Effects of Kyejiinsam-tang in MIA-Induced Osteoarthritis Rats

Soon-sun An; Dong-seok Heo; An, Soon-sun; Heo, Dong-seok

doi:10.13048/jkm.13013

JKM > Volume 34(3); 2013 > Article

An and Heo: Effects of Kyejiinsam-tang in MIA-Induced Osteoarthritis Rats

Original Article

Journal of Korean Medicine 2013; 34(3): 69-85.

Published online: September 30, 2013

DOI: https://doi.org/10.13048/jkm.13013

Effects of Kyejiinsam-tang in MIA-Induced Osteoarthritis Rats

Soon-sun An, Dong-seok Heo

Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Dae-Jeon University

Correspondence to: 허동석 (Dong-seok Heo), 대전광역시 중구 대흥동 22-5 대전대학교 부속 대전한방병원 한방재활의학과교실, Tel: +82-42-229-6813, Fax: +82-42-254-3403, E-mail: hurds@hanmail.net

Received May 22, 2013 Revised June 26, 2013 Accepted June 26, 2013

Abstract

Objectives:

This study investigated the anti-osteoarthritic effects of Kyejiinsam-tang (hereinafter referred to KIT) on the monosodium iodoacetate (MIA)-induced osteoarthritis rats.

Methods:

Anti-oxidative effects of KIT were measured by scavenging activities of DPPH, reactive oxygen species (ROS) and nitric oxide (NO). Scavenging activities of anti-oxidation in lipopolysaccharide (LPS)-treated RAW 264.7 cells were also measured for inhibitory effects against the production of inflammatory mediators (tumor necrosis factor-α, interleukin-1β, interleukin-6).

Osteoarthritis was induced in rats by injecting MIA in the knee joint. Rats were divided into a total of 4 groups (n=6). The normal group were not treated at all without inducing osteoarthritis whereas the control group were induced for osteoarthritis by MIA and oral medicated physiological saline per day. The positive comparison group was injected with MIA and after 7 days, 2 mg/kg of Indomethacin. The experimental group was injected with MIA and after 7 days was medicated with 34 mg/kg of KIT. Indomethacin and KIT were orally-medicated for each substance a total of 4 weeks, once per day.

Weight-bearing on hind legs was measured every week after MIA injection. At the end of the experiment (5 weeks after MIA injection), micro CT (computed tomography)-arthrography and histopathological examinations on the articular structures of knee joint were performed. The effect on inflammatory cytokines and immunological cells in synovial fluid was measured. Volume of cartilage was measured by micro CT-arthrography. Injury to synovial tissue was measured by H & E (hematoxylin and eosin), Safranin-O immunofluorescence.

Results:

Cytotoxicity against hFCs was insignificant.
KIT showed the potent full term for DPPH.

NO was significantly reduced by KIT (at 100, 200 μg/ml) and ROS was also reduced, but not significantly, by KIT (at 200 μg/ml).

IL-6 and IL-1β were significantly reduced by KIT (at 100, 200 μg/ml) and TNF-α was also reduced, but not significantly, by KIT (at 200 μ/ml).

In hind legs weight-bearing measurement, level of weight increased.
Functions of liver and kidney were not affected.
IL-1β was significantly reduced and TNF-α, IL-6 were also reduced but not significantly.
PGE2 (prostaglandin E2), LTB4 (leukotriene B4) were significantly reduced in the KIT group.
MMP-9 (matrix metalloproteinase-9), TIMP-1 (tissue inhibitor of metalloproteinases-1) and Osteocalcin were significantly reduced in the KIT group.
Destruction of cartilage on micro CT arthrography was reduced but had no significant differences.
Histopathologically, injury to synovial membrane of the KIT group was decreased and proteoglycan content of KIT group was increased.

Conclusions:

According to this study, Kyejiinsam-tang has inhibiting effect on the progression of arthritis in MIA-induced osteoarthritis rat. Kyejiinsam-tang has anti-oxidants and anti-inflammation effects, and is related to inhibiting the activity of inflammatory cytokine and injury of volume in cartilage.

Keywords: Kyejiinsam-tang, osteoarthritis, anti-inflammation, anti-oxidation, MIA-induced

Fig. 1.

Effects of KIT on the cell viability, DPPH free radical scavenging activity, the Reactive oxygen species(ROS), the Nitric Oxide(NO) of RAW 264.7 cells.

RAW 264.7 cells were treated with 50, 100, 200 μg/ml of KIT for 24 hours. Cell viability was determined using the WST assay. Extracts were incubated with DPPH solution at 37°C for 30 mins. Activities were determined by measurement of absorbance at 517 nm. The ROS production was analysed following incubation with DCFH-DA by flow cytometry. Significant value was calculated by compared with control group by student’s t-test(***p<0.001).

Fig. 2.

Effects of KIT on LPS-stimulated TNF-α, IL-1β, IL-6 production in RAW 264.7 cells.

TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1β, IL-6: interleukin-6. Cells were treated with LPS(1 μg/ml) and control(0 μg/ml), 50, 100, 200 μg/ml of KIT for 24 hours. The results are expressed as mean ± SD from three independent experiments. Significant value was calculated by compared with control group by student’s t-test(**p<0.01, ***p<0.001).

Fig. 3.

Effects of KIT on weight bearing changes in the hind legs of MIA-induced osteoarthritis rats.

Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. The results are expressed as mean ± SD from three independent experiments. Statistically significant value compared with control by t-test(*p<0.05, **p<0.01).

Fig. 4.

Effect of KIT on the AST and ALT, BUN and creatinine in MIA-induced osteoarthritis rats.

Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. AST: aspatrate aminotransferase, ALT: alanine aminotransterase, BUN: blood urea nitrogen. Values represent the means ± SD.

Fig. 5.

Effects of KIT on levels of TNF-α, IL-1β, IL-6, PGE₂ in the serum of MIA-induced osteoarthritis rats.

Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT, TNF-α: tumor necrosis factor-α, IL-1β: interleukin - 1β, IL-6: interleukin-6, PGE₂: Prostaglandin E₂. Values represent the means ± SD. Statistically significant value compared with control by t-test(*p<0.05, ** p<0.01, ***p<0.001).

Fig. 6.

Effects of KIT on levels of LTB₄, MMP-9, TIMP-1, Osteocalcin in the serum of MIA-induced osteoarthritis rats.

Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT, LTB₄: Leukotriene B₄, MMP-9: Matrix metalloproteinase-9, TIMP-1: Tissue inhibitor of metalloproteinase-1. Values represent the means ± SD. Statistically significant value compared with control by t-test(*p<0.05, **p<0.01, ***p<0.001).

Fig. 7.

Effects of KIT on imaging and volume of cartilage degeneration using micro CT arthrography in joint tissue of MIA-induced osteoarthritis rats.

Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin

KIT; MIA-induced osteoarthritis group treated with KIT

Fig. 8.

Effects of KIT on joint pathology(hematoxylin & eosin staining) from joint tissue of MIA-induced osteoarthritis rats(× 100).

Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. In normal, articular cartilages are sell developed. In control, most of articular cartilage was degenerated. In Indomethacin and KIT, most of articular cartilages are degenerated and appeared homogeneous, but proteoglycan in periphery of degenerated area are remained.

Fig. 9.

Effects of KIT on joint pathology(Safranin-O staining) from joint tissue of MIA-induced osteoarthritis rats(× 100).

Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. Stain intensity of red color increased in proportion to proteoglycan content in normal. The red color disappeared in almost articular cartilage and red color remained in periphery of necrotic area in control. Compare the stain intensity and areas of red color in Indomethacin and KIT with the normal and control.

Table 1.

The Compositions of Kyejiinsam-tang(KIT)

Pharmacognostic nomenclatures	Amount(g)
Cinnamomi Ramulus	8.0
Glycyrrhizae Radix	8.0
Atractylodis macrocephalae Rhizoma	6.0
Ginseng Radix	6.0
Zingiberis Rhizoma	6.0

Total	34.0

References

1.. The Korean Academy of Oriental Rehabilitation Medicine. Oriental Rehabilitation Medicine 3rd. Seoul. KOONJA publishing inc;(2011). p. 84

2.. Braunwald Eugene, Fauci A, Kasper D, Hauser S, Longo D. The Korean Association of Internal Medicine. Harrison’s principles of internal medicine. 16th ed. Seoul. MIP Publisher;(2006). p. 2083-4. p. 2222-7.

3.. Yoh, SB, Sul, JU, & Shin, MS. Research trends on the treatment of knee Osteoarthritis in Korean medicine. The Korean Journal of Acupuncture, (2011). 28(1), 139-55.

4.. Park BH. The commentary sanghanron clinical applications. Kangwon. Uibang publisher;(2004). p. 317-8.

5.. Kim, SJ, Park, SM, Kang, H, Shim, BS, Kim, SH, & Choi, SH, et al. Anti-inflammatory effect of Cinnamomi ramulus on collagen induced arthritis;a model for rheumatoid arthritis in DBA/1J mice and cytokine production in raw 264.7 cells. The Korean Journal of Oriental Physiology & Pathology, (2008). 22(3), 542-7.

6.. Park, EK, Shin, YW, Lee, HU, Kim, SS, Lee, YC, & Lee, BY, et al. Inhibitory effect of ginsenoside Rb1 and compound K on NO and prostaglandin E2 biosynthesis of RAW 264.7 cells induced by lipopolysaccharide. Biol Pharm Bull, (2005). 28, 652-6.

7.. Jung TY. Inhibitory effect of Korean ginseng extract on type II collagen-induced arthritis. Seoul. Graduate school, Yonsei Univ;(2009.

8.. Kim, SH, & Park, YG. Effects of Atractylodis Rhizoma Alba extract on collagen-induced arthritis in mice. The Korean Journal of herbology, (2012). 27(3), 1-6.

9.. Park, JP, Son, JH, Kim, YM, Lee, EY, Lim, KH, & Kim, EH. Suppression of inflammatory macrophage response by Glycyrrhiza Uralensis herbal acupuncture extract. The Korean Journal of Acupuncture, (2011). 28(4), 49-58.

10.. Ryu, HS, & Kim, HS. Effect of Zingiber officinale roscoe extracts on mice immune cell activation. The Korean Journal of Nutrition Korean, (2004). 37(1), 23-30.

11.. Lee SY. The new gobangchancha. Seoul. Koonja publisher;(2012). p. 72

12.. Piscaer, TM, Waarsing, JH, Kops, N, Pavljasevic, P, Verhaar, JAN, & van Osch, GJVM, et al. In vivo imaging of cartilage degeneration using μCT-arthrography. Osteoarthritis and Cartilage, (2008). 16(9), 1011-7.

13.. Murphy, JM, Dixon, K, Beck, S, Fabian, D, Feldman, A, & Barry, f. Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis and Rheum, (2002). 46(3), 704-13.

14.. Yun, JH. Osteoarthritis update. The Korean Journal of Medicine, (2012). 82(2), 170-4.

15.. Goldring, MB, Otero, M, Plumb, DA, Draqomir, C, Favero, M, & El Hachem, K, et al. Roles of inflammatory and anabolic cytokines in cartilage metabolism:signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis. Eur Cell Mater, (2011). 21, 202-220.

16.. Jhun, HJ, Ahn, K, & Lee, SC. Estimation of the prevalence of osteoarthritis in Korean adults based on the data from the fourth Korea national health and nutrition examination survey. Anesth Pain Med, (2010). 5, 201-206.

17.. Frontera Walter R. Muscular skeletal disease. Seoul. Koonja publisher;(2003). p. 401

18.. Liu CC. Anti-Oxidants and Anti-inflammation Effects of Geongangbuja-tang. Daejoen. Graduate school, Daejoen Univ;(2011.

19.. Choi WJ. The antioxidative and anti-inflammatory effects of lophatheri extracts for herbal-acupuncture via reducing iNOS synthesis induced by LPS in RAW 264.7 Cell. Iksan. Graduated school, Wonkwong Univ;(2010.

20.. Park, SD. Effect of Cervi Pantotrichum Cornu herbal acupuncture on protease activities anti-oxidant in rheumatoid arthritis rats. The Journal of Korean Acupuncture and Moxibustion Society, (2002). 19(2), 51-64.

21.. Sung, YS, Choi, HJ, Oh, JM, Ji, JG, Bak, JW, & Kim, DH. The effect of KKBT in papain-induced osteoarthritis mice models. The Journal of Daejeon Oriental Medicine, (2012). 21(1), 33-52.

22.. Textbook Compilation Committee, College of Oriental Medicine, the National sanghanron. Determined sanghanron. Seoul. Ilzungsa;(2003). p. 342-3.

23.. Gil Ik-dong-dong. Yakjing. Seoul. Cheonghong Publisher;(2006). p. 105p. 116p. 129p. 203p. 216

24.. Yoo, YC. Role of nitric oxide in the nickel and cobalt induced cytotoxicity in RAW 264.7 cell. The Korean Journal of Occupational and Environmental Medicine, (2001). 13(3), 274-7.

25.. Matsukawa, A, Ohkawara, S, Maeda, T, Takagi, K, & Yoshinaga, M. Production of IL-1 and IL-1 receptor antagonist and the pathological significance in lipolysis-accharideinduced arthritis. Clin Exp Immunol, (1993). 93(2), 206-11.

26.. Cho, HJ, Jang, JB, Jung, JU, Sung, SC, & Kim, TK. Prevalence of radiographic knee osteoarthritis in elderly koreans. Knee surgery & related research, (2009). 21, 223-31.

27.. Bove, SE, Calcaterra, SL, Brooker, RM, Huber, CM, Guzman, RE, & Juneau, PL, et al. Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis. Osteoarthritis Cartilage, (2003). 11(11), 821-30.

28.. Kim, WY, & Choi, JB. Effects of Bee venom and Cervi Cornu Parvum pharmacoacupuncture in monosodium iodoacerare(MIA)-induced osteoarthritis rat. Journal of Oriental Rehabilitation Medicine, (2010). 20(1), 61-77.

29.. Janusz, MJ, Hookfin, EB, Heitmeyer, SA, Woessner, JF, Freemont, AJ, & Hoyland, JA, et al. Moderation of iodoacetate-induced experimental osteoarthritis rats by matrix metalloproteinase inhibitors. Osteoarthritis Cartilage, (2001). 9, 751-60.

30.. Guzman, RE, Evans, MG, Bove, S, Morenko, B, & Kilgore, K. Mono-iodoacetate-induced histologic changes in subchondralbone and articular cartilage of rat femorotibial joints: an animal model of osteoarthritis. Toxicol Pathol, (2003). 31(6), 619-24.

31.. Kim, NJ, Park, JY, Han, HC, & Chang, SH. Analgesic and anti-inflammatory effects of Ibuprofen, Indomethacin and NS-398 on an acute model of arthritis in rats. Korean Journal of Anesthesiology, (2001). 40, 802-13.

32.. Ahn, SI, Heuing, BJ, & Son, JY. Antioxidative activity and nitrite scavenging abilities of some phenolic compounds. Korean J. Food Cookery Sci, (2007). 23, 19-24.

33.. Kim SJ. DPPH radical scavenging and ACE inhibitory effects of the aerial parts of Fagopyrum esculentum, and isolation of flavonoids. MS thesis.Sunchon. Sunchon National Univ;(2004.

34.. Moncada, S, Palmer, RMJ, & Higgs, EA. Nitric Oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev, (1991). 43, 109-42.

35.. Wink, DA, & Mitchell, JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med, (1998). 25(4–5), 434-56.

36.. Miyasaka, N, & Hirata, Y. Nitric oxide and inflammatory arthritides. Life Sci, (1997). 61(21), 2073-81.

37.. Brune, B, Zhou, J, & von Knethen, A. Nitric oxide, oxidative stress and apoptosis. Kidney Int Suppl, (2003). 84, 4-22.

38.. Delanty, N, & Dichter, MA. Oxidative injury in the nervous system. Acta Neurol Scand, (1998). 98, 53-145.

39.. Iwanami, K, Matsumoto, I, Tanaka-Watanabe, Y, Inoue, A, Mihara, M, & Ohsuqi, Y, et al. Crucial role of IL-6/IL-17 cytokine axis in the induction of arthritis by glucose 6 phosphate isomerase. Arthritis Rheum, (2008). 58, 754-63.

40.. Ji, H, Pettit, A, Ohmura, K, Ortiz-Lopez, A, Duchatelle, V, & Deqott, C, et al. Critical roles for interleukin 1 and tumor necrosis factor alpha in antibody induced arthritis. J Exp Med, (2002). 196, 77-85.

41.. Smolen, JS, Aletaha, D, Koeller, M, Weisman, MH, & Emery, P. New therapies for treatment on rheumatoid arthritis. Lancet, (2007). 370(9602), 1861-74.

42.. Lim, CK, & Yun, YG. Anti-rheumatiod arthritis effects of I-Myo-San water extract. The Korean Journal of Oriental Medical Prescription, (2009). 17(1), 99-111.

43.. Martel-Pelletier, J, Pelletier, JP, & Fahmi, H. Cyclooxygenase-2 and prostaglandins in articular tissues. Semin Arthritis Rheum, (2003). 33(3), 155-67.

44.. Bang, SJ, Kim, IS, Kim, OS, Kim, YJ, & Jung, HJ. IL-6 gene promoter polymorphisms in Korean generalized aggressice periodontitis patients. The Journal of the Korean Academy of Periodontology, (2008). 38, 579-88.

45.. MaryKCShawnOFKwokHSKimJWKimHGJungHSTaeGS. Biochemistry. Fifth Edition. Seoul. Life Science Publisher;(2007). p. 451

46.. Harizi, H, Corcuff, JB, & Gualde, N. Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology. Trends in Molecular Medicine, (2008). 14(10), 461-9.

47.. Horton, JK, Williams, AS, Smith-Phillips, Z, Martin, RC, & O’Beirne, G. Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression. Anal Biochem, (1999). 271(1), 18-28.

48.. Dubois, RN, Abramson, SB, Crofford, L, Gupta, RA, Simon, LS, & Van De Putte, LB, et al. Cyclooxygenase in biology and disease. FASEB J, (1998). 12, 1063-73.

49.. Anguita, J, Samanta, S, Ananthanarayanan, SK, Revilla, B, Geba, GP, & Barthold, SW, et al. Cyclooxygenase 2 activity modulates the severity of murine Lyme arthritis. FEMS Immunol Med Microbiol, (2002). 34(3), 187-91.

50.. Harizi, H, Corcuff, JB, & Gualde, N. Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology. Trends in Molecular Medicine, (2008). 14(10), 461-9.

51.. Laufer, S. Role of eicosanoids in structural degradation in osteoarthritis. Curr Opin Rheumatol, (2003). 15(5), 623-724.

52.. Griffiths, RJ, Smith, M, Roach, ML, Stock, JL, Stam, EJ, & Milici, AJ, et al. Collagen-induced arthritis is reduced in 5-lipoxygenase-activating protein-deficient mice. J Exp Med, (1997). 185, 1123-9.

53.. Bresnihan, B. Pathogenesis of joint damage in rheumatoid arthritis. J Rheumatol, (1999). 26, 717-9.

54.. Nagase, H, & Woessner, JF. Matrix metalloproteinases. J. Biol. Chem, (1999). 274, 21491-4.

55.. Halliwell, B. Oral inflammation and reactive species: a missed opportunity. Oral Dis, (2000). 6, 137.

56.. Lambert, E, Dasse, E, Haye, B, & Petitfrere, E. TIMPs as multifacial proteins. Crit Rev Oncol Hematol, (2004). 49(3), 187-98.

57.. Garnero, P, Piperno, M, Gineyts, E, Christigau, S, Delmas, PD, & Vignon, E. Cross sectional evaluation of biochemical markers of bone, cartilage and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage. Ann Rheum Dis, (2001). 60, 619-26.