N-Acetylcysteine causes analgesia in a mouse model of painful diabetic neuropathy

Abstract

N-Acetylcysteine, one of the most prescribed antioxidant drugs, enhances pain threshold in rodents and humans by activating mGlu2 metabotropic glutamate receptors. Here, we assessed the analgesic activity of N-acetylcysteine in the streptozotocin model of painful diabetic neuropathy and examined the effect of N-acetylcysteine on proteins that are involved in mechanisms of nociceptive sensitization. Mice with blood glucose levels ≥250 mg/dl in response to a single intraperitoneal (i.p.) injection of streptozotocin (200 mg/kg) were used for the assessment of mechanical pain thresholds. Systemic treatment with N-acetylcysteine (100 mg/kg, i.p., either single injection or daily injections for seven days) caused analgesia in diabetic mice. N-acetylcysteine-induced analgesia was abrogated by the ${\text{Sx}}_{\text{c}}^{-}$ inhibitors, sulfasalazine (8 mg/kg, i.p.), erastin (30 mg/kg, i.p.), and sorafenib (10 mg/kg, i.p.), or by the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.). Repeated administrations of N-acetylcysteine in diabetic mice reduced ERK1/2 phosphorylation in the dorsal region of the lumbar spinal cord. The analgesic activity of N-acetylcysteine was occluded by the MEK inhibitor, PD0325901 (25 mg/kg, i.p.), the TRPV1 channel blocker, capsazepine (40 mg/kg, i.p.), or by a cocktail of NMDA and mGlu5 metabotropic glutamate receptor antagonists (memantine, 25 mg/kg, plus MTEP, 5 mg/kg, both i.p.). These findings offer the first demonstration that N-acetylcysteine relieves pain associated with diabetic neuropathy and holds promise for the use of N-acetylcysteine as an add-on drug in diabetic patients.

Keywords: N-acetylcysteine; Neuropathic pain; diabetes; nociceptive sensitization; streptozotocin.

Figure 1.

NAC-induced analgesia in the STZ model of painful diabetic neuropathy. Blood glucose levels in mice receiving a single injection of saline or STZ (200 mg/kg, i.p.) are shown in (a), where values are means ± S.E.M. of 7–10 mice. *p < 0.05 (Student’s t-test; t = −8.279). Reductions of mechanical pain thresholds in the same mice at 14 and 21 days following STZ injection are shown in (b), where values are also means ± S.E.M. *p < 0.05 (one-way ANOVA for repeated measures + Duncan method; F_(1,13) = 77.224). The effect of a single injection of NAC or pregabalin on mechanical pain thresholds in diabetic mice and non-diabetic control mice are shown in (c), where values are means ± S.E.M. of 7–10 mice. *p < 0.05 (one-way ANOVA + Duncan method applied only to the groups of diabetic mice; F_(2,18) = 4.562). The effect of repeated administrations of saline or NAC (100 mg/kg, i.p., once a day for 7 days starting from 21 days after STZ injection) on mechanical pain thresholds are shown in (d and e). In (d), all groups of mice were injected once with saline, LY341495, or sulfasalazine at the end of the chronic treatment with saline or NAC (see Methods). Values are means ± S.E.M. of 6–7 mice per group. p < 0.05 versus the control group receiving repeated injections of saline followed by a single injection of saline (*); or versus the group treated with NAC and a single injection of saline (#) (one-way ANOVA + Duncan method; F_(5,35) = 3.364). In (e), all groups of mice were injected once with vehicle, erastin, sorafenib, PD0325901, capsazepine, and mementine+MTEP at the end of the chronic treatment with saline or NAC. Values are means ± S.E.M. of 4–10 mice per group. p < 0.05 versus the control group receiving repeated injections of saline followed by a single injection of vehicle (*); or versus the group treated with NAC and a single injection of vehicle (#); or versus the control group receiving repeated injections of saline followed by a single injection of vehicle ($) (one-way ANOVA + Duncan method; F_(11,73) = 7.947). Blood glucose levels in mice receiving a single injection of saline or NAC (100 mg/kg, i.p.) are shown in (f), where values are means ± S.E.M. of 9 mice. In (g), four groups of mice were injected once with vehicle or JNJ47965567 (30 mg/kg) at the end of the chronic treatment with saline or NAC. Values are means ± S.E.M. of 7–10 mice per group. p < 0.05 versus all other groups (one-way ANOVA + Duncan method; F_(3,28) = 18.643). NAC: N-acetylcysteine; STZ: streptozotocin.

Figure 2.

Expression of proteins targeted by NAC or involved in mechanisms of nociceptive sensitization in the dorsal region of the lumbar spinal cord of diabetic and non-diabetic mice receiving repeated administrations of NAC. Mice were treated i.p. with either saline or NAC for seven days (100 mg/kg) once a day, starting from 21 days following a single injection of saline or STZ. Densitometric values are expressed as % values obtained in the group of STZ mice treated with saline for seven days (this group is common to two–three different immunoblots used for the analysis). Only one representative blot is shown. Densitometric values are means ± S.E.M. of 2–7 mice. p < 0.05 (two-way ANOVA + Dancan’s method; P2X7: F_(1,13) = 11.996 for pathological model (control and diabetic mice) and F_(1,13) = 4.717 for drug treatment; P-ERK1: F_(1,17) = 13.312 for pathological model (control and diabetic mice) and F_(1,17) = 17.514 for drug treatment; and P-ERK2: F_(1,16) = 6.652 for pathological model (control and diabetic mice) and F_(1,16) = 19.678 for drug treatment)). NAC: N-acetylcysteine.

Figure 3.

NAC-induced analgesia in the CFA model of chronic inflammatory pain. Mechanical pain thresholds in mice seven days following CFA injection in the plantar paw after acute systemic treatment with saline or NAC (25-100 mg/kg, i.p.) are shown in (a), where values are means ± S.E.M. of 6–7 mice per group. p < 0.05 (one-way ANOVA + Duncan’s method; F_(4,28) = 3.834) versus saline treated-mice (*) or versus CFA-injected mice after saline treatment (#). The effect of repeated administrations of saline or NAC (100 mg/kg, i.p., once a day for seven days starting from seven days after CFA injection) on mechanical pain thresholds is shown in (b). Values are means ± S.E.M. of seven mice per group. *p < 0.05 (Student’s t-test; t = −1.841). NAC: N-acetylcysteine; CFA: Complete Freund’s Adjuvant.

Figure 4.

Expression of proteins targeted by NAC or involved in mechanisms of nociceptive sensitization in the dorsal region of the ipsilateral lumbar spinal cord or in the plantar paw of CFA-injected mice receiving repeated administrations of NAC. Mice were treated i.p. with either saline or NAC for seven days (100 mg/kg) once a day, starting from seven days following CFA injection in the plantar paw. Densitometric values are means ± S.E.M. of 3–5 mice. *p < 0.05 (Student’s t-test). NAC: N-acetylcysteine.