Characterization and genomic insight of surfactin-producing Bacillus velezensis and its biocontrol potential against pathogenic contamination in lettuce hydroponics.

Due to food borne pathogen, maintaining the viability of fresh fruits and vegetable is a great concern. Several strategies including microbial and plant-based formulations to reduce their infection and maintain quality of the fresh food are in practice. Currently, Bacillus has gained significant traction as a biocontrol agent for regulating diseases affecting a variety of agricultural and horticultural crops. Food-grade citric acid and plant growth-promoting rhizobacteria (PGPR) were used as antimicrobial agent, MIC results showed that PGPR (14.87 mm) and CA (20.25 mm) exhibited notable antimicrobial activity against E. coli. Lettuce treated with PGPR showed reduction in E. coli contamination, E. coli was detected at 3.30, 3.68 in control, and 2.7 log CFU/g in random root injury lettuce inoculated with PGPR KACC 21110 respectively. Random root injury showed a trend toward increasing E. coli internalization. The strains exhibited resistance to multiple antibiotics, including Imipenem, tetracycline, ampicillin, cefotaxime, cefoxitin, and ceftriaxone. Comprehensive data analysis revealed the presence of ten putative bacteriocin or bacteriocin-like gene clusters. The structure of lipopeptide homologs was characterized by using QTOF-MS/MS. The mass ion peaks attributed to surfactin homologs, surfactin A ion at m/z 1008.66, surfactin B, C at m/z 1022.67 and 1036.69. In addition to surfactin, a polyketide oxydifficidin and lipopeptide NO were extracted and detected from the extract of B. velezensis. Both isolates are key biocontrol agents and have significant potential in combating foodborne pathogens and can be utilized to explore novel antibacterial products for preventing pathogens in fresh produce.

High-Quality Genome Assembly of Peronospora destructor, the Causal Agent of Onion Downy Mildew.

Peronospora destructor is an obligate biotrophic oomycete that causes downy mildew on onion (Allium cepa). Onion is an important crop worldwide, but its production is affected by this pathogen. We sequenced the genome of P. destructor using the PacBio sequencing platform, and de novo assembly resulted in 74 contigs with a total contig size of 29.3 Mb and 48.48% GC content. Here, we report the first high-quality genome sequence of P. destructor and its comparison with the genome assemblies of other oomycetes. The genome is a very useful resource to serve as a reference for analysis of P. destructor isolates and for comparative genomic studies of the biotrophic oomycetes.

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.

Long-term effectiveness of bee venom acupuncture and physiotherapy in the treatment of adhesive capsulitis: a one-year follow-up analysis of a previous randomized controlled trial.

OBJECTIVE: An earlier randomized controlled trial (RCT) study showed that bee venom acupuncture (BVA) in combination with physiotherapy can be more effective in functional improvement and pain reduction in patients with adhesive capsulitis (AC). The objective of the current study was to examine the long-term effect of BVA in combination with physiotherapy on AC of the shoulder. DESIGN: Retrospective 1-year follow-up analysis of a previous RCT using a telephone interview method. SETTING: Outpatient joint center at the Gang Dong Kyung Hee University Hospital of Seoul, Republic of Korea. PATIENTS: A total of 192 patients had been enrolled in the previous study, and 124 of these were excluded from the current study. Sixty-eight patients who had been treated with combined BVA and physiotherapy for AC of the shoulder for 2 months were interviewed at approximately 1 year after treatment by telephone. INTERVENTION: Sixty of 68 patients were included in the follow-up analysis. Twenty received BV 1 treatment (1:10,000 concentration BVA plus physiotherapy), 22 received BV 2 treatment (1:30,000 concentration BVA plus physiotherapy), and 18 received control treatment (normal saline injection plus physiotherapy). OUTCOME MEASURES: The primary outcome measure was Shoulder Pain And Disability Index (SPADI) score. Secondary outcome measure was score on verbal rating scale for pain and patient satisfaction. RESULTS: Baseline characteristics of the groups did not significantly differ. SPADI scores at 1 year significantly differed between the BV 1 group and the control group (p=0.043). No significant differences were found in pain verbal rating scores after 1 year. Treatment satisfaction with therapy was also assessed, and the BV 1 and BV 2 groups showed significantly greater satisfaction compared with the control group. CONCLUSIONS: BVA combined with physiotherapy remains clinically effective 1 year after treatment and may help improve long-term quality of life in patients with AC of the shoulder.

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.

Clinical effectiveness of bee venom acupuncture and physiotherapy in the treatment of adhesive capsulitis: a randomized controlled trial.

BACKGROUND: Bee venom acupuncture (BVA) has been used in the treatment of adhesive capsulitis (AC) in the clinical field. This study aimed to investigate whether the addition of BVA to physiotherapy (PT) would be more effective in the management of AC, and whether BVA would have a dose-dependent effect. MATERIALS AND METHODS: Sixty-eight patients diagnosed with AC were recruited into 3 groups; BV 1 (1:10,000 BVA plus PT), BV 2 (1:30,000 BVA plus PT), and group 3 (normal saline (NS) injection, as a control, plus PT). PT was composed of 15 minutes of transcutaneous electrical nerve stimulation (TENS), transcutaneous infrared thermotherapy (TDP), and manual PT. Treatments were given in 16 sessions within 2 months. Shoulder pain and disability index (SPADI), pain visual analogue scale (VAS), and 3) active/passive range of motion (ROM) were measured before treatment and at 2, 4, 8, and 12 weeks after the treatment. RESULTS: All 3 groups showed statistically significant improvements in SPADI, pain VAS scores, and active/passive ROM. The BV 1 group showed significantly better outcomes in SPADI at 8 and 12 weeks, in pain VAS (at rest) at 8 weeks, and in pain VAS (during exercise) at 12 weeks than the NS group. No significant differences were found in active/passive ROM among all the groups. CONCLUSION: BVA in combination with PT can be more effective in improving pain and function than PT alone in AC. However, the effectiveness of BVA was not shown in a dose-dependent manner.

Analgesic effect of electroacupuncture on inflammatory pain in collagen-induced arthritis rats: mediation by alpha2- and beta-adrenoceptors.

This study was designed to investigate whether alpha- and beta-adrenergic systems mediate the analgesic effect of electroacupucture (EA) on inflammatory pain in a rat model of collagen-induced arthritis (CIA). To induce CIA, male Sprague-Dawley rats were immunized with bovine type II collagen emulsified in Freund's incomplete adjuvant, followed by a booster injection 14 days later. After induction of arthritis, the inflammatory pain threshold by the tail flick latency decreased as time passed and reached the minimum value at 4th week. Four weeks after the first immunization, low-frequency EA stimulation (2 Hz, 0.07 mA, 0.3 ms) delivered to Zusanli (ST(36)) for 30 min showed the analgesic effect. And also, the analgesic effect of EA was blocked by pretreatment with yohimbine (alpha2-adrenoceptor antagonist, 2 mg/kg, i.p.), but not by pretreatment with prazosin (alpha1-adrenoceptor antagonist, 1 mg/kg, i.p.) and propranolol (non-selective beta-adrenoceptor antagonist, 10 mg/kg, i.p.). These results suggest that the low-frequency EA can relieve the inflammatory pain in CIA, and the analgesic effect of EA can be mediated by alpha2- and beta-adrenoceptor, but not by alpha1-adrenoceptor.

The analgesic effect of electroacupuncture on inflammatory pain in the rat model of collagenase-induced arthritis: mediation by opioidergic receptors.

Electroacupuncture (EA) is widely practiced for the treatment of osteoarthritic (OA) pain, but its therapeutic mechanisms have not yet been fully studied, especially in the experimental OA rat model. In order to induce collagenase-induced arthritis (CIA) in rats, male Sprague-Dawley rats were intra-articularly injected with 0.05 ml of 4 mg/ml collagenase solution in the left knee of the hind limb, followed by a booster injection 4 days later. Maximal gross, histopathological features and biomarker activity changes consistent with human OA characteristics were observed four weeks after the first collagenase injection. In the exploratory preliminary study of EA stimulation parameters, low-frequency train pulse EA stimulation (2 Hz, 0.07 mA, 0.3 ms) delivered to the Zusanli (ST36) acupoint exerted an antinociceptive effect with acupoint specificity in a rat model of CIA. The antinociceptive effect of Zusanli EA was blocked by intraperitoneal pretreatment with naloxone (µ-opioid receptor antagonist, 2 mg/kg) and naltrindole (δ-opioid receptor antagonist, 1 mg/kg), but not with norbinaltophimine (κ-opioid receptor antagonist, 20 mg/kg). The synergistic antinociceptive effects of Zusanli EA were achieved with statistical significance by i.p. pretreatment with DAMGO (µ-opioid receptor agonist, 1 mg/kg) and with [D-Ala2]-Deltorphin II (δ-opioid receptor agonist, 6 mg/kg), but not with (±)-U-50488 (κ-opioid receptor agonist, 3 mg/kg). These results suggest that the 2-Hz EA can attenuate the osteoarthritic pain in CIA, and the analgesic effects of EA can be mediated by µ-opioid and δ-opioid, but not by κ-opioid receptors.

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.

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).

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.

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.

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.

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.

The analgesic and anti-inflammatory effect of WIN-34B, a new herbal formula for osteoarthritis composed of Lonicera japonica Thunb and Anemarrhena asphodeloides BUNGE in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Lonicera japonica Thunb and Anemarrhena asphodeloides BUNGE have been used for the treatment of a variety of inflammatory diseases, cold and infective diseases in many countries, including Korea and China. AIM OF THE STUDY: This study aimed to assess the anti-nociceptive and anti-inflammatory activities of n-butanol fraction (WIN-34B) prepared from dried flowers of Lonicera japonica and dried roots of Anemarrhena asphodeloides as potential novel treatment of osteoarthritis. MATERIALS AND METHODS: Anti-nociceptive activities of WIN-34B (100, 200 and 400 mg/kg, p.o.) were measured using acetic acid-induced writhing response, formalin-induced paw licking, hot plate, radiant heat tail-flick, carrageenan-induced paw pressure, and Hargreaves tests, respectively. Anti-inflammatory activities of WIN-34B (100, 200 and 400 mg/kg, p.o.) were assessed using acetic acid-induced vascular permeability, carrageenan-induced paw edema, and croton oil-induced ear edema. Anti-osteoarthritis effect of WIN-34B was analyzed using monosodium iodoacetate (MIA)-induced osteoarthritis animal model. RESULTS: WIN-34B exhibited better anti-inflammatory activity than that of celecoxib in carrageenan at the dose of 200 mg/kg and croton oil-induced paw edema and ear edema at the doses of 200 and 400 mg/kg. WIN-34B exhibited significant anti-inflammatory effects on vascular permeability. WIN-34B also exhibited significant anti-nociceptive activities in the late phase of formalin-induced paw licking and writhing response model in mice. In radiant heat tail-flick and carrageenan-induced paw pressure tests, WIN-34B at the dose of 400 mg/kg and at the doses of 200 and 400 mg/kg presented similar activities to indomethacin and celecoxib. Compared to indomethacin WIN-34B at 400mg/kg showed similar or better anti-nociceptive activities after 1 and 2h of theraphy in the hot plate test and better anti-nociceptive activity than that of celecoxib in Hargreves test. In the MIA-induced osteoarthritis animal models, WIN-34B at 400 mg/kg exhibited similar or better anti-nociceptive property than that of celecoxib throughout the pain measurement periods. CONCLUSION: When compared to celecoxib, WIN-34B exhibited similar or better anti-nociceptive and anti-inflammatory activities in osteoarthritic animal models, which may become a potential novel treatment for osteoarthritis.

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.

Therapeutic effect of Aralia cordata extracts on cartilage protection in collagenase-induced inflammatory arthritis rabbit model.

AIM OF THIS STUDY: Aralia cordata has been used to alleviate symptoms of osteoarthritis (OA) in traditional medicine. However, there is no in vivo study related with the therapeutic effects and mechanisms of Aralia cordata. On the basis of this background, our study was designed to examine the cartilage protective and proliferative effects of Aralia cordata by using a collagenase-induced osteoarthritis (CIA) rabbit model. MATERIALS AND METHODS: The right knees of rabbits were injected intra-articularly with collagenase, and rabbits were orally administered with distilled water (vehicle), Aralia cordata (200mg/kg) and celecoxib (100mg/kg) once a day for 28 days after the initiation of the CIA. RESULTS: In histopathologic studying by using H&E and Safranin O staining, Aralia cordata showed a cartilage protective effect in CIA rabbit femoral condyle. However, celecoxib had no effect on cartilage protection in CIA. The inflammatory mediators involved in cartilage destruction, such as COX-2 and PGE(2), were inhibited in the Aralia cordata-treated group. Aralia cordata also showed an anti-apoptotic effect through suppression of caspase-3 activity and chondrocyte proliferation induction in both in vivo and in vitro. CONCLUSION: These results indicate that Aralia cordata showed cartilage protective effects through the down-regulations of COX-2 expression, PGE(2) production, caspase-3 activity, and chondrocyte proliferation in the CIA rabbit model.