Joint application of N-Acetylcysteine and allopurinol reduces spinal cord TNF-alpha expression and attenuates mechanical allodynia in diabetic rats

Abstract

Diabetic Neuropathy is a frequent and painful complication of diabetes that leads to progressive and length-dependent Diabetic Peripheral Neuropathic Pain (DPNP). Studies have indicated the involvement of oxidative stress and the activation spinal pro-inflammatory TNF-alpha in the development of diabetic neuropathy. Joint application of the antioxidants N-Acetylcysteine (NAC) and allopurinol (ALP) has been shown to confer superior protection against diabetic cardiomyopathy to their individual administration (Free Radic Biol Med. 2013; 63: 291–303). However, the potential effects and mechanism NAC and ALP combination on DPNP has not been explored. We hypothesized that NAC and ALP may reduce diabetes induced increase in central nervous system (CNS) TNF-alpha expression and attenuate DPNP.Control or streptozotocin (STZ, 65mg/kg)-induced diabetic Sprague Dawley rats (n=5 per group) received vehicle or NAC (1.5g/kg/day) plus ALP (100mg/kg/day) in drinking water for three weeks starting one week after STZ injection. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured weekly. At the end of the experiments (4 weeks after STZ injection), L3–L5 segments of spinal cord and somatosensory cortex were extracted for detecting TNF-alpha protein expression by western blotting assay.The results showed that the thresholds for PWMT and PWTL were reduced significantly in diabetic rats respectively at 1 and 2 weeks after diabetic induction as compared to non-diabetic control (all p<0.05), which was associated with significant increase of spinal cord TNF-alpha protection expression (p<0.05 diabetic vs. control) and moderate increase in cortex TNF-alpha protection expression (P>0.05). Joint application of NAC and ALP significantly attenuated the reduction of thresholds for PWMT starting at two weeks of treatment and onwards (P<0.01 diabetic vs. Control) that was associated significant reduction and normalization of spinal cord TNF-alpha expression (P<0.05, NAC+ALP vs. diabetes; p>0.05, NAC+ALP vs. control). However, NAC and ALP combination did not affect diabetic induced reduction in the threshold for PWTL, nor did it affect cortex TNF-alpha protein expression.It is concluded that reduction of spinal cord TNF-alpha may represent a mechanism by which antioxidants NAC and ALP reduce mechanical allodynia in diabetic rats, and that diabetic thermal hyperalgesia may be mediated by mechanisms other than spinal cord TNF-alpha at a relatively early stage of diabetes.