Rat Plantar Fascia Stem/Progenitor Cells Showed Lower Expression of Ligament Markers and Higher Pro-Inflammatory Cytokines after Intensive Mechanical Loading or Interleukin-1ß Treatment In Vitro.

The pathogenesis of plantar fasciitis is unclear, which hampers the development of an effective treatment. The altered fate of plantar fascia stem/progenitor cells (PFSCs) under overuse-induced inflammation might contribute to the pathogenesis. This study aimed to isolate rat PFSCs and compared their stem cell-related properties with bone marrow stromal cells (BMSCs). The effects of inflammation and intensive mechanical loading on PFSCs' functions were also examined. We showed that plantar fascia-derived cells (PFCs) expressed common MSC surface markers and embryonic stemness markers. They expressed lower Nanog but higher Oct4 and Sox2, proliferated faster and formed more colonies compared to BMSCs. Although PFCs showed higher chondrogenic differentiation potential, they showed low osteogenic and adipogenic differentiation potential upon induction compared to BMSCs. The expression of ligament markers was higher in PFCs than in BMSCs. The isolated PFCs were hence PFSCs. Both IL-1ß and intensive mechanical loading suppressed the mRNA expression of ligament markers but increased the expression of inflammatory cytokines and matrix-degrading enzymes in PFSCs. In summary, rat PFSCs were successfully isolated. They had poor multi-lineage differentiation potential compared to BMSCs. Inflammation after overuse altered the fate and inflammatory status of PFSCs, which might lead to poor ligament differentiation of PFSCs and extracellular matrix degeneration. Rat PFSCs can be used as an in vitro model for studying the effects of intensive mechanical loading-induced inflammation on matrix degeneration and erroneous stem/progenitor cell differentiation in plantar fasciitis.

Human mast cells induce osteoclastogenesis through cell surface RANKL.

OBJECTIVES: We employed the co-culture of CD34+ stem cell-derived human mast cells (HMC) and human monocyte-derived osteoclast precursors to evaluate if mast cells contribute to the pathogenesis of osteoporosis through regulation of osteoclast proliferation and activation. METHODS: Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate. RESULTS: Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC. CONCLUSION: Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

Anti-inflammatory, antipyretic efficacy and safety of inhaled Houttuynia cordata thunb. essential oil formulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata Thunb. (H. cordata) is a well-known folk traditional Chinese medicine that is renowned for its use in the management of inflammatory respiratory diseases and pneumonia. Its essential oils have demonstrated their anti-inflammatory efficacy in vitro, however, their in vivo biological effects via inhalation have not been elucidated. AIM OF THE STUDY: This study aims to evaluate the anti-inflammation and toxicology of H. cordata essential oil-containing formulation, H16 aerosol in vivo. MATERIALS AND METHODS: A laser diffraction particle size analyser and a Next Generation Impactor were used to measure the mass median aerodynamic diameter (MMAD) of the H16 aerosol. The anti-inflammatory and antipyretic effects of the H16 aerosol were evaluated in the xylene-evoked ear oedema and Brewer's yeast-induced fever models, respectively. The biological safety of the H16 aerosol was evaluated by acute toxicity and local toxicity tests in animal models. RESULTS: Our data showed that the MMAD of the bioactive aerosol was 3-5 µm, which implied tracheal and pharyngeal deposits. Significant anti-inflammatory and antipyretic effects were also observed in the animal models treated with H16 aerosol. The maximum tolerable dose of H16 in rats was >2.5 mL/kg. Irritation was not found on respiratory tract mucosa in the local toxicity test. CONCLUSIONS: Taken together, the present study suggested that H16 could be delivered in the form of aerosol and possessed its antipyretic and anti-inflammatory effects. This study provides a new perspective for the development of a new herbal aerosol therapy and herbal modernization.

Green tea extract synergistically enhances the effectiveness of an antiresorptive drug on management of osteoporosis induced by ovariectomy in a rat model.

Antiresorptive drugs are effective for reducing bone loss in postmenopausal women, but their long-term application may be associated with adverse effects. The present study aimed to investigate the potential in vivo synergistic effects of green tea extract (GTE) and alendronate or raloxifene on the management of osteoporosis. Ovariectomized rats were fed orally with GTE, alendronate and raloxifene at different concentrations and various combinations for 4 weeks. Bone mineral density (BMD) at the lumbar spine, femur and tibia was monitored weekly using peripheral quantitative computed tomography. Bone microarchitecture in the left distal femur was analyzed using micro-CT, while serum biochemical levels were measured using ELISA kits at the end of the study. GTE alone effectively mitigated BMD loss and improved bone microarchitecture in rats. The co-administration of GTE and alendronate increased total BMD in the lumbar spine, femur and tibia. Particularly, GTE synergistically enhanced the effect of alendronate at a low dose on bone microarchitecture and decreased serum tartrate-resistant acid phosphatase. These findings imply that the dosage of certain antiresorptive agents could be reduced when they are administrated simultaneously with GTE, so that their adverse effects are minimized. The findings may be used to support the development of a new synergistic intervention between food therapy and pharmacotherapy on the management of osteoporosis in a long-term basis.

Topical application of Chinese herbal medicine DAEP relieves the osteoarthritic knee pain in rats.

BACKGROUND: The potential adverse effects of conventional oral pharmacotherapy of osteoarthritis (OA) restrict their long-term use. Topical application of a Chinese herbal paste for relieving OA knee pain can be effective and safe. However, evidence-based scientific research is insufficient to support its application worldwide. The aim of this study was to investigate the in vivo efficacy of a topical Chinese herbal paste on relieving OA knee pain and its underlying mechanism. METHODS: An OA rat model was developed by anterior cruciate ligament transection (ACLT) followed by treadmill running. A herbal paste including Dipsaci Radix, Achyranthis Bidentatae Radix, Eucommiae Cortex and Psoraleae Fructus, named as DAEP, was applied topically on the knee joint of the rats (DAEP). The rats without DAEP treatment served as Control. Rats with surgery but without ACLT, treadmill running and DAEP treatment acted as Sham. The morphologic change of the knee joint was observed radiographically. Nociception from the knee of the rats was assessed using Incapacitent test and CatWalk gait system. The therapeutic mechanism was investigated by analyzing the gene and protein expression of inflammatory markers via qPCR and Western blot, respectively. RESULTS: Radiographic images showed less destruction at the posterior tibial plateau of the DAEP group compared with the Control after 2 weeks of treatment. The static weight ratio and the gait parameters of the Control were reduced significantly via Incapacitance test and CatWalk gait analysis, respectively. DAEP treatment increased the Print Area and Maximum Intensity significantly compared with the Control. DAEP significantly suppressed the upregulation of gene expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS). CONCLUSIONS: DAEP exhibited its effect via the nuclear factor (NF)-κB pathway by suppressing the phosphorylation of IκB kinase αß (p-IKKαß) and cyclooxygenase-2 (COX-2) protein expression. This study provides scientific evidence to support the clinical application of the Chinese herbal paste on reliving OA pain.

GSP-2, a polysaccharide extracted from Ganoderma sinense, is a novel toll-like receptor 4 agonist.

Ganoderma sinense is a Chinese unique medicinal fungus that has been used in folk medicine for thousands of years. Polysaccharides are considered to be biologically active ingredients due to their immune-modulating functions. Previously we found that GSP-2, a new polysaccharide isolated from Ganoderma sinense, exerts an immunomodulatory effect in human peripheral blood mononuclear cells but the underlying mechanism is unclear. The present study aimed to investigate how GSP-2 triggers immunologic responses and the implicated signaling pathways. GSP-2 effects were investigated both in a macrophagic cell line, RAW264.7, and in primary macrophages. Moreover, the molecular basis of GSP-2 recognition by immune cells, and the consequent activation of signaling cascades, were explored by employing recombinant human HEK293-TLR-Blue clones, individually overexpressing various Toll-like receptors. GSP-2 dose-dependently induced the overexpression of Toll-like receptor 4 (TLR4) but did not affect the expression of other TLRs. Moreover, GSP-2 induced TNFα secretion in primary macrophages from wild-type, but not TLR4-knockout mice. In addition, GSP-2 upregulated TLR4 protein expression and activated the MAPK pathway in RAW246.7 macrophages. Finally, GSP-2 induced the production of the cytokines TNFα, IL1ß, and IL6. Our data demonstrated that GSP-2 was specifically recognized by TLR4, promoting cytokine secretion and immune modulation in macrophages.

Chinese topical herbal medicine gives additive effect on pharmaceutical agent on fracture healing.

OBJECTIVE: To investigate the efficacy on the combination of oral strontium ranelate (SrR) with a topical Chinese herbal paste on facilitation of fracture healing. METHODS: An open fracture was created at the mid-shaft of the right tibia of rat. A herbal paste called CDR containing Honghua (Flos Carthami), Chuanxuduan (Radix Dipsaci Asperoidis) and Dahuang (Radix Et Rhizoma Rhei Palmati) was prepared. The rats were treated with either CDR topically on the fracture site, or SrR orally, or their combinations. Bone turnover biochemical markers in serum were measured. Microarchitecture of the fracture was analyzed using micro-CT after 14 and 28 d, followed by histomorphometrical analysis. RESULTS: Micro-computed tomography analysis revealed that the combined treatment of CDR with 600 mg/g SrR significantly increased the total callus density, mineralized callus volume fraction, mineralized callus mineral content and mineralized callus density of the callus after 28 d of treatment. This result was consistent with the histomorphometrical analysis on the osteoid volume. Analysis of biochemical markers showed that the combined treatments reduced the bone resorption that occurs temporarily after fracture. CONCLUSION: This study demonstrated that the combined treatment of oral SrR and topical CDR is effective to promote fracture healing by their additive effect on promoting bone formation and retarding bone resorption.

Inhibitory effect of different Dendrobium species on LPS-induced inflammation in macrophages via suppression of MAPK pathways.

Dendrobii Caulis (DC), named 'Shihu' in Chinese, is a precious herb in traditional Chinese medicine. It is widely used to nourish stomach, enhance body fluid production, tonify "Yin" and reduce heat. More than thirty Dendrobium species are used as folk medicine. Some compounds from DC exhibit inhibitory effects on macrophage inflammation. In the present study, we compared the anti-inflammatory effects among eight Dendrobium species. The results provided evidences to support Dendrobium as folk medicine, which exerted its medicinal function partially by its inhibitory effects on inflammation. To investigate the anti-inflammatory effect of Dendrobium species, mouse macrophage cell line RAW264.7 was activated by lipopolysaccharide. The nitric oxide (NO) level was measured using Griess reagent while the pro-inflammatory cytokines were tested by ELISA. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and mitogen-activated protein kinases (MAPKs) phosphorylation were evaluated by Western blotting analysis. Among the eight Dendrobium species, both water extracts of D. thyrsiflorum B.S.Williams (DTW) and D. chrysotoxum Lindl (DCHW) showed most significant inhibitory effects on NO production in a concentration-dependent manner. DTW also significantly reduced TNF-α, MCP-1, and IL-6 production. Further investigations showed that DTW suppressed iNOS and COX-2 expression as well as ERK and JNK phosphorylation, suggesting that the inhibitory effects of DTW on LPS-induced macrophage inflammation was through the suppression of MAPK pathways. In conclusion, D. thyrsiflorum B.S.Williams was demonstrated to have potential to be used as alternative or adjuvant therapy for inflammation.

The natural agent 4-vinylphenol targets metastasis and stemness features in breast cancer stem-like cells.

BACKGROUND AND PURPOSE: Cancer stem-like cells (CSC) are regarded as the source of tumour origins, metastasis and drug resistance, and limits current treatment regimens. Previously, we reported the first study of the anti-angiogenic and anti-tumour activities of 4-vinylphenol. To further examine the therapeutic role of 4-vinylphenol, the inhibitory effects of 4-vinylphenol on cancer stemness, drug resistance and metastasis in breast cancer were investigated in the present study. STUDY DESIGN AND METHODS: We enriched parental MDA-MB-231 cells with CSCs in serum-free medium to give spheroids. The effects of 4-vinylphenol on cancer stemness, metastasis and drug resistance in CSC-enriched MDA-MB-231 cells were studied in vitro and in vivo. RESULTS: CSC-enriched MDA-MB-231 cells demonstrated higher tumorigenic and metastatic potential. 4-Vinylphenol reduced spheroid formation and ALDH1 expression in CSC-enriched cultures, revealing its inhibitory effects on the traits of CSCs. 4-Vinylphenol suppressed colony formation and cell proliferation. 4VP also inhibited in vitro invasion and in vivo metastasis in zebrafish model. Our results showed that it reduced vimentin expression, suppressed cell migration, affected the expression and/or activity of MMPs, TIMPs and uPA. In addition, the expressions of caspases 3 and 9 were increased upon its treatment, and surprisingly, prolonged treatment did not confer cancer cells with drug resistance to 4-vinylphenol. 4-Vinylphenol probably exhibited its anti-cancer activities via beta-catenin, EGFR and AKT signaling pathways. CONCLUSION: 4-Vinylphenol was shown to inhibit metastasis and cancer stemness in CSC-enriched breast cancer cells. Since conventional therapies not targeting CSCs possibly lead to failure to eliminate cancer, 4-vinylphenol is a highly potential therapeutic agent for breast cancer patients.

Neuroprotective effect of Da Chuanxiong Formula against cognitive and motor deficits in a rat controlled cortical impact model of traumatic brain injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuanxiong Formula (DCXF) is one of the famous herb pairs that contains dried rhizomes of Ligusticum chuanxiong Hort. and Gastrodia elata Bl. in the mass ratio of 4:1. This classic representative traditional Chinese medicine has been widely used to treat brain diseases like headache and migraine caused by blood stasis and wind pathogen. However, the therapeutic effect of DCXF on traumatic brain injury (TBI) has not been reported yet. AIM OF STUDY: The present study was performed to investigate the neuroprotective effects of DCXF and its underlying mechanisms in the controlled cortical impact (CCI)-induced TBI rat model. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into four groups: Sham, TBI control, 1X DCXF (520.6 mg/kg) and 5X DCXF (2603.0 mg/kg). Two treatment groups (1X and 5X DCXF) were intragastrically administered daily for 7 days before CCI-induced TBI and then DCXF treatments were continued post-TBI until the animal behavioral tests, including Morris water maze test, acceleration rotarod motor test and CatWalk quantitative gait analysis test, were done. The brain water content and blood brain barrier (BBB) integrity were measured by wet-dry weight method and Evans blue method, respectively. The number of neuron cells, neural stem cells (NSCs), GFAP positive cells (astrocyte) as well as Iba-1 positive cells (microglia) were determined by histology and immunohistochemistry. RESULTS: Treatment with DCXF significantly improved the learning ability and memory retention in Morris water maze test, and remarkably enhanced motor performances in acceleration rotarod motor test and catwalk quantitative gait analysis test after TBI. Moreover, DCXF treatment was able to reduce BBB permeability, brain edema, microglia and astrocyte activation, improve the proliferation of NSCs and decrease neurons loss in the brain with TBI. CONCLUSIONS: The present study demonstrated that DCXF treatment could decrease BBB leakage and brain edema, reduce neuron loss, microglia and astrocyte activation, and increase NSCs proliferation, which may contribute to the cognitive and motor protection of DCXF in the TBI rats. It is the first time to provide potentially underlying mechanisms of the neuroprotective effect of DCXF on TBI-induced brain damage and functional outcomes.

The roles of cellular and molecular components of a hematoma at early stage of bone healing.

Bone healing is a complex repair process that commences with the formation of a blood clot at the injured bone, termed hematoma. It has evidenced that a lack of a stable hematoma causes delayed bone healing or non-union. The hematoma at the injured bone constitutes the early healing microenvironment. It appears to dictate healing pathways that ends in a regenerative bone. However, the hematoma is often clinically removed from the damaged site. Conversely, blood-derived products have been used in bone tissue engineering for treating critical sized defects, including fibrin gels and platelet-rich plasma. A second generation of platelet concentrate that is based on leukocyte and fibrin content has also been developed and introduced in market. Conflicting effect of these products in bone repair are reported. We propose that the bone healing response becomes dysregulated if the blood response and subsequent formation and properties of a hematoma are altered. This review focuses on the central structural, cellular, and molecular components of a fracture hematoma, with a major emphasis on their roles in regulating bone healing mechanism, and their interactions with mesenchymal stem cells. New angles towards a better understanding of these factors and relevant mechanisms involved at the beginning of bone healing may help to clarify limited or adverse effects of blood-derived products on bone repair. We emphasize that the recreation of an early hematoma niche with critical compositions might emerge as a viable therapeutic strategy for enhanced skeletal tissue engineering.

Dihydrofisetin exerts its anti-inflammatory effects associated with suppressing ERK/p38 MAPK and Heme Oxygenase-1 activation in lipopolysaccharide-stimulated RAW 264.7 macrophages and carrageenan-induced mice paw edema.

Dihydrofisetin is a flavanonol derived from some edible wild herbs and traditional Chinese medicines. It has been found to possess many biological activities. However, the anti-inflammatory potential of Dihydrofisetin remains uncharacterized. The aim of the present study was to investigate the anti-inflammatory activity of Dihydrofisetin and its underlying mechanisms. We found that Dihydrofisetin dose-dependently inhibited lipopolysaccharide-induced productions of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages, probably through suppressing the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The expressions of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and monocyte chemotactic protein (MCP-1) were also suppressed. We further demonstrated that Dihydrofisetin inhibited the activation of mitogen-activated protein kinases (MAPKs) pathway and phosphorylation of IκB-α whereas upregulated the expression of heme oxygenase-1 (HO-1). The in vivo carrageenan-induced mice paw edema study also indicated that treatment with 100 mg/kg of Dihydrofisetin could significantly inhibit carrageenan induced paw edema, decrease the levels of TNF-α, IL-6 and MDA, and increase the activity of GSH-Px in paw tissues. Taken together, Dihydrofisetin may act as a natural agent for treating inflammatory diseases by targeting MAPK, NF-κB and HO-1 pathways.

Evaluating Efficacy and Safety of Combination Medication of Atorvastatin and a Herbal Formula Containing Salvia miltiorrhiza and Pueraria lobata on Hyperlipidemia.

Despite being a potent hypolipidemic drug, atorvastatin (AS) possesses certain adverse effects. Using AS and an herbal formula (Danshen and Gegen, DG) in combination may achieve potentiated hypolipidemic effects and also reduce its adverse effects. Hence, this study aimed to investigate the efficacy and safety of an AS and DG combination on high-fat diet-induced hyperlipidemia. Treatment outcomes were assessed by measuring parameters including body weight, adipose tissue, liver, total cholesterol, triglyceride, and low-density and high-density lipoprotein cholesterol. Measurements of adverse effects were achieved by determining aspartate aminotransferase (AST), alanine transaminase (ALT), and creatine kinase (CK). Danshen and Gegen, as well as AS alone, reduced body weight, adipose tissue, liver weight, liver fat vacuoles, total liver lipids, total cholesterol, triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in high-fat diet-fed mice but increased AST, ALT, and CK. A combination of AS and DG was able to enhance reduced effects on the aforementioned parameters in relation to hyperlipidemia over AS or DG alone. It also reduced the elevation of AST, ALT, and CK induced than by AS or DG alone. Results demonstrated that an AS and DG combination resulted in stronger hypolipidemic effects than with AS or DG alone. Additionally, DG might attenuate adverse effects of AS on the liver and skeletal muscle. Copyright © 2017 John Wiley & Sons, Ltd.

Polysaccharides of Dendrobium officinale Kimura & Migo protect gastric mucosal cell against oxidative damage-induced apoptosis in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium officinale Kimura & Migo (DO) is a valuable Traditional Chinese Medicine to nourish stomach, in which polysaccharides are identified as active ingredients. However, limited scientific evidences have been reported on the gastroprotective efficacy of DO. The aim of the current study was to investigate the protective effects and underlying mechanism of polysaccharides from DO(DOP) on gastric mucosal injury. MATERIAL AND METHODS: For in vitro study, HFE145 cells were pretreated with DOP before induction of cell apoptosis by H2O2. Cell apoptosis and related proteins expression were detected. In the in vivo study, absolute ethanol was administered orally to induce gastric mucosal injury in rat. The gastric mucosal injury area and histological examination were used to evaluate the effects of DOP treatment on the recovery of the gastric mucosal injury. RESULTS: H2O2 treatment for 6h significantly induced cell apoptosis in HFE145 cells. However, the destructive effects of H2O2 on HFE 145 cells could be reversed by the pretreatment with DOP. The increased ROS level induced by H2O2 for 4h was reduced after DOP pretreatment. The number of apoptotic cells in both early and late apoptosis stages decreased significantly and the nuclei morphology changes were improved with DOP pretreatment. Furthermore, DOP inhibited caspase 3 activation and PARP cleavage, downregulated Bax expression and upregulated Bcl2 expression in cell model. Further study revealed that pretreatment of DOP inhibited p -NF-κBp65/NF-κBp65 level, indicating DOP inhibited H2O2-mediated apoptosis via suppression of NF-κB activation. In addition, DOP treatment could ameliorate gastric mucosal injury and inhibit mucin loss induced by ethanol in animal model. DOP treatment also interfered with ethanol-induced apoptosis process by downregulating Bax/Bcl2 ratio in gastric mucosa. CONCLUSIONS: The present study was the first one to demonstrate the gastroprotective effect of DOP through inhibiting oxidative stress-induced apoptosis. This study provided a solid evidence for the potential use of DO as a therapy or health supplement for gastric mucosal diseases.

Whole extracts of Radix Achyranthis Bidentatae and Radix Cyathulae promote angiogenesis in human umbilical vein endothelial cells in vitro and in zebrafish in vivo.

Although Radix Achyranthis Bidentatae (RAB) and Radix Cyathulae (RC) are from two different medicinal plants, they are both used as 'Niu-Xi', a widely used traditional Chinese medicine that is believed to stimulate menstruation and affect bone injury. Angiogenesis is actively involved in treating these illnesses. The aim of the present study was to investigate whether the whole extracts of RAB and RC possess pro-angiogenic effects. In order to examine this idea whole extracts of RAB and RC were extracted with boiling water followed by ethanol, respectively. Results from the MTT, wound healing and tube formation assays in human umbilical vein endothelial cells (HUVECs) in vitro revealed that the whole extracts of RAB and RC did not increase cell proliferation or tube formation, but enhanced cell migration. Their angiogenic effects were also confirmed in zebrafish in vivo via increasing the sprout numbers in the sub-intestinal vessel. As determined by quantitative polymerase chain reaction, the whole extracts of RAB and RC both regulated the expression of cell migration-related genes in zebrafish. It is concluded that the whole extracts of RAB and RC induced angiogenesis in HUVECs in vitro and in zebrafish in vivo via increasing cell migration.

Evaluation of the combined use of metronomic zoledronic acid and Coriolus versicolor in intratibial breast cancer mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Coriolus versicolor (CV) is a mushroom traditionally used for strengthening the immune system and nowadays used as immunomodulatory adjuvant in anticancer therapy. Breast cancer usually metastasizes to the skeleton, interrupts the normal bone remodeling process and causes osteolytic bone lesions. The aims of the present study were to evaluate its herb-drug interaction with metronomic zoledronate in preventing cancer propagation, metastasis and bone destruction. MATERIALS AND METHODS: Mice inoculated with human breast cancer cells tagged with a luciferase (MDA-MB-231-TXSA) in tibia were treated with CV aqueous extract, mZOL, or the combination of both for 4 weeks. Alteration of the luciferase signals in tibia, liver and lung were quantified using the IVIS imaging system. The skeletal response was evaluated using micro-computed tomography (micro-CT). In vitro experiments were carried out to confirm the in vivo findings. RESULTS: Results showed that combination of CV and mZOL diminished tumor growth without increasing the incidence of lung and liver metastasis in intratibial breast tumor model. The combination therapy also reserved the integrity of bones. In vitro studies demonstrated that combined use of CV and mZOL inhibited cancer cell proliferation and osteoclastogenesis. CONCLUSIONS: These findings suggested that combination treatment of CV and mZOL attenuated breast tumor propagation, protected against osteolytic bone lesion without significant metastases. This study provides scientific evidences on the beneficial outcome of using CV together with mZOL in the management of breast cancer and metastasis, which may lead to the development of CV as adjuvant health supplement for the control of breast cancer.

Integrative Approach to Facilitate Fracture Healing: Topical Chinese Herbal Paste with Oral Strontium Ranelate.

Strontium ranelate (SrR) is one of the pharmaceutical agents reported to be effective on the promotion of fracture healing. This study aimed to evaluate the integrative effect of the oral SrR with a topical Chinese herbal paste, namely, CDR, on facilitation of bone healing. The in vivo efficacy was evaluated using rats with tibial fracture. They were treated with either CDR topically, or SrR orally, or their combined treatments. The in vivo results illustrated a significant additive effect of CDR on SrR in increasing the yield load of the fractured tibia. The in vitro results showed that neither SrR nor CDR exhibited a cytotoxic effect on UMR106 and bone-marrow stem cell (BMSC), but both of them increased the proliferation of BMSC at low concentrations. The combination of CDR at 200 µg/mL with SrR at 200 or 400 µg/ml also showed an additive effect on increasing the ALP activity of BMSC. Both SrR and CDR alone reduced osteoclast formation, and the effective concentration of SrR to inhibit osteoclastogenesis was reduced in the presence of CDR. This integrative approach by combining oral SrR and topical CDR is effective in promoting fracture healing properly due to their additive effects on proosteogenic and antiosteoclastogenic properties.

Seropharmacological study on osteogenic effects of post-absorption ingredients of an osteoprotective herbal formula.

OBJECTIVE: To further investigate the {ptin vitro} effects of an osteoprotective herbal formula "ELP" (Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae) using seropharmacological approach. METHODS: Rats were fed with ELP or its individual component herbs for 2 days. The serum containing the postabsorbed ingredients of the herbal items were collected for cell culture using UMR106 cell, RAW264.7 cell and mesenchymal stem cell (MSC) isolated from the bone marrow of the rats. The effects of the herbal-containing serum on cell toxicity were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay; bromodeoxyuridine assay was conducted to measure the cell proliferation of UMR106 cell and MSC; cell activity was measured using colorimetric method, and mRNA expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteopontin (OPN) of UMR106 and MSC as well as matrix metalloproteinase 9 (MMP-9), tartrate-resistant acid phosphatase (TRAP) and cathepsin K of RAW264.7 were analyzed using real-time reverse-transcription polymerase chain reaction. RESULTS: ELP and its component serum exhibited no cytotoxic effects on the cells. The ELP-containing serum increased the proliferation of UMR106 cell and MSC by 25.7% and 14.4 %, respectively and the alkaline phosphatase activity of MSC was increased by 42.6%. On the contrary, it inhibited the RAW264.7 cell differentiation by 29.2 %. ELP serum upregulated the Runx2 expression of UMR and MSC by 1.18 fold and 1.27 fold, respectively. It also upregulated ALP and OPN expression in MSC by 1.69- and 2.12-fold, respectively. On the other hand, ELP serum down-regulated MMP-9 and cathepsin K expression of RAW264.7 cell by 0.46- and 0.36-fold, respectively. CONCLUSIONS: The serum of the animals fed with ELP contains active ingredients which are effective in promoting osteogenesis and inhibiting osteoclastogenesis.

A traditional Chinese formula composed of Chuanxiong Rhizoma and Gastrodiae Rhizoma (Da Chuanxiong Formula) suppresses inflammatory response in LPS -induced RAW 264.7 cells through inhibition of NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuanxiong Formula (DCXF) which origins from Jin Dynasty is a famous classical 2-herb Chinese medicinal prescription. It is composed of dried rhizomes of Ligusticum chuanxiong (Chuanxiong Rhizoma, CR) and Gastrodia elata (Gastrodiae Rhizoma, GR) at the ratio of 4:1 (w/w). It has been used to treat headache which is caused by wind pathogen and blood stasis for thousands of years in China. AIM OF STUDY: The present study was performed to investigate the anti-inflammatory effect of DCXF and elucidate its underlying molecular mechanisms using LPS-stimulated RAW 264.7 cells. MATERIALS AND METHODS: The anti-inflammatory effect of DCXF was evaluated using LPS-stimulated RAW 264.7 cells. Generation of nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. The gene expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the effect of DCXF on NF-κB activation was measured by western blot assay. RESULTS: Treatment with DCXF significantly suppressed the productions of NO and PGE2 through inhibitions of iNOS and COX-2 expressions in LPS-stimulated RAW 264.7 cells. DCXF significantly decreased IκBα phosphorylation, inhibited p65 expression and reduced p-p65 level. These results suggested the anti-inflammatory effect of DCXF was associated with the reduction of inflammatory mediators through inhibition of NF-κB pathway. CONCLUSIONS: These results indicated that DCXF inhibited inflammation in LPS-stimulated RAW 264.7 cells through inactivation of NF-κB pathway.

In vivo and in vitro anti-inflammatory effects of Zao-Jiao-Ci (the spine of Gleditsia sinensis Lam.) aqueous extract and its mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Zao-Jiao-Ci (ZJC), as the spine of Chinese Honey locust (Gleditsia sinensis Lam.), is traditionally used as Chinese medicine to reduce inflammation. AIM OF THE STUDY: The present study aimed to investigate an anti-inflammatory effect of ZJC aqueous extract both in vitro and in vivo, as well as its underlying mechanisms. MATERIALS AND METHODS: Anti-inflammatory effect of ZJC aqueous extract was evaluated by using carrageenan-induced paw edema in rats. In addition, the inhibitory effects of ZJC on nitric oxide production, intracellular reactive oxygen species production, pro-inflammatory mediator expression and prostaglandin E2 (PGE2) production were determined by using LPS-activated RAW 264.7 cells. The anti-oxidant activity of ZJC was assessed using 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid assay. RESULTS: ZJC aqueous extract showed significant suppressive effect on paw edema in rats at 100mg/kg. Moreover, ZJC aqueous extract decreased the expression of cyclooxygenase (COX)-2 and significantly decreased the PGE2, tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 production in LPS-activated macrophages in dose-dependent manners. ZJC aqueous extract inhibited the mRNA expression of these inflammatory cytokines as well. Furthermore, ZJC aqueous extract was found as an anti-oxidant and could inhibit ROS production in the LPS-induced cells. CONCLUSIONS: These findings show the potential of ZJC aqueous extract as a naturally occurring COX-2 inhibitor to reduce inflammation.