Tese
Elevação da concentração plasmática de TNF pelo diabetes mellitus altera a atividade de canais de sódio dependentes de voltagem, efeito atenuado pelo tratamento com o composto A
Fecha
2018-08-31Autor
Flávio Henrique Pequeno de Macedo
Institución
Resumen
Peripheral Diabetic Neuropathy (PDN) is the most common complication of diabetes, affecting approximately 50% of diabetic patients and being the main cause of foot ulcer morbidity and amputation. Among patients who develop PDN, between 20 and 30% suffer from Diabetic Neuropathic Pain (DNP). Individuals affected by DNP describe episodes of pain such as tingling sensations, burning, numbness and/or drilling by electrified needles. Clinical and preclinical studies have shown that diabetic patients with DNP show an increase in serum TNF concentration, whereas studies with diabetic animals have shown that TNF induces increased expression of Nav 1.7 sodium channels, which would result in sensitization of terminals periphery of nociceptive neurons, a factor that would contribute to the development of DNP. Currently, treatments available for DNP result in unsatisfactory pain relief for most of these patients. Therefore, the present study verified whether the treatment of diabetic rats with Compound A, a natural agent with proven anti-inflammatory activity, is able to prevent the development of hyperalgesia in these animals. For this, 28-day-old Wistar rats were induced to diabetes via intraperitoneal injection of streptozotocin (65 mg/kg). During the 60 day period, these rats had their mechanical threshold assessed by the Randall-Selitto method. After collection of blood samples, serum TNF concentration was measured in these rats on the day prior to diabetes induction and at day 60 after induction by the ELISA method. At day 60 after induction to diabetes, the rats were euthanized to obtain their DRGs and dissociate their neurons. From these neurons the total sodium current was measured by the Patch Clamp technique, in Whole Cell mode. These protocols were approved by the Ethics Committee on the Use of Animals (CEUA) under the protocol number 233/2013. The rats of the Diabetic group had sustained hyperglycemia in comparison to control rats (550.7 ± 10.6 mg/dl, n=22 and 117.9 ± 1.7 mg/dl, n=24, respectively, day 60 after induction to diabetes). In this parameter, diabetic rats treated with Compound A (Diabetic + CA) showed glycemic values similar to those of the Diabetic group (539.4 ± 30.1 mg/dl, n=7). The Diabetic group also showed a reduction of the mechanical threshold when compared to the Control group (116.1 ± 5.8 g, n=16 and 212.1 ± 5.9 g, n=18, respectively, day 60 after induction to diabetes). This reduction in the mechanical threshold of the diabetic group was attenuated in the Diabetic + CA group (143.9 ± 5.6 g, n=11). Concomitant with these data, the rats of the Diabetic group exhibited a high serum TNF concentration compared to the Control group rats (624.9 ± 97.8, n=7 and 340.3 ± 16, n=7,
respectively), while in the Diabetic + CA rats the values verified were similar to those in the Control group (320.8 ± 10, n=7). Regarding to the cellular electrophysiology data, the Diabetic group presented increase of total sodium current density in comparison with the Control group (231.4 ± 25.1 pA/pF, n=10 and 189.4 ± 10.7 pA/pF, n=18, respectively). The Diabetic + CA group, on the other hand, showed values of total sodium current density similar to those in the Control group (159.2 ± 14.7 pA/pF, n=12). These data indicate that treatment with Compound A was able to attenuate, but not completely prevent, the development of hyperalgesia. This attenuation is possibly a result of the reduction in serum TNF levels of the rats from the Diabetic + CA group.
The present work also verified the effects of the exposure of neurons dissociated from DRG to TNF at a concentration of 700 pg/ml (concentration similar to that found in the blood of rats from the Diabetic group) over both TTX-sensitive sodium currents (TTXs) and resistant to TTX (TTXr) by the Patch Clamp technique. After 6 hours exposure, TNF induced the increase in both TTX sodium current density (≈89%) and TTX sodium current density (≈65%). However, it was found that TNF sensitizes both the fast stationary activation kinetics and the fast inactivation kinetics of the TTXs sodium current, only, causing hyperpolarization in the V1/2 values in the order of ≈5 mV and ≈3 mV, respectively. With these data, it is noticed that the serum concentration of TNF found in the rats of the Diabetic group is able to induce a change in the sodium current TTXs and TTXr, in order to contribute with the excitability increase of these neurons. Thus, the reduction in serum TNF levels in the Diabetic + CA group rats would attenuate the development of hyperalgesia.