Effects of Drynariae rhizoma on the proliferation of human bone cells and the immunomodulatory activity

Pharmacological Research 51 (2005) 125–136

Effects of Drynariae rhizoma on the proliferation of human bone cells and the immunomodulatory activity Ji-Cheon Jeonga , Boo-Tae Leea , Cheol-Ho Yoona , Hyung-Min Kimb , Cheorl-Ho Kima,∗ a

Department of Internal Medicine, Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University and National Research Laboratory for Glycobiology, Sukjang-Dong 707, Kyungju, Kyungbuk 780-714, Republic of Korea b Department of Pharmacology, Kyung-Hee University College of Oriental Medicine, Dongdaemun-Gu, Seoul 130-701, Republic of Korea Accepted 14 June 2004

Abstract Drynariae Rhizoma (DR), a traditional Korea medicine, which is known for its effect to strengthen myoskeletal systems, frequently appears as the main ingredient in prescriptions for bone injuries. However, it is unclear how it pharmacologically contributes to the reformation of bone. In this study, the effect of DR on bone cells was investigated in vitro for the first time. The human osteoprecursor cells (OPC-1) were incubated in the medium with different concentrations of DR and the cell proliferation was studied. When the concentration of DR was ≤120 ␮g ml−1 , the proliferation of OPC-1 was enhanced. However, the proliferation of OPC-1 was inhibited by DR with the concentrations of >250 ␮g ml−1 . Under most treatments, the cells presented very pale expression for cyclooxygenase-2 (Cox-2) protein; slightly intensified band showed at the highest DR concentration, 120 ␮g ml−1 during the course of culture. On the other hand, we investigated the immunomodulatory activity of DR on cellular and humoral immunity. When different doses of ethanolic and water extracts of DR was administerd to mice, it was dose-dependently potentiated the delayed-type hypersensitivity reaction induced by both sheep red blood cells (SRBC) and oxazolone. It significantly enhanced the production of circulating antibody titre in mice in response to SRBC. But, DR did not any effect on macrophage phagocytosis. Prolonged administration of DR significantly ameliorated the total white blood cell count and also restored the immunosuppressive effects induced by cyclophosphamide. The present investigation reveals that DR possesses immunomodulatory activity. From the results, it was concluded that DR directly stimulated the proliferation, alkaline phosphatase activity, protein secretion and particularly type I collagen synthesis of OPC-1 at dose-dependent manner, and stimulated both the cellular and the humoral immunity. © 2004 Elsevier Ltd. All rights reserved. Keywords: Drynariae rhizoma; Human osteoprecursor cells; Alkaline phosphatase; Collagen type I; Immunomodulation; Phagocytosis

1. Introduction The immune system is involved in the etiology as well as pathophysiologic mechanisms of many diseases. Modulation of the immune responses to alleviate the diseases has been of interest for many years [1]. The primary cause of postmenopausal osteoporosis is an estrogen deficiency resulting in the decrease in bone mass. An ovariectomized rat model, which artificially produces the depleted state of estrogen, has been used for the study of postmenopausal osteoporosis. Both aged rats and mature rats have been used as animal models ∗

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1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2004.06.005

to study experimental osteoporosis and the mature rat model has characteristics that are comparable with those of early postmenopausal trabecular bone loss [2]. Several medications have been reported to be effective for curing osteoporosis based upon the results obtained using these animal models. Estrogen [1,3], bisphosphonates [2], calcitonin [2], calcium products [2,4], ipriflavone [5] and anabolic steroids [6] are clinically employed as effective medications. Oriental traditional medicines have been reevaluated by clinicians [7], because these medicines have fewer side effects and they are more suitable for a long-term use as compared to chemically synthesized medicines. About forty kinds of traditional Korean medicines are claimed to be effective for gynecological diseases such as climacteric psychosis,

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feeling of cold, menstrual disorders, dysmenorrhea, and low back pain. From ancient times in China and Korea, women who have had low back pain in climacteric and senescent periods have been treated with traditional Korean medicines. For example, some formula have been used for treating ovary function failure, low back pain during the climacteric period, and also used after oophorectomies because of malignant tumors [8,9]. Traditional Korean medicinal plants are a rich source of substances, which are claimed to induce paraimmunity, the non-specific immunomodulation of essentially granulocytes, macrophages, natural killer cells and complement functions [10]. Traditional Korean medicine lays emphasis on the promotion of health by strengthening host defenses against diseases. These medicines have been endowed with multiple properties like delaying the onset of senescence and improving mental functions by strengthening the psychoneuro-immune axis. The isolation, purification and chemical characterisation of the immunoactive moieties have been carried out in some of these plants [11]. However, few data are available concerning the recovery of bone mass by these traditional Korean medicines. Drynariae rhizoma (DR) is a woody climber found throughout Korea. The roots of this plant have been regarded as alterative and tonic, and are said to be useful in rheumatism and diseases of the nervous system. Phytochemical analysis of the plant has shown the presence of lipids, flavonoids, triterpenes and phenylpropanoids. DR is effective for the treatment of deficient kidney manifested by a lower back pain, weakness of the legs, tinnitus or toothache presumably, by functioning in tonifying the kidney, invigorating blood and stopping bleeding according to the traditional Korean medicinal literature [12]. Previously, it was shown that DR prevented the development of bone loss induced by ovariectomy in rats, and preserved the fine particle surface of the trabecular bone [13]. It was also demonstrated that the interaction between PGE2 and its cell surface receptor results in activation of the PKA signaling pathway. And DR strongly inhibited inflammation through LPS-stimulated IL-1␤ production. DR was useful for preventing both the postmenopausal osteoporosis and osteoporosis associated with the ovary function failure. DR extracts was also shown to be potent inhibitors of the degradation of denatured collagen by cathepsin K (cat K) and bone resorption in an in vitro model. Treatment of the DR extracts to cat K strongly inhibited cat K activity and bone resorption activity, suggesting that cat K involves the osteoporosis pathway. The naturally occuring DR which contains phenolic compounds, possesses estrogenic activity [14]. Thus, it was suggested that DR influences the bone tissue to heal promptly, and stimulates directly the bone formation [13,14]. But, much more scientific research on this mechanism is needed. This study focuses on the direct cellular-level effect of DR on bone cells. It was tested whether DR stimulates the proliferation and protein production, particularly type I collagen synthesis of human osteoprecursor cells, a cell line of

osteoblast suited for in vitro culture. Whether different concentrations of DR can induce cytotoxicity during the course of culture was also examined. In addition, during the evaluation of immunomodulatory activity of native DR to identify plant-based immunomodulators, the ethanolic and water extracts of DR were found to stimulate the immune system. The present study was aimed at exploring the influence of DR the on humoral and cell-mediated immune responses and the phagocytic function of the cells of the reticuloendothelial system.

2. Materials and methods 2.1. Drynariae rhizoma, materials and chemicals DR was kindly supplied by the Oriental Medical Hospital of Dongguk University (Kyungju, Korea). Its identity was confirmed by a comparison with the descriptions of characteristics in the literature and authenticated based on its microscopic and macroscopic characteristics, and the appropriate monograph in Korea Pharmacopoeia [15]. Ten grams of DR was finely cut, and added to distilled water (100 ml) in a flask with a condensation apparatus on the top allowing evaporated steam to reenter the system and heated at 100 ◦ C for 24 h in an oil bath, using an electric hot plate as a heat source. After the solution cooled, precipitated residue was filtered out and put into water for a secondary extraction. The water extracts were mixed and evaporated to dryness under reduced pressure with a rotary evaporator at 40 ◦ C. The dried residue was dissolved in distilled water and 1% DR water extract (DRWE) was used for the experiments. The water-extract preparation is essentially described in our previous paper [14]. For the ethanolic extracts, DR was exhaustively extracted using 95% ethanol (1000 ml) in a Soxhlet extractor for 3 h. The extract was concentrated under reduced pressure and low temperature (40 ◦ C) to yield a syrupy mass (yield: 12.8%). An aqueous suspension of DR ethanolic extract (DREE, prepared in 0.1% sodium carboxymethylcellulose) was used for the experiments. The vehicle alone served as control. Previous phytochemical analysis of the DR has shown presence of a disubstituted tetrahydrofuran, caffeic acid-derivatives and steroids. All chemicals and laboratory materials were from Sigma (St. Louis, MO) or Gibco BRL (Grand Island, NY) unless otherwise stated. Drugs used were cyclophosphamide (Sigma Co., USA) and D-penicillamine (Nakarai Pure Chemicals, Kyoto, Japan). For dosing, the drugs were dissolved in saline. Tissue culture media and reagents, fetal bovine serum (FBS) were from Gibco (Chagrin Falls, OH). Human osteoprecursor cells (OPC-1) was described previously [14] and obtained as described by Winn et al. [16].

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2.2. Animals

2.5. Evaluation of alkaline phosphatase (ALP) activity

Inbred Swiss albino mice (between 18 and 25 g) were used. The animals were housed under standard conditions of temperature (23 ± 1 ◦ C), relative humidity (55 ± 10%) and 12/12 h light/dark cycles, and fed with standard pellet diet (Daehan Experimental Animals Ltd., Seoul, Korea) and tap water ad libitum.

Sheep red blood cells (SRBC) were obtained from Sigma Ltd., USA. SRBC, collected in Alsever’s solution, were washed three times with large volumes of pyrogen-free, sterile saline. Oxazolone was purchased from Sigma Chemical Co., USA.

Because OPC-1 cells produce strongly ALP-positive secretions, the activity of alkaline phosphatase was evaluated as another mark of bone cell proliferation. The activities of alkaline phosphatase were measured by the method of Ishaug et al. [17]. Briefly, on the fifth day of culture, 100 ␮l medium from each dish was taken out and mixed with 1 ml of pnitrophenyl phosphate solution (16 mM, Diagnostic Kit 245, Sigma) at 30 ◦ C for up to 5 min. The light absorbance at 405 nm of p-nitrophenol product formed as a result of ALP reaction on p-nitrophenyl phosphate was measured on a microplate reader (Bio-Tek Elx 800, Fisher Scientific, USA) and compared with serially diluted standards. The activity of the enzyme was expressed as nanomole of p-nitrophenol per minute per dish.

2.4. Osteoblasts isolation and culture

2.6. Cell harvest and protein extraction

Cells were grown to confluence in 75 cm2 culture flasks (Falcon) in Dulbesso’s modification of Eagel’s medium (DMEM) supplemented with antibiotics (penicillin and streptomycin) and 10% fetal calf serum (FCS; Gibco, BRL, Bethesda, MD, USA). Incubations were carried out at 37 ◦ C in a humidified atmosphere of 5% CO2 /95% air; the medium was changed every 2–3 days. Cells were grown to confluence at 37 ◦ C and split in duplicate or triplicate wells for an additional 24 h in serum-free medium supplemented with Polymixin B sulfate to prevent endotoxin effects prior to treatment. Initially, human osteoprecursor cell line (OPC-1) was cultured in 250 ml tissue culture flask (Becton Dickinson, England) without the interference of herbal extract using 15 ml F12 tissue culture medium with 10% fetal calf serum and antibiotics (1% penicillin and streptomycin) at 37 ◦ C with 5% CO2 and 95% air. The medium was changed on the third day. The cells showed complete adhesion to the bottom of the flask after 2 days. On the seventh day of culture, the cells were harvested with 5 ml Trypsin-EDTA (Gibco, USA) and diluted with 35 ml of fresh medium. Newly harvested OPC-1 cells were split into culture dishes (ϕ = 8.5 mm) for subculture under the influence of DRWE. Each dish consisted of 2 ml of harvested cells, 0–800 ␮l of 1% DRWE and a corresponding amount of F12 medium so the total volume of culture medium was 8 ml with a plating cell density 1 × 105 cells ml−1 . For the control group, 800 ␮l of distilled water and 5.2 ml of F12 medium were used without DRWE. Three sets of eight samples with final DR concentrations 0, 12.5, 31.3, 62.5, 125, 250, 500, 750 and 1000 ␮g ml−1 were examined. The cells were cultured in the conditions described above for 5 days. The growth of cells was monitored under a light microscope every 12 h and the cell numbers in each dish were counted via microscopic observation. The cells were fixed in 2.5% paraformaldehyde and 2% glutaraldehyde in PBS for 30 min and microscopic pictures were taken on the fourth day.

After 5 days of culture (approximately 120 h), the cells in each dish were harvested with 2.5 ml trypsin. After the cells completely detached from the bottom of the dish, the mixture was diluted with 6.5 ml of F12 medium and transferred to 10 ml tubes to be centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were rinsed once with 1× phosphate buffer solution (PBS: 8 g NaCl, 0.2 g KCl, 0.14 g Na2 HPO4 , 0.24 g KH2 PO4 in 1.0 l of H2 O; pH 7.3). Each tube was added 200 ␮l of lysis buffer/CLAP solution (Lysis buffer: 0.187 g HEPES, 0.4235 g NaCl, 0.001 g MgCl2 and 0.19 g EGTA dissolved in 50 ml PBS. CLAP solution: 4 ␮l each of chymostatin, leupeptin, antipain and pepstatin A in 100 ␮l PBS. Lysis buffer/CLAP solution: 100 ␮l CLAP solution added to 6.6 ml Lysis buffer). To assure complete rupture of the cells, the tubes were stored in −20 ◦ C for 12 h. Supernatants after centrifugation of the lysed cells were taken. Total protein concentration was quantified using the bovine serum albumin (BSA) protein assay kit (Fisher Scientific), which measured the light absorbance at 562 nm on a microplate reader.

2.3. Antigens

2.7. Western blot analysis for type I collagen and cyclooxygenase-2 (Cox-2) The protein was obtained as described above and the sample volume that would contain 15 ␮g total protein was calculated according to the protein concentration. The samples with 15 ␮l loading buffer (loading buffer: 2.4 ml of 1 M Tris–HCl; pH 6.8, 3 ml of 20% SDS, 3 ml of 10% glycerol, 1.6 ml of ␤-mercaptoethanol and 6 mg bromophenol blue) were then boiled at 100 ◦ C for 3 min and subjected to sodium dodecyl sulfate-polyacrylamide 10% gel electrophoresis (SDS-PAGE) for 110 min at 125 V [18,19]. Electrophoresed proteins were transferred onto Imobilon-P membrane (Nippon Millipore) using a semidry blotting apparatus (Sartorius, USA) for 60 min at 2.0 mA/cm2 . The membrane

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was rinsed with deionized water, placed into 1× PBS-Tween containing 5% fat-free milk, 1% fetal bovine serum (FBS) to shake overnight. To ensure that equal amount of total protein was loaded and transferred to the membrane, glycerealdehyde3-phosphate dehydrogenase (GAPDH) was detected using rat-anti-human GAPDH antibody (ICN Biochemicals, USA) followed by peroxidase-conjugated rabbit-anti-rat IgG antibody (E.Y. Laboratories, USA). Next, Western blotting membranes were prepared in the same method as above for the detection of type I collagen and cyclooxygenase2 (Cox-2), a dioxygen and peroxidative enzyme acting as the inflammatory factor of cells which was tested here for the cytotoxicity of DR. Type I collagen was detected using goat-anti-human collagen I antibody (ICN) followed by peroxidase-conjugated rabbit-anti-goat IgG antibody (E.Y. Laboratories). Cox-2 was detected using mouse-anti-human Cox-2 antibody (ICN) and goat-anti-mouse IgG antibody (E.Y. Laboratories). Molecular weights of the proteins were determined using prestained molecular weight standards (14,300–200,000 molecular weight range; Gibco BRL). The lanes were scanned by DensitoScanner CBS (Core Bio System, Seoul, Korea) and the intensity of the protein bands were analyzed using NIH Image software (Wayne Rasband, National Institute of Health, USA). 2.8. Delayed type hypersensitivity (DTH) reaction using SRBC as an antigen The method described by Doherty [20] was used. Mice of either sex were divided into six groups of six each. DREE (50, 100 and 200 mg kg−1 , p.o.), DRWE (10, 20 and 50 mg kg−1 , p.o.) and d-penicillamine (20 mg kg−1 , p.o.) were administered on day 0 and continued until the day of challenge. The mice were primed with 0.1 ml of SRBC suspension containing 1 × 108 cells, i.p., on day 7 and challenged on day 14 with 0.05 ml of 2 × 108 SRBC in the right hind foot pad. The contralateral paw received equal volume of saline. The thickness (diameter) of the foot pad was measured at 0, 23, 24, 25, 48 and 72 h after challenge using Mitutoyo Dial Caliper (Mitutoyo manufacturing Company, Japan). Measurement was made with a dial thickness gauge (Ozaki Sangyou, Tokyo, Japan) and the results were expressed as the difference in thickness. Also, the percentage of swelling is calculated by the following formula: (the thickness of footpad after the boosting)/(the thickness of foodpad before the boosting) × 100%. The difference in the thickness of the right hind paw and the left hind paw was used as a measure of DTH reaction. 2.9. DTH reaction using oxazolone as an antigen The method described by Griswold et al. [21] was used. Mice of either sex were divided into six groups of six each. The treatment schedule was similar to that of SRBC-induced DTH reaction. On day 7, mice were sensitized by applying 0.1 ml of 3% oxazolone in absolute ethanol to the skin of

shaved abdomen. They were challenged on day 14 for contact hypersensitivity using 0.1 ml of 3% oxazolone in absolute ethanol by applying it to both sides of the right hind paw, the contralateral paw received equivalent volume of ethanol. The thickness of the foot pad was measured at 0, 23, 24, 25, 48 and 72 h as described earlier. 2.10. Humoral antibody response to SRBC Mice of either sex were divided into four groups of six each. DREE (50, 100 and 200 mg kg−1 , p.o.) and DRWE (10, 20 and 50 mg kg−1 , p.o.) was administered on day 0 and continued until the day of the experiment. Cyclophosphamide (50 mg kg−1 , p.o.) was administered 2 days before the experiment. On day 7, the mice were immunized with 0.1 ml of 1 × 108 SRBC, i.p. Blood samples were collected from the orbital plexuses of individual animals on day 14 and the antibody titres were determined using the method described by Puri et al. [22]. Briefly, an aliquot (25 ␮l) of 2-fold-diluted sera in saline was challenged with 25 ␮l of 0.1% (v/v) SRBC suspension in microtitre plates. The plates were incubated at 37 ◦ C for 1 h and then observed for haemagglutination. The highest dilution giving haemagglutination was taken as the antibody titre. The antibody titres were expressed in a graded manner, the minimum dilution 1/2 being ranked as 1. The mean ranks of different groups were statistically compared. 2.11. Macrophage phagocytosis by carbon clearance method Mice of either sex were divided into four groups of six each. DREE at doses of 50, 100 and 200 mg kg−1 , p.o. and DRWE (at doses of 10, 20 and 50 mg kg−1 , p.o.) was administered 15 days prior to injection of carbon particles. On day 16, mice were injected with 0.1 ml of carbon suspension (Pelikan Tuschea Ink, Germany), i.v., through the tail vein by Biozzi et al. [23]. Blood samples (25 ␮l) were collected from the orbital plexuses of individual animals immediately before and at 3, 6, 9 and 12 min after the injection of carbon suspension, lysed with 2 ml of 0.1% glacial acetic acid and the absorbance was measured spectrophotometrically at 675 nm by Atal and Sharma [24]. The rate of carbon clearance, termed as phagocytic index, was calculated as the slope of time–concentration curve. 2.12. Effect of chronic administration of DREE and DRWE on peripheral blood count The method used was as described by Ziauddin et al. [25]. Mice of either sex were divided into four groups of 10 each. The animals received DREE at the doses of 50, 100 and 200 mg kg−1 , p.o., and DRWE (at doses of 10, 20 and 50 mg kg−1 , p.o.) for 15 days. On day 16, blood was collected from orbital plexuses of the individual animals and total WBC, RBC, HCT, MCV and haemoglobin were determined using Erma PC-607 cell counter (Erma Inc., Japan).

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The differential leukocyte count was performed by fixing the blood smears and staining with Field Stains A and B, and percent neutrophils in each sample was determined. 2.13. Effects of DREE and DRWE on cyclophosphamide-induced immunosuppression The method used was as described by Ziauddin et al. [25]. Mice were divided into five groups, each containing 10 mice. Groups III, IV and V received DREE at doses of 50, 100 and 200 mg kg−1 , p.o., and DRWE (at doses of 10, 20 and 50 mg kg−1 , p.o.), respectively, for 15 days prior to administration of cyclophosphamide. Groups II–V received cyclophosphamide at a dose of 30 mg kg−1 , p.o., for the next 3 days. On day 19, blood was collected from retro-orbital plexuses of individual animals and white blood cell count was determined using Erma PC-607 cell counter (Erma Inc., Japan).

3. Statistics Data were obtained from three to five measurements and were expressed as the means ± S.D. The calculation for ALP activity and total protein concentration from optical density was performed on SPFT max Pro program (Molecular Devices, USA). Other statistical analyses were carried out on Microsoft Excel program. All quantitative data reported here are expressed as means of samples for each treatment with or without DR water extract. The results are also presented as means ± S.D. Statistical significance between the groups was analysed by student’s t-test. P < 0.05 was considered to be statistically significant.

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control group (P < 0.05). The concentration of DR for the best cell growth was 120 ␮g ml−1 . On the other hand, at concentrations over 120 ␮g ml−1 , DR had suppressing effect on OPC-1 cells. Cell growth started to slow down compared to the control group from the second day of culture. This was most evident at the highest concentration of 120 ␮g ml−1 (P < 0.05). 4.2. Human osteoprecursor cells (OPC-1) on the 5th day of culture as affected by the DR concentrations As shown in Fig. 1, OPC-1 cells on the 5 day of culture in different culture were observed to be in different proliferations under the light microscope. In the control dish, the cells held high density and well-developed inter-cellular collagen networks. Cell density increased as the concentration of DR increased in the medium at first (0–120 ␮g ml−1 ), but decreased as the concentration of DR (higher than 120 ␮g ml−1 .) increased in the medium. Well-developed matrix connections were also observed in the DR concentration of 60 ␮g ml−1 (Fig. 1D). Occasionally, over-extended matrix networks were present in DR medium (Fig. 1E). However, generally, the shrinkage of cell body and many dead cells, as indicated by the bright spots, were evident in Fig. 1E, F and G. Precipitations of chemical component crystals from DR were also observed at higher concentrations. The cell growth was often irregular in higher concentrations of DR medium, showing both the crowded and completely empty spots on the dishes. When water was added to the medium instead of DR extract, the cells demonstrated less development of matrix connection compared to the dishes with the lower concentrations of DR (Fig. 1A, C and D) but had a higher cell density than the cells cultured in the medium with higher concentrations of DR water extract (Fig. 1A, E, F and G).

4. Results 4.1. The effect of DR on human osteoprecursor cells

4.3. Effect of DR on the activity of alkaline phosphatase secreted by the OPC-1 cells

The growth of human osteoprecursor cells (OPC-1) over the 5-day culture period is shown in Table 1. The control group cultured without DR expressed a steady increase and was confluent by the fifth day. At concentrations of 10, 30, 60 and 120 ␮g ml−1 , DR accelerated cell growth over the

Fig. 2 shows the effect of DR on the activity of alkaline phosphatase secreted by the OPC-1 cells. The regression line for ALP was y = 0.0149x+0.0118, where y-axis represented optical absorbance (OD) at 405 nm and x-axis represented the activity of alkaline phosphatase (nanomole of p-nitrophenol

Table 1 The effect of DR on the growth of human osteoprecursor cells (OPC-1) over the 5-day culture period, expressed here as the cell numbers in the culture dish Concentration of DR (␮g ml−1 ) 0

10

Cell proliferation (×105 cells per well) Day-1 20.4 ± 2.3 21.4 ± 3.6 Day-2 40.5 ± 5.3 43.5 ± 5.7 Day-3 60.3 ± 8.2 64.6 ± 3.7 Day-4 80.5 ± 6.7 85.5 ± 8.2 Day-5 100.5 ± 15.4 102.1 ± 9.3

30

60

120

250

500

24.6 ± 1.6 45.6 ± 3.4 70.3 ± 9.3 90.3 ± 10.8 103.2 ± 8.3

27.5 ± 4.2 50.3 ± 3.6 75.6 ± 8.3 93.2 ± 5.7 105.3 ± 9.3

30.3 ± 2.3 54.3 ± 6.3 80.3 ± 5.7 96.4 ± 7.2 106.3 ± 8.9

23.4 ± 1.4 41.4 ± 3.6 67.8 ± 7.8 86.3 ± 9.6 90.4 ± 9.5

20.2 ± 1.6 38.4 ± 4.3 55.3 ± 6.4 75.4 ± 8.2 81.2 ± 4.6

The cells were treated as described in Section 2 and the values are means ± S.D. of three samples.

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Fig. 1. Human osteoprecursor cells (OPC-1) on the 5 days of culture as affected by different concentrations of DR in the medium. The cells were treated and the figures taken as described in Section 2. Magnification: original 40× under light microscope. (A) Control; (B) 10 ␮g ml−1 ; (C) 30 ␮g ml−1 ; (D) 60 ␮g ml−1 ; (E) 120 ␮g ml−1 ; (F) 250 ␮g ml−1 ; (G) 500 ␮g ml−1 .

per minute). The correlation coefficient of the standard curve was 0.9989 (Fig. 2A). ALP activity was very low in the control medium where 800 ␮l of water was added in place of DR extract. At concentrations ≤120 ␮g ml−1 , DR stimulated the secretion of ALP enzyme to approximately 2.5 times higher than that in control medium (P < 0.05 for concentrations 10 and 31.3 ␮g ml−1 ; P < 0.01 for concentrations 60 and 120 ␮g ml−1 ). 4.4. Effect of DR on protein production of human osteoprecursor cells at the end of the 5-day culture period Fig. 3 showed the concentration of total intracellular soluble protein produced by OPC-1 cells at the end of 5-day culture period. When DR water extract was added to the

medium, protein production in the bone cells was increased. However, at the highest concentration of DR, 1200 ␮g ml−1 , protein production was reduced to 30% of that in control medium (P < 0.01) (data not shown). On the other hand, the cells grown in the medium with lower concentration of DR water extract synthesized and secreted more proteins than that in control medium (P < 0.05 for concentrations 34.2 and 67.2 ␮g ml−1 ). The highest protein production was observed in the cells grown in the medium with 120 ␮g ml−1 water extract of DR (P < 0.01). 4.5. Western blot analysis for type I collagen and Cox-2 protein expression in human osteopercursor Western blot analysis showed the influence of different concentrations of DR water extract on the growth of OPC-1.

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Fig. 3. Effect of DR on protein production of human osteoprecursor cells at the end of the 5-day culture period, expressed as the concentration of total protein (␮g ml−1 ). The values are means ± S.D. for different four samples.

4.7. Effect of DREE and DRWE on DTH reaction using oxazolone as an antigen DREE (50–200 mg kg−1 , p.o.) and DRWE (10, 20 and 50 mg kg−1 , p.o.) produced significant, dose-related increases in DTH reactivity in mice. A peak of edema was obtained at 24 h, after which it subsided. d-Penicillamine produced a significant inhibition of edema as compared to the control at 24 h (Fig. 6). Fig. 2. (A) Standard curve for the alkaline phosphatase activity showed the regression line of y = 0.0149x + 0.0118. In the equation, y represented optical absorbance (OD) at 405 nm and x was the activity of alkaline phosphatase (nanomole of p-nitrophenol per minute). The correlation coefficient of the standard curve was 0.9989. (B) Effect of DR on the alkaline phosphatase secreted by human osteoprecursor cells (OPC-1) at the end of a 5-day culture period, expressed as an amount of p-nitrophenol produced per minute per dish (8 ml of medium). The samples were treated as described in Section 2. The values are means ± S.D. of five samples.

GAPDH control showed an equal protein load (Fig. 4B). The expression of type I collagen was increased by the addition of DR in the medium until the concentration of 120 ␮g ml−1 and then declined (Fig. 4A). Under most treatments, the cells showed very low expression of Cox-2 protein. A higher expression of Cox-2 was seen at the highest DR concentration of 500 ␮g ml−1 (Fig. 4C).

4.8. Effect of DREE and DRWE on humoral antibody response to SRBC A significant, dose-related increase in humoral antibody titres was observed in mice treated with DREE (50–200 mg kg−1 , p.o.) and DRWE (10, 20 and 50 mg kg−1 , p.o.) (Table 2). DRWE was more superior than DREE for inducing humoral antibody response.

4.6. Effect of DREE and DRWE on delayed type hypersensitivity reaction using SRBC as an antigen DREE (50–200 mg kg−1 , p.o.) and DRWE (10–50 mg kg−1 , p.o.) produced significant, dose-related increases in DTH reactivity in mice. A peak of edema was seen at 24 h, after which it subsided. d-Penicillamine produced a significant inhibition of edema as compared to the control at 24 h (Fig. 5).

Fig. 4. Western blot analysis for GAPDH, type I collagen and Cox-2 protein expression in human osteopercursor cells at the end of 5th day culture period affected by the concentration of DR in medium. (A) Collagen type I; (B) GAPDH; (C) Cox-2. The result was one of three independent experiments. Each band intensity has been calculated densitometrically.

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Fig. 5. Effects of DREE (A) and DRWE (B) on SRBC (1 × 108 cells, i.p.)induced DTH at 22, 24, 26, 48 and 72 h. Percent edema for the control group was 36.5%. Each value represents the means ± S.D. of six observations. ∗ P < 0.05 (student’s t-test).

the macrophage phagocytic activity as evident by the phagocytic index (Table 2).

4.9. Effect of DREE and DRWE on macrophage phagocytosis by carbon clearance method DREE (50–200 mg kg−1 , p.o.) and DRWE (10, 20 and 50 mg kg−1 , p.o.) did not show any significant potentiation of Table 2 Effects of the DREE and DRWE on humoral antibody titre and macrophage phagocytosis Treatment

Dose (mg kg−1 , p.o)

Humoral antibody titre

Phagocytic index

– 50

5.6 ± 0.35 4.1 ± 0.43∗

0.054 ± 0.04 –

DREE

50 100 200

6.3 ± 0.65 7.8 ± 0.73∗ 8.5 ± 0.56∗

0.056 ± 0.03 0.057 ± 0.04 0.058 ± 0.03

DRWE

10 20 50

7.1 ± 0.6∗ 8.0 ± 0.5∗ 8.7 ± 0.3∗

0.062 ± 0.04 0.063 ± 0.03 0.058 ± 0.04

Control Cyclophosphamide

Values are expressed as means ± S.D. of six observations. ∗ P < 0.05 as compared to control.

Fig. 6. Effects of DREE (A) and DRWE (B) on oxazolone (0.1 ml of 3% oxazolone)-induced DTH at 22, 24, 26, 48 and 72 h. Percent edema for the control group was 24.25%. Each value represents the means ± S.D. of six observations. ∗ P < 0.05 (student’s t-test).

4.10. Effect of DREE and DRWE on peripheral blood count A significant, increase in the WBC count was observed in mice treated with DREE (50–200 mg kg−1 , p.o.) and DRWE (10, 20 and 50 mg kg−1 , p.o.) for 15 days. However, no significant changes were observed in the other haematological parameters. Also, neutrophils were increased significantly (Table 3). 4.11. Effect of DREE and DRWE on cyclophosphamide-induced immunosuppression Cyclophosphamide at the dose of 30 mg kg−1 , p.o. caused a significant reduction in the white blood cell count. Combined treatments of cyclophosphamide, and DREE or DRWE resulted in the restorations of bone marrow activity as compared with ones of cyclophosphamide treatment alone

J.-C. Jeong et al. / Pharmacological Research 51 (2005) 125–136 Table 3 Effect of prolonged administration of DREE and DRWE on peripheral blood count Treatment

Dose

RBC (×106 cells ␮l−1 )

WBC (×103 cells ␮l−1 )

Neutrophils (%)

–

10.23 ± 0.43

12.45 ± 1.56

23.65 ± 4.6

DREE

50 100 200

9.65 ± 0.51 11.12 ± 0.46 10.43 ± 0.65

15.32 ± 2.34∗ 15.87 ± 1.23∗ 16.55 ± 3.24∗

26.43 ± 2.1 26.95 ± 6.3 36.76 ± 4.3∗

DRWE

10 20 50

10.12 ± 0.64 10.45 ± 0.52 11.01 ± 0.57

15.72 ± 5.04∗ 16.68 ± 2.03∗ 16.53 ± 3.04∗

26.35 ± 6.3 35.34 ± 3.5∗ 38.52 ± 5.4∗

Control

Values are expressed as means ± S.D. of six observations. ∗ P < 0.05 as compared to control.

(Table 4). Observations for the recovery period indicated that cyclophosphamide-induced immunosuppression was not restored completely back to the normal, even after discontinuous use of the cyclophosphamide. It was observed that the animals treated with DR showed more normal activity as compared to the cyclophosphamide-treated group. 5. Discussion Traditional Korean and Chinese medicines, which have been developed over the period of 3000 years, are known to have low toxicity, and may offer advantages for a longertime usage over the synthetic agent medication [7]. Although the preventive mechanism of these agents remains to be explained, this initial study does show that DR may be effective for the gynecological diseases [8,9] and also can be administered for the prevention of osteoporosis. DR frequently appears in the traditional prescriptions for bone and tendon injuries. For example, 56 of the 73 fracture prescriptions contain DR as one of the main ingredients, as collected in the Encyclopedia of Esoteric Prescriptions in Traditional Chinese Medicine [26]. Clinical data has shown that these prescriptions had significantly reduced the time needed for the injured bones to heal. When the Golsebo-Tang, mainly consisting of DR, was applied to 112-closed fracture cases of people raging from age 1 to 40, the patients regained health

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on average in 31.6 days, much shorter than the normal healing time of 8–10 weeks. Medicines prepared with water or wine stir-baking technique for oral intake also yielded similar results. X-ray images presented the formation of new bone tissue at fracture sites within 7–10 days of injury. This report further proved the advantage of traditional prescriptions over conventional Western surgical treatment [11]. Recently, Lee [13] investigated weeks whether DR could prevent the progression of bone loss induced by ovariectomy in rats. As a result, the bone mineral density of tibia in ovariectomized rats decreased by 22% from those in sham-operated rats, with the decrease completely inhibited by DR or 17␤-estradiol. When DR or 17-␤-estradiol was administered to ovariectomized rats, the fine particle surface of the trabecular bone was normally preserved. DR strongly inhibited PGE2 and LPS-stimulated IL-1␤ production. Pretreatment of DR also suppressed the IL-1␤ production. DR was as effective as 17-␤-estradiol in preventing the development of bone loss induced by ovariectomy in rat and he suggested that DR is effective for anti-bone resorptive action in bone cells [13]. DR is likely to directly stimulate the bone formation, but scientific mechanisms were not well understood and elucidated. Thus, we have done this study. From quantitative and morphological observation on the human osteoprecursor cells during the 5-day culture period, this study suggested that low concentrations of DR had an effect in accelerating the proliferation of bone cells. In contrast, at high concentrations of 250–500 ␮g ml−1 , the addition of DR to culture medium had suppressive effect on the cell proliferation and differentiation. These results also suggested that lower dose of DR increased the total amount of protein produced in OPC-1 cells and stimulated the production of alkaline phosphatase and type I collagen, two important proteins synthesized by bone cells, particularly osteoblasts, during osteogenesis, the formation of bone. Human bone is composed of mineralized organic matrix and bone cells. Osteoblasts are the active mature bone cells that synthesize the organic matrix and regulate its mineralization. Osteogenesis starts with osteoblasts producing and secreting type I collagen, which makes about 90% of the organic bone matrix, or the osteoid. Osteoblasts also

Table 4 Effects of prolonged administration of DREE and DRWE on cyclophosphamide (30 mg kg−1 , p.o.) induced immunosuppression RBC (×106 cells ␮l−1 )

WBC (×103 cells ␮l−1 )

Neutrophils (%)

– –

10.54 ± 0.87 10.02 ± 0.32

12.65 ± 3.54 5.43 ± 2.32∗

17.43 ± 2.3 11.89 ± 1.5∗

DREE + CY

50 100 200

9.45 ± 0.32 10.32 ± 3.41 9.87 ± 1.23

5.83 ± 1.44∗ 8.04 ± 2.32# 8.54 ± 1.37#

14.98 ± 2.1 16.12 ± 1.7 16.72 ± 2.4

DRWE + CY

10 20 50

10.32 ± 0.64 10.54 ± 1.32 10.32 ± 0.54

7.63 ± 2.04# 8.73 ± 1.56# 8.87 ± 2.34#

17.32 ± 5.2 18.84 ± 3.4 18.23 ± 3.7

Treatment Control Cyclophosphamide (CY)

Dose

Values are expressed as means ± S.D. of six observations. ∗ P < 0.05 as compared to control. # P < 0.05 as compared to cyclophosphamide control.

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become high in alkaline phosphatase, a phosphate-splitting enzyme. Alkaline phosphatase is released into the osteoid to initiate the deposit of minerals. Calcium hydroxyapatite, Ca10 (PO4 )6 (OH)2 , which comprises 70% of the bone mass, crystallizes along the cavities in the three-dimensional collagen fibrils. After mineralization, the complete bone becomes hard and rigid with necessary mechanical properties to withstand the external forces, to support the body and to protect the internal organs [27]. Three components are thus essential to bone formation: bone cells, type I collagen production, and sufficiency of mineral deposits. Therefore, on the micro scale, in an environment with adequate supply of calcium, phosphorus and other mineral elements, the proliferation and activity of osteoblasts control the speed of bone formation. Accelerated osteoblast growth and protein synthesis are the key factors for efficient bone repair. While DR at low concentrations have prevented the depletion of cell density, shrinkage of cell body, and increased total protein concentration and Cox-2 expression, DR above 120 ␮g ml−1 caused slight cytotoxicity to OPC-1 cells during in vitro culture. Probably, some chemical components of DR dissolved in the water extract reached the maximum limit of safe concentration and damaged the cells. DR contains several organic compound reported to be toxic to the cell development, mainly phenol and furocoumarine groups which were present in the water extract used in this study as indicated by the infrared spectra. It is then a contradictory phenomenon that the activity of alkaline phosphatase and the production of type I collagen were the highest at 120 ␮g ml−1 of DR concentration. One possible explanation is that DR at low concentrations might be a stimulant for the cells to generate the specific proteins. However, DR at high concentrations might not have this effect. So that the protein productions were close to those expressed by the cells cultured in control medium. High dose of DR may inhibit the proliferation of the cells. Another factor that must be considered in evaluating the results is that the water extract of DR contained 99% distilled water. It occupied 0.125–10% of the total volume of culture medium and might have changed the osmotic gradient in the cells environment compared to the culture medium alone. For this reason, a control group to which 800 ␮l of distilled water was added instead of DR extract was added. The results suggest that water was indeed another stimulant for the cells. It significantly increased the protein production, collagen type I formation and ALP activity in bone cells until the concentration of 120 ␮l ml−1 . It also had a stimulating effect on collagen I synthesis. Because at higher DR concentrations, the cells behaved in a very different, sometimes even the opposite manner, compared to the cells cultured in the medium with water, possibly DR had overcome the effect of water and acted upon the cells by the stimulating factors it had. To clarify this point, when extraction of DR was done with PBS that is isoosmotic to the cells, the same results as the water control have been observed (data not shown).

This research introduced some insights to the effect of DR on human bone cells. Since water extract of DR improved the activity of alkaline phosphatase, a messenger initiating the calcification, it is possible that DR accelerated mineralization of the organic matrix, thus speeding up bone formation. DR not only enhanced total protein production significantly but also increased type I collagen synthesis, increasing the portion of collagen in total protein. Usually, prescriptions of traditional Chinese medicine consisted of three to ten medical substances; although only one or two are responsible for the central effect, the supplemental ingredients are also important to achieve the goal of remedy. Therefore, some effects of DR may only be seen in combination with other medicines, which may simultaneously lessen its cytotoxicity. Typical length of clinical treatment for bone injuries with DR ranges from a week to a couple of months. Some long-term effects of DR may not have been revealed in the short culture period of this study. In addition, the concentration of DR may change once the medicine is taken into the body because of compound-binding proteins. The concentrations used in this study were only for in vitro experiments. Whether the effect of DR will change in vivo is not known. Further investigation is planned to examine these possibilities. Therefore, the need for safer and effective anti-inflammatory drug and the lack of enough scientific data to support the claims written in ancient literature have prompted the present study. This result also suggested that DR is effective for anti-bone resorptive action in bone cells. DTH is a part of the process of graft rejection, tumor immunity, and most importantly, immunity to many intracellular infectious microorganisms, especially those causing chronic diseases such as tuberculosis [28]. DTH requires the specific recognition of a given antigen by activated T lymphocytes, which subsequently proliferate and release cytokines. These in turn increase vascular permeability, induce vasodilatation, macrophage accumulation [29] and activation, promoting increased phagocytic activity and increased concentrations of lytic enzymes for more effective killing [30]. In the present study, SRBC and oxazolone, a chemical sensitizer, which in combination with skin proteins acquires antigenicity [31], were used to elicit contact hypersensitivity reaction in mice. It was found that DREE and DRWE potentiated the DTH reaction induced by both SRBC and oxazolone. Increase in DTH reaction in mice in response to thymus-dependent antigen by the stimulatory effect of DREE and DRWE might indicate the involvement of T lymphocytes and accessory cell types, which are required for the DTH reaction [32]. The humoral immunity involves the interaction of B cells with the antigen and their subsequent proliferation and differentiation into antibody-secreting plasma cells. The antibody functions as the effector of the humoral response by binding to antigen and neutralizing it or facilitating its elimination by cross-linking to form clusters that are more readily ingested by phagocytic cells. To evaluate the effect of DR on humoral response, its influence was tested by measuring sheep erythrocyte-specific haemagglutination antibody titre

J.-C. Jeong et al. / Pharmacological Research 51 (2005) 125–136

in mice. Cyclophosphamide at a dose of 30 mg kg−1 , p.o., showed a significant inhibition of antibody responses, while DREE and DRWE significantly enhanced the production of circulating antibody. This indicates that the enhanced responsiveness of macrophages, and T and B lymphocyte subsets might be involved in antibody synthesis [33]. The role of phagocytosis is primarily the removal of microorganisms and foreign bodies, and also the elimination of dead or injured cells. Phagocytic defects are associated with many pathological conditions in humans [34]. In view of the pivotal role played by the macrophages in coordinating the processing and presentation of antigen to B cells, DREE and DRWE were evaluated for its effect on the phagocytic activity of macrophage. When the carbon particles are injected intravenously, the rate of clearance of carbon from the blood by macrophage is expressed by an exponential equation. This seems to be the general way in which inert particulate matter is cleared from the blood. In this study, however, DREE and DRWE failed to show any effect on the phagocytic activity of macrophage. Since DREE and DRWE augmented the circulating antibody titre, it was thought worthwhile to evaluate its effect on peripheral blood count and cyclophosphamide-induced immunosuppression. Chronic administration of DREE and DRWE significantly ameliorated the total white blood cell count and also restored the myelosuppressive effects induced by cyclophosphamide. The present investigation suggests that DR may stimulate both the cellular and the humoral immunity. Further studies to elucidate the exact immunostimulatory mechanism of DR are needed.

Acknowledgments This study was in part supported by Dongguk University Research Fund and National Research Laboratory Program (2002–2007) from Korean Ministry of Science and Ministry.

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