Inducible nitric oxide synthase mediates complete Freund's adjuvant-induced inflammatory pain aggravated by short-term high-fat diet

Abstract

INTRODUCTION: Chronic inflammatory pain has remained a therapeutic challenge due to the lack of safe and potent analgesic agents. This problem has been accentuated by the worldwide prevalence of a high-fat dietary lifestyle, which has pro-inflammatory effects and predisposes to worse inflammatory pain problems. This study aimed at investigating pathways potentially mediating the chronic inflammatory pain augmented by short-term high-fat diet (HFD) in animals. METHODS: an in-vivo model of persistent inflammatory pain was established on C57BL/6J mice 8 weeks of age by subcutaneous injection of 20 μL of Complete Freund’s Adjuvant (CFA) on the plantar surface of left hind paws, compared to subcutaneous intraplantar normal saline injection in control group. Behavioral tests for pain, including manual von Frey test for mechanical hyperalgesia and Hargreaves’ test for thermal hyperalgesia, were performed at pre-injectional baseline and post-injection time points including the 6th hour, 1st, 3rd, 5th, 7th, 10th and 14th day. Short-term dietary modification with ad libitum high-fat diet (HFD) providing 60% total calories commenced 14 days prior to CFA injection in the assigned animal groups and lasted until experimental endpoint. Body weight was measured and random glucose levels were tested on blood samples from tail-tip amputation using a glucometer set. Molecular assays including RT-qPCR ELISA were used to quantify gene expression levels of the selected candidate genes at the CFA or saline-injected hind paws. Subsequent experiments also utilized 1400W, a highly selective inducible nitric oxide synthase (iNOS) inhibitor, administered 10 mg/kg intraperitoneally every 12 hours since 2 hours after CFA injection to demonstrate any downstream effects in body weight, CFA-induced pain behaviors, and mRNA transcription levels after CFA injection in HFD-fed mice. FINDINGS: Intraplantar injection of CFA induced mechanical and thermal hyperalgesia persisting up to 14 days after injection. CFA also upregulated both pro-inflammatory and anti-inflammatory cytokines. Short-term HFD led to significantly higher body weight and random blood glucose levels at pre-injectional baseline, consistent with diet-induced obesity. Pain thresholds were not significantly altered in HFD-fed mice before CFA injection. Subsequently, HFD-fed mice exhibited prolongation of CFA-induced thermal hyperalgesia and further reduction in nociceptive thresholds at selected few time points, consistent with aggravated thermal and mechanical pain profiles in general. Aggravated pain paralleled further upregulation of local mRNA transcription regarding pro-inflammatory cytokine genes. The pattern of differential expression of immune markers induced by CFA differed from that induced by the additional HFD. iNOS was the only tested inflammatory pain mediator upregulated by the additional HFD. 1400W elicited analgesic but pro-inflammatory effects on the inflamed paw on post-injection day 5. CONCLUSION: Short-term HFD aggravated mechanical and thermal pain induced by intraplantar CFA. iNOS linked CFA-induced inflammation to pain in HFD-fed mice and exerted anti-inflammatory effects in this context. iNOS inhibitors could potentially benefit obese patients living with chronic inflammatory pain. The analgesic and pro-inflammatory effects could potentially be mediated via different groups of pathways involving molecular signaling cascades and hormonal influence. The mediating roles of these pathways could be validated by future research.