Alpha-pinene modulates feeding behavior and hypothalamic orexin-A expression in a rat model of painful temporomandibular disorder

Background: Temporomandibular disorders (TMDs) are common conditions involving the temporomandibular joint (TMJ) and masticatory muscles, often presenting with pain and impaired orofacial function. Painful TMD can disrupt jaw motor activities, including chewing and feeding behavior, reflecting alteration in muscle performance and central neuroregulation. The hypothalamic neuropeptide orexin A integrates pain, arousal, and energy balance and may be involved in these disturbances. This study examined whether intracerebroventricular (ICV) administration of alpha-pinene, an anti-inflammatory monoterpene, could modulate pain-related impairments in feeding behavior and orexin A expression in a rat model of inflammatory TMD. Methods: TMJ inflammation was induced in male Wistar rats via Complete Freund’s Adjuvant (CFA) injection. Rats received ICV alpha-pinene (0.1, 0.2, or 0.4 µg/rat). Feeding behavior parameters—including meal frequency, duration, and total intake—were recorded with an automated monitoring system as functional readouts of masticatory muscle activity during food processing. Anxiety-like behavior was evaluated using the elevated plus maze, and hypothalamic orexin A expression was assessed by immunohistochemistry. Results: CFA-treated rats showed reduced pain thresholds, anxiety-like behavior, and impaired feeding behavior, including fewer meals, shorter feeding duration, and reduced intake. Alpha-pinene, particularly at 0.4 µg/rat, significantly improved these behavioral outcomes and restored hypothalamic orexin A expression compared with untreated CFA rats. Conclusions: Alpha-pinene mitigated pain-related disruptions in feeding behavior and restored hypothalamic orexin A expression in a rat model of TMJ inflammation. These findings highlight the interplay between orofacial pain, altered oral motor function, and central neuroregulation. The observed behavioral improvements suggest that alpha-pinene may offer therapeutic benefits for managing functional impairments associated with both muscular and joint-related TMD pain, supporting its potential as a candidate for integrative TMD management.

Temporomandibular joint disorders; Alpha-pinene; Feeding behavior; Orexins; Rats

[1] Jászberényi M, Thurzó B, Bagosi Z, Vécsei L, Tanaka M. The orexin/hypocretin system, the peptidergic regulator of vigilance, orchestrates adaptation to stress. Biomedicines. 2024; 12: 448.

[2] Nasri-Heir C, Epstein JB, Touger-Decker R, Benoliel R. What should we tell patients with painful temporomandibular disorders about what to eat? The Journal of the American Dental Association. 2016; 147: 667–671.

[3] Fagan HA, Huneke NTM, Domschke K, Baldwin DS. The role of the orexin system in the neurobiology of anxiety disorders: potential for a novel treatment target. Neuroscience Applied. 2024; 3: 103922.

[4] Marcelino V, De Rovere S, Paço M, Gonçalves M, Marcelino S, Guimarães AS, et al. Masticatory function in individuals with temporomandibular disorders: a systematic review and meta-analysis. Life. 2023; 13: 472.

[5] Tao J, Wang X, Xu J. Expression of CGRP in the trigeminal ganglion and its effect on the polarization of macrophages in rats with Temporomandibular Arthritis. Cellular and Molecular Neurobiology. 2024; 44: 22.

[6] Sun S, Yin Y, Yin X, Cao F, Luo D, Zhang T, et al. Anti-nociceptive effects of Tanshinone IIA (TIIA) in a rat model of complete Freund’s adjuvant (CFA)-induced inflammatory pain. Brain Research Bulletin. 2012; 88: 581–588.

[7] Zhang W, Lyu J, Xu J, Zhang P, Zhang S, Chen Y, et al. The related mechanism of complete Freund’s adjuvant-induced chronic inflammation pain based on metabolomics analysis. Biomedical Chromatography. 2021; 35: e5020.

[8] Chen Y, Liu K, Qin Y, Chen S, Guan G, Huang Y, et al. Effects of pereskia aculeate miller petroleum ether extract on complete Freund’s adjuvant-induced rheumatoid arthritis in rats and its potential molecular mechanisms. Frontiers in Pharmacology. 2022; 13: 869810.

[9] Son H, Shannonhouse J, Zhang Y, Gomez R, Amarista F, Perez D, et al. Elucidation of neuronal activity in mouse models of temporomandibular joint injury and inflammation by in vivo GCaMP Ca2+ imaging of intact trigeminal ganglion neurons. Pain. 2024; 165: 2794–2803.

[10] de la Puente B, Romero-Alejo E, Vela JM, Merlos M, Zamanillo D, Portillo-Salido E. Changes in saccharin preference behavior as a primary outcome to evaluate pain and analgesia in acetic acid-induced visceral pain in mice. Journal of Pain Research. 2015; 8: 663–673.

[11] Xia L, Liu HY, Wang BY, Lin HN, Wang MC, Ren JX. A review of physiological functions of orexin: from instinctive responses to subjective cognition. Medicine. 2023; 102: e34206.

[12] Hosaini M, Abbasnejad M, Kooshki R, Esmaeili-Mahani S, Raoof M, Naderi R, et al. The involvement of orexin-1 receptors in modulation of feeding and anxiety-like behavior in rats with complete Freund’s adjuvant-induced temporomandibular joint disorder. Odontology. 2025; 113: 764–775.

[13] Jariyasakulroj S, Chang Q, Ko PF, Shu Y, Lin Z, Chen J, et al. Temporomandibular joint pain measurement by bite force and Von Frey filament assays in mice. Journal of Visualized Experiments. 2024; e67203.

[14] Razavi BM, Hosseinzadeh H. A review of the role of orexin system in pain modulation. Biomedicine & Pharmacotherapy. 2017; 90: 187–193.

[15] Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell. 1998; 92: 573–585.

[16] Ten-Blanco M, Flores Á, Cristino L, Pereda-Pérez I, Berrendero F. Targeting the orexin/hypocretin system for the treatment of neuropsychiatric and neurodegenerative diseases: from animal to clinical studies. Frontiers in Neuroendocrinology. 2023; 69: 101066.

[17] Chung MK, Wang S, Alshanqiti I, Hu J, Ro JY. The degeneration-pain relationship in the temporomandibular joint: current understandings and rodent models. Frontiers in Pain Research. 2023; 4: 1038808.

[18] Kotz CM, Teske JA, Levine JA, Wang C. Feeding and activity induced by orexin A in the lateral hypothalamus in rats. Regulatory Peptides. 2002; 104: 27–32.

[19] Kooshki R, Abbasnejad M, Esmaeili-Mahani S, Raoof M, Sheibani V. Activation orexin 1 receptors in the ventrolateral periaqueductal gray matter attenuate nitroglycerin-induced migraine attacks and calcitonin gene related peptide up-regulation in trigeminal nucleus caudalis of rats. Neuropharmacology. 2020; 178: 107981.

[20] Islam J, Rahman MT, Ali M, Kc E, Park YS. Potential hypothalamic mechanisms in trigeminal neuropathic pain: a comparative analysis with migraine and cluster headache. The Journal of Headache and Pain. 2024; 25: 205.

[21] Sakurai T. The role of orexin in motivated behaviours. Nature Reviews Neuroscience. 2014; 15: 719–731.

[22] Tang HD, Dong WY, Hu R, Huang JY, Huang ZH, Xiong W, et al. A neural circuit for the suppression of feeding under persistent pain. Nature Metabolism. 2022; 4: 1746–1755.

[23] Lord MN, Madu GC, Loera-Lopez AL, Aaron AP, Lin J, Noble EE. Cannabinoid-induced hyperphagia is mediated by increased meal frequency and the orexin-1 receptor in male rats. Pharmacology Research & Perspectives. 2025; 13: e70171.

[24] Bakhtazad S, Ghotbeddin Z, Tabandeh MR, Rahimi K. Alpha-pinene ameliorate behavioral deficit induced by early postnatal hypoxia in the rat: study the inflammatory mechanism. Scientific Reports. 2024; 14: 6416.

[25] Rufino AT, Ribeiro M, Judas F, Salgueiro L, Lopes MC, Cavaleiro C, et al. Anti-inflammatory and chondroprotective activity of (+)-α-pinene: structural and enantiomeric selectivity. Journal of Natural Products. 2014; 77: 264–269.

[26] Rahimi K, Zalaghi M, Shehnizad EG, Salari G, Baghdezfoli F, Ebrahimifar A. The effects of alpha-pinene on inflammatory responses and oxidative stress in the formalin test. Brain Research Bulletin. 2023; 203: 110774.

[27] Rahbar I, Abbasnejad M, Haghani J, Raoof M, Kooshki R, Esmaeili-Mahani S. The effect of central administration of alpha-pinene on capsaicin-induced dental pulp nociception. International Endodontic Journal. 2019; 52: 307–317.

[28] Weston-Green K, Clunas H, Jimenez Naranjo C. A review of the potential use of pinene and linalool as terpene-based medicines for brain health: discovering novel therapeutics in the flavours and fragrances of cannabis. Frontiers in Psychiatry. 2021; 12: 583211.

[29] Yang H, Woo J, Pae AN, Um MY, Cho NC, Park KD, et al. α-Pinene, a major constituent of pine tree oils, enhances non-rapid eye movement sleep in mice through GABAA-benzodiazepine receptors. Molecular Pharmacology. 2016; 90: 30–39.

[30] Khan‐Mohammadi‐Khorrami M, Asle‐Rousta M, Rahnema M, Amini R. Neuroprotective effect of alpha‐pinene is mediated by suppression of the TNF‐α/NF‐κB pathway in Alzheimer’s disease rat model. Journal of Biochemical and Molecular Toxicology. 2022; 36: e23006.

[31] Zamyad M, Abbasnejad M, Esmaeili-Mahani S, Mostafavi A, Sheibani V. The anticonvulsant effects of Ducrosia anethifolia (Boiss) essential oil are produced by its main component alpha-pinene in rats. Arquivos de Neuro-Psiquiatria. 2019; 77: 106–114.

[32] Feriotto G, Tagliati F, Costa V, Monesi M, Tabolacci C, Beninati S, et al. α-Pinene, a main component of Pinus essential oils, enhances the expression of insulin-sensitive glucose transporter type 4 in murine skeletal muscle cells. International Journal of Molecular Sciences. 2024; 25: 1252.

[33] Jin L, Xie Z, Lorkiewicz P, Srivastava S, Bhatnagar A, Conklin DJ. Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels. American Journal of Physiology-Heart and Circulatory Physiology. 2023; 325: H1446–H1460.

[34] Chiba K, Tsuchiya M, Koide M, Hagiwara Y, Sasaki K, Hattori Y, et al. Involvement of IL-1 in the maintenance of masseter muscle activity and glucose homeostasis. PLOS ONE. 2015; 10: e0143635.

[35] Anaeigoudari A, Seyedi F, Kooshki R, Poran M, Zamyad M, Abbasnejad M. Methyl jasmonate modulates feeding behaviors and hypothalamic expression of the orexin 1 receptor in rats. Turkish Journal of Pharmaceutical Sciences. 2024; 20: 374–379.

[36] Zamyad M, Abbasnejad M, Esmaeili-Mahani S, Sheibani V, Raoof M. Pain influences food preference and food-related memory by activating the basolateral amygdala in rats. Experimental Brain Research. 2021; 239: 79–93.

[37] Gaspar L, Bartman S, Coppotelli G, Ross JM. Effect of apparatus characteristics on anxiety-like behavior in young adult and old mice of both sexes assessed by the elevated plus maze assay. Frontiers in Behavioral Neuroscience. 2023; 17: 1182661.

[38] Su LY, Jiao L, Liu Q, Qiao X, Xie T, Ma Z, et al. S-nitrosoglutathione reductase alleviates morphine analgesic tolerance by restricting PKCα S-nitrosation. Redox Biology. 2024; 75: 103239.

[39] Wehrle E, Günther D, Mathavan N, Singh A, Müller R. Protocol for preparing formalin-fixed paraffin-embedded musculoskeletal tissue samples from mice for spatial transcriptomics. STAR Protocols. 2024; 5: 102986.

[40] Bancroft JD, Gamble M. Theory and practice of histological techniques. 6th edn. Churchill Livingstone Elsevier: Philadelphia (PA). 2008.

[41] Hashemi P, Ahmadi S. Alpha-pinene moderates memory impairment induced by kainic acid via improving the BDNF/TrkB/CREB signaling pathway in rat hippocampus. Frontiers in Molecular Neuroscience. 2023; 16: 1202232.

[42] Marim GC, Machado BCZ, Trawitzki LVV, de Felício CM. Tongue strength, masticatory and swallowing dysfunction in patients with chronic temporomandibular disorder. Physiology & Behavior. 2019; 210: 112616.