Effects of Clematidis Radix Pharmacopuncture on MIA induced Osteoarthritis Rat

Article information

Acupunct. 2016;33(2):35-49
Dept. of Acupuncture & Moxibustion medicine, College of Korean Medicine, Daegu Haany University
*Corresponding author: Deprtment of Acupuncture & Moxibution Medicine, Daegu Oriental Hospital of Daegu Haany University, 136, Sincheondong-ro, Suseong-gu, Daegu, 706-828, Republic of Korea, Tel: +82-53-770-2115, E-mail: whiteyyou@nate.com
Received 2016 May 08; Revised 2016 May 28; Accepted 2016 June 01.

Abstract

Objectives:

This study was designed to investigate the anti-osteoarthritic effects of clematidis radix pharmacopuncture on the MIA-induced osteoarthritis rats.

Methods:

The subjects were divided into 4 groups; Normal rat group(N, n=5), MIA-induced osteoarthritis control rat group(C, n=5), MIA-induced osteoarthritis rat group injected with normal saline at ST36(NS, n=5), and MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture at ST36(CR, n=5). The experiment was conducted over a period of 21 days after injecting MIA. We analyzed body weight, hind paw weight distribution, liver and renal function, immunocytes, cykokines, inflammatory mediators, inflammatory proteins and mRNA expressions, as well as histopathological changes.

Results:

The CR group showed a significant increase in the hind paw weight distribution, and significant decreases in IL-1β, IL-6, PGE2, LTB4, osteocalcin, deoxypyridinoline level, the protein expression of COX2, arachidonate 5 lipoxygenase, and the mRNA expression of COX2, TNF-α, IL-1β, and NOS-II. In terms of the joint damages induced by osteoarthritis, the CR group showed a greater protective effect than group C in histopathologic observation (H&E, Safranin-O staining).

Conclusion:

These results demonstrated that clematidis radix pharmacopuncture had anti-inflammatory and analgesic effects. In addition, these results showed that it inhibited the progression of osteoarthritis.

Fig. 1

The Changes in Hind Paw Weight Distribution Ratio for 3 Weeks on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 2

The AST, ALT Levels in Serum on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

AST: aspartate aminotransferase.

ALT: alanine aminotransferase.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 3

The Creatinine Level in Serum on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 4

The Total WBC, Eosinophil and Monocyte Count on N, C, NS and CR

All values were showed mean ± SE.

In WBC count, there were no significant differences between each group.

*: compared with N.

†: compared with C.

WBC: white blood cell.

Eos: Eosinophil.

Mono: Monocyte.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 5

The Inflammatory Cytokines and Inflammatory Mediators Level in Serum on N, C, NS and CR

All values were showed mean ± SE.

In TNF-α, there were no significant differences between each group.

*: compared with N.

†: compared with C.

TNF-α: Tumor Necrosis Facter-α IL-1β: Interleukin-1β.

IL-6: Interleukin-6 PGE2: Prostaglandin E2.

LTB4: Leukotriene B4 DPD: Deoxypyridinoline.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 6

The protein expression of COX-2, 5-LOX in joint tissue on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

COX2: Cyclooxyganase-2 5-LOX: Arachidonate 5 lipoxygenase.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 7

The mRNA expression of COX-2, TNF-α, IL-1β, NOS-II in joint tissue on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

COX-2: Cyclooxyganase-2 TNF-α: Tumor Necrosis Facter-α.

IL-1β: Interleukin-1β NOS-II: nitric oxide synthase II.

mRNA: messenger ribonucleic acid RQ: relative quantitative.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 8

The Histological Analysis of synovial tissue on N, C, NS and CR.(H&E staining, ×200)

In the damage of synovial membrane and synovial tissue, CR and NS were suppressed than C.

H&E staining: Hematoxylin & Eosin staining.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 9

The Histological Analysis of articular cartilage on N, C, NS and CR.(Safarin-O staining ×200)

In the damage of articular cartilage, CR and NS were suppressed than C.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

The Body Weight for 3 Weeks on N, C, NS and CR

References

1. Korean Acupuncture & Moxibustion Society. The Acupuncutre and Moxibustion Medicine Seoul: Jibmoondang; 2012. p. 559–64. p. 573–7.
2. The Korean Orthopedic Association. Orthopedics 7th edth ed. Seoul: The newest medical Publishers CO; 2013. p. 317–27.
3. Seo BK, Ryu SR, Lee SS, et al. Systemic review: Herbal Medicines in the Treatment of Osteoarthritis in Pubmed and Chinese Medical Journals. The Acupuncture 2006;21(3):265–82.
4. An HJ, Lee CK, Park JH, et al. Effects of Bee Venom on Papain-induced Osteoarthritis in an Animal Model. Kor J Pharmacogn 2012;43(2):167–72.
5. Kim HS, Seo IB, Park SK, et al. Effects of Aconitum pseudo-laeve on Nitrick Oxide Synthase in the Periaqueductal Gray of Collagenase-induced Rat Osteoarthritis Model. Korean Journal of Acupuncture 2006;23(1):87–94.
6. Yoh SB, Sul JU, Shin MS. Research Trends on the Treatment of Knee Osteoathritis in Korean Medicine. Korean Journal of Acupuncture 2011;28(1):139–55.
7. Korean Herbalogy Society. Herbalogy Seoul: Young-lim; 2008. p. 305–6.
8. Yoo S, Kem DH, Lee MJ. Effect of CR(Clematidis Radix) on anti-inflammatory, analgesic and antipyretic in Animal model. J of Korean rehabilitation medicine 2004;14(2):75–84.
9. Lee SH, Jung SH, Kim SJ, Seo IB. Effect of GCP Treatment on the Monosodium Iodoacetate-Induced Osteoarthritis in Rats. J of Korean rehabilitation medicine 2008;18(1):75–94.
10. Yang KJ, Kim SJ, Seo IB, et al. Effect of Clematis mandshurica Rupr. on Nitric Oxide Synthase in the Periaqueductal Gray of Collagenase-induced Rat Osteoarthritis Model. Korean Journal of Acupuncture 2005;22(4):109–16.
11. Koo ST, Kim SK, Kim EH, et al. Acupuncture Point Locations for Experimental Animal Studies in Rats and Mice. Korean Journal of Acupuncture 2010;27(3):67–78.
12. Sung YS, Choi HJ, Oh JM, Ji JG, Bak JW, Kim DH. The effect of KKBT in Papain-Induced Osteoarthritis Mice Models. Collection of dissertations of Daejeon University 2012. 21(1)33–52.
13. Yoon CH. Osteoarthritis Update. Korean Journal of medicine 2012;82(2):170–4.
14. Longo DL, Kasper D, Jameson JL, Fauci AS, Hauser SL, Joseph L. Harrison’s principles of internal medicine 18th edth ed. New York: The Mc-Graw-Hill Companies, Inc; 2012. p. 2828–36.
15. Kim HR, Kim EJ. Prevalence of Osteoarthritis and Its Affecting Factors among a Korean Population Aged 50 and Over. J of Korean Public Health Nursing 2013;27(1):27–39.
16. Kim HA. Pharmacological Treatment of Osteoarthritis. J of Korean Orthopaedic Research Soc 2010;13(1):16–22.
17. Korean rehabilitation medicine society. Korean rehabilitation medicine Seoul: Gunja; 2011. 84p. 97.
18. Ham KW, Kim EK, Song MY, et al. Protective effect of Radix Clematidis Extract on Streptozotocin-induced Diabetes. Korean J Oriental Physiology & Pathology 2008;22(3):580–4.
19. Park JW, Lim HH. Protective effect of Clematidis Radix extract on CoCl2-induced apoptosis in human neuroblastoma cells. J of korean rehabilitation medicine 2014;24(2):41–50.
20. Song YH, Yoo JE, Lim HJ, Yoo DY. Effects of Clematidis Radix Extract on Osteoclastogenesis and Gene Expression in RANKL-induced RAW 264.7 Cell. The journal of oriental gynecology 2010;23(3):78–90.
21. Kim SJ, Chun JM, Yang WK, Cheon MS, Sung YY, Park JY. Synergistic Effect of Notopterygium incisum with Clematis Manshurica in the Anti-inflammatory Activity. Kor J Herbology 2010;25(4):11–6.
22. Park WT, Jeong SH, Seo IB, Kim SJ. GCP Treatment on the Expression of NOS, C-fos, Serotonin and Substance-P in Central Nerve System of Monosodium Iodoacetate-induced Osteoarthritis Pain Model. Korean J Oriental Physiology&Pathology 2007;21(6):1483–90.
23. Choi WJ, Kim KB, Kim KH, Kim JH. Analgesic effect of Clematidis Radix(CR) herbalacupuncture: a behavioral evaluation on the test rats with induced acute pain. The Acupuncture 2009;26(6):91–100.
24. Lee JS, Kim KH, Lee SD, Kim KS. The effect of clematidis radix herbal-acupuncture solution, on collagen, adjuvant, lipopolysaccharide and phospholipase A2 induced rheumatoid arthritis in mice. The Acupuncture 2012;29(1):127–37.
25. Guingamp C, GeGout-Pottie P, Philippe L, Terlain B, Netter P, Gillet P. Mono-iodoacetate-induced experimental osteoarthritis: a dose-response study of loss of mobility, morthology and biochemistry. Arthritis Rheum 1997;40(9):1670–9.
26. Johanne M, Jean P, Hassan F. Cyclooxygenase-2 and Prostaglandins in Articular Tissues. Semanars in Arthritis and Rheumatism 2003;33(3):155–67.
27. Emanuel R, Howard M. Reisner Essentials of Rubin’s pathology 5th edth ed. Seoul: KoonJa; 2013. p. 21–44.
28. Hong J, Allison P, Koichiro P, et al. Critical Roles for Interlukin 1 and Tumor Necrosis Factor α in Antibody-induced Arthritis. J Exp Med 2002;196(1):77–85.
29. Ji JD. Cytokines in rheumatoid arthritis. Hanyang medical reviews 2005;25(2):43–52.
30. Johanne M, Francois M, Hassan F, et al. Regulation of the Expression of 5-Lipoxygenase-Activation Protein/5-Lipoxygenase and the synthesis of Leukotriene B4 in Osteoarthritis Chondrocytes. Arthritis & Rheumatism 2004;50(12):3925–33.
31. Rousseau JC, Delmas PD. Biological markers in osteoarthritis. Nature clinical practive rheumatology 2007;3(6):346–56.
32. Masaaki T, Kazuhiro K, Hironobu H, et al. Concentrations of pyridinoline and deoxypyridinoline in joint tissues from patients with osteoarthritis or rheumatoid arthritis. Annals of the Rheumatic Diseases 1996;55:324–7.
33. Miguel O, Rocio L, Francisca L, Juan JGR, Oreste G. Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1. Arthritis Research&Therapy 2005;7:R581–R91.

Article information Continued

Fig. 1

The Changes in Hind Paw Weight Distribution Ratio for 3 Weeks on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 2

The AST, ALT Levels in Serum on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

AST: aspartate aminotransferase.

ALT: alanine aminotransferase.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 3

The Creatinine Level in Serum on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 4

The Total WBC, Eosinophil and Monocyte Count on N, C, NS and CR

All values were showed mean ± SE.

In WBC count, there were no significant differences between each group.

*: compared with N.

†: compared with C.

WBC: white blood cell.

Eos: Eosinophil.

Mono: Monocyte.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 5

The Inflammatory Cytokines and Inflammatory Mediators Level in Serum on N, C, NS and CR

All values were showed mean ± SE.

In TNF-α, there were no significant differences between each group.

*: compared with N.

†: compared with C.

TNF-α: Tumor Necrosis Facter-α IL-1β: Interleukin-1β.

IL-6: Interleukin-6 PGE2: Prostaglandin E2.

LTB4: Leukotriene B4 DPD: Deoxypyridinoline.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 6

The protein expression of COX-2, 5-LOX in joint tissue on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

COX2: Cyclooxyganase-2 5-LOX: Arachidonate 5 lipoxygenase.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 7

The mRNA expression of COX-2, TNF-α, IL-1β, NOS-II in joint tissue on N, C, NS and CR

All values were showed mean ± SE.

*: compared with N.

†: compared with C.

COX-2: Cyclooxyganase-2 TNF-α: Tumor Necrosis Facter-α.

IL-1β: Interleukin-1β NOS-II: nitric oxide synthase II.

mRNA: messenger ribonucleic acid RQ: relative quantitative.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 8

The Histological Analysis of synovial tissue on N, C, NS and CR.(H&E staining, ×200)

In the damage of synovial membrane and synovial tissue, CR and NS were suppressed than C.

H&E staining: Hematoxylin & Eosin staining.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Fig. 9

The Histological Analysis of articular cartilage on N, C, NS and CR.(Safarin-O staining ×200)

In the damage of articular cartilage, CR and NS were suppressed than C.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.

Table 1

The Body Weight for 3 Weeks on N, C, NS and CR

0 day 7 days 14 days 21 days
N 298.7 ± 3.55 g 335.3 ± 5.48 g 361.7 ± 7.88 g 369.7 ± 4.91 g
C 302.0 ± 4.46 g 322.0 ± 5.69 g 353.2 ± 7.18 g 360.3 ± 3.79 g
NS 305.2 ± 7.87 g 305.7 ± 17.55 g 345.4 ± 12.09 g 371.0 ± 10.78 g
CR 314.0 ± 7.97 g 311.6 ± 11.00 g 342.4 ± 13.63 g 378.0 ± 13.69 g

All values were showed mean ± SE.

There were no significant differences between each group.

N: Normal rat group.

C: MIA-induced osteoarthritis control rat group.

NS: MIA-induced osteoarthritis rat group injected with normal saline.

CR: MIA-induced osteoarthritis rat group injected with clematidis radix pharmacopuncture.