Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

Abstract

Chronic pain after bone fracture and orthopedic surgery is often refractory to most analgesics currently in use, thus emphasizing the urgent need for improved therapeutic medications. Chemokine-dependent neuroinflammation is critical for excitatory synaptic plasticity and central nociception sensitization. Recent studies have focused on the inhibition of inflammatory responses by artesunate, the first anti-malaria drug extracted from artemisinin. The present study investigated the analgesic effects and potential targets of artesunate in a mouse model of chronic pain induced by tibial fracture and orthopedic surgery. Three injections of artesunate were intrathecally administered on a daily basis from days 4 to 6 after fracture. We reported that repetitive exposure to artesunate (10 and 100 μg but not 1 μg) dose-dependently prevented fracture-induced mechanical and cold allodynia. Moreover, single intrathecal injection of artesunate (100 μg) alleviated the established chronic pain on day 14 after fracture surgery. Intraperitoneal artesunate (10 and 50 mg kg−1) therapy was effective against chronic fracture pain. Intriguingly, artesunate inhibited the upregulation of spinal chemokine CCL21, triggering receptor expressed on myeloid cells 2 (TREM2) and DNAX-activating protein of 12 kDa (DAP12) expressions and microglia activation in fracture mice. Furthermore, spinal CCL21 neutralization attenuated the severity of fracture-associated post-surgical pain. Exogenous CCL21-induced acute inflammatory pain was impaired by artesunate therapy. Additionally, the pharmacological blockage of TREM2 reduced recombinant CCL21-elicited behavioral hypernociception. The present findings demonstrate that artesunate therapy reduces the initiation and maintenance of fracture-associated chronic postoperative pain by inhibiting CCL21-dependent TREM2/DAP12 inflammatory signaling and microglia activation, thus suggesting that artesunate could emerge as a therapeutic strategy for fracture pain management.

Hydrogen-Rich Saline Attenuates Chronic Allodynia after Bone Fractures via Reducing Spinal CXCL1/CXCR2-Mediated Iron Accumulation in Mice

“…In general, expression variations in chemokines and their receptors in the spinal dorsal horn are a key step for the pathophysiology of fracture-caused chronic allodynia developments [ 24 , 25 , 26 ]. Elisa analyses represented a considerable elevation in the protein levels of CXCL1 and its receptor CXCR2 on day 7 following fracture procedures in mice ( p < 0.05, n = 4, one-way ANOVA; Figure 2 A,B).…”

“…An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of CXCL1 (Abcam, UK), and CXCR2 (Wuhan Fine Biotech Co., Wuhan, China) in the L4-5 levels of left spinal cord [ 24 ]. Spinal cord tissues were homogenized in a lysis buffer containing protease and phosphatase inhibitors.…”

Chronic Pain after Bone Fracture: Current Insights into Molecular Mechanisms and Therapeutic Strategies

“…The triggering receptor expressed on myeloid cells 2 (TREM2) forms a receptor complex with DNAX-activating protein of 12 kDa (DAP12) on the plasma membrane of microglia and induces the activation of microglia via DAP12, which, in turn, is involved in the induction and maintenance of neuropathic pain [ 70 ]. Based on the studies described above, we found [ 71 ] that exposure of the spinal cord to CCL21 upregulates TREM2 and DAP12 expression, and pharmacological inhibition of TREM2/DAP12 ameliorates CFP and CCL21-induced acute pain. These results suggest that inhibition of CCL21-dependent TREM2/DAP12 neuroinflammatory signaling may have a previously undescribed role in alleviating CFP.…”

“…Kallikrein is essentially a proteolytic enzyme that produces bradykinin (BK), a related peptide (Lys-BK), and its active metabolites from kininogen substrates [ 72 ]. Kinin is involved in the regulation of inflammatory processes by activating two G protein-coupled receptors, B1 and B2 [ 71 ]. B1R, encoded by the gene BDKRB1, is rarely expressed in healthy tissue, and its expression is only induced under specific conditions such as injury and inflammation, whereas B2R is expressed continuously [ 73 ].…”

“…Emerging research shows that artesunate is effective in suppressing acute pain caused by opioids and chemical irritants [ 130 , 131 ]. Repeated exposure to artesunate prevents mechanical allodynia and cold hyperalgesia from fractures at 10 and 100 μg but not 1 μg, and this effect is dose-dependent [ 71 ]. More interestingly, a single intrathecal injection of 100 μg of artesunate on 14 d after orthopedic surgery relieved the established CFP.…”