Nicotine inhibits rapamycin-induced pain through activating mTORC1/S6K/IRS-1-related feedback inhibition loop

Highlights

• Nicotine at a dose of 1.5 mg/kg relieves rapamycin-induced pain in the naïve mice.

• Nicotine modulates the mTORC1/S6K/IRS1 pathway in ACC of rapamycin-treated mice.

• Nicotine corrects dysregulated neuronal excitability in ACC of rapamycin-treated mice.

• The above effects can be blocked by the antagonists of α4β2 or α7 nAChRs.

Abstract

Mammalian target of rapamycin complex 1 (mTORC1) inhibitors increase the incidence of pain in patients, and this finding has been replicated in animal models. However, reports on possible analgesics for this condition are scant. Accumulating evidence finds that nicotinic acetylcholine receptors (nAChRs) are involved in mediating pain. However, whether nicotine, a full agonist of nAChRs, alleviates mTORC1 inhibition-induced pain and its underlying mechanisms remain unknown. In this study, pain was induced in naïve male C57BL/6J mice by intraperitoneally injecting rapamycin acutely or repeatedly. Subsequently, pain thresholds, including mechanical and thermal pain, were measured. The involving signaling pathway was tested using western blot analysis and immunofluorescent assay. Changes in neuronal excitability caused by different treatments were also analyzed using whole-cell recording. Microinjection into the anterior cingulate cortex (ACC) was used to test the role of nAChRs containing the α4β2 or α7 subtype in this brain region in pain modulation. Our results showed that nicotine significantly reduced hyperalgesia in mice that received acute or repeated rapamycin injections, and reversed the effects of rapamycin on the phosphorylation of S6K, 4E-BP1, insulin receptor substrate-1 (IRS-1) at Ser636/639, AKT at Ser473, and ERK at Thr202/Tyr204. Whole-cell recording results showed that nicotine reduced the firing rates of pyramidal neurons in the ACC, and a pharmacological blockade of nAChRs containing the α4β2 or α7 subtype in ACC inhibited the antinociceptive effects of nicotine in mice with rapamycin-induced pain. Our findings indicate that analgesics targeting nAChRs can be developed to help patients with rapamycin-induced pain.

Introduction

Mammalian target of rapamycin complex 1 (mTORC1) functions as a central regulator for cell proliferation, growth and survival. For clinical use, the natural inhibitor of mTORC1, rapamycin, has been developed as an anticancer and immunosuppressive agent. However, clinical studies have shown that the incidence of pain is increased in patients chronically treated with mTORC1 inhibitors (Budde, 2011; Budde et al., 2011; McCormack et al., 2011), including the possible development of complex regional pain syndrome (Massard et al., 2010; Molina et al., 2008). Preclinical data demonstrated that mTORC1 inhibition induced pain through the insulin receptor substrate-1 (IRS-1)-dependent feedback activation of ERK (Melemedjian et al., 2013). However, reports on possible analgesics for this condition remain scant.

In recent years, increasing scientific data have shed light on nAChRs modulation for pain relief (Lawand et al., 1999; Umana et al., 2013). Because of the serious adverse effects of the frog skin-derived alkaloid epibatidine as an analgesic, which is a non-selective agonist for nAChRs, the research focus has shifted to develop compounds that selectively target specific neuronal nAChR subtypes. However, available evidence has indicated that α4β2-, α7-, α5-, α3β4-, and α9/α10-containing nAChRs are all involved in pain processing (Dineley et al., 2015; Umana et al., 2013). Targeting different subtypes at the same time will probably produce synergistic analgesic effect.

Nicotine is the primary reinforcing agent in tobacco and exerts its effects through activating nAChRs. Except for its performance-enhancing effects on cognition, alertness, and attention, nicotine has been studied as a novel intervention in pain therapeutics. The antinociceptive properties of nicotine were first observed in feline visceral pain models (Davis and Stone, 1932) and subsequently demonstrated in numerous animal and human studies (Matthews et al., 2016; Umana et al., 2013). In animal behavioral studies, nicotine exerted a potent antinociceptive action on thermal stimuli as measured by the tail-flick test (Sahley and Berntson, 1979), and was found to act as a pain reliever in trauma- and chemotherapy-induced neuropathy models (Di Cesare Mannelli et al., 2013). However, whether nicotine alleviates mTORC1 inhibition-induced pain remains unknown.

Despite the crucial role of the spinal cord in mTORC1 inhibition-induced pain, a body of evidence has demonstrated that the anterior cingulate cortex (ACC) is a key region in the brain for pain perception (Bliss et al., 2016; Fuchs et al., 2014; Zhuo, 2008). A positron emission tomography (PET) study showed a high density of nAChRs in the ACC of nonsmoking healthy Caucasian men (Picard et al., 2013). However, whether this brain region is involved in the processing of mTORC1 inhibition-induced pain has not been evidenced.