Papel das enzimas-conversoras de angiotensina na osteoartrite de joelho em ratos

Abstract

The aim of this study was to evaluate the role of angiotensin-converting enzymes (ACEs) in osteoarthritis (OA) of the knee. Firstly, the standardization of the OA model by sodium monoiodoaceto (MIA) in rats, in relation to inflammatory and pathological parameters, was performed. Male Wistar rats (n=4 per group) were submitted to intra articular injection of 2mg (~50L) of MIA in the knee joint in order to induce OA. Control animals were injected with saline alone (~50L). Paw hyperalgesia and swelling of the knee before and after the intervals of 6, 12, 24, 48 and 72 hours and 7, 14, 21 and 28 days were evaluated. After these periods, the animals were euthanized and the knee joint lavage was performed and used to analyze the kinetics of cell migration and cytokines. Also, the knee was collected for histological analysis. The role of the ACEs was evaluated 28 days after induction of OA using the following orally treatment for 8 weeks (n=7): saline; Captopril [ACE inhibitor, 30mg/kg/day]; Losartan [AT1 receptors antagonist, 30mg/kg/day]; [Diminazene aceturate (DIZE) (ACE2 activator, 1mg/kg/day) or [Captopril, 30mg/kg/day + L-NAME (nitric oxide synthase inhibitor), 15mg/kg/day]. The parameters evaluated in the treatment groups were similar to those performed in the standardization protocol added to the measurement of mean arterial pressure (MAP) and immunohistochemistry for IL-10 and TNF-. The results of the standardization of the experimental model showed that the hyperalgesia was significantly higher in MIA animals at all times points (6 hours to 28 days). The kinetics of cell migration revealed that the leukocyte peak occurred at the time of 72 hours and the macrophage was the main cell responsible for this peak. This difference remained at all times evaluated, except to 21 days. Neutrophils peak happened at 24h and, in this time point, there was also a significant increase in the level of IL-10. It was observed that the swelling of the knee occurred at the time of 12h and 24h and the peak of IL-6 happened at the time of 12h. The loss of proteoglycans started in the 7th day, becoming more significant in the days 14, 21 and 28 post-induction. The evaluation of the granulation tissue present between the subchondral bone and cartilage showed that the change in the cellularity began at 72 hours, but only at the time of 21 days it is statistically different. No significant correlation was viewed between the inflammatory infiltrate in the bone and cartilage and the loss of proteoglycans. Furthermore, the evaluation of the morphological changes in the cartilage revealed disorientation of condrons, clustering and atrophy of chondrocytes starting at the day 14 and vertical fissures and hypocellularity starting at the day 21. On the day 28, all these changes were more pronounced. Regarding to the role of the ACEs in OA, the results showed that Captopril and Losartan were able to significantly reduce the number of total leukocytes and macrophages in the knee joint cavity of the MIA animals. Also, the effect of Captopril was partially blocked by L-NAME. There was a marked increase in the area of IL-10 labeling in the cartilage of control animals treated with Captopril. In addition, ACE inhibition led to a small chondroprotective effect in cartilage. However, the inflammatory profile of the DIZE treated MIA animals did not change significantly. Treatment with DIZE reduced the intensity staining of IL-10 and TNF- in the cartilage, subchondral bone and synovium of MIA rats. Nevertheless, the ACE2 activator did not preserve the cartilage of MIA animals. After 12 weeks of the induction of OA, the histopathological analysis showed that the osteoarthritic changes were more pronounced with great loss of cartilage and proteoglycans, deformation of the subchondral bone and presence of fibrocartilaginous tissue in the joint. There was a trend of reduction in MAP in MIA animals, even when treated with saline. In conclusion, inhibition of ACE has anti-inflammatory effects in the knee of OA rats, as well as a small chondroprotective effect. Likely, these results were limited by the dose of MIA used to induce OA and by the duration of the post-induction period before the evaluation of the parameters.