Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice

Abstract

Background: Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process.

Results: Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1beta), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1beta. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-1beta, and IL-10 following CFA, overall corroborating the inhibitor data.

Conclusion: These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

Figure 1

Effects of pretreatment with the NOS inhibitors 7-NINA, AG, L-NIO and L-NAME on CFA-induced thermal hyperalgesia assessed by the paw withdrawal latency (PWL) tests. Following CFA injection, PWLs were markedly decreased within 6 h and continued until 24 h post injection in the ipsilateral hindpaw (A, ***P < 0.001, NS + CFA vs NS + NS). Pretreatment with 7-NINA, AG and L-NAME, but not L-NIO, dramatically attenuated thermal hyperalgesia in mice receiving CFA throughout the observation period (A, ^###P < 0.001, inhibitor + CFA vs NS + CFA). None of the inhibitors altered pain thresholds in mice receiving NS (B, inhibitor + NS vs NS + NS). n = 4 for each group.

Figure 2

Relative gene expression of nNOS (A), iNOS (B) and eNOS (C) in the ipsilateral plantar skin in mice receiving CFA compared to control mice receiving NS (NS + CFA vs NS + NS). NNOS (A) and eNOS (C) gene expression was increased at 6 h (***P < 0.001), followed by a rapid decline to baseline levels at 16 and 24 h, after CFA. INOS gene expression was increased at 6 h, and peaked at 24 h, after CFA (B, **P < 0.01 and ***P < 0.001). At each time point n = 4.

Figure 3

Relative gene expression of TNF (A), IL-1β (B) and IL-10 (C) in the ipsilateral plantar skin in mice receiving CFA compared to control mice receiving NS (NS + CFA vs NS + NS). Note the rapid increase at 6 h, followed by a second rise for TNF and IL-1β at 24 h and a decline for IL-10 at 16 and 24 h, after CFA (**P < 0.01 and ***P < 0.001). At each time point n = 4.

Figure 4

Effects of pretreatment with the NOS inhibitors 7-NINA, AG, L-NIO and L-NAME on cytokine gene expression after CFA. All treatments prevented the increase of TNF (A, *P < 0.05 and ***P < 0.001; inhibitor + CFA vs NS + CFA) and IL-1β (B, **P < 0.01 and ***P < 0.001; inhibitor + CFA vs NS + CFA) gene expression induced by CFA. In contrast to the effects of AG and L-NIO, which reduced the increase of IL-10 gene expression at 6, 16, 24 h and at 6 and 16 h (C, ***P < 0.001; AG + CFA or L-NIO + CFA vs NS + CFA), respectively, both 7-NINA and L-NAME at 6 and 16 h significantly enhanced the increase of IL-10 (C, ^#P < 0.05 and ^###P < 0.001; 7-NINA + CFA or L-NAME + CFA vs NS + CFA), after CFA. At each time point n = 4.

Figure 5

Cytokine gene expression was lower in NOS-KO mice after CFA compared to WT mice. In control mice, there was no significant difference in baseline gene expression of TNF between KO and WT (A); IL-1β in nNOS- and iNOS-KO, but not in eNOS-KO, was significantly higher than in WT (B;^##P < 0.01); IL-10 in KO was significantly lower than in WT (C; **P < 0.01 and ***P < 0.001). After CFA, the nNOS-, iNOS- and eNOS-KO mice displayed lower TNF, IL-1β and IL-10 gene expression (A, B and C respectively; ***P < 0.001; nNOS-, iNOS- or eNOS-KO CFA vs WT CFA), except that eNOS-KO mice had a higher TNF when compared to WT mice (A;^###P < 0.001) and there was no difference in IL-10 between iNOS-KO and WT mice (C), at 24 h. All measured samples were related to WT control. At each time point n = 4.