D. Rajkumar, O. Gudyrev, A. V. Faitelson
2016
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Abstract
The experiment was carried out in female white Wistar rats to investigate the effect of L-norvaline, rosuvastatin and their combination on parameters of blood supply to the bone tissue on models of experimental osteoporosis and osteoporotic fractures. It was found that the studied drugs prevent reduction of microcirculation in osteoporotic bone tissue as well as in callus tissue of experimental osteoporotic fractures and it positively influences the course of reparative regeneration of bone tissue. Keyword: osteoporosis, osteoporotic fracture, microcirculation, endothelial dysfunction, L-norvaline, rosuvastatin, Bivalos. Introduction The World Health Organization defines osteoporosis as “a systemic disease of skeleton, characterized by low density of bone tissue and its microarchitecture defects”, that leads to high bone fragility and high fracture risks. The modifiable factors of osteoporotic disorders are low level of calcium and vitamin D, sedentary life-style, smoking, alcohol abuse, long-term glucocorticoid therapy and rheumatoid arthritis. The non modifiable factors includes advanced age, genetic predisposition, rapid weight loss and menopause [1, 2, 3]. According to the National Osteoporosis Foundation, ten million people in the USA have osteoporosis and 80% of them are women. Fractures are considered one of the main reasons of physical inactivity in elderly women, this significantly reduces their life quality. Russian and foreigner orthopedic trauma specialists, state that fractures of neck of the femur is the most typical osteoporotic complication and the mortality rate is up to 30% during the first six months. The number of surgical procedures on bone fractures using different implants is growing every year, including the use of endoprosthesis for large joints. According to recent investigations, each fifth operation of primary endoprosthesis replacement includes one rereplacement manipulation. This is generally connected with the development of aseptic instability due to osteoporotic disorders in bone tissue. Blood circulation plays a significant role in bone tissue normal functioning. An important feature of the bone microvasculature is that the vessel wall does not have muscle and connective tissue layers, it consists only of the endothelial layer. Consequently, the humoral regulation between the osteocytes and blood is mediated by the endothelium. According to many authors, the imbalance between the processes of bone resorption and osteogenesis is based on endothelial dysfunction [4, 5, 6, 7, 8, 9, 10]. Rajkumar D.S.R., Gudyrev O.S., Faitelson A.V., Stepchenko A.A., Dolzhikov A.A., Povetkin S.V. Study of the influence of L-norvaline, rosuvastatin and their combination on the level of microcirculation in bone tissue in experimental osteoporosis and fractures on its background. Research result: pharmacology and clinical pharmacology. 2016. Vol. 2, No1 (2): 20-24. 21 RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Norvaline (α-aminovaleric acid, propyl glycine) is a unconventional amino acid which is part of some proteins, in animals it is the antagonist of valine and leucine. L-norvaline is an inhibitor of arginase (a pharmacological agent with mechanism of action directed in blocking the arginase activity, thus increasing the concentration of L-arginine and, consequently increasing nitric oxide in serum) an enzyme that catalyses the cleavage of L-arginine, a substrate for nitric oxide, that is a powerful vascular endothelial mediator functioning on ornithine and urea [11, 12, 13, 14, 15]. L-norvaline is one of those substances, however its effect on the bone tissue microcirculation has not previously been investigated. This fact is the reason for the present research. It is widely known that statins have an indirect corrective effect on endothelial dysfunction through the normalization of lipid profile and by direct effect on the endothelium (regardless of changes in lipid profile). Possible mechanisms for improvement of endothelial functioning in the administration of statins is achieved through increased production of nitric oxide (NO), with beneficial effect on the expression of endothelial NO-synthase, decreased secretion of endothelin-1 and increased synthesis of prostacyclin, which positively affects the balance shift vasoconstrictors/vasodilators in favour of vasodilation, restoration of high content of plasma vascular endothelial growth factor (VEGF) and the induction of apoptosis in endothelial cells. Rosuvastatin is a medication from statins group. In the available literature there is a wide variety of works about the role of statins in the correction of endothelial dysfunction, however, data about their effects on the bone tissue microcirculation is not sufficient. Based on the powerful endothelial protective effect of arginase inhibitor, L-norvaline, and selective and competitive inhibitor of HMG-CoA reductase, rosuvastatin. A scientific interest on the study of their combined use has arisen, as implemented in this research. Objectives. To study the effect of L-norvaline, rosuvastatin and their combination in the bone tissue microcirculation after experimental models of osteoporosis and fractures of proximal femur in Wistar rats. Methods and materials. The experiment involved 240 female Wistar rats weighing 200-250g. Manipulations were performed with animals under general anesthesia (intraperitoneal injection of an aqueous solution of chloral hydrate at a dose of 300 mg/kg). The animals were divided into eight groups – 20 rats each. I – Intact – false bilateral oophorectomy surgery (performed abdominal incision without removal of the ovaries, followed by wound closure); II – Control – true bilateral oophorectomy surgery (performed abdominal incision with removal of the ovaries, followed by wound closure) [16, 17, 18]; III – 8 weeks after oophorectomy and development of osteoporosis L-norvaline was daily administrated intragastrically, at a dose of 10mg/kg from the ninth to twelfth week [19]; IV – 8 weeks after oophorectomy, rosuvastatin was daily administered intragastrically, at a dose of 0.86mg/kg from the ninth to twelfth week; V – 8 weeks after oophorectomy, a combination of L-norvaline at a dose of 10mg/kg, and rosuvastatin at a dose of 0.86mg/kg, was daily administered intragastrically from the ninth to twelfth week; VI – 8 weeks after oophorectomy, a drug for comparison, Bivalos (strontium ranelate) was daily administered intragastrically at a dose of 171mg/kg from the ninth to twelfth week; VII – 8 weeks after false oophorectomy, a model fracture of the proximal femoral metaphysis was performed; VIII – 8 weeks after bilateral oophorectomy, a model fracture of the proximal femoral metaphysis was performed; IX – 8 weeks after oophorectomy, a model fracture of the proximal femoral metaphysis was performed with further daily intragastrical administration of L-norvaline at a dose of 10mg/kg from the ninth to twelfth week; X – 8 weeks after oophorectomy, a model fracture of the proximal femoral metaphysis was performed with further daily intragastrical administration of rosuvastatin at a dose of 0.86mg/kg from the ninth to twelfth week; XI – 8 weeks after oophorectomy, a model fracture of the proximal femoral metaphysis was performed with further daily intragastrical administration of a combination of L-norvaline at a dose 10mg/kg and rosuvastatin at a dose 0.86mg/kg from the ninth to twelfth week; XII – 8 weeks after oophorectomy, a model fracture of the proximal femoral metaphysis was performed with further daily intragastrical administration of a drug for comparison, Bivalos, at a dose 171mg/kg from the ninth to twelfth week. Fractures were induced by close method using a cutting blade, the cutting edges were covered with rubber tubes and external force was applied perpendicular to the axis of the limbon the proximal Rajkumar D.S.R., Gudyrev O.S., Faitelson A.V., Stepchenko A.A., Dolzhikov A.A., Povetkin S.V. Study of the influence of L-norvaline, rosuvastatin and their combination on the level of microcirculation in bone tissue in experimental osteoporosis and fractures on its background. Research result: pharmacology and clinical pharmacology. 2016. Vol. 2, No1 (2): 20-24. 22 RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY metaphysis of the femur, the pressure was applied until the characteristic signs of fracture (abnormal motility and crepitations on bone fragments) were present. After that the fractures were stabilized by Kwires (1 mm in diameter) inserted intramedullary from the distal to the proximal part of the femur. For measurement of the level of microcirculation in the proximal femoral metaphysis, conducted after 12 weeks (on the eighty-fifth day), equipment manufactured by Biopac systems were used: namely the polygraph MP150 unit with laser Doppler flowmetry (LDF) module LDF100C and invasive needle probe TSD144. A hole was drilled in the cortical layer of bone tissue with a depth of 23mm, into which the needle of sensor probe was placed. The data of laser Doppler flowmetry was processed and recorded by software AcqKnowledge version 4.1, the values were expressed in microcirculatory perfusion units (PU). To confirm the endothelial vessels dysfunction and the degree of correction of the studied medication after oophorectomy in the I, II, III, IV, V and VI groups, tests were conducted based on the endothelium dependent vasodilation (EDVD) and endothelium non dependent vasodilation (ENVD) in response to intravenous bolus solutions of acetylcholine at a dose of 40μg/kg and sodium nitroprusside at a dose of 30μg/kg, respectively. After that the coefficient of endothelial dysfunction (CED) was calculated based on the LDF on the bone. It is defined as the ratio of the triangular area over recovery curve of microcirculation in response to the administration of sodium nitroprusside to the triangular ares over the recovery curve of microcirculation in response t