Relationship between spinal lncRNA and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain / 中华麻醉学杂志

ABSTRACT

Objective:To investigate the relationship between spinal long chain noncoding RNA (lncRNA) and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain (NP).Methods:The experiment was performed in two parts.Experiment Ⅰ Sprague-Dawley rats of both sexes, aged 7 days, weighing 15-20 g, were selected.Rats were sacrificed, the dorsal horn of spinal cord was removed, and the primary astrocytes were extracted and cultured.Lipopolysaccharide 1 μg/ml was added to induce the activation of astrocytes for 24 h. The lncRNA binding to kindlin-1 was identified using PCR immunoprecipitation method.The localization of lncRNA FOXF1-AS1 in astrocytes was observed by fluorescence in situ hybridization, and the binding between lncRNA FOXF1-AS1 and kindlin-1 was detected by biotin-labeled magnetic bead method.Experiment Ⅱ Thirty clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 5 groups ( n=6 each) using a random number table method: sham operation control group (group C), NP group, lncRNA FOXF1-AS1 overexpression group (group F), lncRNA FOXF1-AS1 overexpression plus kindlin-1 shRNA group (group FK) and lncRNA FOXF1-AS1 overexpression + Wnt inhibitor group (group FW). NP was induced by chronic constrictive injury in anesthetized animals.In group F, lncRNA FOXF1-AS1 overexpression lentivirus 10 μl was intrathecally injected at 28 days before operation, and vector virus 10 μl was intrathecally injected in the other groups.In FK group, kindlin-1 interfering shRNA interference adenovirus 10 μl, and vector virus 10 μl was intrathecally injected in the other groups.In group FW, Wnt inhibitor IWP-2 10 μl was intrathecally injected at 1-3 days after operation, artificial cerebrospinal fluid 10 μl was intrathecally injected at the same time point in the other groups.Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at 1 day before operation, at 4 days and 7 days after operation.The animals were sacrificed at the end of measurement of pain threshold at 7 days after operation, and the spinal cord tissues were taken for determination of the expression of kindlin-1, Wnt3a and glial fibrillary acidic protein (GFAP) (by Western blot) and the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay). Results:ExperimentⅠ lncRNA FOXF1-AS1, which was expressed in the cytoplasm of astrocytes, combined with kindlin-1.Experiment Ⅱ Compared with C group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in group NP ( P<0.05). Compared with NP group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in F group, MWT was increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and the expression of kindlin-1 was up-regulated, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Compared with group F, MWT was significantly increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of spinal kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Conclusion:lncRNA FOXF1-AS1 can up-regulate kindlin-1 expression, activate Wnt3a signaling pathway, promote astrocyte activation, and then regulate inflammatory responses and is involved in the process of neuropathic pain in rats.