{"paper_id":"8f3cd62b-bc2c-4a4d-92cc-d58389f4e5ad","body_text":"This is an Open Access article distributed under the terms of the Creative Commons At-\ntribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) \nwhich permits unrestricted non-commercial use, distribution, and reproduction in any \nmedium, provided the original work is properly cited.\nCopyright © 2018 Korean Society of Exercise Rehabilitation http://www.e-jer.org pISSN 2288-176X\neISSN 2288-1778 731\n*Corresponding author: Ee-Hwa Kim  \n  https://orcid.org/0000-0003-0400-9056\nDepartment of Meridian and Acupoint, College of Korean Medicine,  \nSemyung  University, 65 Semyeong-ro, Jecheon 27136, Korea\nTel: +82-43-649-1348, Fax: +82-43-649-1702, E-mail: kimeh@semyung.ac.kr\nReceived: August 10, 2018 / Accepted: September 5, 2018\nEffect of type II collagen extract on immunosuppression \ninduced by methotrexate in rats\nEe-Hwa Kim1,*, Yong-Min Kim2, Jung-Ho Suh3 \n1Deptartment of Meridian and Acupoint, College of Korean Medicine, Semyung University, Jecheon, Korea\n2Department of Oriental Medical and Herbal Cosmetic Sciences, Semyung University, Jecheon, Korea\n3Gupup Incorporation, Seoul, Korea\nThis study investigated the effect of type II collagen extract on SD rats \nwith deteriorated immunity caused by methotrexate. The test samples \nwere dosed once a day for 28 days by gastric gavage at dosage 250 \nmg/kg and 500 mg/kg after methotrexate treatment, and the changes on \nbody weight, total blood leukocyte numbers, the percentages of B-cells, \nCD4+ T-cells and CD8+ T-cells in the blood and spleen were observed. \nThe changes on body weight, the total blood leukocyte numbers, the to\n-\ntal lymphocyte numbers in the spleen, the ratio of CD4+ and CD8+ \nT-cells in the blood and spleen were increased significantly in type II \ncollagen extract groups as compared with the control group. According \nto the above results, type II collagen extract has an effect of increasing \nimmune responses on rats with deteriorated immunity caused by meth\n-\notrexate. \nKeywords: Type II collagen extract, Methotrexate, CD4+ T-cells, CD8+ \nT-cells\nINTRODUCTION\nImmunity is a term derived from Greek immurus, which \nmeans exemption from heavy burdens or taxes. It can be regarded \nas an action that induces an appropriate bioreactor to effectively \nprevent external invasion. In Korean medicine, this immune effect \nis explained by nutritional and tonic effect. They are biological re-\nsponse modifiers that can directly or indirectly increase the de-\ncreased immune function. It is known to play an important role \nin maintaining homeostasis. This can be closely related to the im-\nmunological concept of modern medicine (Hong et al., 1999).\nThere are many immune cells in the body to perform immune \nfunction, and these cells are located in various tissues and organs \nof the body including blood, lymph nodes, spleen and gastroin-\ntestinal tract. Immune cell members include lymphocytes, mono-\nnuclear cells, polynuclear leukocytes and platelets. Among them, \nthe main cells responsible for the immune response are lympho-\ncytes and mononuclear cells.\nIn particular, the lymphocyte proliferative capacity is consid-\nered to be an immunological index as a result of cell division as \nwell as new DNA synthesis generated by various kinds of stimuli \nincluding various mitogens, antigens, cytokines, and growth fac-\ntors (Baek et al., 1996). Cytokines act as regulatory mediators of \ninnate immunity that stimulate or inhibit inflammatory respons-\nes, produced during the activation and specific stages of natural \nand specific immunity. And, they recognize specific antigens and \nare secreted by T cells (Lee et al., 2000; Lee et al., 2002). It is \nknown that the immune response plays an important role in the \nimmune response and the inflammatory response.\nType II collagen has been classically recognized as the indis-\npensable collagenous component in cartilage, and plays a critical \nrole in the development and maturation process of chondrocytes. \nThus, type II collagen has drawn more attention and interest in \nthe treatment and research of rheumatoid arthritis, osteoarthritis \nand auto immune diseases. Recently, some studies have reported \nthat type II collagen embedded with adipose-derived stem cells or \nchondrocytes can promote cartilage repair in a inflammatory car-\ntilage defect model in vivo (Lazarini et al., 2017). \nhttps://doi.org/10.12965/jer.1836480.240\nOriginal Article\nJournal of Exercise Rehabilitation 2018;14(5):731-738\n\nhttps://doi.org/10.12965/jer.1836480.240\nKim EH, et al.  •   Effect of type II collagen extract on immunosuppression\n732    http://www.e-jer.org\nTherefore, we investigated the effect of type II collagen extract \non the immune function. Methotrexate (MTX) was used to in-\nduce immunosuppression in rats, and type II collagen extract was \nadministered to measure the body weight, total leukocyte, B cell \nratio in the spleen, and the change of CD4+ and CD8+ T cell ra-\ntio in the spleen and blood were observed.\nMATERIALS AND METHODS\nAnimals and reagents\nThe animals were Sprague-Dawley male rats weighing 120±20 \ng and were fed a solid diet (21.1% crude protein, crude fat 3.5%, \ncrude fiber 5.0%, crude protein 8.0%, calcium 0.6%, phosphorus \n0.6%). All experiments were conducted according to guidelines \nof the Animal Use and Care Committee of Semyung University \n(No. Smecae-08-01). The reagent used in this experiment was \ntype II collagen extract obtained by Gupup Inc. (Seoul, Korea).\nClassification of experimental groups\nExperimental groups were divided to normal group, control \ngroup, low dose treated group, and high dose treated group. The \nnormal group fed only solid feed and water. The control group \nwas administered MTX in the same environment as the normal \ngroup. In the low and high dose treated groups, MTX was ad-\nministered in the same manner as the control group, and the type \nII collagen extract was orally administered.\nImmune degradation using MTX\nMTX (Sigma Chemical Co., St. Louis, MO, USA) extract was \ndissolved in physiological saline and injected intraperitoneally \ninto experimental animals. The main dose was 2 mg/kg, and 1 \nmL was injected for once a day for 4 consecutive days.\nReagent administration\nFrom the day after the last day of inducing immune degrada-\ntion by MTX administration, type II collagen extract was orally \nadministered at 250 mg/kg per day in the low dose treated group \nand 500 mg/kg per day in the high dose treated group for 28 con-\nsecutive days. The control group was orally administered the same \namount of saline.\nMeasurement of body weight\nThe body weights of the experimental animals were measured \nfour times, 1st week, 2nd week, 3rd week, and 4rd week after the \nlast day of MTX administration.\nBlood sampling\nThe animals were anesthetized with chloroform, cardiac punc-\nture and blood was added to the bottle containing ethylen di -\namine tetraacetic acid dipotassium salt (EDTA) to prevent coagu-\nlation.\nPreparation of splenocytes\nAfter cardiac blood culture, the abdomen was completely cov-\nered with 70% alcohol, and we took spleen out of the rat’s body, \nand the tissues around the spleen were carefully removed. After \nwashing twice with Rosewell Park Memorial Institute (RPMI)-\n1640 (GibcoBRL, Grand Island, NE, USA) medium at 4˚C, the \nspleen was minced on a petridish containing RPMI-1640 and the \nspleen was carefully rubbed into the sterilized glass membrane to \nfloat the splenocytes. This suspension was filtered through a stain-\nless steel wire mesh (mesh No. 100: Cheonggye Sangong Co., \nSeoul, Korea) to remove tissue pieces and unlabeled cell masses, \nand washed once with RPMI-1640 in Hanks balanced salt solu-\ntion (HBSS, GibcoBRL). After hypotonic shock with the steril-\nized distilled water, the red blood cells were completely hemo-\nlyzed, washed twice with 10× HBSS and once again with RPMI-\n1640 medium, and the spleen cells were resuspended in the \nmixed medium supplemented with 10% fetal bovine serum \n(FBS).\nMeasurement of B cell ratio in the spleen\nThe heart-collected blood was placed in an EDTA tube and 100 \nµL was placed in a 12×75 test tube. After adding 0.1 µL of fluo-\nrescein isothiocyanate (FITC) anti-rat CD4 monoclonal antibody \n(Pharmingen, San Diego, CA, USA), 0.5 µL of PE anti-rat \nCD45R/B220 monoclonal antibody (Pharmingen) was added and \nmixed with vortex mixer. The test samples were centrifuged at \n1,000 rpm for 5 min. The supernatant was discarded, 2 mL of \nwashing solution (phosphate buffered saline, PBS) was added, and \nthe mixture was centrifuged at 1,000 rpm for 5 min. The super-\nnatant was discarded, and the test samples were added 500 µL of \nPBS and mixed well with a vortex mixer, and analyzed using a \nflow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).\nMeasurement of CD4+ and CD8+ T cell ratio in spleen and \nblood\nThe test samples were placed in an EDTA tube and 100 µL was \nplaced in a 12×75 test tube. After adding 0.5 µg of FITC anti-rat \nCD3 monoclonal antibody (Pharmingen), the test samples were \nadded 0.1 μL of PE Anti-Rat CD4 or CD8 monoclonal antibody \n\nhttp://www.e-jer.org    733https://doi.org/10.12965/jer.1836480.240\nKim EH, et al.  •   Effect of type II collagen extract on immunosuppression\n(Pharmingen) and mixed with vortex mixer. The test samples \nwere added 2 mL of lysing solution (FACS lysing solution, Becton \nDickionson) and left for 15 min. The test samples were centri-\nfuged at 1,000 rpm for 5 min. The supernatant was discarded, 2 \nmL of washing solution (PBS) was added, and the mixture was \ncentrifuged at 1,000 rpm for 5 min. The supernatant was discard-\ned, and the test samples were added 500 µL of PBS and mixed \nwell with a vortex mixer, and analyzed using a flow cytometer \n(Becton Dickinson).\nCell culture\nCells of the murine machophage RAW 264.7 were purchased \nfrom the American Type Culture Collection. Cells were cultured \nin Dulbecco’s modified eagle’s medium (Gibco BRL, Grand Is-\nland, NY , USA) supplemented with 10% FBS (Gibco BRL) at \n37˚C in 5% CO2 incubator (MCO-17AIC, Sanyo, Tokyo, Japan).\nCell viability measurement\nCell viability was determined using the 3-(4,5-dimethylthi-\nazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit \n(Boehringer Mannheim GmbH, Mannheim, Germany) as per the \nmanufacturer’s protocols. In order to determine the cytotoxicity of \ntype II collagen extract, cells were treated with 25, 50, 100, and \n200 µg/mL for 24 hr. Cultures of the control group were left un-\ntreated. Ten µL of the MTT labeling reagent was added to each \nwell, and the plates were incubated for 4 hr. Solubilization solu-\ntion of 100 µL was then added to each well, and the cells were in-\ncubated for another 12 hr. The absorbance was then measured \nwith a microtiter plate reader absorbance was then measured with \na microtiter plate reader (Bio-Tek, Winooski, VT, USA) at a test \nwavelength of 195 nm and a reference wavelength of 690 nm. \nOptical density (O.D.) was calculated as the difference between \nthe absorbance at the reference wavelength and that at the test \nwavelength. Percent viability was calculated as (O.D. of drug- \ntreated sample/control O.D.) ×100.\nNitric oxide production measurement\nIn order to determine the effect of type II collagen extract on \nnitric oxide (NO) synthesis, the amount of nitrite in the superna-\ntant was measured using a commercially available NO detection \nkit (Intron Biotech., Seoul, Korea). After collection of 100 µL of \nsupernatant, 50 µL of N1 buffer was added, and the plate was in-\ncubated at room temperature for 10 min. N2 buffer was then \nadded, and the plate was incubated at room temperature for 10 \nmin. The absorbance of the content of each well was measured at \n450 nm. The nitrite concentration was calculated from a nitrite \nstandard curve. \nStatistical analysis\nResults are expressed as mean±standard deviation. Data were \nanalyzed by one-way analysis of variance followed by Duncan post \nhoc test using SPSS ver. 11.0 (SPSS Inc., Chicago, IL, USA). Dif-\nferences were considered statistically significant at P<0.05. \nRESUL TS \nBody weight change measurement\nWe observed the changes of body weight (Table 1). As a result \nof the measurement, there was a significant increase in the low \nand high doses of type II collagen extract treated groups com-\npared to the control group treated with MTX in the 1st and 2nd \nweek. And, there was a significant increase in the low dose of type \nII collagen extract treated groups compared to the control group.\nChanges in total leukocyte counts \nThe changes of total leukocyte counts were observed (Fig. 1). \nThe results were 11.51±3.78 in the normal group, 4.11±1.28 in \nthe control group, 8.03±1.09 in the low dose group and 10.48± \n1.40 in the high dose group. Therefore, a statistically significant \nincrease was observed in the experimental group treated with low \nand high doses type II collagen extract compared to the control \nTable 1. Body weight changes\nGroup 0 Week 1 Week 2 Weeks 3 Weeks 4 Weeks\nNormal 146.75 ± 5.23 165.75 ± 7.45 189.50 ± 12.97 214.25 ± 15.23 239.14 ± 21.96\nControl 145.17 ± 6.04 158.01 ± 6.09 177.41 ± 8.24 197.66 ± 11.05 224.72 ± 17.12\nLow dose 147.75 ± 6.94 162.25 ± 5.72* 183.83 ± 9.11* 203.09 ± 8.60* 225.20 ± 12.35\nHigh dose 144.67 ± 4.57 160.33 ± 2.67* 180.25 ± 5.92* 197.02 ± 8.59 219.36 ± 9.51\nValues are presented as mean ± standard deviation (g).\nNormal, untreated group; Control, treated with methotrexate (MTX) group; Low dose, treated with MTX and type II collagen extract (250 mg/kg) group; High dose, treated with \nMTX and type II collagen extract (500 mg/kg) group. \n*P< 0.05 compared to the control group. \n\nhttps://doi.org/10.12965/jer.1836480.240\nKim EH, et al.  •   Effect of type II collagen extract on immunosuppression\n734    http://www.e-jer.org\ngroup treated with MTX.\nMeasurement of CD4+ cell ratio in blood \nThe changes of CD4+ cell ratio in blood were observed (Fig. 2). \nThe results were 45.10%±6.16% in the normal group, 37.8%± \n4.21% in the control group, 33.66% ±3.05% in the low dose \ngroup and 45.65%±4.07% in the high dose group. In contrast to \nthe control group, no statistically significant increase was ob -\nserved in the low and the high doses type II collagen extract treat-\ned groups.\nMeasurement of CD8 + cell ratio in blood \nThe changes of CD8+ cell ratio in the blood were observed (Fig. \n3). The results were 20.80% ±1.77% in the normal group, \n12.38%±1.25% in the control group, 14.78%±0.72% in the \nlow dose group and 24.54% ±1.93% in the high dose group. \nThere was a statistically significant increase in the high dose type \nII collagen extract treated group compared to the control group.\nMeasurement of B cell ratio in the spleen\nThe changes of splenocyte B cell ratio in the spleen were ob-\nFig. 1. The changes of total leukocyte counts on type II collagen extract in \nmethotrexate (MTX)-induced immunosuppressed rats. Normal, untreated \ngroup; Control, treated with MTX group; Low dose, treated with MTX and type \nII collagen extract (250 mg/kg) group; High dose, treated with MTX and type II \ncollagen extract (500 mg/kg) group. *P< 0.05 compared to the control group. \n14\n12\n10\n8\n6\n4\n2\n0\nNormal Control Low dose High dose\n*\n*\nTotal WBC numbers ( × 103 cells/mm2)\nFig. 2. The changes of CD4+ T lymphocytes on low and high dose of undena-\ntured type II collagen extract in methotrexate (MTX)-induced immunosup-\npressed rats. Normal, untreated group; Control, treated with MTX group; Low \ndose, treated with MTX and type II collagen extract (250 mg/kg) group; High \ndose, treated with MTX and type II collagen extract (500 mg/kg) group.\n60\n50\n40\n30\n20\n10\n0\nNormal Control Low dose High dose\nRatio of CD4+ T lymphocytes (%)\nFig. 3. The changes of CD8+ T lymphocytes on type II collagen extract in meth-\notrexate (MTX)-induced immunosuppressed rats. Normal, untreated group; \nControl, treated with MTX group; Low dose, treated with MTX and type II col-\nlagen extract (250 mg/kg) group; High dose, treated with MTX and type II col-\nlagen extract (500 mg/kg) group. *P< 0.05 compared to the control group. \n30\n25\n20\n15\n10\n5\n0\nNormal Control Low dose High dose\nRatio of CD8+ T lymphocytes (%)\n*\nFig. 4. The changes of B lymphocytes in the spleen on type II collagen extract \nin methotrexate (MTX)-induced immunosuppressed SD rats. Normal, untreated \ngroup; Control, treated with MTX group; Low dose, treated with MTX and type \nII collagen extract (250 mg/kg) group; High dose, treated with MTX and type II \ncollagen extract (500 mg/kg) group. *P< 0.05 compared to the control group. \n45\n40\n35\n30\n25\n20\n15\n10\n5\n0\nNormal Control Low dose High dose\nRatio of B lymphocytes (%)\n*\n*\n\nhttp://www.e-jer.org    735https://doi.org/10.12965/jer.1836480.240\nKim EH, et al.  •   Effect of type II collagen extract on immunosuppression\nserved (Fig. 4). The results were 40.84%±1.41% in the normal \ngroup, 24.62%±1.54% in the control group, 35.50%±4.12% \nin the low dose group and 41.40% ±1.74% in the high dose \ngroup. There was a statistically significant increase in the low and \nthe high dose type II collagen extract treated group compared to \nthe control group.\nMeasurement of CD4+ cell ratio in the spleen\nThe changes of spleen CD4+ cell ratio in the spleen were ob-\nserved (Fig. 5). The results were 39.06%±4.71% in normal group, \n20.83%±2.17% in control group, 25.84%±0.94% in low dose \ngroup and 35.01%±2.91% in high dose group. There was a sta-\ntistically significant increase in the low and the high doses type II \ncollagen extract treated groups compared to the control group.\nMeasurement of CD8+ cell ratio in the spleen\nThe changes of spleen CD8+ cell ratio in the spleen were ob-\nserved (Fig. 6). The results were 14.20%±1.25% in the normal \ngroup, 4.80%±1.09% in the control group, 10.41%±1.13% in \nthe low dose group and 15.65%±0.67% in the high dose group. \nThere was a statistically significant increase in the low and the \nhigh doses type II collagen extract treated groups compared to the \ncontrol group.\nMeasurement of cell viability\nThe cell viability rates were observed (Fig. 7). The cell viability \nrates at 62.5, 125, 250, 500, and 1,000 μg/mL of type II collagen \nextract were 101.15%±4.18%, 101.73%±4.40%, 104.83%± \n2.24%, and 99.54%±6.14% and 98.27%±10.27%, respective-\nly. The results showed that the type II collagen extract showed no \nstatistically significant toxicity in RAW264.7 macrophages at the \nconcentrations of 62.5, 125, 250, 500, and 1,000 μg/mL.\nMeasurement of NO production\nThe inhibitory effect of type II collagen extract on NO produc-\ntion in RAW264.7 macrophages was measured (Fig. 8). The NO \nproduction rate of the group treated with lipopolysaccharides \n(LPS) alone increased to 151.15%±3.30%. In the group treated \nwith LPS and type II collagen extract at the concentrations of \nFig. 5. The changes of CD4+ ratio in the spleen on type II collagen extract in \nmethotrexate (MTX)-induced immunosuppressed SD rats. Normal, untreated \ngroup; Control, treated with MTX group; Low dose, treated with MTX and type \nII collagen extract (250 mg/kg) group; High dose, treated with MTX and type II \ncollagen extract (500 mg/kg) group. *P< 0.05 compared to the control group. \n50\n45\n40\n35\n30\n25\n20\n15\n10\n5\n0\nNormal Control Low dose High dose\nRatio of CD4+ T lymphocytes (%)\n*\n*\nFig. 6. The changes of CD8+ cell ratio in the spleen on type II collagen extract \nin methotrexate (MTX)-induced immunosuppressed SD rats. Normal, untreated \ngroup; Control, treated with MTX group; Low dose, treated with MTX and type \nII collagen extract (250 mg/kg) group; High dose, treated with MTX and type II \ncollagen extract (500 mg/kg) group. *P< 0.05 compared to the control group. \n18\n16\n14\n12\n10\n8\n6\n4\n2\n0\nNormal Control Low dose High dose\nRatio of CD8+ T lymphocytes (%)\n*\n*\nFig. 7. The effect of collagen type II extract on the cell viability of RAW264.7 \ncells. Con, untreated group; 62.5, 125, 250, 500, and 1,000, treated with each \nconcentration (µg/mL) of collagen type II extract.\n100\n80\n60\n40\n20\n0 Con 62.5 125 250 500 1,000\nCell viability (%)\n\nhttps://doi.org/10.12965/jer.1836480.240\nKim EH, et al.  •   Effect of type II collagen extract on immunosuppression\n736    http://www.e-jer.org\n62.5, 125, 250, 500, and 1,000 μg/mL, they were 137.81%± \n5.38%, 131.64%±6.08%, 124.47%±3.66%, 111.86%±7.93%, \nand 102.45%±7.22%, respectively. They were significantly de-\ncreased compared with the LPS-treated group.\nDISCUSSION\nThere are two types of immunity: innate and acquired (or adap-\ntive). Acquired immunity requires a prior exposure to an antigen \nand involves antibodies and lymphocytes. It is dependent on \nmemory and recognition of prior pathogens with antigen-specific \nmemory cells generating a more forceful responses on re-exposure. \nThis system is therefore typically slower in its initial response to \nattack with 72–96 hr required to generate specific T cells and an-\ntibodies (Ahn et al., 2015; Matarese et al., 2003). \nThese acquired immune responses can be divided into humoral \nimmune responses by B lymphocytes and cell mediated immune \nresponses by T lymphocytes (Ahn et al., 2015). B lymphocytes are \nprecursor cells of antibody-producing plasma cells. Mature B lym-\nphocytes roughly account for 10%–15% of peripheral blood lym-\nphocytes, 50% of splenic lymphocytes, and 10% of bone marrow \nlymphocytes. T lymphocytes act as mediators in cellular immune \nresponses and mature T lymphocytes account for 70%–80% of \nperipheral blood lymphocytes, 90% of thymocyte lymphocytes, \n30%–40% of lymph node cells, and 20%–30% of splenic lym-\nphocytes. T-lymphocytes are composed of helper T-lymphocytes, \ncytotoxic T-lymphocytes, suppressor T-lymphocytes and de -\nlayed-type hypersensitivity T-lymphocytes (Ahn et al., 2015). \nHelper T lymphocytes are the cells that regulate the immune \nresponse. They are also called CD4 cells because they have CD4 \non the cell surface. All blood cells and immune cells are differenti-\nated from a single primitive cell called hematopoietic stem cells. \nAs the hematopoietic stem cells differentiate, each surface of im-\nmune cells expresses a unique type of cell surface protein. We refer \nto this as the CD, which refers to a group of monoclonal antibod-\nies specific for various markers of lymphocyte differentiation \n(Kralickova and Vetvicka, 2015; Paul Dmowski and Braun, 2004). \nCD4 is a cell surface protein that is a marker of helper T lympho-\ncyte. It plays an important role in recognizing antigenic peptides \nbound to the type 2 major histocompatibility complex (MHC) of \nT cell receptors. It binds to the side of the type II MHC and acts \nas a coreceptor. CD8 is a cell surface protein that is part of cyto-\ntoxic T lymphocytes and inhibitory T lymphocytes, and plays an \nimportant role in recognizing antigenic peptides bound by T cell \nreceptors to type I MHC. It binds to the side of the type 1 MHC \nand acts as a co-receptor (Berkkanoglu and Arici, 2003).\nThe antigen receptor on the surface of T lymphocytes is called \nT-cell receptor. It reacts only with MHC/peptide complexes that \nhave been treated by antigen-presenting cells and bound to the \nsurface of antigen-presenting cells with the types 1 and 2 MHC. \nCD4 is an adjunct to the formation of type 2 MHC and CD8 is \nalso associated with type 1 MHC (Senturk and Arici, 1999).\nCD4 increases adhesion to antigen presenting cells (APCs) or \ntarget cells by specific affinity of MHC T lymphocytes for mole-\ncules in T lymphocyte activation. It is involved in the initial sig-\nnal transduction pathway that occurs when T lymphocytes recog-\nnize MHC/peptides in APC (Berkkanoglu and Arici, 2003). \nThus, the association of the attachment and signal transduction \nfunctions of CD4 coreceptors greatly enhances the efficiency of \nmature T lymphocytes and antigen stimulation. Immunity can be \nquantified using antibodies against the labeling protein on the \nsurface of lymphocytes that play such roles (Anderson et al., 1990; \nHofmann et al., 1996). It is also widely used as a marker of gener-\nal immunity (Chiappelli et al., 1991; Everaus, 1992).\nMTX is currently being used as an anticancer drug, rheumatoid \ndrug, and immunosuppressive agent, and has an excellent effect \non choriocarcinoma. The mechanism of MTX inhibits DNA syn-\nthesis by inhibiting folic acid reductase. In the immunosuppres-\nsion model by MTX, it is generally known to evaluate the efficacy \nof immunologically active substances by observing changes in \nbody weight, changes in B lymphocytes in spleen cells and blood, \nchanges in CD4+, CD8+ cells (Artym et al., 2004).\nInflammation is a defense mechanism of the host and this re-\nFig. 8. Effects of type II collagen extract on the nitric oxide (NO) production in \nRAW264.7 cells. Control, untreated group; Lipopolysaccharides (LPS), treated \nwith LPS (1 µg/mL); 62.5, 125, 250, 500, 1,000, treat with LPS and each con-\ncentration (µg/mL) of type II collagen extract. Values are relative to the control. \n*P< 0.05 indicate a significant different from the LPS.\n180\n160\n140\n120\n100\n80\n60\n40\n20\n0 Control LPS 62.5 125 250 500 1,000\nNO production (%)\n* * * * *\n\nhttp://www.e-jer.org    737https://doi.org/10.12965/jer.1836480.240\nKim EH, et al.  •   Effect of type II collagen extract on immunosuppression\nsponse is known to be the most important response in the macro-\nphage-mediated immune response (Kim and Kim, 2015). Nucle-\nar transcription factor-kappa B (NF-κB), an important factor in \ninflammatory responses, is a transcription factor that regulates the \nsynthesis of various cytokines. NF-κB enters the nucleus and acts \nas a transcription factor to induce inflammatory cytokines such as \ninducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), IL-\n1α, and IL-1β. And various inflammatory mediators such as NO \nare produced (Lee et al., 2012). NO has various physiological \nfunctions such as body defense function, signal transduction func-\ntion, neurotoxicity, and vasodilation. NO is synthesized by three \ntypes of NOS: neuronal nitric oxide synthase, endothelial nitric \noxide synthase, and iNOS. Among these NOS, NO production \nby iNOS is absolutely high, and it plays an important pathologi-\ncal role. It has been reported that iNOS is expressed in various \ncells such as macrophage when exposed to external stimuli to pro-\nduce large amounts of NO. Prostaglandin E2 promotes the ex-\npansion of blood vessels and permeability of blood vessel walls \nand stimulates the secretion of inflammatory cytokines such as IL-\n6, as well as aggregating immune cells into inflammatory sites \n(Lee et al., 2012).\nIn order to investigate the effect of type II collagen extract on \nthe immune function, we investigated the changes of the body \nweight, leukocyte count, blood CD4+, CD8+ cell ratio, spleen \nCD4+, and CD8+ cell ratio in MTX-induced immunosuppres-\nsive rats. Cell viability and NO production of type II collagen ex-\ntract were also observed.\nBody weight was significantly increased at 1st and 2nd weeks \nafter type II collagen extract administration, but not at 3rd and \n4rd weeks. Leukocyte counts were significantly increased in the \nexperimental group treated with low and high doses type II colla-\ngen extract treated group compared to the control group induced \nwith MTX treatment. As a result of the measurement of CD4+ \ncell ratio in the blood, the experimental group administered with \nhigh dose type II collagen extract was significantly increased com-\npared to the control group induced by MTX administration. As a \nresult of the CD8+ cell ratio in the blood, the experimental group \nadministered with the low and high doses type II collagen extract \nwas significantly increased compared with the control group in-\nduced by MTX administration. As a result of the spleen B cell ra-\ntio, the experimental groups administered with low and high dos-\nes type II collagen extract were significantly increased compared \nto the control group induced with MTX administration. As a re-\nsult of measuring the CD4+ cell ratio in the spleen, the experi-\nmental groups administered with the low and high doses type II \ncollagen extract were significantly increased as compared with the \nMTX-induced experimental group. In addition, the concentration \nof CD8+ cells ratio in the spleen was significantly increased in the \nexperimental groups administered with the low and high doses \ntype II collagen extract compared to the experimental group in-\nduced with the MTX administration.\nThese results suggest that the type II collagen extract may con-\ntribute to changes in leukocyte counts caused by MTX and \nchanges in CD8+ cell ratio in the blood. It was also confirmed \nthat there was a significant effect on the changes of B cell ratio, \nCD4+ and CD8+ cell ratio in the spleen. In addition, the type II \ncollagen extract did not show cytotoxicity, and it was also con-\nfirmed that the inhibition of NO production was significant. \nTherefore, it is thought that type II collagen extract may help to \nrestore the immune function in the living body caused by im-\npaired immune function. \nCONFLICT OF INTEREST\nNo potential conflict of interest relevant to this article was re-\nported.\nACKNOWLEDGMENTS\nThis work was supported by the Strat-up Growth Technology \nDevelopment Program (S2402431) funded by Korea Technology \nand Information Promotion Agency for SMEs.\nREFERENCES\nAhn SH, Monsanto SP, Miller C, Singh SS, Thomas R, Tayade C. Patho-\nphysiology and immune dysfunction in endometriosis. 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