This mouse study looked at how removing a protein called complement factor D affects joint pain and cartilage damage after a knee injury. While the knockout mice had protected cartilage, they still experienced pain, and their blood showed different pain‑related lipid molecules. The research does not involve the peptide palmitoyl‑dipeptide‑6 and offers no direct advice for human health or supplementation.

Osteoarthritis (OA) is the leading cause of pain worldwide. However, clinical discordance between pain and cartilage damage presents challenges in determining the mechanisms of OA pain, thus creating a need for well-controlled models that probe the separable mechanisms of structural damage and knee pain. We previously identified that deletion of complement factor D (FD) results in increased pressure-pain hyperalgesia despite cartilage protection after destabilization of the medial meniscus (DMM) surgery. However, how these discordant OA phenotypes manifest is not understood. We employed a novel targeted lipidomics approach to elucidate the role of eicosanoids in FD-mediated pain. We hypothesize that the absence of Cfd (FD^-/-) will protect cartilage but cause increased pressure-pain hyperalgesia and eicosanoid dysregulation persisting throughout OA development. Male and female FD^-/- and wild-type (WT) mice were challenged with DMM or remained naïve at 16 weeks old. Pressure-pain hyperalgesia was measured every two weeks for 8 weeks post-DMM. A second cohort was evaluated at 2 weeks post-DMM to investigate DMM injury response. Structural damage was scored using the Modified Mankin system. Eicosanoid profiles were characterized via liquid chromatography-mass spectrometry (LC-MS) on serum and synovial fluid samples. Statistical analysis was performed with unpaired t-test or two-way ANOVA with Sidak's posthoc test, p < 0.05. Unlike WT mice, FD^-/- mice exhibited no differences in Modified Mankin scores 8 weeks post-DMM in both sexes. As expected, FD^-/- and WT hyperalgesia was present at 2 weeks, persisted through 8 weeks, and was not associated with knee structural changes. Despite both sexes exhibiting similar levels of hyperalgesia, eicosanoid profiles differed. Male FD^-/- demonstrated greater pain-driving (12-HETE, 13-HODE) and lower pain-driving (15-HETE) and pain-suppressing (14-HDHA) abundances of eicosanoids compared to WT. Paradoxically, female FD^-/- exhibited bi-directional differences in pain-suppressive factors (palmitoyl ethanolamide, EPA, 14-HDHA) and lower abundances of pro-inflammatory arachidonic acid compared to WT. The absence of Cfd protects cartilage but does not prevent hyperalgesia after DMM. Changes in eicosanoid profiles suggests that loss in FD drives pain acutely and creates a hyperalgesia phenotype early in response to DMM. Eicosanoid profiling is a novel tool to mechanistically determine pain drivers in osteoarthritis.