Shikonin alleviates bone cancer pain through suppression of spinal IGF2-associated p38 MAPK signaling

Abstract

Background Bone cancer pain (BCP) is a debilitating complication of advanced cancer that substantially impairs physical function and emotional wellbeing. Current pharmacological treatments often provide incomplete analgesia and may be limited by adverse effects. Shikonin, a bioactive naphthoquinone derived from Lithospermum erythrorhizon, exhibits analgesic and anti inflammatory activities, but its effects and underlying mechanisms in BCP remain unclear. Methods A rat model of BCP was established by intratibial inoculation of luciferase labeled MADB106 cells. The analgesic and behavioral effects of intrathecal Shikonin were evaluated using mechanical and thermal nociceptive assays, CatWalk gait analysis, the open field test (OFT), elevated plus maze (EPM), light/dark box (LDB) test, and rotarod testing. Bioluminescence imaging and hematoxylin eosin staining were used to characterize tumor development and bone pathology. Spinal dorsal horn transcriptomic sequencing was performed to identify candidate molecular pathways. Western blotting, quantitative PCR, and ELISA were used to examine IGF2 associated p38 MAPK signaling and proinflammatory cytokines. The functional involvement of IGF2 and p38 associated signaling was further evaluated using intrathecal IGF2 siRNA and the p38 inhibitor Adezmapimod (SB203580). Results Intrathecal Shikonin produced dose and time dependent antinociceptive effects in BCP rats, with an estimated ED50 of 29.80 g/kg, and did not produce an apparent impairment of rotarod performance. Shikonin also improved gait abnormalities and pain related anxiety like behaviors. Transcriptomic analysis identified Igf2 as a candidate gene responsive to Shikonin treatment, and spinal IGF2 and p38 phosphorylation were increased during BCP development. Shikonin significantly reduced IGF2 expression and p38 phosphorylation in the spinal dorsal horn, accompanied by decreased IL 6, IL 1{beta};, and TNF ; expression in spinal tissue. IGF2 knockdown reproduced the analgesic and behavioral effects of Shikonin and reduced p38 phosphorylation, whereas p38 inhibition reproduced key behavioral effects without a marked change in IGF2 expression. Combined treatment with Shikonin and either IGF2 knockdown or p38 inhibition produced no significant additional behavioral benefit. Conclusion Intrathecal Shikonin alleviates nociceptive hypersensitivity, functional impairment, and pain related anxiety like behaviors in rats with MADB106 induced BCP. The phenocopying and non additive effects of IGF2 knockdown and p38 inhibition support the involvement of an IGF2 associated p38 MAPK signaling axis in the analgesic actions of Shikonin.