MiR-187-3p mimic alleviates ischemia-reperfusion-induced pain hypersensitivity through inhibiting spinal P2X7R and subsequent mature IL-1ß release in mice.

BACKGROUND: Ischemia-reperfusion (IR)-induced pain hypersensitivity shares features of neuroinflammation and neuropathic pain, accompanied by overproduction of interleukin (IL)-1ß. Multiple microRNAs (miRs) are dysregulated during IR; among these miRs, miR-187-3p was recently reported to drive IL-1ß release in retinal disease by activating members of the purinergic receptor family. However, the roles of miR-187-3p in the spinal cord are unclear. Thus, we investigated whether miR-187-3p is involved in the pathogenesis of IR-induced pain hypersensitivity by regulating the P2X7R signal and subsequent IL-1ß release. METHODS: A mouse model was established by 5-min occlusion of the aortic arch. Pain hypersensitivity was assessed by the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). MiR-187-3p, P2X7R, cleaved caspase-1 and mature IL-1ß expression levels were measured by RT-PCR and Western blotting. The in vivo roles of miR-187-3p, P2X7R and IL-1ß were explored by intrathecal treatment with synthetic miRs, selective agonists and antagonists in separate experiments. Double immunofluorescence staining was performed to delineate the cellular distribution of P2X7R and IL-1ß. RESULTS: IR-induced progressively decreased PWT and PWL values were closely related to decreases in miR-187-3p and increases in P2X7R expression levels over time. The functional miR-187-3p/P2X7R pair was preliminarily predicted by a bioinformatic database and confirmed in vivo by quantitative analysis, as mimic-187 greatly increased miR-187-3p but decreased P2X7R expression levels, whereas inhibitor-187 reversed these changes. In contrast, downregulating P2X7R by mimic-187 or A-438079 treatment comparably increased PWT and PWL values in IR-injured mice, while upregulating P2X7R by inhibitor-187 or BzATP treatment decreased PWT and PWL values in sham-operated mice. Moreover, P2X7R and IL-1ß immunoreactivities in each group were changed in the same patterns. This finding was further supported by results showing that downregulating IL-1ß by A-438079 and IL-1ß-neutralizing antibody similarly decreased P2X7R, cleaved caspase-1 and mature IL-1ß expression levels, whereas BzATP treatment increased these levels. Expectedly, mimic-187 treatment preserved PWT and PWL values, with decreased cleaved caspase-1 and mature IL-1ß expression levels, whereas inhibitor-187 reversed these effects. CONCLUSIONS: The spinal miR-187-3p/P2X7R pair functioned in a mouse IR model. Increasing miR-187-3p protected against pain hypersensitivity and mature IL-1ß overproduction, partially through inhibiting P2X7R activation.

Down-Regulation of CXCL12/CXCR4 Expression Alleviates Ischemia-Reperfusion-Induced Inflammatory Pain via Inhibiting Glial TLR4 Activation in the Spinal Cord.

Toll-like receptor 4 (TLR4) is important for the pathogenesis of inflammatory reactions and the promotion of pain processing after ischemia/reperfusion (IR) in spinal cord. Recently, C-X-C chemokine ligand 12 (CXCL12) and its receptor, C-X-C chemokine receptor 4 (CXCR4), were demonstrated to be simultaneously critical for inflammatory reactions, thereby facilitating glial activation. However, whether CXCL12/CXCR4 expression can contribute to IR-induced inflammatory pain via spinal TLR4 remained unclear. A rat model was established by 8 min of aortic arch occlusion. The effects of CXCL12/CXCR4 expression and TLR4 activation on inflammatory hyperalgesia were investigated by pretreatments with CXCL12-neutralizing antibody, CXCR4 antagonist (AMD3100) and TLR4 antagonist (TAK-242) for 5 consecutive days before surgery. The results indicated that IR induced significant and sustained inflammatory pain, observed as decreases in paw withdrawal threshold (PWT) and paw withdrawal latency (PWL), throughout the post-injury period. The increased levels of TLR4 and proinflammatory chemokine CXCL12, as well as its receptor, CXCR4, were closely correlated with the PWT and PWL trends. Double immunostaining further suggested that TLR4, which is mainly expressed on astrocytes and microglia, was closely co-localized with CXCL12 and CXCR4 in spinal dorsal horn. As expected, intrathecal pretreatment with the TLR4 antagonist, TAK-242 markedly ameliorated pain by inhibiting astrocytic and microglial activation, as shown by decreases in TLR4 immunoreactivity and the percentage of double-labeled cells. These protective effects were likely due in part to the reduced production of the downstream cytokines IL-1ß and TNF-α, as well as for the recruitment of CXCL12 and CXCR4. Additionally, intrathecal pretreatment with CXCL12-neutralizing antibody and AMD3100 resulted in similar analgesic and anti-inflammatory effects as those receiving TAK-242 pretreatment. These results suggest that intrathecal blockade of CXCL12/CXCR4 expression may attenuate IR-induced pain sensation and the release of inflammatory cytokines by limiting glial TLR4 activation in spinal cord.

miR-320a affects spinal cord edema through negatively regulating aquaporin-1 of blood-spinal cord barrier during bimodal stage after ischemia reperfusion injury in rats.

BACKGROUND: Spinal cord edema is a serious complication and pathophysiological change after ischemia reperfusion (IR) injury. It has been demonstrated closely associated with bimodal disruption of blood-spinal cord barrier (BSCB) in our previous work. Aquaporin (AQP)1 plays important but contradictory roles in water homeostasis. Recently, microRNAs (miRs) effectively regulate numerous target mRNAs during ischemia. However, whether miRs are able to protect against dimodal disruption of BSCB by regulating perivascular AQP1 remains to be elucidated. RESULTS: Spinal water content and EB extravasation were suggested as a bimodal distribution in directly proportion to AQP1, since all maximal changes were detected at 12 and 48 h after reperfusion. Further TEM and double immunofluorescence showed that former disruption of BSCB at 12 h was attributed to cytotoxic edema by up-regulated AQP1 expressions in astrocytes, whereas the latter at 48 h was mixed with vasogenic edema with both endothelial cells and astrocytes involvement. Microarray analysis revealed that at 12 h post-injury, ten miRs were upregulated (>2.0 fold) and seven miRs were downregulated (<0.5 fold) and at 48 h, ten miRs were upregulated and eleven were downregulated compared to Sham-operated controls. Genomic screening and luciferase assays identified that miR-320a was a potential modulator of AQP1 in spinal cord after IR in vitro. In vivo, compared to rats in IR and negative control group, intrathecal infusion of miR-320a mimic attenuated IR-induced lower limb motor function deficits and BSCB dysfunction as decreased EB extravasation and spinal water content through down-regulating AQP1 expressions, whereas pretreated with miR-320a AMO reversed above effects. CONCLUSION: These findings indicate miR-320a directly and functionally affects spinal cord edema through negatively regulating AQP1 of BSCB after IR.

Sevoflurane inhibits nuclear factor-κB activation in lipopolysaccharide-induced acute inflammatory lung injury via toll-like receptor 4 signaling.

BACKGROUND: Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR4) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms. METHODS: In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR4 and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR4 and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards. RESULTS: The mRNA and protein levels of NF-κB and TLR4 in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR4 and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol. CONCLUSIONS: This study demonstrates the crucial role of TLR4 activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo and in vitro via inhibition of TLR4/NF-κB pathway.

Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.

Ischemic preconditioning has been reported to protect against spinal cord ischemia-reperfusion (I-R) injury, but the underlying mechanisms are not fully understood. To investigate this, Japanese white rabbits underwent I-R (30 min aortic occlusion followed by reperfusion), ischemic preconditioning (three cycles of 5 min aortic occlusion plus 5 min reperfusion) followed by I-R, or sham surgery. At 4 and 24 h following reperfusion, neurological function was assessed using Tarlov scores, blood spinal cord barrier permeability was measured by Evan's Blue extravasation, spinal cord edema was evaluated using the wet-dry method, and spinal cord expression of zonula occluden-1 (ZO-1), matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor-α (TNF-α) were measured by Western blot and a real-time polymerase chain reaction. ZO-1 was also assessed using immunofluorescence. Spinal cord I-R injury reduced neurologic scores, and ischemic preconditioning treatment ameliorated this effect. Ischemic preconditioning inhibited I-R-induced increases in blood spinal cord barrier permeability and water content, increased ZO-1 mRNA and protein expression, and reduced MMP-9 and TNF-α mRNA and protein expression. These findings suggest that ischemic preconditioning attenuates the increase in blood spinal cord barrier permeability due to spinal cord I-R injury by preservation of tight junction protein ZO-1 and reducing MMP-9 and TNF-α expression.