Peroxynitrite and protein nitration in the pathogenesis of diabetic peripheral neuropathy

Abstract

Background: Peroxynitrite, a product of the reaction of superoxide with nitric oxide, causes oxidative stress with concomitant inactivation of enzymes, poly(ADP-ribosylation), mitochondrial dysfunction and impaired stress signalling, as well as protein nitration. In this study, we sought to determine the effect of preventing protein nitration or increasing peroxynitrite decomposition on diabetic neuropathy in mice after an extended period of untreated diabetes.

Methods: C57Bl6/J male control and diabetic mice were treated with the peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS, 10 mg/kg/day) or protein nitration inhibitor (-)-epicatechin gallate (20 mg/kg/day) for 4 weeks, after an initial 28 weeks of hyperglycaemia.

Results: Untreated diabetic mice developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia and loss of intraepidermal nerve fibres. Both FeTMPS and epicatechin gallate partially corrected sensory nerve conduction slowing and small sensory nerve fibre dysfunction without alleviation of hyperglycaemia. Correction of motor nerve conduction deficit and increase in intraepidermal nerve fibre density were found with FeTMPS treatment only.

Conclusions: Peroxynitrite injury and protein nitration are implicated in the development of diabetic peripheral neuropathy. The findings indicate that both structural and functional changes of chronic diabetic peripheral neuropathy can be reversed and provide rationale for the development of a new generation of antioxidants and peroxynitrite decomposition catalysts for treatment of diabetic peripheral neuropathy.

Keywords: antioxidants; diabetic neuropathy; nitrosative stress; oxidative stress; peroxynitrite; superoxide.

Figure 1

Representative images of intraepidermal nerve fiber profiles, magnification x 400 (A, C), and intraepidermal nerve fiber densities (B, D), in experimental groups at 28 weeks (before treatments with FeTMPS or epicatechin gallate), (A and B) and after 4 weeks of treatment (C and D). Mean ± SEM, the number of mice for each study group for C and D are the same as described in Table 2. Along the X axis C represents control mice; D, diabetic mice; E, epicatechin gallate treated mice; and Fe, FeTMPS treated mice. ** p < 0.01 vs non-diabetic mice; ^# p < 0.05 vs untreated diabetic mice.

Figure 2

Nitrated proteins in dorsal root ganglion neurons (DRG), sciatic nerve and spinal cord isolated from control (C) and diabetic (D) mice treated with or without FeTMPS (Fe) or epicatechin gallate (E) for 4 weeks after 28 weeks of untreated diabetes. Mean ± SEM, the number of mice for each study group is the same as described in Table 2. Along the X axis C represents control mice; D, diabetic mice; E, epicatechin gallate treated mice; and Fe, FeTMPS treated mice. * p < 0.05 compared to control; ** p < 0.01 compared to control; # p < 0.05 compared to untreated diabetic; ## p < 0.01 compared to untreated diabetic

Figure 3

4-Hydroxynonenal adducts in dorsal root ganglion neurons (DRG), sciatic nerve and spinal cord isolated from control (C) and diabetic (D) mice treated with or without FeTMPS (Fe) or epicatechin gallate (E) for 4 weeks after 28 weeks of untreated diabetes. Mean ± SEM, the number of mice for each study group is the same as described in Table 2. Along the X axis C represents control mice; D, diabetic mice; E, epicatechin gallate treated mice; and Fe, FeTMPS treated mice. * p < 0.05 compared to control; ** p < 0.01 compared to control; ## p < 0.01 compared to untreated diabetic