Morphometric Effect of Erythropoietin Used as Doping and Swimming Exercise in Female Rats in Puberty on Humerus and Femur Bones

In this study, the effect of human erythropoietin (rhEPO) with aerobic exercise on the femur and humerus bone of female rats in the pubertal period was investigated by morphometric method. The study was performed on 40 female rats of the Spraque – Dawley genus. The rats were divided into four groups as erythropoietin, swimming exercise with erythropoietin, swimming and sedentary. For 4 weeks, every other day all rats were injected with rhEPO (100 IU / kg, IP) 4 days a week. After the injection, the swimming group with rhEPO and the solo swimming group were swam for 30 minutes. At the end of 4 weeks, the rats were euthanized, and corpus, height and cortex and cavum medulla measurements of humerus and femur bones were done. Statistical evaluation also indicated that there were no differences (p>0.05) between rhEPO group, swimming group and rhEPO + swimming group femur length and femur cavum medulla da sedentary group. Sedentary group was found to be thicker (p<0.05) in femoral Corpus than swimming and rhEPO group, while there was no statistical difference in femoral cortex but a numerical difference was found. While Humerus bone length and Corpus were not significant in our study (p>0.05), although the numerical values of rhEPO + swimming group in Cortex and Cavum medullada were different compared to other groups, no statistical difference was found (p > 0.05). As a result, it is thought that long-term use of erythropoietin may cause bone development disorders.


Introduction
It is well known that blood and bone share a unique, regulatory relationship with one another. But the details of the relationship between these two are still unanswered (McGee et al., 2012). Erythropoietin (EPO) is a hematopoietic growth factor that stimulates the formation of red blood cells. EPO is known as a doping agent in high-performance sports, and especially in cycling. In the clinical setting, this erythropoiesis stimulating agent is used to treat anemia, especially if it is caused by a lack of endogenous EPO production due to chronic kidney failure. In recent years, the non-hematopoietic functions of EPO, also known as pleiotropic functions, have been extensively investigated. Of interest for orthopedics and musculoskeletal tissue engineering, EPO's nonhematopoietic capabilities include osteogenic and angiogenic potencies (Rölfing, 2014).
The differential role of EPO on different organs indicates tissue-specific functions of EPO (Haroon, et al., 2003 ). The finding that EPO has pleiotropic roles in non-hematopoietic tissues has led to research into the role of EPO in bone formation and homeostasis Wilson & Trumpp, 2006;Yin & Li, 2006 ). Therefore, combining hematopoiesis with skeletal homeostasis via Epo signaling makes important sense (Foldes et al., 1989 ;Gazit et al., 1990 ;Bab et al., 1992 ;Bab & Einhorn, 1993 ;Bab, 1995;Greenberg et al., 1995 ). In their studies, Lee et al., (1991) showed that Epo therapy can increase cortical thickness.
Studies have repeatedly shown the morphological effect of EPO on bone, but the mechanisms that regulate the Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.9, 2020 58 process remain unclear. One suggestion is that EPO may play an important role in regenerating newly absorbed bone by stimulating JAK-STAT signaling pathways through Epo-R in HSCs ( Shiozawa et al., 2010 ).
Among studies that observed bone formation in response to EPO, there are several notable major differences between those that did not. The first is the doses used. Supraphysiological doses are used in most studies showing bone formation. Those who used supraphysiological doses often used EPO doses that fell within a normal range. There may also be age-related differences in the animals ' response to EPO. We recorded that the reaction of young animals to EPO was more robust than the reaction in older animals (unpublished observations). Also, there are well-known differences in the hormonal response of different bones. For example, Singbrant et al. He studied the effects of EPO on the proximal tibia ( Singbrant et al., 2011 ).
Recent and ongoing studies show that hematopoietic stimulation improves bone formation in the context of both early ossification and fraction recovery and advances in mechanical strength (Ferguson et al., 1999;Brager et al., 2000;Bozler et al., 2006;Holstein et al., 2007).
The debate about the effects of EPO on bone formation still needs to be resolved. Also, there is much to learn about the molecular mechanisms of EPO and bone formation; is EPO's effects on the skeleton coupled with hematopoiesis or secondary to EPO's effects on hematopoiesis? If there is a direct effect on the skeleton, the HSC or MSC is the target or both, and both activities need to be present simultaneously for bone formation to occur (McGee et al., 2012).text text text text text

1-Subject Selection: 40 female Sprague-Dawley type rats from the Selçuk University Experimental Medicine
Research and Application Center were used in the study.
Rats were housed in polycarbonate cages (Tecniplast, Italy) in a light-dark cycle of 14:10 hours at 21±2 0C, with 1 rat in 250 cm2 area, adlibitum was fed with standard rat feed (Purina, Canada) and water (normal tap water in glass bottles). The study was continued for 4 weeks.
Forming groups: the groups in the study were formed as follows.     Vol.10, No.9, 2020 groups, no statistical difference was found (p > 0.05).

4.Discussion
The bone that forms the basis of skeletal mobility is a dynamic organ that plays a variety of roles in the body ( Anjos-Afonso & Bonnet, 2007 ). EPO bone creation may also include a hetereceptor that protects tissue (Brines, et al., 2004 ). EPO supplementation may increase cortical thickness in bone (Lee et al., 1991 ). EPO can improve bone formation (Ferguson et al., 1999;Brager et al., 2000;Bozler et al., 2006;Holstein et al., 2007). In their study, Sakunya et al., (2019) states that EPO therapy makes thickening in the corpus by regulating low bone mass in long bones. In our study, it was determined that the sedentary group was thicker (p<0.05) in femoral Corpus than in swimming and rhEPO group, while there was no statistical difference in femoral cortex but a numerical difference was found. Özdemir, (2013) reports that the widest corpus femoris (p<0.05) measurement was determined in the sedentary group.
In another study, Röfling, (2014) in his study on bone fracture recovery in mice, states that EPO increases bone volume and decreases bone marrow cavity. After methanolone enanthate application to adolescent rats, Bozkurt et al., (2011) found that there was a significant (p<0.05) thinning of the corpus femorist while finding that there was a significant (p < 0.05) shortening of the femur length.
In our study, the length and Corpus of humerus bone were not significant (p>0.05), although the numerical values of rhEPO + swimming group in Cortex and Cavum medullada were different compared to other groups, no statistical difference was found (p>0.05). In their study, Lavoie et al., (1998) examined the effect of anaerobic exercise on rat metabolism by using rhEPO supplementation and found that rhEPO contributes to bone tissue in a low proportion. Mohammadian et al., (2003) noted that the increase in iron load made growth retardation in bone particularly apparent in the age of puberta. Similarly, Low., (1997) stated that it inhibits growth in bone in his study looking at growth functions in beta-thalassemia patients.
Low bone mineral density in some elite athletes suggests that intense exercise may have negative effects (Hatun, 2000). There are studies that show that high intensity exercises performed before and during puberta have positive or negative effects on growth and skeletal development. Studies on this subject show that girls with rhythmic gymnastics are shorter and weaker than their peers in other sports and non-sports (Benardot & Czerwinski 1991, Damsgaard et al., 2000. Akin et al., (2004) reported that in their study with rhythmic gymnasts before puberta, intense exercise is shortening in the neck of gymnasts and thinning in bone thickness.

5.Conclusion
As a result, while rhEPO, commonly used as a doping agent in recent years, not only improves performance but provides positive effects in bone forty repairs in the long term, its negative effects on healthy bone tissue have been tried to be explained by rat models. Although the effects of rhEPO and intense exercise on bone development have not been demonstrated, further studies are needed to reveal their effects on other organs.