Suppression of bone resorption by Mori Radicis Cortex through NFATc1 and c-Fos signaling-mediated inhibition of osteoclast differentiation.

BACKGROUND: Mori Radicis Cortex (MRC) is the root bark of the mulberry family as Morus alba L. In Korea, it is known as "Sangbaegpi". While MRC has demonstrated anti-inflammatory and antioxidant effects, its specific mechanisms of action and impact on osteoporosis remain poorly understood. METHODS: To investigate the anti-osteoporosis effect of MRC, we examined the level of osteoclast differentiation inhibition in receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced-RAW 264.7 cells and animal models of ovariectomy (OVX) with MRC. Serum analysis in OVX animals was investigated by enzyme-linked immunosorbent assay (ELISA), and bone density analysis was confirmed by micro-computed tomography (micro-CT). The expression analysis of NFATc1 was confirmed by immunohistochemistry (IHC) in femur tissue. In addition, osteoclast differentiation inhibition was measured using tartrate-resistant acid phosphatase (TRAP). mRNA analysis was performed using reverse transcription polymerase chain reaction (RT-PCR), and the protein expression analysis was investigated by western blot. RESULTS: Micro-CT analysis showed that MRC effectively inhibited bone loss in the OVX-induced rat model. MRC also inhibited the expression of alkaline phosphatase (ALP) and TRAP in serum. Histological analysis showed that MRC treatment increased bone density and IHC analysis showed that MRC significantly inhibited the expression of NFATc1. In RANKL-induced-RAW 264.7 cells, MRC significantly reduced TRAP activity and actin ring formation. In addition, MRC significantly inhibited the expression of nuclear factor of activated T cells 1 (NFATc1)/ and c-Fos, and suppressed the mRNA expression. CONCLUSION: Based on micro-CT, serum and histological analysis, MRC effectively inhibited bone loss in an OVX-induced rat model. In addition, MRC treatment suppressed the expression of osteoclast differentiation, fusion, and bone resorption markers through inhibition of NFATc1/c-Fos expression in RANKL-induced RAW 264.7 cells, ultimately resulting in a decrease in osteoclast activity. These results demonstrate that MRC is effective in preventing bone loss through inhibiting osteoclast differentiation and activity.

Ursodeoxycholic acid alleviates atopic dermatitis-associated inflammatory responses in HaCaT and RBL-2H3 cells and DNCB/DFE-treated mice.

AIMS: Ursodeoxycholic acid (UDCA) is a hydrophilic dihydroxy bile acid used for cholestatic liver disease and exhibits antioxidant, antitumor, and anti-inflammatory effects. However, its potential effects on atopic dermatitis (AD) have not been elucidated. This study aimed to evaluate the efficacy of UDCA in inhibiting the inflammatory response and alleviating lesions in AD-like mice. MAIN METHODS: To investigate the efficacy of UDCA in AD-like inflammatory responses, tumor necrosis factor-alpha (TNF-α)- and interferon-gamma (IFN-γ)-stimulated HaCaT cells and anti-dinitrophenyl immunoglobulin E (DNP-IgE)- and human serum albumin (HSA)-stimulated RBL-2H3 cells were used to investigate the levels of inflammatory factors and their mechanisms. AD-like lesions were induced by applying DNCB/DFE to mice. The effect of UDCA administration in AD-like mice was analyzed by assessing organ weight, serum IgE and inflammatory cytokine levels, and histopathological changes using immunohistochemical and immunofluorescent staining. KEY FINDINGS: In HaCaT cells, UDCA significantly diminished TARC, MDC, MCP-1, and IL-6 expression by inhibiting the phosphorylation of nuclear NF-κB and cytoplasmic IκB, and also increased the levels of skin barrier protein. In RBL-2H3 cells, UDCA reduced ß-hexosaminidase and IL-4 levels. In AD-like mice, UDCA suppressed organ hypertrophy, ear edema, SCORAD index, DFE-specific IgE levels, inflammatory cytokine levels, skin hypertrophy, mast cell invasion, skin barrier loss, and thymic stromal lymphopoietin-positive areas. SIGNIFICANCE: UDCA suppressed the expression of pro-inflammatory cytokines by keratinocytes and mast cells. It also alleviated atopy by suppressing symptoms without organ toxicity in AD-like mice. UDCA may be an effective and safe treatment for AD.

Exploring the Anti-Osteoporotic Potential of Daucosterol: Impact on Osteoclast and Osteoblast Activities.

Osteoporosis is a debilitating condition characterized by reduced bone mass and density, leading to compromised structural integrity of the bones. While conventional treatments, such as bisphosphonates and selective estrogen receptor modulators (SERMs), have been employed to mitigate bone loss, their effectiveness is often compromised by a spectrum of adverse side effects, ranging from gastrointestinal discomfort and musculoskeletal pain to more severe concerns like atypical fractures and hormonal imbalances. Daucosterol (DC), a natural compound derived from various plant sources, has recently garnered considerable attention in the field of pharmacology. In this study, we investigated the anti-osteoporosis potential of DC by characterizing its role in osteoclasts, osteoblasts, and lipopolysaccharide (LPS)-induced osteoporosis. The inhibitory effect of DC on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation by fluorescent staining, and bone resorption by pit formation assay. In addition, the calcification nodule deposition effect of osteoblasts was determined by Alizarin red S staining. The effective mechanisms of both cells were verified by Western blot and reverse transcription polymerase chain reaction (RT-PCR). To confirm the effect of DC in vivo, DC was administered to a model of osteoporosis by intraperitoneal administration of LPS. The anti-osteoporosis effect was then characterized by micro-CT and serum analysis. The results showed that DC effectively inhibited osteoclast differentiation at an early stage, promoted osteoblast activity, and inhibited LPS-induced bone density loss. The results of this study suggest that DC can treat osteoporosis through osteoclast and osteoblast regulation, and therefore may be considered as a new therapeutic alternative for osteoporosis patients in the future.

Dolichos Lablab Linné Inhibits Bone Density Loss and Promotes Bone Union in Senile Osteoporosis through Osteogenesis.

As populations continue to age, osteoporosis has emerged as an increasingly critical concern. Most advancements in osteoporosis treatment are predominantly directed toward addressing abnormal osteoclast activity associated with menopause, with limited progress in developing therapies that enhance osteoblast activity, particularly in the context of aging and fractures, and serious side effects associated with existing treatments have highlighted the necessity for natural-product-based treatments targeting senile osteoporosis and fractures. Dolichos lablab Linné (DL) is a natural product traditionally used for gastrointestinal disorders, and its potential role in addressing bone diseases has not been extensively studied. In this research, we investigated the anti-osteoporosis and bone-union-stimulating effects of DL using the SAMP6 model, a naturally aged mouse model. Additionally, we employed MC3T3-E1 cells to validate DL's osteoblast-promoting effect and to assess the involvement of core mechanisms such as the BMP-2/Smad and Wnt/ß-catenin pathways. The experimental results revealed that DL promoted the formation of osteoblasts and calcified nodules by upregulating both the BMP-2/Smad and Wnt/ß-catenin mechanisms. Based on its observed effects, DL demonstrated the potential to enhance bone mineral density in aged osteoporotic mice and promote bone union in fractured mice. These findings indicate the promising therapeutic potential of DL for the treatment of osteoporosis and bone-related conditions, thus warranting further investigation and potential clinical applications.

A study on specific factors related to inflammation and autophagy in BEAS-2B cells induced by urban particulate matter (PM, 1648a) and histological evaluation of PM-induced bronchial asthma model in mice.

As particulate matter (PM) poses an increasing risk, research on its correlation with diseases is active. However, researchers often use their own PM, making it difficult to determine its components. To address this, we investigated the effects of PM with known constituents on BEAS-2B cells, examining cytokine levels, reactive oxygen species ROS production, DNA damage, and MAPK phosphorylation. Additionally, we evaluated the effects of PM on normal and OVA-induced asthmatic mice by measuring organ weight, cytokine levels, and inflammatory cells in bronchoalveolar lavage fluid, and examining histological changes. PM markedly increased levels of IL-6, GM-CSF, TNF-α, ROS, nitric oxide, and DNA damage, while surprisingly reducing IL-8 and MCP-1. Moreover, PM increased MAPK phosphorylation and inhibited mTOR and AKT phosphorylation. In vivo, lung and spleen weights, IgE, OVA-specific IgE, IL-4, IL-13, total cells, macrophages, lymphocytes, mucus generation, and LC3II were higher in the asthma group. PM treatment in asthmatic mice increased lung weight and macrophage infiltration, but decreased IL-4 and IL-13 in BALF. Meanwhile, PM treatment in the Nor group increased total cells, macrophages, lymphocytes, and mucus generation. Our study suggests that PM may induce and exacerbate lung disease by causing immune imbalance via the MAPK and autophagy pathways, resulting in decreased lung function due to increased smooth muscle thickness and mucus generation.

Gleditsiae fructus regulates osteoclastogenesis by inhibiting the c­Fos/NFATc1 pathway and alleviating bone loss in an ovariectomy model.

Medical and economic developments have allowed the human lifespan to extend and, as a result, the elderly population has increased worldwide. Osteoporosis is a common geriatric disease that has no symptoms and even a small impact can cause fractures in patients, leading to a serious deterioration in the quality of life. Osteoporosis treatment typically involves bisphosphonates and selective estrogen receptor modulators. However, these treatments are known to cause severe side effects, such as mandibular osteonecrosis and breast cancer, if used for an extended period of time. Therefore, it is essential to develop therapeutic agents from natural products that have fewer side effects. Gleditsiae fructus (GF) is a dried or immature fruit of Gleditsia sinensis Lam. and is composed of various triterpenoid saponins. The anti­inflammatory effect of GF has been confirmed in various diseases, and since the anti­inflammatory effect plays a major role in inhibiting osteoclast differentiation, GF was expected to be effective in osteoclast differentiation and menopausal osteoporosis; however, to the best of our knowledge, it has not yet been studied. Therefore, the present study was designed to examine the effect of GF on osteoclastogenesis and to investigate the mechanism underlying inhibition of osteoclast differentiation. The effects of GF on osteoclastogenesis were determined in vitro by tartrate­resistant acid phosphatase (TRAP) staining, pit formation assays, filamentous actin (F­actin) ring formation assays, western blotting and reverse transcription­quantitative PCR analyses. Furthermore, the administration of GF to an animal model exhibiting menopausal osteoporosis allowed for the analysis of alterations in the bone microstructure of the femur using micro­CT. Additionally, assessments of femoral tissue and serum were conducted. The present study revealed that the administration of GF resulted in a reduction in osteoclast levels, F­actin rings, TRAP activity and pit area. Furthermore, GF showed a dose­dependent suppression of nuclear factor of activated T­cells cytoplasmic, c­Fos and other osteoclastogenesis­related markers.

Persicae Semen Promotes Bone Union in Rat Fractures by Stimulating Osteoblastogenesis through BMP-2 and Wnt Signaling.

Fractures cause extreme pain to patients and impair movement, thereby significantly reducing their quality of life. However, in fracture patients, movement of the fracture site is restricted through application of a cast, and they are reliant on conservative treatment through calcium intake. Persicae semen (PS) is the dried mature seeds of Prunus persica (L.) Batsch, and in this study the effects of PS on osteoblast differentiation and bone union promotion were investigated. The osteoblast-differentiation-promoting effect of PS was investigated through alizarin red S and Von Kossa staining, and the regulatory role of PS on BMP-2 (Bmp2) and Wnt (Wnt10b) signaling, representing a key mechanism, was demonstrated at the protein and mRNA levels. In addition, the bone-union-promoting effect of PS was investigated in rats with fractured femurs. The results of the cell experiments showed that PS promotes mineralization and upregulates RUNX2 through BMP-2 and Wnt signaling. PS induced the expression of various osteoblast genes, including Alpl, Bglap, and Ibsp. The results of animal experiments show that the PS group had improved bone union and upregulated expression of osteogenic genes. Overall, the results of this study suggest that PS can promote fracture recovery by upregulating osteoblast differentiation and bone formation, and thus can be considered a new therapeutic alternative for fracture patients.

Effect of Taraxaci Herba on Bone Loss in an OVX-Induced Model through the Regulation of Osteoclast Differentiation.

Bone is a dynamic tissue that maintains homeostasis with a balance of osteoclasts for bone resorption and osteoblasts for bone formation. Women are deficient in estrogen after menopause, which promotes bone resorption due to excessive activity of osteoclasts, leading to osteoporosis. TH (also known as dandelion) is native to warm regions and has traditionally been used to treat gynecological diseases and inflammation. Menopause is a major cause of osteoporosis as it causes abnormal activity of osteoclasts, and various studies have shown that anti-inflammatory drugs have the potential to treat osteoporosis. We analyzed the effect of TH on osteoclast differentiation and the relevant mechanisms using RANKL. After administration of TH in a menopause-like rat model in which ovariectomy of the was rats carried out, changes in bone microstructure were analyzed via micro-CT, and the antiosteoporosis effect of TH was verified by a histological analysis. In addition, the pharmacological effects of TH in an animal model of osteoporosis were compared and analyzed with osteoporosis medications (17ß-estradiol (E2) and alendronate (ALN)). TH significantly inhibited the initial osteoclast differentiation via the NFATc1/c-Fos mechanism. In addition, bone density in the femur of osteoporotic rats was increased, and the expression of osteoclast-related factors in the serum and tissues was controlled. The results of this study provide objective evidence of the inhibitory effect of TH on osteoclastogenesis and OVX-induced bone loss.

Dendrobium nobile Lindley Administration Attenuates Atopic Dermatitis-like Lesions by Modulating Immune Cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that can significantly affect daily life by causing sleep disturbance due to extreme itching. In addition, if the symptoms of AD are severe, it can cause mental disorders such as ADHD and suicidal ideation. Corticosteroid preparations used for general treatment have good effects, but their use is limited due to side effects. Therefore, it is essential to minimize the side effects and study effective treatment methods. Dendrobium nobile Lindley (DNL) has been widely used for various diseases, but to the best of our knowledge, its effect on AD has not yet been proven. In this study, the inhibitory effect of DNL on AD was confirmed in a DNCB-induced Balb/c mouse. In addition, the inhibitory efficacy of inflammatory cytokines in TNF-α/IFN-γ-induced HaCaT cells and PMACI-induced HMC-1 cells was confirmed. The results demonstrated that DNL decreased IgE, IL-6, IL-4, scratching behavior, SCORAD index, infiltration of mast cells and eosinophils and decreased the thickness of the skin. Additionally, DNL inhibited the expression of cytokines and inhibited the MAPK and NF-κB signaling pathways. This suggests that DNL inhibits cytokine expression, protein signaling pathway, and immune cells, thereby improving AD symptoms in mice.

Lycopus lucidus Turcz ameliorates DNCB­induced atopic dermatitis in BALB/c mice.

Atopic dermatitis (AD) is a chronic inflammatory allergic skin disease, characterized by pruritic and eczematous skin lesions. Lycopus lucidus Turcz (LLT) is a perennial herb that has been reported to have various biological properties, including effects on blood circulation, as well as anti­inflammatory, antioxidant, anti­vascular inflammation and wound­healing effects. However, whether LLT improves dermatitis and the underlying mechanisms has yet to be determined. The aim of the present study was to determine whether LLT can improve 2,4­dinitrochlorobenzene (DNCB)­induced dermatitis and to verify the inhibitory effect of LLT on the expression of chemokines and pro­inflammatory cytokines in the HaCaT immortalized keratinocyte cell line. In addition, the anti­inflammatory function of LLT in RAW264.7 mouse macrophages was investigated. In the DNCB­induced AD mouse model, LLT inhibited infiltration by mast cells, eosinophils and CD8+ cells in the dorsal skin tissue of AD mice, and suppressed the expression of IgE and IL­6 in serum. In addition, LLT inhibited the phosphorylation of ERK and JNK, as well as NF­κB in skin tissue. In the HaCaT cell model induced by TNF­α/IFN­Î³, LLT inhibited the expression of thymus and activation­regulated chemokine, granulocyte­macrophage colony­stimulating factor, monocyte chemoattractant protein­1, TNF­α and IL­1ß, whilst inhibiting the phosphorylation of NF­κB. In addition, in the lipopolysaccharide­induced RAW 264.7 cell inflammation model, LLT inhibited the expression of TNF­α and IFN­Î³, the nuclear translocation of NF­κB and the phosphorylation of ERK and JNK. These results suggested that LLT may be a promising candidate for the treatment of inflammatory dermatitis.

Albiflorin Promotes Osteoblast Differentiation and Healing of Rat Femoral Fractures Through Enhancing BMP-2/Smad and Wnt/ß-Catenin Signaling.

Fracture healing is related to osteogenic differentiation and mineralization. Recently, due to the unwanted side effects and clinical limitations of existing treatments, various natural product-based chemical studies have been actively conducted. Albiflorin is a major ingredient in Paeonia lactiflora, and this study investigated its ability to promote osteogenic differentiation and fracture healing. To demonstrate the effects of albiflorin on osteoblast differentiation and calcified nodules, alizarin red S staining and von Kossa staining were used in MC3T3-E1 cells. In addition, BMP-2/Smad and Wnt/ß-catenin mechanisms known as osteoblast differentiation mechanisms were analyzed through RT-PCR and western blot. To investigate the effects of albiflorin on fracture healing, fractures were induced using a chainsaw in the femur of Sprague Dawley rats, and then albiflorin was intraperitoneally administered. After 1, 2, and 3 weeks, bone microstructure was analyzed using micro-CT. In addition, histological analysis was performed by staining the fractured tissue, and the expression of osteogenic markers in serum was measured. The results demonstrated that albiflorin promoted osteoblastogenesis and the expression of RUNX2 by activating BMP-2/Smad and Wnt/ß-catenin signaling in MC3T3-E1 cells. In addition, albiflorin upregulated the expression of various osteogenic genes, such as alkaline phosphatase, OCN, bone sialoprotein, OPN, and OSN. In the femur fracture model, micro-CT analysis showed that albiflorin played a positive role in the formation of callus in the early stage of fracture recovery, and histological examination proved to induce the expression of osteogenic genes in femur tissue. In addition, the expression of bone-related genes in serum was also increased. This suggests that albiflorin promotes osteogenesis, bone calcification and bone formation, thereby promoting the healing of fractures in rats.

Effects of chloroform fraction of Fritillariae Thunbergii Bulbus on atopic symptoms in a DNCB-induced atopic dermatitis-like skin lesion model and in vitro models.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae thunbergii Bulbus (FT), knowns as "Jeolpaemo ()" in Korean traditional medicine, is a perennial plant belonging to the Liliaceae family and has been used to treat symptoms such as cough, sputum formation, and purulent pneumonia. Owing to its effects of lowering heat, removing sputum, and reducing swelling, the plant has also been used as an external prescription medicine to treat inflammation. AIM OF THE STUDY: To analyze the anti-inflammatory effects of FT-ethanol extract (FT-Et) and FT-chloroform fraction extract (FT-Cl) on 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD) in vivo and in vitro. MATERIALS AND METHODS: The effect of FT-Et and FT-Cl on AD was observed using an AD-like skin lesion model induced by DNCB in vivo. HaCaT and RBL2H3 cells were used to determine the effects of FT-Et and FT-Cl in vitro. After inducing AD-like skin lesions in vivo, FT was topically applied to the skin lesion for 35 days. Epidermal thickness, dermal thickness, scratching behavior, infiltration of inflammatory cells, and expression of skin barrier proteins were measured. TARC, MDC, and IL-4 levels were analyzed using ELISA in HaCaT cells. Beta-hexosaminidase and IL-4 levels were measured in RBL2H3 cells. The expression of filaggrin (FLG), loricrin (LOR), involucrin (INV), and aquaporin-3(AQP-3) was measured by PCR. Phosphorylation of MAPKs was analyzed using Western blot technique. RESULTS: FT-Cl significantly reduced ear swelling, scratching behavior, SCORAD index, epidermal thickness, infiltration of inflammatory cells, and loss of skin barrier proteins. FT-Et inhibited the infiltration of mast cells and CD8+ cells and decreased the loss of skin barrier proteins. In TNF-α/IFN-γ-stimulated HaCaT cells, FT-Cl inhibited TRAC, MDC, and IL-4 expression and upregulated the expression of FLG, INV, and AQP-3, whereas FT-Et inhibited the expression of TRAC and MDC and increased the expression of FLG, INV, and AQP-3 at high concentrations. In RBL2H3, FT-Cl downregulated ß-hexosaminidase and IL-4 expression. In addition, FT-Cl inhibited the phosphorylation of ERK and p-38 in HaCaT and RBL2H3 cells. CONCLUSIONS: Collectively, FT-Cl showed better effect than FT-Et in vivo and in vitro. These results suggest that a specific component present in FT-Cl acted against AD. Future research should focus on the analysis of components contained in FT-Cl and the anti-inflammatory effects of the active ingredient.

Lycii radicis cortex inhibits glucocorticoid­induced bone loss by downregulating Runx2 and BMP­2 expression.

Lycii radicis cortex (LRC) has been used to regulate high blood pressure, body temperature, pain and bone disorders in East Asia. Glucocorticoids (GCs), also known as steroids, are potent immunity regulators widely used in the treatment of inflammatory diseases. However, despite their effectiveness, GC usage is strictly controlled due to severe side­effects, such as osteoporosis. However, further research is required as to date, at least to the best of our knowledge, there is no appropriate model to overcome secondary osteoporosis as a side­effect of GC use. Thus, the aim of the present study was to establish an experimental model of osteoporosis induced by GC. Furthermore, the present study aimed to establish the research methodology for medical evaluations of the effectiveness and side­effects of GCs. A secondary osteoporosis animal model was established, and the animals were divided into two groups as follows: The allergic contact dermatitis (ACD)­induced group and the non­ACD­induced group. In the ACD­induced group, a GC topical application group was compared with a GC subcutaneous injection group. The results revealed that the presence of ACD affected the induction of GC­mediated osteoporosis. Therefore, the group exhibiting induced ACD that was treated with a topical application of GC was selected for examining the side­effects of GCs. The effects of LRC on secondary osteoporosis were confirmed in vivo and in vitro. The results indicated that LRC regulated dexamethasone­induced osteoblast apoptotic markers, including caspase­6, caspase­9, X­linked inhibitor of apoptosis, apoptosis inhibitor 1 and apoptosis inhibitor 2, and increased the expression of osteoblast differentiation­related genes, such as Runt­related transcription factor 2 and bone morphogenetic protein 2 in the MC3T3E­1 cell line. LRC also significantly reduced GC­induced osteoporosis and exerted anti­inflammatory effects in vivo. In addition, LRC inhibited the reduction of calbindin­D28k in the kidney. Overall, the results of the present study suggest that the use of LRC alleviates GC­induced secondary osteoporosis.

Solanum nigrum Line inhibits osteoclast differentiation and suppresses bone mineral density reduction in the ovariectomy­induced osteoporosis model.

Bone homeostasis is maintained by osteoclasts that absorb bone and osteoblasts that form bone tissue. Menopausal osteoporosis is a disease associated with aging and hormonal changes due to menopause causing abnormal activation of osteoclasts, resulting in a decrease in bone density. Existing treatments for osteoporosis have been reported to have serious side effects, such as jawbone necrosis and breast and uterine cancer; therefore, their use by patients is decreasing, whilst studies focusing on alternative treatments are increasingly popular. Solanum nigrum Line (SL) has been used as a medicinal plant that possesses several pharmacological effects, such as anti­inflammatory and hepatotoxic protective effects. To the best of our knowledge, however, its effects on osteoporosis and osteoclasts have not been demonstrated previously. In the present study, the anti­osteoporotic effect of SL was investigated using a postmenopausal model of osteoporosis in which Sprague­Dawley rat ovaries were extracted. In addition, the inhibitory effects on osteoclast differentiation and function of SL was confirmed using an osteoclast model treated with receptor activator of NF­κB ligand (RANKL) on murine RAW 264.7 macrophages. In vivo experiments showed that SL reduced the decrease in bone mineral density and improved changes in the morphological index of bone microstructure, such as trabecular number and separation. In addition, the number of tartrate resistant acid phosphatase­positive cells in the femur and the expression levels of nuclear factor of activated T­cells cytoplasmic 1 (NFATc1) and cathepsin K protein were inhibited. In vitro, SL suppressed RANKL­induced osteoclast differentiation and bone resorption ability; this was mediated by NFATc1/c­Fos, a key transcription factor involved in osteoclast differentiation, ultimately inhibiting expression of various osteoclast­associated genes. These experimental results show that SL may be an alternative treatment for osteoporosis caused by abnormal activation of osteoclasts in the future.

Effects of Melandrium firmum Rohrbach on RANKL­induced osteoclast differentiation and OVX rats.

Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density (BMD), which results in an increased risk of fracture. Melandrium firmum (Siebold & Zucc.) Rohrbach (MFR), 'Wangbulryuhaeng' in Korean, is the dried aerial portion of Melandrii Herba Rohrbach, which is a member of the Caryophyllaceae family and has been used to treat several gynecological conditions as a traditional medicine. However, to the best of our knowledge, the effect of MFR on osteoclast differentiation and osteoporosis has not been assessed. To evaluate the effects of MFR on osteoclast differentiation, tartrate­resistant acid phosphatase staining, actin ring formation and bone resorption assays were used. Additionally, receptor activator of nuclear factor­κB ligand­induced expression of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) and c­Fos were measured using western blotting and reverse transcription­PCR. The expression levels of osteoclast­related genes were also examined. To further investigate the anti­osteoporotic effects of MFR in vivo, an ovariectomized (OVX) rat model of menopausal osteoporosis was established. Subsequently, the femoral head was scanned using micro­computed tomography. The results revealed that MFR suppressed osteoclast differentiation, formation and function. Specifically, MFR reduced the expression levels of osteoclast­related genes by downregulating transcription factors, such as NFATc1 and c­Fos. Consistent with the in vitro results, administration of MFR water extract to OVX rats reduced BMD loss, and reduced the expression levels of NFATc1 and cathepsin K in the femoral head. In conclusion, MFR may contribute to alleviate osteoporosis­like symptoms. These results suggested that MFR may exhibit potential for the prevention and treatment of postmenopausal osteoporosis.

The Role of Skin Mast Cells in Acupuncture Induced Analgesia in Animals: A Preclinical Systematic Review and Meta-analysis.

While mast cells (MCs) are previously well-known as a pathological indicator of pain, their role in alleviating pain is recently emerged in acupuncture research. Thus, this study systematically reviews the role of MC in acupuncture analgesia. Animal studies on MC changes associated with the acupuncture analgesia were searched in PubMed and EMBASE. The MC number, degranulation ratio and pain threshold changes were collected as outcome measures for meta-analyses. Twenty studies were included with 13 suitable for meta-analysis, most with a moderate risk of bias. A significant MC degranulation after acupuncture was indicated in the normal and was significantly higher in the pain model. In the subgroup analysis by acupuncture type, manual (MA) and electrical (EA, each P < .00001) but not sham acupuncture had significant MC degranulation. Meta-regression revealed the linear proportionality between MC degranulation and acupuncture-induced analgesia (P < .001), which was found essential in MA (P < .00001), but not in EA (P = .45). MC mediators, such as adenosine and histamine, are involved in its mechanism. Taken together, skin MC is an essential factor for acupuncture-induced analgesia, which reveals a new aspect of MC as a pain alleviator. However, its molecular mechanism requires further study. PERSPECTIVE: This systematic review synthesizes data from studies that examined the contribution of skin MC in acupuncture analgesia. Current reports suggest a new role for skin MC and its mediators in pain alleviation and explain a peripheral mechanism of acupuncture analgesia, with suggesting the need of further studies to confirm these findings.

Crataegus pinnatifida Bunge Inhibits RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells and Prevents Bone Loss in an Ovariectomized Rat Model.

Osteoporosis is characterized by a decrease in bone microarchitecture with an increased risk of fracture. Long-term use of primary treatments, such as bisphosphonates and selective estrogen receptor modulators, results in various side effects. Therefore, it is necessary to develop alternative therapeutics derived from natural products. Crataegus pinnatifida Bunge (CPB) is a dried fruit used to treat diet-induced indigestion, loss of appetite, and diarrhea. However, research into the effects of CPB on osteoclast differentiation and osteoporosis is still limited. In vitro experiments were conducted to examine the effects of CPB on RANKL-induced osteoclast differentiation in RAW 264.7 cells. Moreover, we investigated the effects of CPB on bone loss in the femoral head in an ovariectomized rat model using microcomputed tomography. In vitro, tartrate-resistant acid phosphatase (TRAP) staining results showed the number of TRAP-positive cells, and TRAP activity significantly decreased following CPB treatment. CPB also significantly decreased pit formation. Furthermore, CPB inhibited osteoclast differentiation by suppressing NFATc1, and c-Fos expression. Moreover, CPB treatment inhibited osteoclast-related genes, such as Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2. In vivo, bone mineral density and structure model index were improved by administration of CPB. In conclusion, CPB prevented osteoclast differentiation in vitro and prevented bone loss in vivo. Therefore, CPB could be a potential alternative medicine for bone diseases, such as osteoporosis.

Effect of Cone of Pinus densiflora on DNCB-Induced Allergic Contact Dermatitis-Like Skin Lesion in Balb/c Mice.

Cone of Pinus densiflora (CP), or Korean red pinecone, is a cluster of Pinus densiflora fruit. CP has also been verified in several studies to have anti-oxidation, anti-fungal, anti-bacterial, and anti-melanogenic effects. However, anti-inflammatory effects have not yet been confirmed in the inflammatory responses of pinecones to allergic contact dermatitis. The purpose of this study is to prove the anti-inflammatory effect of CP on allergic contact dermatitis (ACD) in vitro and in vivo. CP inhibited the expression of TSLP, TARC, MCP-1, TNF-α, and IL-6 in TNF-α/IFN-γ-stimulated HaCaT cells and MCP-1, GM-CSF, TNF-α, IL-6, and IL-8 in PMACI (phorbol-12-myristate-13-acetate plus A23187)-stimulated HMC-1 cells. CP inhibited the phosphorylation of mitogen-activated protein kinase (MAPKs), as well as the translocation of NF-κB on TNF-α/IFN-γ stimulated in HaCaT cells. In vivo, CP decreased major symptoms of ACD, levels of IL-6 in skin lesion, thickening of the epidermis and dermis, infiltration of eosinophils and mast cells, and the infiltration of CD4+ T cells and CD8+ T cells. This result suggests that CP represents a potential alternative medicine to ACD for diseases such as chronic skin inflammation.

Anti-inflammatory effects of Fritillaria thunbergii Miquel extracts in LPS-stimulated murine macrophage RAW 264.7 cells.

The aim of the present study was to demonstrate that Fritillaria thunbergii Miquel extract exerts anti-inflammatory and antioxidant effects on lipopolysaccharide-stimulated RAW 264.7 cells. To confirm the inhibitory effect of ethyl acetate fraction of FTM (EAFM) on inflammation, the expression of nitric oxide (NO) and inflammatory cytokines was assessed by performing ELISA. Expression of intracellular mRNA and protein was confirmed by reverse transcription PCR and western blotting. In addition, the anti-inflammatory and anti-oxidant mechanisms of NF-κB, MAPK and heme oxygenase-1 (HO-1) were also investigated. EAFM significantly inhibited the expression of inflammatory factors including NO, IL-6 and TNF-α at non-toxic concentrations. EAFM also inhibited the mRNA and protein expression of inducible nitric oxide synthase in a concentration-dependent manner, but did not alter the expression of cyclooxygenase-2. Pre-treatment with EAFM inhibited the nuclear translocation of NF-κB, and suppressed the phosphorylation of ERK and JNK. In addition, EAFM induced 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity and an increase in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. The results indicated that EAFM inhibited the expression of pro-inflammatory cytokines by inhibiting ERK/JNK phosphorylation and NF-κB translocation. EAFM also exerted antioxidant effects via Nrf2/HO-1 stimulation. Collectively, the results of the present study indicated that EAFM may be a valuable alternative for the treatment of a variety of inflammatory diseases.

Effects of Sparganii Rhizoma on Osteoclast Formation and Osteoblast Differentiation and on an OVX-Induced Bone Loss Model.

Postmenopausal osteoporosis is caused by an imbalance between osteoclasts and osteoblasts and causes severe bone loss. Osteoporotic medicines are classified into bone resorption inhibitors and bone formation promoters according to the mechanism of action. Long-term use of bisphosphonate and selective estrogen receptor modulators (SERMs) can cause severe side effects in postmenopausal osteoporosis patients. Therefore, it is important to find alternative natural products that reduce osteoclast activity and increase osteoblast formation. Sparganii Rhizoma (SR) is the dried tuberous rhizome of Sparganium stoloniferum Buchanan-Hamilton and is called "samreung" in Korea. However, to date, the effect of SR on osteoclast differentiation and the ovariectomized (OVX)-induced bone loss model has not been reported. In vitro, tartrate-resistant acid phosphatase (TRAP) staining, western blots, RT-PCR and other methods were used to examine the effect of SR on osteoclast differentiation and osteoblasts. In vivo, we confirmed the effect of SR in a model of OVX-induced postmenopausal osteoporosis. SR inhibited osteoclast differentiation and decreased the expression of TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells 1 (NFATc1) and c-Fos pathway. In addition, SR stimulates osteoblast differentiation and increased protein expression of the bone morphogenetic protein 2 (BMP-2)/SMAD signaling pathway. Moreover, SR protected against bone loss in OVX-induced rats. Our results appear to advance our knowledge of SR and successfully demonstrate its potential role as a osteoclastogenesis-inhibiting and osteogenesis-promoting herbal medicine for the treatment of postmenopausal osteoporosis.