Efficacy and safety of electrical moxibustion for knee osteoarthritis: study protocol for a randomized controlled trial.

BACKGROUND: Knee osteoarthritis (KOA) is a significant health issue because it causes pain and functional limitation. Many studies have reported that moxibustion, a treatment in traditional Korean medicine, is effective in treating KOA. However, conventional moxibustion produces smoke, harmful gases, and odors that can adversely affect the eyes, skin, and throat. It is also difficult to control the intensity of stimulation in conventional moxibustion. An electrical moxibustion device was developed to circumvent these problems, but there are few studies of that device. We will evaluate the efficacy and safety of electrical moxibustion as a treatment for KOA, and compare it with traditional indirect moxibustion and usual care. METHODS: This is a multicenter, randomized, open, assessor-blinded, parallel-group clinical trial. A total of 138 eligible participants with KOA will be randomly allocated into three groups (electrical moxibustion, traditional indirect moxibustion, or usual care) with a 1:1:1 ratio. Participants in each moxibustion group will receive 12 sessions of moxibustion treatment at 6 acupoints (ST36, ST35, ST34, SP9, EX-LE4, SP10) plus up to 2 points of "ashi", if needed, over a period of 6 weeks (2 sessions per week). A specifically designed device that provides thermal stimulation using electrical energy will be used for the electrical moxibustion group. Participants in the usual care group will receive usual treatment and self-care. The primary outcome measure is change in pain on a numerical rating scale (NRS) from week 1 to week 6. The secondary outcome measures are pain assessed on a visual analog scale (VAS), the Korean version of the Western Ontario and McMaster osteoarthritis index (K-WOMAC), patient global assessment (PGA), and the European quality of life five dimension five level scale (EQ-5D-5 L). Safety will be assessed by monitoring adverse events at each visit. Follow-up measurements will be performed at 12 weeks after baseline measurements. DISCUSSION: This trial will provide evidence on the efficacy and safety of electrical moxibustion as a treatment for KOA. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03287570 . Registered on 19 September 2017.

Single-dose Toxicity of Guseonwangdo-go Glucose 5% Intravenous Injection in a Rat Model.

OBJECTIVES: The purpose of this study was to examine the single-dose intravenous toxicity of Guseonwangdo-go glucose 5% pharmacopuncture (GWG5). METHODS: Forty Sprague-Dawley rats were divided into four groups of five males and five females per group: an intravenous (IV) injection of 1.0 mL of normal saline solution per animal was administered to the control group; IV injections of 0.1, 0.5, and 1.0 mL of GWG5 per animal were administered to the experimental groups (G: 0.1, G: 0.5, and G: 1.0). Observation of clinical signs and body weight measurements were carried out for 14 days following the injections. At the end of the observation period, hematological, biochemical, and histopathological tests, as well as necropsy examinations, were performed on the injected parts. RESULTS: No mortalities or adverse clinical signs were observed in any of the groups. The body weights of all groups continuously increased. In the hematological and the biochemical tests, females in G-0.1 had minimal changes, but those changes were not dose dependent. On necropsy examination, no abnormalities were observed. In the histopathological test, focal inflammatory cell infiltrations were observed in two female rats, one in the control group and one in G-1.0. Also, one female rat in the control group had an epidermis crust. These changes were concluded to have been caused by the insertion of the needle into a vein. CONCLUSION: The above findings suggest that the lethal dose of GWG5 administered via IV injection is more than 1.0 mL per animal in both male and female rats. Further studies are needed to establish more detailed evidence of its toxicity.

Study of Single-dose Toxicity of Guseonwangdo-go Glucose Intramuscular Injection in Sprague-Dawley Rats.

OBJECTIVES: This study was performed to analyze single-dose intramuscular toxicity of Guseonwangdo-go glucose pharmacopuncture. METHODS: Eighty six-week-old Sprague-Dawley rats were divided into two large groups of forty rats; Guseonwangdo-go glucose 5% and Guseonwangdo-go glucose 20% groups. Each group was sub-divided into four smaller groups of five males and five females, with the following dosages of pharmacopuncture being administered by intramuscular (IM) injection in each group: group 1 (G1, control group): 1.0 mL of normal saline solution, group 2 (G2, low-dose group): 0.1 mL, group 3 (G3, mid-dose group): 0.5 mL, and group 4 (G4, high-dose group): 1.0 mL. RESULTS: No mortalities or clinical signs were observed in any group. Also, no significant changes in body weights or in hematological/biochemical analyses were observed between the control and the experimental groups during necropsy or histopathology. CONCLUSION: The above findings suggest that the lethal dose of Guseonwangdo-go glucose 5% and 20% pharmacopuncture administered via IM injection is more than 1.0 mL per animal in both male and female rats. Further studies on the repeated-dose toxicity of Guseonwangdo-go glucose should be conducted to yield more concrete data.

Single-dose Toxicity of Guseonwangdo-go Glucose 20% Intravenous Injection in Sprague-Dawley Rats.

OBJECTIVES: This study was performed to evaluate the single-dose intravenous toxicity of Guseonwangdo-go glucose 20% pharmacopuncture. METHODS: Forty Sprague-Dawley rats were divided into four groups of five males and five females per group: an intravenous (IV) injection of 1.0 mL of normal saline solution per animal was administered to group 1 (G1, control group); an IV injections of 0.1, 0.5, and 1.0 mL of Guseonwangdo-go glucose pharmacopuncture per animal were administered to experimental groups 2, 3, and 4 (G2, G3, and G4), respectively. General symptoms, body weights, hematological and biochemical test results, and necropsy histopathological observation were recorded in all groups. In the statistical analyses, significance was determined by using the one-way analysis of variance (ANOVA). The significance level was 0.05 in all comparisons. RESULTS: For 14 days, no deaths or abnormalities were observed in any of the 4 groups. The body weights of all groups continuously increased during the observation period. In the hematological test, the WBC count was significantly increased in female rats of G4 compared to the control group, but this difference was considered not to be statistically meaningful. No significant biochemical changes were observed. On necropsy, crust formation was observed in one rat of the control group, and granulation tissues were observed around the injection site in one rat of G4; these changes were concluded to have been caused by injection of the needle into a vein. CONCLUSION: The findings suggest that the lethal dose of Guseonwangdo-go glucose pharmacopuncture is more than 1.0 mL per animal in both male and female rats. Thus, we can conclude that Guseonwangdo-go glucose pharmacopuncture injection is relatively safe to use in acute toxicity tests. Further studies are needed to establish more detailed evidences of its toxicity.

Comparing the effects of individualized, standard, sham and no acupuncture in the treatment of knee osteoarthritis: a multicenter randomized controlled trial.

BACKGROUND: Acupuncture is an effective yet complex therapy, integrating syndrome differentiation, selection of appropriate acupoints and skillful needling techniques. Clinicians carefully tailor acupuncture treatment to each patient. However, most clinical trials of acupuncture have been based on a standardized formula of points for every patient without properly accounting for individualdifferences and, as a result, have not been reflective of the true efficacy of clinical practice. To determine the efficacy of meridian-based syndrome differentiation and Sa-am acupuncture, we have designed a simple pragmatic trial providing individualized treatments while working within a general framework. METHODS/DESIGN: The study is designed to be a parallel, patient- and assessor-blind, randomized controlled trial (RCT). A total of250 patients with knee osteoarthritis (OA) will be recruited from two independent hospitals, Semyung University Oriental Medicine Hospital in Chung-ju and Dongguk University Oriental Hospital in Ilsan, South Korea. Patients will be randomly allocated into four treatment groups: 1. individualized, meridian-based syndrome differentiation and Sa-am acupuncture treatment;2. standard acupuncture treatment;3. sham acupuncture treatment; and 4. no acupuncture treatment. Patients in groups 1 to 3 will be treated by certified oriental medicine doctors twice a week for 6 weeks. The primary outcome measure will be the self-reported total Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score change. The trial will also include secondary outcome measures. DISCUSSION: This trial is designed to determine the efficacy of individualized acupuncture treatment in patients with knee OA by comparing the differences between individualized, standard, sham and no acupuncture treatments. The results of this trial may validate the efficacy of individualized acupuncture therapy, encouraging its widespread use. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01569230.

Study to establish Ojeok-San (Five Accumulation Powder: wu ji san) administration criteria and a questionnaire to evaluate the holistic effects of Ojeok-San on patients with low back pain.

OBJECTIVES: The study objectives were to establish ojeok-san (Five Accumulation Powder: wu ji san) administration criteria and a questionnaire to evaluate the holistic effects of ojeok-san on patients with low back pain (LBP). METHODS: Texts and literatures, recommended by specialists, were searched to gather ojeok-san-related symptoms. Then, the opinions of Oriental medicine doctors (OMDs) practicing in Seoul were surveyed to ask which symptoms they consider the most in clinical practice. Based on the survey, selection of potential items for the questionnaire was made. The final version was established based on the results of the survey and Delphi process of musculoskeletal diseases specialists. In order to evaluate the reliability and validity of the newly developed assessment tool (Ojeok-san Low Back Questionnaire: OLQ), patients with chronic LBP were recruited. OLQ and other tools such as visual analogue scale, numeric rating scale, Roland-Morris Disability Questionnaire, Modified-Modified Schober test, and 36-Item Short Form Health Survey were applied to the subjects in a 2-week interval. Test-retest reliability, internal consistency, and convergent and discrimination validity were assessed. RESULTS: A total of 90 potential items were generated by the research team. One hundred and two (102) OMDs fully replied to the survey. Based on the survey results, 34 items were initially selected as potential items. Through Delphi method of experts, 10 top items, rated more than 5 points on a scale of 10, were finally established. The 10 items were each established as a response scale of 0-10 (0 as no symptom and 10 as the most excessive form of symptom). Based on the above stages, an initial OLQ was established and used in the evaluation phase. The validity and reliability of OLQ assessment results showed high test-retest reliability, intraclass correlation coefficient, and internal consistency. CONCLUSIONS: The newly developed Ojeok-san administration criteria and questionnaire may be a promising tool for future Oriental medicine clinical study protocols.

Efficacy of pharmacopuncture using root bark of Ulmus davidiana Planch in patients with knee osteoarthritis: a double-blind randomized controlled trial.

The aim of this study was to determine whether pharmacopuncture is a clinically effective and safe method for the treatment of knee osteoarthritis. Patients were recruited between August 2008 and December 2008 at the Ilsan Hospital associated with Dongguk University. Patients were randomly assigned to one of the two groups. The experimental group (n = 30) received pharmacopuncture using root bark of Ulmus davidiana Planch (UDP) twice a week for 6 weeks; the control group (n = 30) received normal saline injections. Fifty-three patients completed the trial. After the seventh treatment, we found that UDP pharmacopuncture was more effective in pain improvement using a Visual Analog Scale than was normal saline injection. However, the two interventions were not significantly different as measured by the Western Ontario and McMaster Universities pain score and total pain scores, 36-Item Short Form Health Survey, and Korean Health Assessment Questionnaire. No subject showed any serious adverse effects. The effects of pharmacopuncture treatment were a combination of placebo, needle stimulation, mechanical effect of the solution, and a chemical effect of UDP. However, normal saline used as the control intervention displayed the first three effects, and thus its effect was not inert. This may have influenced the results of the trial, which was statistically insignificant between the two groups, except following the seventh treatment session.

Effects of TGFbeta1 and extracts from Cervus korean TEMMINCK var. mantchuricus Swinhoe on acute and chronic arthritis in rats.

AIM OF THE STUDY: To elucidate the pharmacological activities of deer antler acupuncture and TGF61538;1 on the acute and chronic phases of rheumatoid arthritis diseases. MATERIALS AND METHODS: Polyarthritis rats were administered with TGF61538;1 and water extract of deer antler acupunture (DAA), prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe. TGF61538; (0.1 to 2 61549;g/animal) and DAA (5-100 61549;g/kg animal) were initiated 1 day before an arthritogenic dose of streptococcal cell wall fragments to see the effects on the joint swelling and distortion during the acute phase and the chronic phase of the disease. Arthritic index suppression of rat arthritis model was examined by TGF61538; and DAA administrations. RESULTS: TGF61538;1 and DAA diminished the polyarthritis development in rats. TGF61538; and DAA eliminated the joint swelling and distortion observed during the acute phase and the chronic phase of the disease. The TGF61538; and DAA suppressed the arthritis progress when administration was begun after acute phase of arthritis. DISCUSSION: Consistent with the inhibition of inflammatory cell recruitment into the synovium, TGF61538;1 and DAA reversed the leukocytosis associated with the chronic phase of the arthritis, respectively.

Inhibition of mouse osteoblast proliferation and prostaglandin E2 synthesis by Ulmus davidiana Planch (Ulmaceae).

Ulmus davidiana Planch (Ulmaceae) (UD) is a widely used Korean herbal medicine that has been used historically in anti-inflammatory and anticancer therapy. Since UD has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions, this study was undertaken to address whether the water extract of the bark of UD could modulate proliferation of mouse osteoblasts in vitro and to investigate its effect on cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandin E2 (PGE2). Mouse osteoblasts were tested in vitro for growth inhibition, proliferating cell nuclear antigen (PCNA) expression, and COX-2 activity and expression after treatment with UD extract. Its effects were compared with those of indomethacin (a nonselective COX inhibitor) and celecoxib (a selective COX-2 inhibitor). UD demonstrated a strong growth inhibition in tested mouse osteoblasts. The IC50s were 10microg/ml for UD, 6microM for celecoxib and 42microM for indomethacin. UD, as well as celecoxib and indomethacin, suppressed PCNA expression and PGE2 synthesis in osteoblasts. UD inhibited COX-2 expression, whereas celecoxib inhibited COX-2 activity directly. UD selectively and effectively inhibits osteoblasts cell growth in vitro. Inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anti-inflammatory activity.

Suppressive effects of bee venom on the immune responses in collagen-induced arthritis in rats.

The effect of bee venom (BVA) on the development of type II collagen (CII)-induced arthritis (CIA) in rats has been studied. Male rats were immunized with an emulsion of 200 microg of CII and complete Freund's adjuvant (CFA). The rats were then given intraperitoneally (i.p.) injection of a suspension of BVA or saline during the experiment. The effect of BVA on cellular responses to CII was examined. In the control rats, the onset of arthritis was observed at the 24th day after the CII-immunization, and the severity of CIA was developed gradually. As compared with rats treated with saline, BVA i.p. injected at doses of more than 20 microl/100g mouse once a day for 14 days inhibited the ability of inguinal lymph node cells to produce T cell cytokines interleukin-1beta, -2, -6, tumor necrosis factor-alpha and interferon-gamma when the cells were obtained from rats 24 days after immunization and cultured in vitro with CII. When rats were injected i.p. with sheep red blood cells, hemagglutination titers in BVA-treated and control rats did not differ significantly when low doses of BVA was given to rats. However, i.p. injection of BVA at doses of more than 10 microl/100g/day suppressed antibody production. Pretreatment of rats with BVA could inhibit the development of collagen arthritis even when 10-20 microl/100g/day of the BVA were used for pretreatment. Interestingly, higher doses than 10 microlBVA/100g mouse were much effective for arthritis incidence. Treatment of rats with BVA prevented the development of collagen arthritis in a dose-dependent manner. Doses of BVA (15 and 20 microl/100g) resulted in decreased incidence of arthritis. In conclusion, therapeutic i.p injection with BVA improved the clinical course of the disease and the immune response to CII.

Inhibition of Ulmus davidiana Planch (Ulmaceae) on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts.

Ulmus davidiana Planch (Ulmaceae) (UD) has long been known to be antiinflammatory in traditional Korean medicine. This experiment investigated the effects of UD on bone resorption using bone cell culture. Different concentrations of crude extract of UD were added to mouse bone cell culture. The mitochondrial activity of the bone cells after exposure of UD was determined by colorimetric 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). It was demonstrated that UD has potential effects on bone cell culture without cytotoxicity. The most effective concentration of UD in bone cells was 100 microg/mL. Cathepsin K (Cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. When mouse long bone cells including osteoclasts and osteoblasts were treated with UD, UD prevented the osteoclast-mediated intracellular processing of Cat K, suggesting that UD may disrupt the intracellular transport of pro Cat K. Since secreted proenzymes have the potential to reenter the cell via the mannose-6-phosphate (M6P) receptor, to prevent this possibility, UD was tested in the absence or presence of M6P. Inhibition of Cat K processing by UD was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of UD. UD dose-dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of Cat K processing.

Effect of Cervus korean TEMMINCK var. mantchuricus Swinhoe on protease activities, antioxidant and free radical damages in rheumatis arthritis rats.

The effect of deer antler extracts (DAA) of Cervus korean TEMMINCK var. mantchuricus Swinhoe on protease activities, oxidant and free radical damages in synovial fluid from rheumatoid arthritis in rats was studied. Rats were i.p. administered with DAA. We have compared (using the same series of experimental samples) the levels of activity of a comprehensive range of cytoplasmic, lysosomal and matrix protease types, together with the levels of free radical induced protein damage (determined as protein carbonyl derivative) and total antioxidant in synovial fluid from rheumatoid arthritis (RA) and DAA-treated rats. Many proteases activities were shown to be significantly increased in RA compared to normal rats. Protease activities (including those enzyme types putatively involved in the immune response, such as dipeptidyl aminopeptidase IV) in plasma were not significantly different between RA and normal rats. DAA treatment at dose of 100 microg/kg suppressed the production of the proteases of cytoplasmic, lysosomal and matrix protease types. The level of free radical induced damage to synovial fluid proteins was approximately 2-fold lower in DAA rats compared to RA rats, although there was no significant difference in total antioxidant status in synovial fluid or plasma between RA and DAA rats. It was concluded that DAA treatment reduces the activation of proteolytic enzymes and free radicals, which are likely to be of equal potential importance as protein damaging agents in the pathogenesis of RA.

Anti-inflammatory effect of Ulmus davidiana Planch (Ulmaceae) on collagen-induced inflammation in rats.

Ulmus davidiana Planch (Ulmaceae) extract (UD) has long been known to have anti-inflammatory and anticancer activities. UD has been also known to have protective effects on damaged tissue, inflammation and bone among other functions. Effects of UD on inflammatory and immune responses and its mechanisms in collagen-induced inflammation (CII) rat were studied. Hind paw volumes of rats were measured by volume meter; lymphocyte proliferation, interleukin (IL)-1, IL-2, tumor necrosis factor (TNF)-α level was determined by 3-(4,5-2dimethylthiazal-2yl)2,5-diphenyltetrazoliumbromide assay. Antibodies to collagen type II (BC-II) were determined by enzyme-linked immunosorbent assay. There was a marked secondary inflammatory response in CII model, which accompanied with the decrease of body weight and the weight of immune organs simultaneously. The administration of UD (20, 80, 150mg/kg, intragastrically×10 days) inhibited the inflammatory response and restored body weight and the weight of immune organs of CII rats. Lymphocyte proliferation and IL-2 production of CII rats increases, together with IL-1 and TNF-α in peritoneal macrophages and synoviocytes. The administration of UD (20, 80, 150mg/kg, 10 days) reduced above changes significantly. UD had no effect on the concentration of antibodies to BC-II. From the results, it was concluded that UD possesses anti-inflammatory and immunoregulatory activities and has a therapeutic effect on CII rats.

Prevention of collagen-induced arthritis in mice by Cervus korean TEMMINCK var. mantchuricus Swinhoe.

The effect of water extract of deer antler (DAA) prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe (Nokyong) on collagen-induced mouse rheumatoid arthritis (RA) model was studied. Identification of common DAA capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. DAA has been shown to possess anti-inflammatory and anti-carcinogenic properties in experimental animals. In this study, we determined the effect of DAA-injection on collagen-induced arthritis in mice. In three independent experiments, mice given DAA in water exhibited significantly reduced incidence of arthritis (30-45%) as compared with mice not given DAA in water (86-98%). The arthritis index also was significantly lower in DAA-injected animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-γ, and tumor necrosis factor α in arthritic joints of DAA-injected mice. The neutral endopeptidase activity was approximately six-fold higher in arthritic joints of non-DAA-injected mice in comparison to non-arthritic joints of unimmunized mice, whereas it was only two-fold higher in the arthritic joints of DAA-injected mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of DAA-injected mice. Taken together our studies suggest that DAA may be useful in the prevention of onset and severity of arthritis.

Immunomodulatory activity of Ulmus davidiana Planch (Ulmaceae) water and ethanolic extracts on bone cells: Stimulation of proliferation, alkaline phosphatase activity and type I collagen synthesis.

Ulmus davidiana Planch (Ulmaceae) (UD) frequently appears as the main ingredient in prescriptions for bone injuries, however, the action mechanism is unclear. In the present study, (i) the effect of the aqueous extract of UD on bone cells was investigated in vitro and (ii) the immunomodulatory activity of UD was investigated with regard to cellular and humoral immunity. The osteoprecursor cells (OPC) were incubated in the medium with different concentrations of the UD and the cell proliferation was studied. When the concentration of UD was <100µg/ml, the proliferation of OPC was enhanced. However, the proliferation of OPC was inhibited by UD with the concentrations >180µg/ml. Under most treatments, the cells presented low expression for cyclooxygenase-2 (Cox-2) protein. On the other hand, oral administration of the ethanolic and water extracts of UD, at the doses of 20, 50, 100 and 200mg/kg in mice, dose-dependently potentiated the delayed-type hypersensitivity reaction induced both by sheep red blood cells (SRBC) and oxazolone. It significantly enhanced the production of circulating antibody titers in mice in response to SRBC. UD had no any effect on macrophage phagocytosis. Chronic administration of UD significantly ameliorated the total white blood cell counts and also restored the myelosuppressive effects induced by cyclophosphamide. From the results, it was concluded that UD directly stimulates the proliferation, alkaline phosphatase activity, protein secretion and particularly type I collagen synthesis of OPC in a dose-dependent manner, and that UD possesses immunomodulatory activity.

Stimulative effects of Ulmus davidiana Planch (Ulmaceae) on osteoblastic MC3T3-E1 cells.

Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions. To treat rheumatoid arthritis (RA), a herbal medicine, Ulmus davidiana Planch (Ulmaceae) extract (UD) is being used in traditional oriental medicine. The effect of UD on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. UD dose-dependently increased DNA synthesis (significant at 5-20 microg/ml). UD increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (5-20 microg/ml). Antiestrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1, which was induced by UD. UD at concentrations ranged from 30 to 100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that UD directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and UD is effective for bone anti-resorptive action in bone cells.

Effects of bee venom on protease activities and free radical damages in synovial fluid from type II collagen-induced rheumatoid arthritis rats.

The effect of bee venom acupuncture (BVA) (api-toxin) on the development of type II collagen (CII)-induced arthritis (CIA) in rats has been studied. We have compared the levels of activity of a comprehensive range of cytoplasmic, lysosomal and matrix protease types, together with the levels of free radical-induced protein damage (determined as protein carbonyl derivative) in synovial fluid from CIA-treated, BVA-treated and normal rats. Many protease types showed significantly increased activity in CIA compared with normal rats. BVA (5 and 10 microl/100g) significantly reduced these enzyme activities by some 80% each, but levels of plasma proteases activity (including those enzyme types putatively involved in the immune response, such as dipeptidyl aminopeptidase IV and proline endopeptidase) in CIA, BVA (5 microl/100g)-treated and normal plasma samples were not significantly different. The level of free radical induced damage to synovial fluid proteins was approximately three-fold higher in CIA compared with normal rats. However, BVA (5 microl/100g) significantly decreased the level of reactive oxygen free radical species (ROS) induced oxidative damage to synovial fluid proteins. It was concluded that activation of proteolytic enzymes and free radicals are likely to be of equal potential importance as protein damaging agents in the pathogenesis of rheumatoid arthritis (RA), and the development of novel therapeutic strategies for the latter disorder should include both protease inhibitory and free radical scavenging elements. In addition, the protease inhibitory element should be designed to inhibit the action of a broad range of enzymatic mechanistic types (cysteine, serine, metallo proteinases and peptidases). In conclusion, BVA is considered to be an effective RA modulator, inhibiting protease activities and removing ROS.

Effects of Ulmus davidiana planch on mineralization, bone morphogenetic protein-2, alkaline phosphatase, type I collagen, and collagenase-1 in bone cells.

Ulmus davidiana Planch (Ulmaceae) (UD) long has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation, and bone among other functions. The herbal medicine also is being used in Oriental medicine to treat osteoporosis. In a preliminary study, treatment of osteoclasts containing long bone cells with the water extract of UD bark prevented the intracellular maturation of cathepsin K (cat K), and thus, it was considered that UD is a pro-drug of a potent bone-resorption inhibitor. To further clarify the role of UD in ossification, we investigated the effects of UD on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, we assessed the effects of UD on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. UD enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the UD was observed at relatively low doses (significant at 5-50 microg/ml and maximal at 50 microg/ml). Northern blot analysis showed that UD (100 microg/ml) increases in bone morphogenic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. UD slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that UD has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.

Effect of Buthus martensi Karsch on aromatase activity and cytokine-inducted NOS and NO production in osteoblasts and leukaemic cell line FLG 29.1.

Among the different scorpion species, Buthus martensi Karsch, a widely distributed scorpion species in Asia especially in Korea, has received a lot of attention. Indeed, over the past decade, more than 70 different peptides, toxins, or homologues have been isolated. It may prove a valuable resource for identifying potential anti-inflammatory and analgesic drugs. The recent observation has suggested that the aromatase is a possible local modulator of bone remodeling in osteoarthritis and osteoporosis. In the present study, therefore, the effect of Buthus martensi Karsch (BMK) extract, traditional immunosuppressive Korean aqua-acupuncture water, on the bone function of human osteoblastic cells was studied. To provide insights into the effect of BMK on aromatase activity in bone-derived cells, we examined the human leukaemic cell line FLG 29.1, which is induced to differentiate toward the osteoclastic phenotype by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and transforming growth factor (TGF)-beta1, and the primary first-passage osteoblastic cells (hOB). Gene expression of the aromatase was not affected by Buthus martensi Karsch in FLG 29.1 and hOB cells. However, enzyme activity was stimulated in a time-dependent fashion by 10.0 microg/ml BMK and by either 1-50 nM TPA or 0.01-0.5 ng/ml TGF-beta1, with maximal responses after 2-3 hr exposure. On the other hand, BMK strongly inhibited interleukin-1beta (IL-1beta)- and tumor necrosis factor (TNF)alpha-induced Nitricoxide (NO) synthase expression with little effect on constitutive NO synthase expression. BMK extracts (10 microg/ml) inhibited cytokine-induced iNOS and nNOS expression. BMK (10 microg/ml) did not affect the ecNOS expression, indicating the extracts are not working on the constitutive NOS expression. BMK strongly inhibited the cytokine-induced NO production (p < 0.01). BMK also showed significant inhibition on NO production in both induced by TNF-alpha+IL-1beta. NO donors, sodium nitroprusside, and NONOate dose-dependently elevated alkaline phosphatase activity. These results suggest that NO directly facilitates osteoblastic differentiation. This result also suggests that BMK is effective for bone resorptive action in bone cells.

Immunosuppressive activity of deer antler extracts of Cervus korean TEMMINCK var. mantchuricus Swinhoe, on type II collagen-induced arthritis.

Unossified horn or pilose antler cut from deer, which belong to the Cervidae generally is termed Nokyong. Nokyong is one of the most famous Korean traditional medicines and has been considered to possess sexual-reinforcing and antiaging actions. In this study, water extract of deer antler extract (DAA) prepared from the growing antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe was used to investigate the efficacy of the DAA on the development of type II collagen (CII)-induced arthritis (CIA) in rats. Male rats were immunized with an emulsion of 200 microg of CII and complete Freund's adjuvant (CFA). The rats then were administered by injection a suspension of DAA or phosphate-buffered saline. The effect of DAA on cellular responses to CII was examined. The injection of DAA suppressed the CII-specific secretion of interferon (IFN)-gamma from splenocytes ex vivo. The influence of DAA also was evaluated on the incidence and development of arthritis in rat CIA. Rats were immunized twice at a 3-wk interval with bovine CII, with DAA being given by injection once a d for 14 d with four different regimens. A 14-d course of DAA treatment at a daily dose of 100 microg/kg, which began on the d of the first CII immunization, suppressed the development of arthritis, as well as antibody formation and delayed-type hypersensitivity to CII. Treatment with DAA resulted in inhibition of development of arthritis and immune responses to CII.