The Effect of Super-Repressor IkB-Loaded Exosomes (Exo-srIκBs) in Chronic Post-Ischemia Pain (CPIP) Models

Abstract

Complex regional pain syndrome (CRPS) is a condition associated with neuropathic pain that causes significant impairment of daily activities and functioning. Nuclear factor kappa B (NFκB) is thought to play an important role in the mechanism of CRPS. Recently, exosomes loaded with super-repressor inhibitory kappa B (Exo-srIκB, IκB; inhibitor of NFκB) have been shown to have potential anti-inflammatory effects in various inflammatory disease models. We investigated the therapeutic effect of Exo-srIκB on a rodent model with chronic post-ischemia pain (CPIP), a representative animal model of Type I CRPS. After intraperitoneal injection of a vehicle, Exo-srIκB, and pregabalin, the paw withdrawal threshold (PWT) was evaluated up to 48 h. Administration of Exo-srIκB increased PWT compared to the vehicle and pregabalin, and the relative densities of p-IκB and IκB showed significant changes compared to the vehicle 24 h after Exo-srIκB injection. The levels of several cytokines and chemokines were reduced by the administration of Exo-srIκB in mice with CPIP. In conclusion, our results showed more specifically the role of NFκB in the pathogenesis of CRPS and provided a theoretical background for novel treatment options for CRPS.

Keywords: NFκB; allodynia; chronic post-ischemic pain model; complex regional pain syndrome (CRPS); exosome loaded with super-repressor IκB; exosomes; inflammation; neuropathic pain.

Figure 1

Experimental protocol. (A) Mechanical allodynia and rotarod tests; (B) Western blot measurement for sham and CPIP models; (C) Western blot measurement for four groups at 24 h and Western blot measurement for four groups at 48 h after drug injection; (D) cytokine and chemokine measurement for four groups at 24 h after drug injection.

Figure 2

Chronic post-ischemia pain model. (A) An O-ring was placed on the upper part of the left ankle for 3 h; (B) the tied O-ring was removed after 3 h of ischemia to induce reperfusion. There was a period of hyperemia after perfusion.

Figure 3

Characterization of Exo-srIκB. Exo-Naïve (vehicle) is non-engineered exosome and Exo-srIκB is engineered exosome. A vehicle used as negative control of Exo-srIκB. (A) Size distribution and concentration of the vehicle (left) and Exo-srIκB (right) were determined by a Nanosight NS 300 (Malvern Panalytical, United Kingdom); (B) Western blot at producing cell and exosome to confirm the expression of target protein (srIκB, CRY2, and CD9 (CD9-CIBN)), exosome-positive markers (endogenous CD9, CD81, TSG101, Alix, and GAPDH) (The original western blot images are in Figure S1); and (C) exosome-negative markers (cell organelle markers; GM130 (Golgi), Lamin B1 (Nuclear), Prohibitin (mitocondria), and Calnexin (ER)) (The original western blot images are in Figure S2).

Figure 4

CPIP model and NFκB. (A) Paw withdrawal thresholds. * p < 0.05 vs. sham, ^† p < 0.05 vs. contralateral; (B) Western blot data showing the relative densities of p-IκB and IκB to tubulin at 48 h after ischemia and reperfusion injury. ** p < 0.01, *** p < 0.001 vs. sham. IκB: inhibitory kappa B, p-IκB: phospho inhibitory kapp B (The original western blot images are in Figure S3).

Figure 5

Antiallodynic effects of Exo-srIκB. (A) Ipsilateral paw withdrawal threshold; (B) contralateral paw withdrawal threshold; (C) rotarod test. * p < 0.05 vs. baseline, ^† p < 0.05 vs. vehicle, ^‡ p < 0.05 vs. pregabalin.

Figure 6

Western blot after drug injection. (A) Western blot at 24 h after drug injection. Western blot data showing the relative densities of p-IκB and IκB to tubulin at 24 h after drug injections (The original western blot images are in Figure S4); (B) Western blot at 48 h after injection. Western blot data showing the relative densities of p-IκB and IκB to tubulin at 48 h after drug injections. * p < 0.05, *** p < 0.001 vs. vehicle (The original western blot images are in Figure S5).

Figure 7

Cytokine array in the paw 24 h after drug injection. Cytokine profiles and plots of endoglin, myeloperoxidase (MPO), osteopontin (OPN), pentraxin 3, and serpin E1/PAI-1. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CPIP vehicle. Cytokine profiles and plots of CCL21, CXCL2, IGFBP-5, IL-10, IL-33, and LDL R s. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CPIP Exo-srIκB (The original Cytokine array images are in Figure S6).

Figure 8

Chemokine array in the paw 24 h after drug injection. Chemokine profiles and plots of C10, the complement component C5/C5a, MCP-2, MIP-1 γ, IL-16, MCP-5, and SDF-1. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CPIP vehicle (The original Chemokine array images are in Figure S7).