T-cells, cytoplasmic 1. In addition, WEUS suppressed the bone resorbing activity of mature osteoclasts without affecting their survival. Furthermore, oral administration of WEUS suppressed RANKL-induced bone loss with a significant amelioration of trabecular bone micro-structures. WEUS also reduced RANKL-induced increase in serum TRAP5b activity and C-terminal cross-linked telopeptide of type I collagen levels. CONCLUSION: The present study demonstrates that WEUS has a pharmacological activity that inhibits osteoclast-mediated bone destruction by suppressing osteoclast differentiation and function. These results suggest that U. sinensis could be a promising herbal candidate for preventing and treating bone diseases such as osteoporosis.

Artemisia capillaris Alleviates Bone Loss by Stimulating Osteoblast Mineralization and Suppressing Osteoclast Differentiation and Bone Resorption.

Artemisia capillaris has been used to treat jaundice and relieve high liver-heat in traditional medicine. In this study, we found that the administration of a water extract from A. capillaris (WEAC) to the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced bone loss model significantly prevents osteoporotic bone loss, increasing bone volume/trabecular volume by 22% and trabecular number by 24%, and decreasing trabecular separation by 29%. WEAC stimulated in vitro osteoblast mineralization from primary osteoblasts in association with increasing expression of osterix, nuclear factor of activated T cells cytoplasmic 1, and activator protein-1, as well as phosphorylation of extracellular signal-regulated kinase. In contrast to the anabolic effect of WEAC, WEAC significantly suppressed in vitro osteoclast formation from bone marrow macrophages by inhibiting the RANKL signaling pathways and bone resorption by downregulating the expression of resorption markers. Therefore, this study demonstrated that WEAC has a beneficial effect on bone loss through the regulation of osteoblast mineralization, as well as osteoclast formation and bone resorption. These results suggest that A. capillaris may be a promising herbal candidate for therapeutic agents to treat or prevent osteoporotic bone diseases.

Alpinia officinarum Stimulates Osteoblast Mineralization and Inhibits Osteoclast Differentiation.

Alpinia officinarum rhizome has been used as a traditional herbal remedy to treat inflammatory and internal diseases. Based on the previously observed inhibitory effect of A. officinarum rhizome in an arthritis model, we evaluated whether a water extract of A. officinarum rhizome (WEAO) would enhance in vitro osteoblast mineralization using calvarial osteoblast precursor cells or would inhibit in vitro osteoclast differentiation and bone resorption using bone marrow derived macrophages. In osteoblasts, WEAO enhanced the mRNA levels of transcription factor (runt-related transcription factor 2, smad1, smad5, and junB) and marker (bone morphogenetic protein-2, collagen type 1alpha1, and osteocalcin) genes related to osteoblast mineralization, consistent with increased alizarin red S staining intensity. WEAO markedly inhibited osteoclast differentiation by suppressing the receptor activator for nuclear factor-[Formula: see text]B ligand-induced downregulation of inhibitor of DNA binding 2 and V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and the phosphorylation of c-Jun N-terminal kinase, p38, nuclear factor-[Formula: see text]B, c-Src, and Bruton's tyrosine kinase to induce nuclear factor of activated T cells cytoplasmic 1 expression. WEAO also suppressed the resorbing activity of mature osteoclasts by altering actin ring formation. Therefore, the results of this study demonstrate that WEAO stimulates osteoblast mineralization and inhibits osteoclast differentiation. Thus, WEAO may be a promising herbal candidate to treat or prevent pathological bone diseases by regulating the balance between osteoclast and osteoblast activity.

A water extract of Malva verticillata seeds suppresses osteoclastogenesis and bone resorption stimulated by RANK ligand.

BACKGROUND: Malva verticillata seeds are used as a therapeutic medicine to treat kidney dysfunction in traditional Chinese medicine (TCM). TCM has suggested that herbal medicine tonifying kidney function may have beneficial effect on bone metabolism. METHODS: Osteoclastogenesis was examined in bone marrow macrophages by measuring tartrate-resistant acid phosphatase (TRAP) activity and counting the number of TRAP-stained multinuclear cells. The activation of receptor activator of nuclear factor-kB (RANK) ligand signaling, and the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) were investigated by western blot analysis. Transcription factor and bone resorption marker mRNA levels were evaluated using real-time quantitative polymerase chain reaction. The bone resorption activity of mature osteoclast was examined in osteoclasts cultured on a hydroxyapatite-coated culture plate. RESULTS: A water extract of M. verticillata seeds (WEMV) inhibited osteoclastogenesis stimulated by RANKL. WEMV also strongly inhibited expression of c-Fos and NFATc1 as well as phosphorylation of c-Jun N-terminal kinase, p38, I-kBα, and phospholipase γ2. Furthermore, WEMV significantly attenuated osteoclast resorption activity and downregulated mRNA expression of resorption markers. CONCLUSION: These results demonstrate that WEMV inhibits osteoclastogenesis and bone resorption by suppressing the RANKL signaling pathway and suggest that M. verticillata seeds may be used as a therapeutic candidate in complementary alternative medicine to treat pathological bone diseases.

Orostachys japonicus Suppresses Osteoclast Differentiation by Inhibiting NFATc1 Expression.

The herb Orostachys japonicus has been traditionally used to treat chronic diseases, such as hepatitis, hemorrhoids, and cancer, in Asia. In this study, we investigated the effect of Orostachys japonicus water extract (OJWE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone loss. We found that OJWE inhibited RANKL-induced osteoclast differentiation in a dose-dependent manner without affecting bone resorption in bone marrow-derived macrophage cells. Interestingly, OJWE significantly reduced serum levels of C-terminal telopeptide of type 1 collagen and tartrate-resistant acid phosphatase (TRAP) 5b, markers of bone resorption and osteoclast number, respectively, in an animal model of bone loss. Furthermore, OJWE suppressed the RANKL-induced up-regulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) expression, and activation of the p38 signaling pathway, but prevented the RANKL-mediated down-regulation of interferon regulatory factor-8 (IRF-8), which is known to be an anti-osteoclastogenic factor that represses NFATc1 expression. We also identified gallic acid and quercetin-3-O-ß-D-glucoside as the OJWE components that inhibit RANKL-induced osteoclast differentiation. These results suggest that OJWE inhibits osteoclast differentiation by inhibiting RANKL-induced NFATc1 expression, which prevents osteoclast differentiation and bone loss. The present study elucidated a mechanism of action underlying the inhibitory effect of OJWE on osteoclast differentiation. Our findings suggest that O. japonicus has therapeutic potential for use in the treatment of bone diseases.