Protectin DX resolves fracture-induced postoperative pain in mice via neuronal signaling and GPR37-activated macrophage efferocytosis
Yize Li, Sangsu Bang, Jasmine Ji, Jing Xu, Min Lee, Sharat Chandra, Charles N. Serhan, Ru-Rong Ji
Yize Li, Sangsu Bang, Jasmine Ji, Jing Xu, Min Lee, Sharat Chandra, Charles N. Serhan, Ru-Rong Ji
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Research Article Cell biology Neuroscience

Protectin DX resolves fracture-induced postoperative pain in mice via neuronal signaling and GPR37-activated macrophage efferocytosis

Abstract

Protectin DX (PDX) is a member of the superfamily of specialized proresolving mediators and exerts anti-inflammatory actions in animal models; however, its signaling mechanism remains unclear. Here, we demonstrate the analgesic actions of PDX in a mouse model of tibial fracture–induced postoperative pain (fPOP). Intravenous early- and late-phase treatment of PDX (100 ng/mouse) effectively alleviated fPOP. Compared with protectin D1 (PD1)/neuroprotectin D1, DHA, steroids, and meloxicam, PDX provided superior pain relief. While dexamethasone and meloxicam prolonged fPOP, PDX shortened the pain duration. The analgesic effects of PDX were abrogated in Gpr37−/− mice, which displayed deficits in fPOP resolution. PDX was shown to bind GPR37 and induce calcium responses in peritoneal macrophages. LC-MS/MS–based lipidomic analysis revealed that endogenous PDX levels were approximately 10-fold higher than those of PD1 in muscle at the fracture site. PDX promoted macrophage polarization via GPR37-dependent phagocytosis and efferocytosis through calcium signaling in vitro, and it further enhanced macrophage viability and efferocytosis in vivo via GPR37. Finally, PDX rapidly modulated nociceptor neuron responses by suppressing C-fiber–induced muscle reflex in vivo and calcium responses in DRG neurons ex vivo and by reducing TRPA1/TRPV1-induced acute pain and neurogenic inflammation in vivo. Our findings highlight multiple benefits of PDX to manage postoperative pain and promote perioperative recovery.

Authors

Yize Li, Sangsu Bang, Jasmine Ji, Jing Xu, Min Lee, Sharat Chandra, Charles N. Serhan, Ru-Rong Ji

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Figure 10

PDX reduces TRPA1-mediated spontaneous pain and neurogenic inflammation in CD1 mice.

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PDX reduces TRPA1-mediated spontaneous pain and neurogenic inflammation ...
(A) Experimental scheme: mice received PDX 30 min before intraplantar AITC (100 μg); spontaneous pain was recorded, followed by i.v. Evans blue to assess neurogenic inflammation. (B) PDX reduced AITC-evoked spontaneous pain (5 min). (C) PDX decreased AITC-induced mechanical allodynia (von Frey, 10 min). (D) Representative hind paw images showing AITC-induced edema. (E and F) Quantification of edema by paw weight (E) and Evans blue extravasation (F) with and without PDX (100 ng, i.v.). (G) Schematic of ex vivo Ca2+ imaging in AAV-MaCPNS.2-hSyn-Gcamp6f–labeled neurons. (H and I) Representative Ca2+ responses (H) and heatmaps (I) showing AITC-evoked neuronal activation. Each group includes 51 DRG neurons. Scale bar: 100 μm. (J) PDX decreased the proportion of AITC-responsive DRG neurons. (K) Bulk RNA-seq revealed reduced Trpa1 expression in DRG after fracture in PDX-treated mice. (L) Left, subcluster annotation map of mouse DRG. Right, UMAP plot showing Gpr37 (red) and Trpa1 (blue) expression. Published scRNA-seq data of mouse DRG (60) shows the percentages of Gpr37- and Trpa1-expressing neurons and their colocalization. (M) Spatial transcriptomics of human DRG showing colocalization of GPR37 with TRPA1 and TRPV1 in human sensory neurons. TRPA1 expression was confined to TRPV1+ neurons (1,062 neurons from 2 donors). Scale bar: 20 μm. (N) Micro-CT of hind limbs 3 days after fracture showed that PDX reduced edema and improved bone integrity. Coronal and 3D reconstructions revealed reduced soft-tissue swelling and enhanced tibial bone density at the fracture site. The rightmost PBS and PDX images show 3D constructed tibial bones, and the white and red arrows indicate the fracture sites. Scale bars: 6 and 3 mm. (O and P) Quantification of diameters (O), indicated by white lines in the leftmost PBS and PDX images in N, and coronal section muscle area (P), indicated in the middle PBS and PDX images in N. Data are presented as mean ± SEM. Statistics: unpaired t test (B, E, F, J, K, O, and P) and 1-way ANOVA with Bonferroni’s post hoc test (C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 6 mice/group for B, C, E, F, J, and K; n = 3 males for O and P; triangles, male; circles, female.