Hericenone C Exhibits Anti-Nociceptive Effects through RORα-Mediated Suppression of TLR4 Transcription

Junhao Li¹Kengo Hamamura^1*Yuya Yoshida¹Shimpei Kawano¹Shohei Uchinomiya²Jiahongyi Xie³Damiana Scuteri⁴Yang Ruan^5,6Tomohito Tanihara¹Kohei Fukuoka¹Orion Zaitsu¹Fumiaki Tsurusaki¹Yuma Terada¹Ryotaro Tsukamoto¹Takumi Nishi¹Taiki Fukuda¹Tsukasa Hamasaki¹Kosuke Oyama⁷Giacinto Bagetta⁸Akio Ojida²Kuniyoshi Shimizu³Chaofeng Zhang^5,6Shigehiro Ohdo¹Naoya Matsunaga^1*

• ¹Department of Clinical Pharmacokinetics, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
• ²Department of Medical Chemistry and Chemical Biology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
• ³Department of Agro-Environmental Sciences, Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan
• ⁴Department of Health Sciences, Magna Graecia University of Catanzaro, Catanzaro, Italy
• ⁵Joint International Research Laboratory of Target Discovery and New Drug Innovation, Ministry of Education, School of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing, China
• ⁶Sino-Jan Joint Lab of Natural Health Products Research, School of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing, China
• ⁷Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
• ⁸Pharmacotechnology Documentation and Transfer Unit, Preclinical and Translational Pharmacology, Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy

Abstract Introduction: Hericenone C exhibits antinociceptive effects in inflammatory pain; however, its molecular target and underlying mechanism remain unclear. Methods and Results: We assessed the effect of hericenone C on formalin-induced nociceptive behavior in mice and explored its molecular target using in vitro experiments. Based on competitive affinity proteomics, we identified direct interactions between RORα and hericenone C; functional assays confirmed the role of hericenone C as a RORα antagonist that suppresses RORE-mediated transcriptional activity. Integrated bioinformatics and experimental validation indicated hericenone C-mediated suppression of TLR4 expression via inhibited RORα binding to the TLR4 promoter, which attenuates NF-κB signaling. This mechanism was further validated through pharmacological and genetic approaches, revealing that hericenone C and RORα antagonist SR3335 synergistically modulate TLR4 expression in RORα-modified macrophages. In the formalin-induced nociceptive pain model mice, formalin activated NF-κB through TLR4-dependent P65 phosphorylation, while macrophage depletion selectively suppressed phase 2 nociception. Critically, adoptive transfer of RORα-overexpressing or SR1078-pretreated monocyte-enriched PBMCs exacerbated pain, which was effectively reversed by hericenone C. Notably, hericenone C pretreatment reduced CD11c+ cell infiltration and decreased TLR4 expression in inflamed paw tissues. Conclusion: Overall, these findings establish hericenone C as a novel RORα antagonist that alleviates inflammatory pain through inhibition of the RORα-TLR4-NF-κB axis in CD11c+ cells, offering a promising therapeutic strategy for pain management.