The Effect of Acupuncture on Oxidative Stress in Rats with Femur Fractured

Öz

The effect of acupuncture on the healing of fractures and oxygen free radicals (ROS) in fractured femur rats has been studied. In this study, 54 female Wistar albino rats (250-300 g in weight) were randomly divided into 10 groups including, control, sacrificed on days 7, 14 and 21 of acupuncture, sacrificed on days 7, 14 and 21 of fracture, and sacrificed on days 7, 14 and 21 of fracture+acupuncture application. The rats in the control group received no treatment.  The 0.5 mm thick kirschner wire was advanced intramedullary as retrograde from the fracture line of the proximal part of the bone and fracture detection was performed. After fracture formation, acupuncture (50 Hz, 20 qA, 25 minutes) was applied to the rats. This treatment was applied for 7, 14 and 21 days as 4 sessions per week. The rats were sacrificed 7, 14 and 21 days after fracture formation and plasma malondialdehyde (MDA), whole blood glutathione (GSH) levels, erythrocyte catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glucose-6-phosphate dehydrogenase (G6PD) activities were measured. Plasma MDA and GSH levels were statistically significant in all fracture groups compared to the control group (p<0.05). It was observed statistically significant reduction in the fracture+acupuncture groups according to the fracture group (p<0.05). While blood CAT activities in 7 day of fracture was observed statistically significant no change compared to the control group, It showed a statistically significant reduction in 14 and 21 days of fracture compared to the control group. There was a statistically significant decrease in blood SOD, GSH-Px and G6PD activities compared to the control group in fracture groups. CAT, SOD, GSH-Px and G6PD activities showed a significant increase in the fracture+acupuncture groups compared to the fracture group (p<0.05). As a result, the application of acupuncture can positively influence the healing process of the fracture by suppressing the harmful effects of ROS and regulating the activities of antioxidant enzymes.

Anahtar Kelimeler

• Acupuncture,Antioxidant Enzyme,Glutathione,Fracture Healing,Malondialdehyde

Femur Kırığı Oluşturulmuş Ratlarda Oksidatif Stres Üzerine Akupunkturun Etkisi

Öz

Femur kırığı oluşturulan ratlarda kırık iyileşmesi ve serbest oksijen radikalleri (SOR) üzerine akupunkturun etkisi araştırıldı. Çalışmada; 54 adet dişi Wistar albino rat (250-300 g ağırlığında), kontrol, 7, 14 ve 21. gün süreyle akupunktur uygulanan gruplar, kırık oluşumunun 7, 14 ve 21. günlerinde sakrifiye edilen gruplar ve 7, 14 ve 21 gün boyunca kırık+akupunktur uygulanan gruplar olmak üzere rastgele 10 gruba ayrıldı. Kontrol grubu ratlara tedavi uygulanmadı. Ratların femurlarında gigli testeresi ile transvers bir kırık hattı oluşturuldu. Kirschner teli kemiğin proksimal parçasının kırık hattından retrograd olarak intramedüller ilerletildi ve kırık tespiti yapıldı. Kırık oluşumunun ardından ratlara akupunktur (50 Hz, 20 qA, 25 dakika) uygulaması yapıldı. Bu tedavi haftada 4 seans olacak şeklinde 7, 14 ve 21 gün süre ile uygulandı. Ratlar kırık oluşturulmasından 7, 14 ve 21 gün sonra sakrifiye edilerek plazma malondialdehit (MDA), tam kan glutatyon (GSH) seviyeleri, eritrosit katalaz (KAT), süperoksit dismütaz (SOD),  glutatyon peroksidaz (GSH-Px) ve glukoz-6-fosfat dehidrogenaz (G6PD) aktiviteleri ölçüldü. Plazma MDA ve kan GSH düzeylerinde tüm kırık gruplarında kontrol grubuna göre istatistiksel olarak önemli bir artma saptandı (p<0,05). Kırık+akupunktur gruplarında kırık gruplarına göre istatistiksel olarak önemli ölçüde azalma gözlendi (p<0,05). Kan KAT aktivitelerinde 7. gün kesilen kırık grubunda kontrol grubuna göre istatistiksel olarak önemli bir değişiklik gözlenmezken, 14 ve 21. günlerde sakrifiye edilen kırık gruplarında kontrol grubuna göre önemli bir azalma saptandı. Kan SOD, GSH-Px ve G6PD aktiviteleri kırık gruplarında kontrol grubuna göre istatistiksel olarak önemli bir azalma saptandı (p<0,05). KAT, SOD, GSH-Px ve G6PD aktivitelerinde kırık+akupunktur gruplarında kırık grubuna göre önemli bir artma gözlendi (p<0,05). Sonuç olarak akupunktur uygulamasının SOR’nin zararlı etkilerini baskılayarak ve antioksidan enzim aktivitelerini düzenleyerek kırık iyileşme sürecini olumlu yönde etkilediği söylenebilir. 

Anahtar Kelimeler

• Akupunktur,Antioksidan Enzim,Glutatyon,Kırık İyileşmesi,Malondialdehid

Kaynakça

1. Acosta-Olivo C, Siller-Adame A, Tamez-Mata Y, Vilchez-Cavazos F, Peña-Martinez V (2017). Laser Treatment on Acupuncture Points Improves Pain and Wrist Functionality in Patients Undergoing Rehabilitation Therapy after Wrist Bone Fracture. A Randomized, Controlled, Blinded Study. Acupunct Electrother Res, 42(1), 11-25.
2. Adrian W (2012). Acupuncture in Medicine. BMJ Group Subscriptions Department, Northwich, Cheshire.
3. Aebi H (1974). Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. 2nd English ed. Weinheim: Verlag Chemie; 673-678.
4. Avitabile M, Rasa R, Campagna NE, et al. (1996). Calcium release from the mineral matrix of the mandibular bone due to hydrogen peroxide exposure. Minerva Stomatol, 45, 401-403.
5. Basu S, Michaelsson K, Olofsson H, Johansson S, Melhus H (2001). Association between oxidative stress and bone mineral density. Biochem Biophys Res Commun, 288, 275-279.
6. Beutler E (1975). Red cell metabolism. In: A manual of biochemical methods. 67–69, New York: Grune Strottan.
7. Bukata SV (2011). Systemic administration of pharmacological agents and bone repair: What can we expect. Injury, 42, 605-608.
8. Cetinus E, Kilinç M, Uzel M, et al. (2005). Does long-term ischemia affect the oxidant status during fracture healing? Arch Orthop Trauma Surg, 125, 376-380.

9. Cheeseman KH (1993). Mechanisms and effects of lipid peroxidation. Mol Aspects Med, 14, 191-197.
10. Claes L, Gebhard F, Ignatius A, et al. (2017). The effect of a combined thoracic and soft-tissue trauma on blood flow and tissue formation in fracture healing in rats. Arch Orthop Trauma Surg, 137(7), 945-952.
11. Cohen ME, Meyer DM (1993). Effects of dietary vitamin E supplementation and rotational stress on alveolar bone loss in rice rats. Arch Oral Biol, 38, 601-606.
12. Collin-Osdoby P, Li L, Rothe L, et al. (1998). Inhibition of avian osteoclast bone resorption by monoclonal antibody 121F: A mechanism involving the osteoclast free radical system. J Bone Miner Res, 13, 67-78.
13. Comeli CN, Lane JM (1992). New. st Factors in fraeture healing. Orthop, 277, 297-311.
14. Durak K, Bilgen ÖF, Kaleli T, et al. (1996). Antioxidant effect of alfa-tocopherol on fracture haematoma in rabbit. J Int Med Res, 24, 419-424.
15. Ellman G (1959). Tissue sulphydryl groups. Arch Biochem Biophys, 2, 70-77.
16. Engle WA, Yoder MC, Baurley JL, Yu P (1998). Vitamin E decreases superoxide anion production by polymorphonuclear leucocytes. Pediatric Res, 23, 245-248.
17. Eyres KS, Saleh M, Kanis JA (1996). Effect of pulsed electromagnetic fields on bone formation and bone loss during limb lengthening. Bone, 18(6), 505-509.
18. Frankel S, Reitman S, Sonnen AC (1970). A textbook on laboratory procedure and their interpretation. Ch 20. In: Grand-Wohl’s Clinical Laboratory Methods and Diagnosis. The CV Mosby Comp, 1(7), 403-404.
19. Frantz AL, Regner GG, Pflüger P, et al. (2017). Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice. Neurosci Lett, 661, 33-40.
20. Frost HM (1989). The biology of fraeture healing. An overview for c1inieians. Part II. Elin Orthop, 248, 294-308.
21. Gao C, Zhao J, Yang, D (2017). Efficacy of acupuncture in pain management of chronic diseases of bone and joint: a review of literature. Int J Clin Exp Med, 10(6), 8788-8800.
22. Garrett IR, Boyce BF, Oreffo ROC, et al. (1990). Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo. J Clin Invest, 85, 632-639.
23. Göktürk E, Turgut A, Bayçu C, et al. (1995). Oxygen-free radicals impair fracture healing in rats. Acta Orthop Scand, 66, 473-475.
24. Göktürk E (1997). Sıçanlarda serbest oksijen radikallerinin kırık iyileşmesine etkisi. Acta Orthop Traumatol Turc, 3, 353-356.
25. Greenwald RA, Moy WW (1979). Inhibition of collagen gelation by action of the superoxide radical. Arthritis Rheum, 22 (3), 251-259.
26. Halıcı M, Öner M, Güney A, et al. (2010). Melatonin promotes fracture healing in the rat model. Eklem Hastalık Cerrahisi, 21, 172-177.
27. Harmankaya A, Özcan A (2017). Effect of different doses of mistletoe lectin-I on the levels of tumor necrosis factor-α, nitric oxide, total antioxidant and oxidant capacity in rabbits. Van Vet J, 28(1), 41-45.
28. Histing T, Marciniak K, Scheuer C, et al. (2011). Sildenafil accelerates fracture healing in mice. J Orthop Res, 29, 867-873.
29. Hunter J (2011). Acupuncture for keloid scar. Acupunct Med, 29(1), 2.
30. Huo MH, Troiano NW (1991). The influence of ibuprofen on fracture repair: biomechanical, biochemical, histologic, and histomorphometric parameters in rats. J Orthop Res, 9(3), 383–390.
31. Keskin D, Karsan O, Ezirmik N, Çiftçioğlu A (1999). Tavşanlarda kırık iyileşmesi üzerine alfa-tokoferolün etkisi. Artroplasti-Artroskopik Cerrahi, 10(2), 207-210.
32. Kim HY, Sohn BY, Seo UK, et al. (2009). An exploratory study of gold wire implantation at acupoints to accelerate ulnar fracture healing in rats. J Physiol Sci, 59(4), 329-333.
33. Leveille SG, LaCroix AZ, Koepsell TD, et al. (1997). Do dietary antioxidants prevent postmenopausal bone loss? Nutr Res, 17(8), 1261-1269.
34. Li CF, Wang JR, Zeng Y, Sun P, Zhao L (2008). Clinical observation on acupuncture combined with inductance coupling for treatment of delayed union and nonunion. Zhongguo Zhen Jiu, 28(5), 334-336.
35. Mammi GI, Rocchi R, Cadossi R, Massari L, Traina GC (1993). The electrical stimulation of tibial osteotomies. Double-blind study. Clin Orthop Relat Res, 288, 246-253.
36. McCord JM (1985). Oxygen-derived free radicals and tissue injury. N Engl J Med, 312(3), 159.
37. McKibbin M (1978). The biology of fraeture healing in long bones. J Bone Joint Surg, 60, 150-162.
38. Nakajima M, Inoue M, Hojo T, et al. (2010). Effect of electroacupuncture on the healing process of tibia fracture in a rat model: a randomised controlled trial. Acupunct Med, 28(3), 140-143.
39. Norazlina M, Ima-Nirwana S, Gapor MTA, Khalid BAK (2002). Tocotrienols are needed for normal bone calcification in growing female rats. Asia Pacific J Clin Nutr, 11(3), 194-199.
40. Paley CA, Bennett MI, Johnson MI (2011). Acupuncture for cancer-induced bone pain?. Evid Based Complement Alternat Med. 671043.
41. Placer ZA, Cushmann LL, Johnson BC (1966). Estimation of products of lipid peroxidation in biochemical systems. Anal Biochem, 16, 359-364.
42. Schulman RA (2001). Tibial shin splint treated with a single acupuncture session: case report and review of the literature. Med Acup, 13(1), 7-9.
43. Serin E, Yılmaz E, Yılmaz S, Ünsaldı E, Durmuş AS (1998). İskemi-reperfüzyon hasarında serbest oksijen radikalleri (ratlarda deneysel çalışma). Artroplasti Artroskopik Cerrahi, 9(1), 36-39.
44. Seyama A (1993). The role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal ischemia/reperfusion injury. Jpn J Surg, 23, 1060.
45. Shandall AA, Williams OT, Hallett MB, Young HL (1986). Colonic healing: a role for polymorphonuclear leucocytes and oxygen radical production. Br J Surg, 73(3), 225-228.
46. Spiro AS, Khadem S, Jeschke A, et al. (2013). The SERM raloxifene improves diaphyseal fracture healing in mice. J Bone Miner Metab, 31, 629-36.
47. Suda N (1991). Role of free radicals in bone resorption. Kokubyo Gakkai Zasshi, 58, 603-612.
48. Sun Y, Oberley LW, Li Y (1988). A simple method for clinical assay of superoxide dismutase. Clin Chem, 34, 497-500.
49. Toledo-Pereyra LH, Lopez-Neblina F, Toledo AH (2004). Reactive oxygen species and molecular biology of ischemia/ reperfusion. Ann Transplant, 9, 81-83.
50. Turgut A, Göktürk E, Köse N, et al. (1999). Oxidant status increased during fracture healing in rats. Acta Orthop Scand, 70, 487-490.
51. Wang WZ, Fang XH, Stephenson LL, et al. (2005). Microcirculatory effects of melatonin in rat skeletal muscle after prolonged ischemia. J Pineal Res, 39, 57-65.
52. Yeler H, Tahtabas F, Candan F (2005). Investigation of oxidative stress during fracture healing in the rats. Cell Biochem Funct, 23, 137-139.
53. Yılmaz S, Atessahin A, Sahna E, Karahan I, Ozer S (2006). Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity. Toxicology, 218 (2-3), 164-171.
54. Yılmaz S, Yılmaz E (2006). Effects of melatonin and vitamin E on oxidative-antioxidative status in rats exposed to irradiation. Toxicology, 222 (1-2), 1-7.
55. Yılmaz S, Issi M, Kandemir MF, Gul Y (2014). Malondialdehyde and total antioxidant levels and hematological parameters of beef cattle with coccidiosis. YYU Vet. Fak. Derg, 25, 41-45.