Analgesic Effects of Diluted Bee Venom Acupuncture Mediated by δ-Opioid and α2-Adrenergic Receptors in Osteoarthritic Rats.

Context • Pain from osteoarthritis is associated with peripheral nociception and central pain processing. Given the unmet need for innovative, effective, and well-tolerated therapies, many patients, after looking for more satisfactory alternatives, decide to use complementary and alternative modalities. The analgesic mechanism of subcutaneous injections of diluted bee venom into an acupoint is thought to be part of an anti-inflammatory effect and the central modulation of pain processing. Objectives • Using the rat model of collagenase-induced osteoarthritis (CIOA), the study intended to investigate the analgesic effects of bee venom acupuncture (BVA) as they are related to the acupuncture points and dosage used and to determine whether the analgesic mechanisms of BVA for pain were mediated by opioid or adrenergic receptors. Design • Male Sprague-Dawley rats were randomly assigned to one of 19 groups, with n = 10 for each group. Setting • The study was conducted at the East-West Bone and Joint Research Institute at Kyung Hee University (Seoul, South Korea). Intervention • All rats were intra-articularly injected with collagenase solution in the left knee, followed by a booster injection performed 4 d after the first injection. For the groups receiving BVA treatments, the treatment was administered into the ST-36 acupoint, except for 1 group that received the treatment into a nonacupoint. Three BVA intervention groups received no pretreatment with agonists or antagonists; 1 of them received a dose of 1 mg/kg of bee venom into acupoint ST-36, 1 received a dose of 2 mg/kg into acupoint ST-36, and 1 received a dose of 1 mg/kg into a nonacupoint location. For the intervention groups receiving pretreatments, the opioid-receptor or adrenergic-receptor agonists or antagonists were injected 20 min before the 1-mg/kg BVA treatments. Outcome Measures • Changes in the rats' pain thresholds were assessed by evaluation of pain-related behavior, using a tail flick latency unit. Results • The pain reached its maximum value after 4 wk of CIOA induction. The 1-mg/kg ST-36 BVA treatment resulted in a more significant analgesic effect than nonacupoint BVA. Pain-related behavior was more effectively improved by treatment with 1 mg/kg of BVA than with 2 mg/kg of BVA. The analgesic effects of the BVA were not synergistic with the agonist pretreatments with the µ-, δ-, or κ-opioid receptors or with the α1-, α2-, and ß-adrenergic receptors. The analgesic effects of the BVA were not decreased by the antagonist pretreatments for the µ- or κ-opioid receptors or for the α1- or ß-adrenergic receptors. The ST-36-BVA-induced analgesia was inhibited by the antagonist pretreatments for the δ-opioid receptor and the α2-adrenergic receptor. Conclusion • The ST-36 BVA treatment exerted an analgesic effect on CIOA-induced pain through the partial involvement of the δ-opioid and α2-adrenergic receptors.

Dual effect of WIN-34B on osteogenesis and osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells.

ETHNOPHARMACOLOGICAL RELEVANCE: As an n-butanol fractionated extracted mixture of Lonicera japonica Thunb, dried flowers and Anemarrhena asphodeloides Bunge, root, WIN-34B has been reported the analgesic, anti-inflammatory, cartilage-repairing and protective effects in previous studies. AIM OF THE STUDY: To investigate the effect of WIN-34B on osteogenesis and osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells. MATERIALS AND METHODS: To examine the effect of WIN-34B on osteogenic differentiation, human mesenchymal stem cells (hMSCs) were treated with WIN-34B (1µg/mL and 10µg/mL). Alkaline phosphatase (ALP) activity was evaluated and Von Kossa staining was conducted. Mice bone marrow macrophages (BMMs) were obtained and treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony stimulating factor (m-CSF) to induce osteoclastogenesis. To investigate osteoclast differentiation, tartrate-resistant acid phosphatase (TRAP) staining was conducted after treatment with WIN-34B. Osteoclastogenic conditions were induced by stimulating the cells with interleukin (IL)-1α, IL-17, and tumor necrosis factor (TNF-α) in hMSCs and BMMs co-culture systems. The expression levels of osteoprotegerin (OPG), RANKL, runt-related transcription factor 2 (RUNX2), IL-17, c-Fos, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured by reverse transcription polymerase chain reaction (RT-PCR). The expression levels of nuclear factor-kappaB (NF-κB), inhibitory kappa B-α (IκBα), phospho-NF-κB, phospho-IκBα, ß-actin, p38 MAPK, phospho-c-Jun N-terminal kinase (JNK), phospho-extracellular-signal regulated kinase (ERK), phospho-p38 mitogen-activated protein kinase (MAPK), phospho-JNK, and phospho-ERK were measured by western blot analysis. RESULTS: WIN-34B promoted ALP activity and mineralization of hMSCs. In TRAP-stained BMMs, the number of multinucleated cells decreased after WIN-34B treatment. WIN-34B increased the OPG/RANKL ratio and the expression of RUNX2, and suppressed the expression of IL-17, c-Fos, and TNF-α. It also suppressed the activation of NF-κB, IκBα, p38 MAPK, and JNK in a dose-dependent manner. CONCLUSIONS: These results demonstrated that WIN-34B increased osteogenesis and decreased osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells via inhibition of the NF-κB, JNK, and p38 MAPK pathways.

The standardized BHH10 extract, a combination of Astragalus membranaceus, Cinnamomum cassia, and Phellodendron amurense, reverses bone mass and metabolism in a rat model of postmenopausal osteoporosis.

Jasin-hwan-gagambang (BHH10), a modified prescription of Jasin-hwan, contains Astragalus membranaceus, Cinnamomum cassia, and Phellodendron amurense, and it has been traditionally used to treat osteoporosis and other inflammatory diseases. In this study, we systematically investigated the protective effects of BHH10 in ovariectomy (OVX)-induced rats. Sprague-Dawley rats were randomly divided into sham and OVX subgroups. The rats in the OVX group were treated with vehicle, BHH10, alendronate (ALN), and 17ß-estradiol (E2). BHH10 treatment significantly inhibited OVX-induced increases in body weight and uterus atrophy. In addition, it significantly increased the bone mineral density (BMD) and prevented a decrease in trabecular bone volume, connectivity density, trabecular number, thickness, and separation at the total femur and femur neck. The OVX rats showed significant decreases in the serum levels of calcium and phosphorous and significant increases in the serum levels of cholesterol, low-density lipoprotein cholesterol, alkaline phosphatase, osteocalcin, C-telopeptide type 1 collagen, and bone morphogenetic protein-2. These changes were significantly reduced to near sham levels by administration of BHH10 to OVX rats. BHH10-treated rats had a greater bone mass, a better structural architecture of the bone, and higher levels of biochemical markers of the bone than did the ALN-treated or E2-treated rats. These results suggest that BHH10 reverses osteoporosis in OVX rats by stimulating bone formation or regulating bone resorption and is not associated with toxicity.

Formononetin attenuates osteoclastogenesis via suppressing the RANKL-induced activation of NF-κB, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1 signaling pathway.

Formononetin (1), a plant-derived phytoestrogen, possesses bone protective properties. To address the potential therapeutic efficacy and mechanism of action of 1, we investigated its antiosteoclastogenic activity and its effect on nuclear factor-kappaB ligand (RANKL)-induced bone-marrow-derived macrophages (BMMs). Compound 1 markedly inhibited RANKL-induced osteoclast differentiation in the absence of cytotoxicity, by regulating the expression of osteoprotegerin (OPG) and RANKL in BMMs and in cocultured osteoblasts. Compound 1 significantly inhibited RANKL-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKL-induced activation of the NF-κB p65 subunit, degradation of inhibitor κBα (IκBα), induction of NF-κB, and phosphorylation of AKT, extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). NF-κB siRNA suppressed AKT, ERK, JNK, and p38 MAPK phosphorylation. Furthermore, 1 significantly suppressed c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key transcription factors during osteoclastogenesis. SP600125, a specific inhibitor of JNK, reduced RANKL-induced expression of phospho-c-Jun, c-Fos, and NFATc1 and inhibited osteoclast formation. These results suggested that 1 acted as an antiresorption agent by blocking osteoclast activation.

Effect of Gambisan on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes.

This study was conducted to explore the antiadipogenic effect and possible mechanism of Gambisan on 3T3-L1 cells. For quality control, Gambisan was standardized by HPLC and the standard compounds ephedrine, epigallocatechin-3-gallate, and caffeine were screened. Cultured 3T3-L1 cells that had been induced to differentiate were treated with various concentrations of Gambisan or its major component extracts (Ephedra intermedia Schrenk, Atractylodes lancea DC., and Thea sinensis L.) for 72 hours for MTT assay to determine cell viability or 10 days for LDH assay, triglyceride assay, DNA content measurement, Oil red O staining, RT-PCR, and western blot. Gambisan significantly inhibited adipogenesis in 3T3-L1 cells by reducing triglyceride contents and lipid accumulation in a dose-dependent manner without obvious cytotoxicity. Viability and DNA content in 3T3-L1 cells treated with Gambisan were significantly higher than cells treated with the major component extracts at every concentration. The anti-adipogenic effects of Gambisan appeared to be mediated by a significant downregulation of the expression of lipoprotein lipase mRNA and PPAR γ , C/EBP α , and SREBP-1 protein apart from the expression of hormone-sensitive lipase. Gambisan could act as a possible therapeutic agent for obesity. However, further studies including in vivo assays and clinical trials are needed to confirm the efficacy, safety and mechanisms of the antiobesity effects of Gambisan.

Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone.

We sought to determine the cartilage repair capacity of WIN-34B in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral bone. The cartilage protective effect of WIN-34B was measured by clinical and histological scores, cartilage area, and proteoglycan and collagen contents in the collagenase-induced osteoarthritis rabbit model. The efficacy of chondrogenic differentiation of WIN-34B was assessed by expression of CD105, CD73, type II collagen, and aggrecan in vivo and was analyzed by the surface markers of progenitor cells, the mRNA levels of chondrogenic marker genes, and the level of proteoglycan, GAG, and type II collagen in vitro. Oral administration of WIN-34B significantly increased cartilage area, and this was associated with the recovery of proteoglycan and collagen content. Moreover, WIN-34B at 200 mg/kg significantly increased the expression of CD105, CD73, type II collagen, and aggrecan compared to the vehicle group. WIN-34B markedly enhanced the chondrogenic differentiation of CD105 and type II collagen in the progenitor cells from subchondral bone. Also, we confirmed that treatment with WIN-34B strongly increased the number of SH-2(CD105) cells and expression type II collagen in subchondral progenitor cells. Moreover, WIN-34B significantly increased proteoglycan, as measured by alcian blue staining; the mRNA level of type II α 1 collagen, cartilage link protein, and aggrecan; and the inhibition of cartilage matrix molecules, such as GAG and type II collagen, in IL-1 ß -treated progenitor cells. These findings suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage repair as well as cartilage protection via enhancement of chondrogenic differentiation in the collagenase-induced osteoarthritis rabbit model and progenitor cells from subchondral bone.

Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes.

BACKGROUND: WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes. METHODS: The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1ß-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed. RESULTS: WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1ß-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1ß. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic acid or mangiferin in IL-1ß-stimulated chondrocytes. CONCLUSIONS: WIN-34B is potentially valuable as a treatment for OA by virtue of its suppression of MMPs, ADAMTSs, and inflammatory mediators, and it's up-regulation of TIMP-1 and TIMP-3 involved in the MAPK pathway.

WIN-34B, a new herbal medicine, inhibits the inflammatory response by inactivating IκB-α phosphorylation and mitogen activated protein kinase pathways in fibroblast-like synoviocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE have been used for the treatment of a variety of inflammatory diseases in traditional Korean medicine. OBJECTIVE: The aim of the study is to evaluate the anti-inflammatory effects of WIN-34B, a new herbal medicine, in fibroblast-like synoviocytes (FLS) obtained from patients with osteoarthritis (OA). MATERIALS AND METHODS: WIN-34B is isolated from the n-butanol fraction of dried flowers of L. japonica and dried roots of A. asphodeloides. The anti-inflammatory effects of WIN-34B on cell viability, the production and release of inflammatory mediators, matrix metalloproteinases (MMPs), aggrecanases, tissue inhibitor of matrix proteinases (TIMP) is compared with celecoxib in IL-1ß-stimulated human OA FLS. Furthermore, the effect of WIN-34B on inhibitory kappa B-α (IκB-α) phosphorylation and mitogen-activated protein kinases (MAPK) in the IL-1ß-stimulated OA FLS was also evaluated. RESULTS: WIN-34B significantly inhibited the IL-1ß-induced cell viability in human OA FLS without cytotoxicity. Compared to celecoxib, WIN-34B exhibited similar or better anti-inflammatory effects through significant suppression of inflammatory mediators (IL-1ß, TNF-α, PGE2 and NO), MMPs (MMP-1, MMP-3 and MMP-13) and aggrecanases (ADAMTS-4 and ADAMTS-5), and enhancement of TIMPs (TIMP-1 and TIMP-3). Moreover, WIN-34B reduced the phosphorylation of IκB-α, ERK1/2, p38 and JNK1/2 in IL-1ß-stimulated OA FLS. CONCLUSIONS: WIN-34B exhibited similar or better anti-inflammatory properties in IL-1ß-stimulated OA FLS compared to celecoxib. The anti-inflammatory effects of WIN-34B are due to inhibition of inflammatory mediators (IL-1ß, TNF-α, PGE2 and NO) and regulation of MMPs, ADAMTSs and TIMPs via the inhibition of IκB-α and MAPK phosphorylation in IL-1ß-stimulated OA FLS.

Polygoni Rhizoma inhibits inflammatory response through inactivation of nuclear factor-kappaB and mitogen activated protein kinase signaling pathways in RAW264.7 mouse macrophage cells.

The objective of this study was to determine the antiinflammatory effects of Polygoni Rhizoma (PR), an Oriental medicinal herb, in interleukin 1 beta (IL-1ß) and lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. PR significantly reduced the production of pro-inflammatory cytokines such as IL-6, tumor necrosis factor alpha (TNF-α) and pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) even at a concentration of 1 µg/mL in the cells. In addition, PR inhibited the transcriptional activity of NF-κB as well as the degradation and phosphorylation of inhibitory kappa B alpha (IκBα). Furthermore, PR suppressed the phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase 1/2 (JNK1/2) in IL-1ß and LPS-treated RAW264.7. The results suggest that PR exerts an antiinflammatory property by inhibiting iNOS, COX-2, TNF-α and IL-6 production in association with inactivation of the NF-κB and MAPK signaling pathways in RAW 264.7 cells.

Gastroprotective and safety effects of WIN-34B, a novel treatment for osteoarthritis, compared to NSAIDs.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried flowers of Lonicera japonica, also known as Japanese honeysuckle, and the dried root of Anemarrhena asphodeloides, the component herbs of WIN-34B, are traditionally used in Eastern medicine to treat various inflammatory conditions including arthritis. OBJECTIVE: To study the acute and chronic toxicities of WIN-34B and to compare its effects on gastric mucosa with those of diclofenac, a widely used NSAID, and celecoxib, a selective COX-2 inhibitor. MATERIALS AND METHODS: To investigate acute toxicity, we orally administered a single dose of 5,000 mg/kg WIN-34B to rats. To investigate chronic toxicity, we orally administered 500, 1000 or 2,000 mg/kg WIN-34B to rats daily for 13 weeks. To assess its effects on gastric mucosa, rats received either a single dose or repeated doses of WIN-34B (400, 1000, or 2,000 mg/kg), diclofenac (10, 40, or 80 mg/kg), celecoxib (100 or 1,000 mg/kg), or vehicle, after which samples of gastric mucosa were assessed grossly and histologically. We also measured tissue activity of myeloperoxidase and synthesis of eicosanoids, including prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)). To further assess its effects, we administered WIN-34B to rats either intraperitoneally or orally, measured gastric injury scores using a rat model of diclofenac-induced gastric injury, and measured eicosanoid synthesis. RESULTS: WIN-34B showed no signs of acute or chronic toxicity in terms of general behavior, gross appearance of the internal organs, blood chemistry, or mortality. WIN-34B did not cause significant gastric mucosal damage after single or repeated doses. In contrast, diclofenac and celecoxib both caused gastric damage. In terms of eicosanoid synthesis, WIN-34B significantly suppressed LTB(4) synthesis while both diclofenac and celecoxib increased LTB(4) synthesis. WIN-34B slightly reduced PGE(2) production, while both diclofenac and celecoxib significantly reduced PGE(2) production. In a rat model of diclofenac-induced gastric injury, WIN-34B significantly suppressed LTB(4) synthesis and restored PGE(2) release. CONCLUSIONS: These results demonstrate that WIN-34B did not cause acute or chronic toxicity in male or female rats. In addition, WIN-34B did not cause significant gastric mucosal damage, instead appearing to protect the mucosa from diclofenac-induced gastric damage through the regulation of PGE(2) and LTB(4).

Combined prescription (OAH19T) of Aralia cordata Thunb and Cimicifuga heracleifolia Komar and its major compounds inhibit matrix proteinases and vascular endothelial growth factor through the regulation of mitogen-activated protein kinase pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: OAH19T, a new herbal extract from a mixture of Aralia cordata Thunb and Cimicifuga heracleifolia, is traditionally used for the treatment of arthritis in far East Asia. To investigate the chondroprotective effects of OAH19T on osteoarthritis was examined and compared with its major compounds pimaradienoic acid (PA) and ferulic acid (FA) of human osteoarthritis (OA) chondrocytes. MATERIALS AND METHODS: Chondrocytes, alone or in the presence of IL-1ß, were cultured with or without OAH19T, PA or FA (10, 20, 40 µg/ml). The release of sulfated glycosaminoglycan (GAG) was measured by colorimetric assay using 1,9-dimethylmethylene blue (DMB) reagent from the cultured media. The level of aggrecanases and VEGF was measured by reverse transcription polymerase chain reaction (RT-PCR). The expression of MMP-1 and MMP-3 analyzed by real time RT-PCR and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of mitogen-activated protein kinases was performed by immunoblotting in OA chondrocytes. The proliferation was examined by the BrdU assay. RESULTS: OAH19T markedly inhibited the release of proteoglycan and the degradation of aggrecan, in a dose-dependent manner in OA chondrocytes. OAH19T also inhibited the level of aggrecanase-1, aggrecanase-2, MMP-1, MMP-3, and VEGF in OA chondrocytes. PA and FA also inhibited the level of aggrecanase-2, MMP-3 and VEGF, while did not significantly affect the levels of aggrecanase-1, MMP-3 in OA chondrocytes. OAH19T exhibited the down-regulation of p38 MAP kinase unlike PA and FA in OA chondrocytes without cytotoxicity. In addition, p38 inhibitor SB203580 abolished the antiproliferative activity and proteoglycan degradation by OAH19T, while had no effect by PA or FA. CONCLUSION: OAH19T have shown the chondroprotective effect by inhibiting cell proliferation, expression of cartilage-specific matrix proteinases and release of VEGF, but bigger than PA or FA, through down-regulation of p38 MAP kinase in human OA chondocyte. These results provide pharmacological basis for use in treatment of OA.

Anti-inflammatory and anti-nociceptive effect of Betula platyphylla var. japonica in human interleukin-1ß-stimulated fibroblast-like synoviocytes and in experimental animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine has widely been used Betula platyphylla var. japonica to treat various inflammatory diseases including arthritis. AIM OF THE STUDY: To determine the anti-inflammatory, anti-nociceptive, and anti-arthritic effects of Betula platyphylla in interleukin-1ß (IL-1ß)-stimulated fibroblast-like synoviocytes from human rheumatoid arthritis and in nociceptive and inflammatory animal model. MATERIALS AND METHODS: The inflammatory mediators such as IL-6, tumor necrosis factor (TNF)-α matrix metalloproteinase (MMP)-1, MMP-13, inducible nitric oxide synthesis (iNOS), nitrites, prostaglandin E(2) (PGE(2)) and cyclo-oxygenase 2 (COX-2) activity of Betula platyphylla were tested in IL-1ß-stimulated fibroblast-like synoviocytes. Tail withdrawal in response to thermal stimulation in tail flick test or paw flinching and shaking in response to sc hind paw formalin injection was measured 1h after oral administration of Betula platyphylla. The former was evaluated with a paw pressure test, and the latter was measured using the squeaking score, and paw volume in inflammatory arthritis tests. RESULTS: Betula platyphylla significantly inhibited proliferation of IL-1ß-induced synoviocytes. Betula platyphylla reduced the levels of inflammatory mediators, such as IL-6, TNF-α, MMP-1, MMP13, and PGE(2). In particular, Betula platyphylla significantly inhibited the releases of nitrites and iNOS, as well as release of NFκB, into the nucleus of IL-1ß-treated synoviocytes, even at concentrations as low as 1µg/ml. Oral administrant of Betula platyphylla at 400mg/kg significantly decreased about 27.8% of tail flick withdrawal and inhibited about the number of paw flinches in both phases 1 and 2 of the formalin test. In the carrageenan-induced acute pain and arthritis model, Betula platyphylla dose dependently reduced the nociceptive threshold and the arthritic symptoms at day 8, respectively, and Betula platyphylla at 400mg/kg markedly reduced the inflammatory area about 48% in the ankle joints. This capacity of Betula platyphylla at 400mg/kg was similar to that of the celecoxib-2 inhibitor in carrageenan-induced nociceptive and inflammatory arthritis model. CONCLUSIONS: These results suggest that Betula platyphylla has anti-nociceptive and anti-inflammatory effects in IL-1ß-stimulated RA FLS and in an animal model of arthritis. Thus, the use of Betula platyphylla as a pharmaceutical candidate for the treatment of arthritis should be further studied.

Effect of Phellodendron amurense in protecting human osteoarthritic cartilage and chondrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine has been widely using Phellodendron amurense Rupr. (Rutaceae) to treat various inflammatory diseases including arthritis. AIM OF THE STUDY: This study investigated the effects of Phellodendron amurense in protecting cartilage, including regulating the levels of aggrecanases, matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMP), proinflammatory cytokines and signaling of the mitogen activated protein kinase (MAPK) pathway in human osteoarticular cartilage and chondrocytes. MATERIALS AND METHODS: Explants from human osteoarthritis cartilage were cultured alone or in IL-1α for 7 days with or without Phellodendron amurense ethanol extract or celecoxib (40, 100, 200µg/ml). The effect of Phellodendron amurense on matrix degradation induced by IL-1α in human articular cartilage was assessed by staining, and the quantities of sulfated glycosaminoglycan (GAG) and type II collagen were calculated from the culture media. The levels of aggrecanases, MMPs, TIMP, and PGE(2) in the culture media were investigated using an enzyme-linked immunosorbent assay (ELISA). In addition, reverse transcription polymerase chain reaction (RT-PCR) evaluated the mRNA expression of aggrecanases, MMPs and TIMP. Furthermore, Western blot analysis was performed to identify the roles that Phellodendron amurense played in the ERK, JNK and p38 signaling pathways. RESULTS: Phellodendron amurense showed no evident cytotoxicity on human articular cartilage. Phellodendron amurense significantly inhibited the IL-1α-induced degradation of GAG and type II collagen from human osteoarticular cartilage in a concentration-dependent manner. Celecoxib did not significantly inhibit IL-1α-induced release of GAG and only slightly reduced type II collagen. Phellodendron amurense also dose-dependently decreased the levels of aggrecanase-1 and -2, MMP-1, -3, and -13, whereas it increased TIMP-1 expression in human osteoarticular cartilage. Celecoxib only decreased MMP-1 and MMP-13 levels in human osteoarticular cartilage. In addition, Phellodendron amurense reduced the phosphorylation of extracellular signal regulated kinase (ERK)1/2, Jun NH2-terminal kinase (JNK) and activated phospho-p38 MAPK in a dose-dependent manner in human osteoarthritic chondrocytes. CONCLUSIONS: Phellodendron amurense inhibited osteoarticular cartilage and chondrocyte destruction by inhibiting proteoglycan release and type II collagen degradation, down-regulating aggrecanases, MMP activities and phospho-ERK1/2, JNK and p38 MAP kinase signaling, and up-regulating TIMP-1 activity. Therefore, our results suggest that Phellodendron amurense is a potential therapeutic agent to protect cartilage against OA progression.

Formononetin accelerates wound repair by the regulation of early growth response factor-1 transcription factor through the phosphorylation of the ERK and p38 MAPK pathways.

Formononetin, a phytoestrogen from the root of Astragalus membranaceus, is used as a blood enhancer and to improve blood microcirculation in complementary and alternative medicine. The present study investigated the influence of formononetin on the expression of early growth response factor-1 (Egr-1) and growth factors contributing to wound healing. Formononetin significantly increased growth factors such as transforming growth factor-beta 1 (TGF-ß1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) in human umbilical vein endothelial cells (HUVECs). Formononetin also increased the expression of Egr-1 transcription factor by 3.2- and 10.5-fold, compared with recombinant VEGF(125) in HUVECs. The formononetin-mediated 12%-43% increase induced endothelial cell proliferation and recovered the migration of wounded HUVECs. In an ex vivo angiogenesis assay, formononetin produced a larger capillary sprouting area than produced using recombinant VEGF(125). Cell proliferation and migration of HUVECs were also greater in the presence of formonectin than VEGF(125). Western blot analysis of scratch-wounded confluent HUVECs showed that formononetin induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slightly inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The formononetin-mediated sustained activation of Egr-1 was suppressed by the ERK inhibitor PD98059 and the p38 inhibitor SB203580. PD98059 inhibited the formononetin-induced endothelial proliferation and repair in scratch-wounded HUVECs, SB203580 increased the cell proliferation and wound healing. Formononetin accelerate wound closure rate as early as day 3 after surgery and consistently observed until day 10 after in wound animal model. These data suggest that formononetin promotes endothelial repair and wound healing in a process involving the over-expression of Egr-1 transcription factor through the regulation of the ERK1/2 and p38 MAPK pathways.

Biphasic positive effect of formononetin on metabolic activity of human normal and osteoarthritic subchondral osteoblasts.

Osteoarthritis is a multifactorial disease characterized by loss of articular cartilage and subchondral plate thickening. Therefore, biochemical analysis of the underlying bone tissue has provided important information for treatment of osteoarthritis. In this study, we determined the potential role of formononetin, a phytoestrogen isolated from Astragalus membranaceus to alter the expression of metabolic markers and cytokine production of human normal osteoblasts (Obs) and osteoarthritis subchondral osteoblasts (OA Obs). Human OA Obs and normal Obs were cultured for 3days, 7days or 14days in the present medium only or were treated with various doses of formononetin. Cells were analyzed for viability by WST-8 assay, alkaline phosphatase (ALP) activity, osteogenic markers (osteocalcin (OCN) and type I collagen (Col I)) and cytokines (interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), bone morphogenic protein-2 (BMP-2)). The level of IL-6, VEGF, BMP-2, OCN and Col I was increased in OA Obs compared with normal Obs. Formononetin dose-dependently decreased ALP, IL-6, VEGF, BMP-2, OCN and Col I in OA Obs, while markedly increased ALP, VEGF, BMP-2, OCN and Col I in normal Obs. Interestingly, formononetin markedly increased the expression of VEGF and BMP-2 for 3days of culture and significantly increased OCN and Col I at 14days in human normal Obs. The remodeling effect of formononetin on osteogenic markers and cytokines of inflammatory mediators was more striking in OA Obs as well. Taken together, these results could suggest that formononetin has biphasic positive effects on normal Obs and OA Obs by modifying their biological synthetic capacities.

Formononetin promotes early fracture healing through stimulating angiogenesis by up-regulating VEGFR-2/Flk-1 in a rat fracture model.

Plant-derived phytoestrogens have bone protective effects, but the molecular mechanism behind these effects remains unclear. This study is aimed at fully characterizing the fracture healing process of formononetin, and investigating the mechanism underlying angiogenesis in calluses of a rat fracture model. Femoral fractures were produced in 2-month-old Sprague-Dawley rats. A 20 microg/kg or 200 microg/kg dose of formononetin was orally administrated once a day during the healing period of 21 days. The results showed that in the early stage of chondrogenesis (days 3), formononetin significantly increased the number of vessels, and expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2/flk-1) compared with control. However, the larger dose of formononetin had no significant difference on expression of VEGF and VEGFR-2/Flk-1 compared with that of the smaller dose of formononetin. After 7 days of administration, formononetin markedly induced differentiation of mesenchymal stem cells in the fracture site. After 14 days, gene expression of mesenchymal progenitors such as alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN) and collagen type I (Col I), indicating osteogenic differentiation, was markedly stimulated by formononetin compared with control. These results suggest that formononetin promotes early fracture healing through angiogenesis activation in the early stage of fracture repair, and osteogenesis acceleration in the later stages, and thus may be beneficial for fracture healing.

Stimulatory effect of Cinnamomum cassia and cinnamic acid on angiogenesis through up-regulation of VEGF and Flk-1/KDR expression.

Complementary and alternative medicine, Cinnamomum cassia is one of the medicinal plants that have been used to improve various diseases caused by insufficient blood circulation. However, relatively little work has been carried out on the angiogenic responses of C. cassia and its active compound cinnamic acid (CA), despite its excellent pharmacological action. In this study, we study the effect of the ethanol extract of C. cassia (CCE) and its active compound CA, on angiogenic processes in in vitro and in vivo. In the Matrigel plug assay in vivo, CCE and CA dose dependently increased von Willebrand Factor (vWF) antigen expression, and hemoglobin contents, whose elevation paralleled the onset of angiogenesis and was considered an early indicator of endothelial activation. CCE and CA induced endothelial cells proliferation, migration and tubule-like structure in vitro. The 25-50% increase observed with CCE (at low doses of 1 or 10 ng/ml) in HUVEC and BAEC proliferation was similar to that observed with CA. The migration and tubule-like structure effect were observed with HUVEC and BAEC. However, the effect of CCE, CA and VEGF on cell proliferation, migration and tubule-like structure in HUVEC were bigger than the effect of CCE, CA and VEGF in BAEC. In addition, CCE and CA each induced 2.2-fold and 2.5-fold increases the production of VEGF, the mRNA expression of VEGF and Flk-1/KDR, the receptor involved in proliferation, migration, and tubule-like structure of endothelial cells. These data suggest that CCE and its active compound CA induce angiogenesis in vivo and in vitro, and this pathway is related with VEGF and Flk-1/KDR expression of endothelial cells.

The efficacy and mechanism action of RvCSd, a new herbal agent, on immune suppression and cartilage protection in a mouse model of rheumatoid arthritis.

The aim of the present study is to investigate the potential therapeutic action of RvCSd, an oriental herbal mixture, in an experimental model of rheumatoid arthritis (RA). DBA/1J mice were immunized with type II collagen. After a second collagen immunization, mice were treated with RvCSd or methotrexate (MTX) orally once a day for 35 days, and the incidence, clinical score, and joint histopathology were evaluated. The inflammatory response cytokines and cartilage protection effect were determined by measuring the levels in the joints and sera. The Th1/Th2-mediated auto-reactive response was evaluated by determining the proliferative response and cytokines of drained spleen cells stimulated with type II collagen. RvCSd treatment significantly reduced the incidence and severity of CIA, markedly abrogating joint swelling, synovial hyperplasia, and cartilage destruction. RvCSd significantly inhibited the production of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-6, IL-2, interferon (IFN)-gamma, and matrix metalloproteinases (MMP)-1 and up-regulated anti-inflammatory cytokines IL-4, IL-10, and metalloproteinase (TIMP)-1 in mice with CIA. In conclusion, RvCSd has therapeutic effects exerted through inhibition of inflammatory and Th1 responses, regulation of MMP/TIMP, and induction of regulatory T cells in CIA; these effects make RvCSd an outstanding candidate for use as an immune suppressive and cartilage protective medicine in RA patients.

Protective effects of butanol fraction from Betula platyphyla var. japonica on cartilage alterations in a rabbit collagenase-induced osteoarthritis.

AIM OF THIS STUDY: Many cartilage protective agents have been developed from natural products, and they have resulted in the development of treatments for osteoarthritis. In this study, we determined the osteoarthritic efficacy and mechanism of butanol fraction from the bark of Betula platyphylla var. japonica (BFBP) in collagenase-induced rabbit model of osteoarthritis (CIA). MATERIALS AND METHODS: The right knees of rabbits were injected intra-articularly with collagenase, and rabbits were orally administrated with distilled water (vehicle), BFBP (50, 100 and 200 mg/kg) or celecoxib (100 mg/kg) once a day for 28 days after the initiation of the CIA. RESULTS: Oral administration of BFBP dose-dependently suppressed the stiffness and global histologic score. Proteoglycan intensity was considerably increased in a dose-dependent manner. As well, the mRNA expression of MMP-1, and MMP-3 was decreased. On the contrary, the level of TIMP-1 in the synovial fluids was significantly increased in the BFBP treated group. The pathologic inflammatory molecules such as PGE2 and COX-2 were inhibited by BFBP, but COX-1 expression not affected. CONCLUSION: We suggest that BFBP has shown the protective effect on cartilage alternations through balance of MMPs/TIMP-1 and regulates inflammatory-related molecules in vivo model of osteoarthritis, and great candidate for development of osteoarthritis treatment.

Efficacy and mechanism of action of KHBJ-9B, a new herbal medicine, and its major compound triterpenoids in human cartilage culture and in a rabbit model of collagenase-induced osteoarthritis.

KHBJ-9B has been formulated by n-butanol fraction from 2 herbs known to have cartilage protection and anti-inflammatory effects. We elected to determine the osteoarthritic efficacy and mechanism of KHBJ-9B on human osteoarthritis cartilage explants culture and in a rabbit model of collagenase-induced osteoarthritis (CIA). The major chemical composition and quantification of KHBJ-9B was determined by high performance liquid chromatography. The efficacy of KHBJ-9B and its major compounds on cartilage protective effects such as inhibition of GAG release and type II collagen degradation, and their cytotoxicity in IL-1beta-treated human cartilage culture were examined. The mechanism of action of KHBJ-9B and its major compounds were evaluated by measuring inflammatory cytokines (IL-1beta and TNF-alpha) and matrix proteinases (ADAMTS-4, ADAMTS-5, MMP-1, MMP-13 and TIMP-3) in IL-1beta-treated human cartilage cultures. Also, the therapeutic effect of KHBJ-9B was confirmed using a collagenase-induced osteoarthritis (CIA) rabbit model. KHBJ-9B and 3 combined triterpenoids potently inhibited the release of proteoglycan and type II collagen in a dose dependent manner without cytotoxicity in IL-1beta-treated human cartilage explants culture, whereas its single major compounds (betulin, pimaradienoic acid and betulinic acid) and COX-2 inhibitor (NS398) showed little inhibition even at high concentrations. KHBJ-9B and the combination of 3 triterpenoids markedly inhibited the level of IL-1beta and TNF-alpha, and down-regulated the level of aggrecanases, ADAMTS-4, ADAMTS-5, MMP-1 and MMP-13, and up-regulated TIMP-3 in human cartilage explants culture. However, standard compounds and NS398 do not much affect the level of TNF-alpha, aggrecanases, and TIMP-3 in cartilage explants culture. In in vivo studies, KHBJ-9B significantly suppressed the stiffness level and global histologic score. Cartilage loss was significantly inhibited in the knee joint in a dose dependent manner, and this was associated with the finding that loss of proteoglycan, degradation of aggrecan and type II collagen was markedly reduced. These results suggest that the effect of KHBJ-9B is bigger than the effects of its single major compounds of triterpenoids or celecoxib inhibitors on cartilage protection and anti-inflammation in human cartilage and in in vivo model of osteoarthritis, and thus has potential for use in osteoarthritis treatment.