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Increasing evidence indicates that neuropeptide FF (NPFF) produces analgesic effects and augments opioid-induced analgesia at the spinal level. However, our recent research demonstrated that NPFF exerted complex opioid-modulating effects in an inflammatory pain model after intrathecal (i.t.) injection. Consistent with previous findings, we found that i.t. NPFF dose-dependently attenuated complete Freund’s adjuvant-induced pain hypersensitivity. Interestingly, pharmacological results illustrated that NPFF exhibited opposite opioid-modulating effects at the spinal level depending on its administration dosage, wherein i.t. NPFF potentiated morphine-induced anti-allodynia at the dose of 10 nmol, while attenuated morphine analgesia at an ultra-low-dose of 10 pmol. Behavioral results obtained from neuropeptide FF receptor 2 (NPFFR2) knockout animals suggested that both pro- and anti-opioid effects of NPFF were mediated by NPFFR2. Moreover, these modulating effects of spinal NPFFR2 were selectively targeting mu-opioid receptor, had no effect on delta- and kappa-opioid receptor agonist-induced analgesia. Finally, the opioid-modulating effects of NPFF were further verified using in vitro calcium imaging assay, demonstrating that pretreated with NPFF in primary-cultured spinal neurons significantly attenuated the inhibitory effects of morphine on high-K+-induced neuronal excitability. Taken together, our results suggested that NPFF exhibited dual modulating effects on morphine-induced analgesia after i.t. administration, which provides a possible mechanism to explain the complex opioid-modulating effects of endogenous NPFF systems.
Neuropeptide FF (NPFF, FLFQPQRFamide) is an endogenous amidated neuropeptide originally isolated from the medulla oblongata of bovine, which plays important roles in several physiological processes, including food consumption, cardiovascular function, and pain perception. Numerous studies proved that NPFF produced different opioid modulating effects at the spinal and supraspinal levels. Intracerebroventricular injection of NPFF and its analogs was reported to elicit significant hyperalgesic effects and markedly reduced opioid-induced analgesia in rodents. Similarly, NPFF also exerted anti-opioid activities after dorsal raphe, parafascicular nuclei, or ventral tegmental area injection. In contrast, intrathecal (i.t.) NPFF exhibited effective analgesic effects in different preclinical pain models, such as radiant-induced acute pain, carrageenan-induced inflammatory pain, and chronic constriction nerve injury-induced neuropathic pain. Furthermore, NPFF strongly augmented opioid-induced analgesia at the spinal level. Therefore, traditional opinion considered that NPFF produced anti-opioid activities at the supraspinal level, while exhibited pro-opioid effects at the spinal level.
Notably, the inconsistent pharmacological results obtained from our recent studies demonstrated that intrathecally injected the neuropeptide FF receptor (NPFFR) antagonist RF9 significantly enhanced the spinal analgesic effects induced by DN-9, a multifunctional agonist of opioid and NPFF receptors, implying that the NPFF moiety of DN-9 might exert anti-opioid activities and attenuate its opioid antinociception at the spinal level. In line with our findings, previous studies demonstrated that NPFF selectively prevented the inhibition of C-fibre evoked activity induced by i.t. DAMGO. Furthermore, our recent research also showed that pharmacological blockade or genetic knockout spinal NPFFRs markedly augmented fentanyl-induced analgesia in the mouse complete Freund’s adjuvant (CFA)-induced inflammatory pain model. Taken together, these findings suggested that NPFF might also produce anti-opioid activities at the spinal level.
In the present study, to further elucidate the opioid-modulating roles of NPFF at the spinal level, the effects of i.t. injection of NPFF on pain threshold and morphine-induced analgesia were investigated in a mouse model of CFA-induced inflammatory pain. Moreover, the NPFFR knockout animals were used to pharmacologically characterize the opioid-modulating action of NPFF in this model. Finally, in primary-cultured spinal neurons, the opioid-modulating action of NPFF was evaluated and pharmacologically characterized by calcium imaging assays.
Kunming strain (n=165, 18–22 g) and wild-type (WT, n=77, 18–22 g) mice were obtained from the Experimental Animal Center of Lanzhou University, while the heterozygous mice with knockout of NPFFR2 genes (C57BL/6 J background, 18–22 g) were obtained from Cyagen Biosciences Inc. (Suzhou, China). To minimize the influence of hormonal changes underlying estrous cycle on NPFF-related opioid modulating effects, only male mice were used in behavioral tests in this study. All mice were group-housed
The acute antinociceptive effects of i.t. administration of NPFF and morphine were investigated in a mouse model of CFA-induced inflammatory pain. As shown in Fig. 1A and B, both NPFF (10, 20, and 40 nmol) and morphine (0.1, 0.3, and 1 nmol) produced dose- and time-dependent anti-allodynic effects following i.t. administration (NPFF: Dose, F 3, 144=35.9, P< 0.001; Time, F 5, 144=199.8, P< 0.001; Dose×Time Interaction, F 15, 144=6.7, P< 0.001; Morphine: Dose, F 3, 140=21.3, P< 0.001;
NPFF is a neuromodulator showing anti-opioid activity such as counteracting morphine-induced analgesia, tolerance, and rewarding effect. The present study, for the first time, indicated the dual modulating effects of NPFF on morphine-induced analgesia at the spinal level. We found that i.t. injection of 10 nmol NPFF significantly potentiated morphine-induced analgesia, while 10 pmol NPFF attenuated it. Both the pro- and anti-opioid effects of NPFF were mediated by
The experiment complied with the European Community guidelines for using experimental animals (2010/63/EU) and was approved by the Ethics Committee of Lanzhou University (Approval No: Lzujcyxy20240210).
Dan Chen: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Mengna Zhang: Writing – review & editing, Validation, Data curation. Yongtao He: Writing – review & editing, Validation, Investigation, Formal analysis, Data curation. Shuyuan Wu: Validation, Investigation, Formal analysis. Junzhe Kuang: Validation. Zixin Zhang: Methodology, Formal analysis. Biao Xu: Writing – review & editing, Supervision, Methodology,
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
This study was supported by grants from the National Natural Science Foundation of China (82301392, 82473760), and the Major Science and Technology Project of Gansu Province (23ZDNA007), and the Natural Science Foundation of Gansu Province (22JR5RA545).
