Anti-inflammatory effects of Stephania tetrandra S. Moore on interleukin-6 production and experimental inflammatory disease models

Deregulation of interleukin-6 (IL-6) expression caused the synthesis and release of many inflammatory mediators. It is involved in chronic inflammation, autoimmune diseases, and malignancy. Stephania tetrandra S. Moore is a Chinese medicinal herb which has been used traditionary as a remedy for neuralgia and arthritis in China. To investigate the anti-inflammatory effects of S. tetrandra S. Moore in vitro and in vivo, its effects on the production of IL-6 and inflammatory mediators were analysed. When human monocytes/macrophages stimulated with silica were treated with 0.1–10 μg/ml S. tetranda S. Moore, the production of IL-6 was inhibited up to 50%. At these concentrations, it had no cytotoxicity effect on these cells. It also suppressed the production of IL-6 by alveolar macrophages stimulated with silica. In addition, it inhibited the release of superoxide anion and hydrogen peroxide from human monocytes/macrophages. To assess the anti-fibrosis effects of S. tetrandra S. Moore, its effects on in vivo experimental inflammatory models were evaluated. In the experimental silicosis model, IL-6 activities in the sera and in the culture supernatants of pulmonary fibroblasts were also inhibited by it. In vitro and in vivo treatment of S. tetrandra S. Moore reduced collagen production by rat lung fibroblasts and lung tissue. Also, S. tetrandra S. Moore reduced the levels of serum GOT and GPT in the rat cirrhosis model induced by CCL4, and it was effective in reducing hepatic fibrosis and nodular formation. Taken together, these data indicate that it has a potent anti-inflammatory and antifibrosis effect by reducing IL-6 production.


Introduction
Inflammation is initiated by the infiltration of inflammatory, immune and mesenchymal cells into the inflamed sites. Monocytes/macrophages involved in the influx of these cells into the inflamed site produce soluble mediators. Once these cells have entered the inflammatory sims, they begin to proliferate and initiate the synthesizing of inflammatory and fibrogenic factors such as collagen. The repair of tissue injury results in replacement of the original tissue by collagen. This collagen production not only serves to repair the damaged tissue, but may also cause damage to the organ. [2][3][4] Silicosis is an occupational lung disease resulting from the inhalation of silica dust which causes chronic inflammation and progressive pulmonary fibrosis. Inhaled silica particles are first ingested by alveolar monocytes/macrophages, which then release oxygen free radicals and lysosomal enzymes which damage lung tissue. 5 They also release pro-inflammatory cytokines which recruit more inflammatory cells to secrete these fibrogenic mediators inducing the proliferation of fibroblasts and collagen synthesis. 6'7 Hepatic fibrosis is one of the chronic inflammatory liver diseases. It is a complex process that involves the deposition of extracellular matrix components, activation of cells capable of producing inflammatory mediators, cytokines, and tissue remodelling. Unbalanced production of pro-inflammatory cytokines and inflammatory mediators has been 9 reported in chronic liver diseases.' In cirrhotic liver, levels of IL-1, TNF, and IL-6 are increased. TGF-[3 is also involved in collagen synthesis and chronic liver diseases.
As is well known, IL-6 is a regulatory factor which participates in the growth, differentiation and activation of cells. It is produced and secreted by various organ cells, and plays an important role in defensive mechanisms of the human body. 10 IL-6, first discovered in the culture supernatants of monocytes, has been reported to induce the production of antibodies by B cells. Since the successful cloning of the cDNA of IL-6,12 IL-6 has been reported to serve as a growth factor for B cell hybridoma and plasmacytoma, and as a factor participating in haematopoiesis. 4 Further, IL-6 has been reported to have the functions of stimulating the activation and growth of T cells; 15 inducing the acute phase response of liver cells; regulating cell differentiation in the nerve system; 17 stimulating the growth of keratinocytes; regulating bone metabolism; stimulating the growth of kidney mesangial cells; and inhibiting the growth of melanoma and breast cancer cells, etc. As has been reported, various diseases may be the results of the improper regulation of IL-6 production. Examples of the diseases repor- 18 19 ted are rheumatoid arthritis, hepatocirrhosis, psoriasis, 2 multiple mveloma, 2 cardiac myxoma, 22 AIDS 23 and other autoimmune diseases. These observations have buttressed the importance of regulating IL-6 production for the maintenance of the homeostasis of the immune system in the human body and for the treatment and prophylaxis of diseases. Numerous approaches have been proposed to regulate the production of interleukins. For instance, proliferation of myelocytes in a patient suffering from myeloma caused by an excessive secretion of IL-6 has been suppressed by emlloying antibodies against IL-6 or IL-6 receptor. -24 However, no substance or method has been reported to inhibit specifically the production of IL-6 and, therefore, there has still existed a need for the discovery of specific inhibitors against the production of IL-6.

S. japonica Miers and Sinomenium acutum
Rehd et Wils (Menispermaceae), which are found in the southern part of the Republic of Korea, have been used for a long time as an analgesic and anti-inflammatory agent. On the other hand, S. tetrandra S. Moore (Menispermaceae), which is not found in the Republic of Korea, has been used traditionally as remedies for neuralgia and arthritis, e.g. in China. Especially, the alkaloid tetrandrine has been used as an anti-inflammatory and anti-hypertensive agent. S. tetrandra S. Moore has been reported to have anti-phagocytic and anti-oxidizing effects, and to exhibit effectiveness in clinical and experimental silicosis 25 26 models and is known to have the ability to inhibit the production of interleukin-1 and tumour necrosis factor-a which are secreted by human monocytes. 27 '28 Tetrandrine and its derivatives are reported to promote brain function and have been developed as an antimalarial drug and also a stimulant for hair growth.
In this study, to investigate the potent antiinflammatory inhibitors, extracts from the root of S. tetrandra S. Moore were tested to suppress the production of IL-6 and inflammatory mediators. It successfully inhibited IL-6 production, and showed anti-inflammatory, anti-silicosis, and anticirrhotic effects in vivo and in vitro.

Materials and methods
Isolation of extracts of S. tetrandra S. Moore.. About 4.0 kg of well dried root of S. tetrandra S. Moore was chopped and extracted with about 51 of methanol for 2 days. The extraction procedure was repeated three times and the combined extracts were concentrated under a reduced pressure to obtain about 224 g of the methanol extract (Extract A) with a yield of 5.6%. Two hundred grams of Extract A was partitioned with 500 ml of 90% methanol and 500 ml of nhexane. The 90% methanol layer was separated and concentrated under reduced pressure to remove methanol. The residue was adjusted to pH 10 with 0.1 M NH4OH and partitioned with 600 ml of distilled water: CHiCl2(l:l(v/v)) mixture. The CHIC12 layer, i.e. the alkaloid fraction was then separated and concentrated under a reduced pressure to obtain about 25 g of Extract B with a yield of 0.6%. Extract C for use in a test for the treatment of silicosis was prepared as follows. One kilogram of dried root of S. tetrandra S. Moore was crushed into powder, sieved (60 mesh) and then suspended in distilled water in a concentration of 100 mg/ml. The resulting suspension was heated at 100C for 6 h and filtered. The filtrate was concentrated under reduced pressure to obtain 80g of water extract of S. tetrandra S. Moore (Extract C) with a yield of 8%, which was then stored at-20C. For the purpose of preparing Extract C for the treatment of hepatocirrhosis, 1113.5g of dried root of S. tetrandra S. Moore was introduced in a 31 round-bottomed flask equipped with a cooling apparatus with 21 of distilled water, and the mixture was heated at 95C for 12 h and then filtered. The filtrate was concentrated under a reduced pressure by employing a rotary vacuum evaporator (Buchi 451), frozen in a deepfreeze (Sanyo, Japan) at -84C for 3 h, and then lyophilized for 4h by employing a lyophilizer (Eyela, Japan) to obtain 56.55g of powdery Extract C with a yield of 5.1%. Further, 500 g of dried root of S. tetrandra S. Moore was extracted with about 1.51 of ethanol at room temperature for 3 mg/ml, BM, Indianapolis, IN, USA) and DNase days. The extraction procedure was repeated type I (0.15 mg/ml, Sigma), and incubated at three times and the combined extracts were con-37C for 2 h in a 5% CO2 incubator. Then, 0.5% centrated under reduced pressure to obtain 13 g trypsin-0.2% EDTA was added and the incubation of ethanol extract of S. tetrandra S. Moore with was continued for 30 min. The digested tissue a yield of 2.6% (Extract D). (1) Separation of human monocytes/macroand incubated for 1 week. Thereafter, synovial phages. Heparinized normal human peripheral adherent cells were isolated with trypsin-EDTA, blood was diluted with an equal amount of washed with DMEM and then suspended in Hank's balanced salt solution (HBSS: Ca 2+ and DMEM-5% FBS in a concentration of 105 cells/ Mg 2+ free). The diluted blood was put into a ml. centrifuge tube containing two layers of Ficoll-(4) Treatment of cells with extracts of S. tetra-.
Hypaque (Sigma, St Louis, MO, USA), one with a ndra S. Moore. Extracts of S. tetrandra S. Moore density of 1.077 and other with a density of were added in various concentrations to 1-5 x 1.119, and then centrifuged at 700 x g for 30 105/ml of cells which were obtained in the above min to obtain monocytes from the layer between procedures, and the cells were preincubated at Ficoll-Hypaque layer with a density of 1.077 and 37C for l h in 5% CO2 incubator. Then, l ml serum layer. The separated cells were washed each of silica (100 g/ml) and RPMI 1640 twice with 4C HBSS (Ca 2+ and Mg 2+ free) and medium containing 2% FBS were added and the resuspended in RPMI 1640 medium (Gibco, cells were cultured under the same conditions as Grand Island, NY, USA) containing 10% foetal above for 48 h. The culture supernatant was colbovine serum (FBS, Hyclone, Logan, UT, USA). lected and centrifuged at 1 500 rpm for 10 min to The suspensions were added to the wells of a remove the cells and silica. The obtained super-24-well incubation plate (Costar, Cambridge, MA, natant was dialyzed against PBS and filtered by USA) and incubated at 37C for 2h to obtain 0.2 l.tm filtration syringe, and the filtrate was monocytes/macrophages, stored at-20C.
(2) Separation of alveolar macrophages. Rat alveolar macrophages were obtained by broncho-Assay for cytotoxicities of Extracts of S. tetrandra alveolar lavage. 29 Rats were anaesthetized with S. Moore. The cytotoxicities of the extracts of S. ketamine and their alveolar macrophage were tetrandra S. Moore were determined by the folobtained therefrom by inserting a sterilized thin lowing procedures. Briefly, 5 x 105 cells/ml tube into the bronchia and repeating three times each of monocytes and macrophages were the injection of 10 ml of RPMI 1640 medium treated with 0.1, 1 and 10 l.tg/ml each of Extracts with a 30 ml syringe. The obtained cells were A and B obtained as described above and incucentrifuged at 400 x g for 5 min, suspended in bated under the same conditions. In accordance 50 ml of RPMI 1640 medium containing 10% FBS with the method of Alley et aL, the culture was and then incubated at 37C for 2h to adhere to added to the wells of the incubation plate, and the incubation plate. The plate was washed twice 100 btg of 3-4,5-dimethylthiazol-2,5-diphenyl-tetwith washing buffer (PBS) to remove alveolar razolium bromide (MTT, Sigma)was added to lymphocytes (floating cells) and to obtain alveo-each of the wells. After incubating at 37C for lar macrophages. 4 h, the culture was centrifuged to remove super-The alveolar macrophages (2 x 105 cells/ natant. One hundred each of acidified isowell) were added to the wells of a 24-well incu-propanol (0.04 N HCl in isopropanol) was added bation plate and treated with 100 l.tg/ml of silica to the cells in each well to elute formazan proand 10 gg/ml of Extract A for 3 days. The culture duced by the living cells, and optical density was centrifuged to obtain the culture super-(O.D.) was determined at 540 nm by using an natants, which were then dialyzed against PBS.
Cytokine assay.. TNF-z activity was measured by (3) Separation of synoviocytes from patients cytolysis of TNF-0t sensitive murine fibroblast cell with rheumatoid arthritis. Synovial membrane line, L929 cells (ATCC, CCL 1, NCTC clone 929). tissue of a patient suffering from rheumatoid In brief, 3 x 10 4 L929 cells were plated on a 96arthritis was washed three times with cool PBS well tissue culture plate. After 24h incubation, and cut into small pieces, which was then susmedia were removed and various dilutions of pended in Dulbecco's Modified Eagle's Medium samples or various concentrations of recombi-(DMEM; Sigma, USA) containing col!agenase A (5 nant human TNF-cz (Genzyme, Cambridge, MA; specific activity 2 x 107 U/mg) were added in tion (4M guanidinium thiocyanate, 25 mM duplicate to each well containing 1 btg/ml (final sodium citrate, pH 7.0, 0.1 M 2-mercapto-ethanol, concentration) of actinomycin D (Sigma)and 5% 0.5% sarcosine) to obtain RNA. To synthesize a FBS. After 18h incubation at 37C, media were single-strand cDNA from the RNA obtained removed and cells were washed three times and above, a reverse transcription reaction was stained with 0.5% crystal violet solution contain-carried out by employing M-MLV reverse traning 20% methanol. After washing off the excess scriptase (Promega, USA). Then, a polymerase dye, the plates were dried and 33% (v/v) acetic chain reaction (PCR) was carried out to amplify acid was added to each well (100 I.tl/well) and cDNA. Twenty l.tl of reverse transcription reacthe optical density at 570 nm was measured with tion, 8 l.tl of 10 x PCR buffer (100 mM Tris-HCl, an ELISA reader. TNF activity was expressed as pH 8.3, 400 mM KC1, 120 mM DTT, 15 mM U/ml from the extrapolation of the standard MgCl2, 5 btg/ml BSA), 1 l.tl (20 pmol) of 5'-end curve obtained using recombinant human TNF-a primer (5'-ATGAACTCCTTCTCCACAAGCGC-3'), (Genzyme). The activity of IL-6 was determined 1 !1 (20 pmol) of 3'-end primer (5'-GAAGAGby using an IL-6 dependent B9.55 hybridoma cell CCCTCAGGCTGG-ACTG-3'), 69 l.tl of distilled line. B9.55 cells were cultured in RPMI 1640 water and 1 I.tl (2.5 U)of Taq DNA polymerase medium containing 10% FBS with the addition of (Promega, USA) were mixed well and the 2 U/ml of recombinant human IL-6, and the cells mixture was incubated at 95C for 5 min. The were washed three times with serum-free PCR was carried out by repeating 30 times the medium. The cells were suspended in RPMI 1640 thermal cycle consisting of 95C for 1.5 min; medium containing 10% FBS in a concentration 55C for 1 min; 72C for 1.5 min, and the reacof 5 X 10 4 cells/ml, and the suspension was tion mixture as consequently incubated at 95C added to the wells of a 96-well incubation plate for 1.5 min; at 55C for 1 min; and at 72C for 5 in an amount of 100 btl/well. Then the plate was min. Ten of the PCR product was subjected to incubated at 37C under 5% CO2 for 68 h. an electrophoresis on an 1.0% agarose gel at 100 [H]thymidine (0.5 Ci)was added to the wells volts for 30 min. The gel was stained in EtBr in the amount of 50 l.tl/well and incubation was solution for 10 min, washed with distilled water continued for 4h. When the incubation was and photographed. completed, the cells were collected on a glass fibre filter by using a multiple cell harvester IL-6 CAT assay.. The plasmid IL-6-CAT was con-(Inotech) and the amount of incorporated structed by fusing IL-6 promoter (from-1180 to [3H}thymidine was determined by a liquid scintil-+ 13) and the bacterial CAT transcription unit. 31 lation counter (Beckman, Somerset, NJ, USA). On the day before DNA transfection, HeLa cells IL-6 ELISA was performed using IL-6-specific were plated at a density of 5 x 105 cells per 60 monoclonal and polyclonal antibodies obtained mm Petri dishes in DMEM supplemented with from Genzyme (Cambridge, MA). IL-1 activity was 5% FBS. On day 2, 3 h before transfection, fresh determined using thymocyte costimulation assay DMEM containing 1% FBS was added and cells as described. Briefly, thymocytes were prepared were transfected by the calcium phosphate-DNA from female CH/HeJ mice and resuspended in co-precipitation method with a total of 8 btg of IL-RPMI containing 10% FBS and 2 t.tg/ml phytohae-6-CAT DNA. After 18h, cells were washed with magglutinin. 1 x 10 cells (100 tl) were plated serum-free medium and added with fresh DMEM on a 96-well tissue culture plate and various dilucontaining 1% FBS. Then cells were further incutions of samples (100 btl) were added to each bated for 18h in the presence of 10 l.tg/ml of well. After 44h incubation, cells were labelled LPS plus 100 ng/ml of PMA and/or 10 l.tg/ml of with 0.5 l.tCi/well [3H]thymidine for 4h. One unit EXT.B. The cell extracts were heat-inactivated at of cytokine was defined as the amount inducing 65C for 10 min and centrifuged and assayed at the half maximal proliferation. 37C for 2h with the equal amounts of protein (20 l.tg) as measured by using Bio-Rad protein assay kit. The CAT expression was detected by IL-6 gene expression.. The synoviocytes (1.5 x TLC and autoradiography. 10 cells/well) were incubated at 37C under 5% CO. for 24h until it adhered to the wells. One Collagen synthesis: Collagen released from the tg/ml or 10 l.tg/ml of Extract B was added to the fibroblast cells was measured by indirect ELISA wells and the plate was incubated at 37C under using anti-type I collagen antibody (Southern 5% CO2 for I h and stimulated with 100 btg/ml Biotechnology). Collagen synthesized in the lung silica for 12h. Then, the culture medium was tissue of the rats was assayed by measuring the removed, and the cells were washed with PBS, amount of hydroxy proline. 2 To measure the disrupted by adding 500 1 of denaturating soluamount of synthesized collagen by rat pulmonary Mediators of Inflammation Vol 5 1996 283 fibroblasts, collagen (Sigma, type I) as an internal control group was dissolved thoroughly in 1 M acetic acid containing 1 mg/ml of pepsin, and the solution was serially diluted 5-fold with coating buffer (0.05 M carbonate, pH 9.6) in a concentration ranging from 1 g to 16 pg. The diluted solutions were added to the wells of a fiat-bottomed microtiter plate (Dynatech, Cantilly, VA, USA, Immulon 2) in the amount of 100 tl/ well. In addition, 1 ml of the culture supernatants of rat pulmonary fibroblast were concentrated up to 10to 20-fold using a speedvac dryer (Savant, Hicksville, NY, USA) and dissolved in 100 btl of coating buffer (0.1 M NaHCO3, 0.02% NAN3; pH was adjusted to 9.6 with NaiCO), and the solution was added to the wells in an amount of 100 btl/well and then incubated at 4C overnight. The plate was washed three times with washing buffer (PBS, 0.05% Tween 20, pH 7.4), and 2% bovine serum albumin (BSA, Sigma) was added to the wells in an amount of 100 l.tl/well. The plate was incubated at room temperature for 2 h to block the uncoated parts. The plate was washed four times with the same buffer as above, and alkaline phosphatase-conjugated rabbit anti-goat IgG (Cappel, Dunham, NC, USA) which was 1 000-fold diluted with a dilution buffer (0.05 M Tris-HCl, 1 mM MgCli.6H20, 0.15 M NaC1, 0.02% NAN,, 1% BSA, 0.05% Tween 20, pH 8.1) was added to the wells in an amount of 100 Il/well. The plate was incubated at 37C for 2h and then washed three times with the same buffer. To the wells was added 100 l.tl/well of /nitrophenyl phosphate which was diluted with substrate buffer (0.05 M NaHCO, 10 mM MgCl2"6H20, pH 9.8) in a concentration of 1 mg/ml, and the O.D. was determined using an ELISA reader at 405 nm. The amount of produced collagen was calculated from the O.D. value with reference to that of the internal control group.
Production of reactive oxygen species: The amount of H202 was determined by a microassay employing a 96-well microplate as follows. 5 x 10 cells of human monocytes/macrophages were added to each well containing RPMI 1640 medium, and 25 btl of horseradish peroxidase (500 tg/ml; type II, Sigma) and 75 btl of phenol red (1 mg/ml) were added to each of the wells. Thereafter, the cells were treated with 10 l.tg/ml of Extract A for i h, stimulated with 100 t.tg/ml silica and then reacted at 37C for 60 min. When the incubation was completed, 3 M NaOH was added to the wells in an amount of 25 t.tl/well to stop the reaction and O.D. was measured at 620 nm by using an ELISA reader. The amount of H202 was determined by employing a standard curve prepared using the diluted standard H202 (Sigma).
For the purpose of measuring the amount of produced O2-, monocytes/macrophages suspended in RPMI 1640 medium in a concentration of 1 x 10 cells/800 btl was added to the wells of a 24-well plate, and 10 l.tg/ml of superoxide dismutase (SOD, Sigma) was added to the empty wells. The plate was stored at 37C for 2 min, and cytochrome C (3 mg/ml, Sigma) was added to the wells in a concentration of 100 l.d/well. The cells were treated with 10 l.tg/ml of Extract A for i h, and stimulated at 37C for 20 min by adding 100 l.tg/ml silica. The reaction was terminated by adding 1 mM N-ethylmaleimide (Sigma) to the wells and the culture was centrifuged at 1600 x g for 10 min to obtain the supernatants. The change of color caused by the reduction of cytochrome C was measured at 550 nm using an UVvisible spectrophotometer (Kontron Instrument, Milan, Italy). The amount of produced O2-was represented by the concentration of SOD which can suppress the reduction of cytochrome C in 1 x 10 cells for 20 min, by employing the extinction coefficient of cytochrome C (E550nm 1.83 x 10 4 mM cm-1).
Experimental silicosis and cirrhosis model.. To induce the experimental hepatocirrhosis in 4week aged male Sprague-Dawley rats, 0.1 ml/ 100 g of body weight of CC14 solution (50% CC14 + 50% corn oil)was injected intraperitoneally to the rats twice a week, and 0.2 ml each of Extract A, B, C or D was administered orally at the time of the injections of CC14 twice a week. After 13 weeks, each of the rats was anaesthetized with ether and blood samples were obtained from the heart to determine the levels of serum glutamicoxaloacetic transaminase (sGOT) and serum glutamic-pyruvic transaminase (sGPT).
For the pathohistological examination of the livers separated from the rats, the liver was fixed in 10% aqueous solution of neutral formalin and then embedded in paraffin. The embedded tissue was sectioned into 5 mm slices, stained with haematoxylin eosin and Masson's trichrome, and then observed under the microscope.
The cytotoxicity of the extracts of S. tetrandra S. Moore were determined as described Materials and Methods. Figure 1 shows the relative values of optical density of the sample with respect to the concentration of Extract A or B when the optical density of the control group which was not treated with Extract A or B is regarded as  Inhibition of lL-dproduction by extracts of S. tetrandra S. Moore: Human monocytes/macrophages were incubated with 0.1 to 10 Ig/ml of Extract A or B for i h and treated with 100 l.tg/ml of silica for 48 h. The culture was centrifuged to obtain the culture supernatants, which were then dialysed against PBS. IL-6 production was measured by IL-6 bioassay using B9.55 cell line. Then, Extract C (40 mg/injection) was administered orally, or murine IL-6 polyclonal antibody (250 lg/injection) was injected intravenously, into the silicosis rats twice per week for 17 weeks. IL-6 activities in the sara were determined as described in Materials and Methods. (E) The culture supernatants of pulmonary fibroblasts isolated from silicosis rats (D) were analysed for IL-6 activity.  Fig. 2B, it was observed that the IL-6 production by rat alveolar macrophages was also inhibited by Extract A. As blank controls for possible residual effects of compounds and silica on bioassay, the same concentrations of compounds and silica were mixed with fresh media and dialysed as the culture supernatants were prepared. These blank controls had no effects on bioassay (data not shown). The results of IL-6 bioassay were further confirmed by IL-6 ELISA (Fig. 2C). In this case, cells were treated with 10 txg/ml pokeweed mitogen (PWM) and 10 l.tg/ml extracts. Extract A and B showed dramatic inhibitory effects on IL-6 production. Next, the effects of S. tetrandra S. Moore on IL-6 production in vivo were analysed.

Mediators of Inflammation
In the experimental silicosis model, IL-6 activities in the serum (Fig. 2D) and in the culture supernatants of pulmonary fibroblasts isolated from the rats treated with compounds or anti-murine IL-6 monoclonal antibody (Fig. 2E) were determined as described in Materials and Methods. IL-6 activity was inhibited by injecting anti-IL-6 antibody as expected. And its activity was also inhibited by Extract C in both cases, indicating that the extracts of S. tetrandra S. Moore exhibited the inhibitory effects on IL-6 production in the animal silicosis models.
Suppression of IL-6 gene expression by extracts ofS. tetrandra S. Moore.. In addition, to confirm the effects of the extracts of S. tetrandra S.
Moore on the IL-6 gene expression, the effect of Extract B on synoviocytes which were obtained from the patients with rheumatoid arthritis caused by the overproduction of IL-6 s was analysed. As can be seen in Fig. 3A, the expression of constantly expressed adenine phosphoribosyl transferase (APRT) RNA was not influenced by Extract B, while IL-6 RNA expression was significantl inhibited by 10 g/ml of the Extract B.
When IL-6-CAT construcff" was used to see the effects of Extract B on IL-6 promoter activity, the IL-6 promoter activity induced by LPS plus PMA was suppressed about 60% by 10 l.tg/ml of the Extract B. However, acanthoic acid (A), another natural product which had no effect on IL-6 production, 34 did not inhibit the IL-6 expression (Fig. 3B,C). These results indicated that the extracts of S. tetrandra S. Moore suppressed IL-6 production at the transcriptional level, and further studies are required to know the exact suppressive mechanism of S. tetrandra S. Moore on IL-6 gene expression.
Inhibition of silicosis by extracts of S. tetrandra $.
Moore.. IL-6 is known as a cytokine which causes fibrogenesis and induces collagen synthesis in rat fibroblasts. 5 Based on the observation of the inhibitory effects on the IL-6 production, their were preincubated with 10 ig/ml Extract A for h and stimulated with 100 lg/ml silica. After 48h incubation, the culture supernatants were assayed for collagen production by ELISA using anti-collagen type antibody. (B) The experimental silicosis was induced as described in Materials and Methods. Then, Extract C (40 mg/injection) was orally injected into the silicosis rat twice per week for 17 weeks. The lung tissue (0.1 g) was hydrolyzed and assayed for collagen content by measuring hydroxy proline. Data represent the percentages of control (normal lung).
inhibitory effect on the collagen synthesis in rat pulmonary fibroblasts and pulmonary tissues was determined. As a result, it was observed that the amount of synthesized collagen was significantly decreased in the culture supernatants of rat pulmonary fibroblasts which were treated with 10 g/ml of Extract A compared with the control (Fig. 4A). Furthermore, in vivo effects of S. tetrandra S. Moore (Extract C) on the collagen synthesis in rat pulmonary tissues was tested by injecting 500 mg of silica and 40 mg of Extract C orally into the rats twice a week for 17 weeks, and then the amount of hydroxyproline was measured as described in Materials and Methods.
In Fig. 4B, the amount of collagen synthesized by rat pulmonary tissues from the rats treated with silica only (si) or treated with silica and dimethylsulphoxide (si+DMSO) increased about twice compared with the control, while the amount of synthesized collagen decreased by 50% in rat pulmonary tissue treamd with silica, DMSO  Inhibition of hepatocirrhosis by extracts of S. tetrandra S. Moore: Cirrhosis is another fibrotic disease in the liver. Anti-inflammatory effects of S. tetrandra S. Moore were analysed in the experimental cirrhosis model. When compared with the blood sample obtained from the rats treated with CC14, DMSO, and PBS, sGOT values of the blood samples obtained from the rats treated with Extract A or C did not decrease, but those of samples obtained from the rat treated with Extract D or B decreased by 20% and 40%, respectively (Fig. 6A). Further, sGPT in the blood samples obtained from the rats treated with Extract B decreased more than 60% (Fig. 6B).
In the liver of the rats administered with CC14 only (Fig. 7B), the nodule formation of hepatic lobules with the thickened fibrous bands was remarkable compared with the normal liver (Fig.  7A). In the liver of the rats administered with CC14 and Extract B (Fig. 7D), the fibrous bands surrounding the nodule of hepatic lobule were thinner than those of the liver obtained from the rats treated with CC14 only, and many nodules were incomplete. Also, the regenerative change of hepatic cells decreased compared with that of the liver obtained from the rats treated with CC14 only. In the liver of the rats administered with CC14 and Extract A (Fig. 7C), the suppressive effect on the hepatocirrhosis was not dominant as seen in the case of Extract B.

Discussion
Inflammation is a localized or systemic response induced by infection or injury to tissues. Right after exposure to injurious agents, monocytes and macrophages synthesize and release a variety of inflammatory mediators such as oxygen free radicals, proteases, platelet-activating factors, and proinflammatory cytokines. '2 They are major inflammatory mediators that modulate fibroblast proliferation, neutrophil function, and collagen synthesis. Once macrophages infiltrated into inflammatory sites, NADPH-oxidase in the membrane is activated to produce the oxygens. The oxygens are then reduced to superoxide anion by receiving an electron from NADPH, and then dismutated to hydrogen peroxide. These reaction oxygens then damage the tissues in the inflammatory sites. Therefore, an excessive quantity of these cytokines and free radicals caused tissue destruction in the inflammatory sites and they are involved in many immune diseases and inflammatory diseases. To inhibit their action, several protein antagonists such as IL-lra, monoclonal antibodies to proinflammatory cytokines, and these cytokine receptors have been developed to block acute and chronic inflammation. But these protein antagonists have some limitations such as stability and delivery to be used clinically as anti-inflammatory agents.
Two major aspects of the proliferation of fibroblasts and the production of collagen are the hallmarks of fibrosis. IL-6 has been known to be involved in this process in connection with other fibrogenic cytokines. To modulate IL-6 production, S. tetrandra S. Moore, known as antiinflammatory agent, was tested in different inflammatory systems. First, several aspects of IL-6 in chronic inflammation including silicosis, rheumatoid arthritis, and liver cirrhosis were evaluated. Significantly elevated levels of IL-6 activity were observed in silica-treated monocytes/macro-phages, and in the sera of CCl4-treated rats and the patients with rheumatoid arthritis, indicating that .L-6 is a key element in an inflammatory cytokine network. The culture supernatants of human monocytes/macrophages treated with S. tetrandra S. Moore contained far less IL-6 activity compared with those treated with silica alone. In addition, it suppressed the IL-6 gene expression, collagen synthesis, and the production of reactive oxygen from several inflammatory cells, suggesting that it had potent anti-fibrogenic effects. But it cannot be ruled out that S. tetrandra S. Moore may regulate the production of other proinflammator cytokines. Actually, Extract B of S. tetrandra S. Moore showed the inhibitory effects on IL-1 production (29.1% and 45% inhibition at the concentration of 1 I.tg/ml and 10 btg/ml, respectively) and on TNF<z production (5.2% and 18.6% inhibition at the concentration of 1 I.tg/ml and 10 l.tg/ml, respectively). The further fractionation of the extracts to identify the effective components inhibiting each cytokine production is now in progress.
To evaluate the in vivo effects of S. tetrandra S. Moore, two different experimental models, in which IL-6 is known to be involved, were tested. Silicosis is a chronic lung inflammation. By the exposure to stimuli, alveolar macrophages are activated and secrete many fibrogenic mediators. S. tetrandra S. Moore strongly suppressed the experimental silicosis induced by silica treatment. It reduced the collagen production, and granuloma formation and fibrosis in the lung.
Hepatocirrhosis is characterized by the fibrogenesis of the whole liver, complete disruption of liver parenchyma by the fibrous septa, and formation of regenerative nodules. It is derived mostly from a chronic hepatitis or chronic alcoholism, however, the precise causes are unknown. We previously reported that in vivo administration of IL-6 could induce the early stage of liver cirrhosis. 7 In a hepatocirrhosis patient, the amount of cytokines, e.g. IL-6 which is involved in the inflammation and fibrogenesis, is in an increased state. Based on its effects on IL-6 production, these inhibitory effects in vivo might be due to reduction of IL-6 and/or other inflammatory mediators induced by IL-6 such as reactive oxygens free radicals.
Various root extracts of S. tetrandra S. Moore which inhibit the production of interleukin-6, can be used for the preparation thereof and pharmaceutical compositions comprising extracts which are effective for the treatment of immune diseases caused by the overproduction of interleukin-6. Each of Extracts A, B, C and D exhibited an anti-inflammatory effect, inhibited the synthesis of collagen and the production of the reactive oxygen species and reduced GOT and GPT level in the serum. Therefore, they can be employed alone or in combination with each other in a pharmaceutical composition for the treatment of such immune diseases caused by an excessive production of IL-6 as rheumatoid arthritis, hepatocirrhosis, psoriasis, multiple myeloma, cardiac myxoma, and silicosis. These extracts seemed to have the similar effects on IL-6 production, but they showed some different effects on experimental animal models. The Extract B was most effective in suppressing serum GOT and GPT level, and hepatic cirrhosis. Preferably the Extract B may be applied for the treatment of inflammatory disease, arthritis and autoimmune hepatocirrhosis, but the subsequent studies are required to identify the active compounds as mentioned above. In summary, significantly elevated levels of IL-6 activity and concentration were observed in silica-treated human monocytes/macrophages, CC14-or silica-treated rats, and the patients with rheumatoid arthritis. In addition, IL-6 itself augmented fibrogenic events such as fibroblast or synoviocyte proliferation, and collagen synthesis. S. tetrandra S. Moore possesses the. ability to inhibit the production of IL-6 and inflammatory mediators in a concentration-dependent fashion. It was also effective in experimental silicosis and cirrhosis models. Considering its effects on cytokine regulation and in vivo effects, it can be used as an anti-fibrosis agent in the treatment of many inflammatory diseases. Further biological and immunological analysis of its isolated compounds can lead to the development of more specific anti-inflammatory therapeutics for the treatment of various immune diseases caused by an excessive production of IL-6 and other inflammatory mediators.