MiR-204-5p减轻大鼠坐骨神经慢性缩窄损伤引起神经病理性疼痛的作用机制

MiR-204-5p alleviates neuropathic pain in rats with chronic constriction injury

背景:miR-204-5p是神经病理性疼痛的潜在生物标志物,但其在神经病理性疼痛中的作用及作用机制尚未阐明。目的:探讨miR-204-5p对大鼠坐骨神经慢性缩窄性损伤引起的神经病理性疼痛的作用及其分子机制。方法:①将90只雄性SD大鼠随机分为假手术组、模型组、空载体组、miR-204-5p过表达组、杂乱干扰组和AURKB干扰组,每组15只。采用坐骨神经慢性缩窄性损伤方法建立大鼠神经性疼痛动物模型,建模后分别注射以下重组慢病毒(空载体、miR-204-5p过表达载体、杂乱干扰载体、AURKB短发夹RNA),注射量为1μg。术前及术后第7,14天,采用RT-qPCR检测大鼠背根神经节中miR-204-5p和AURKB mRNA的表达水平;术前及术后第3,7,14,21天,通过缩足反应阈值和缩足潜伏期评估机械异位痛和热痛觉过敏;术后第7天,采用酶联免疫吸附法分析大鼠背根神经节中炎性细胞因子水平。②采用100 nmol/L脂多糖诱导大鼠小胶质细胞HAPI产生炎症反应,细胞分为对照组、脂多糖组、脂多糖+NC mimic组、脂多糖+miR-204-5p mimic组、脂多糖+miR-204-5p mimic+空载体组、脂多糖+miR-204-5p mimic+AURKB过表达组。采用酶联免疫吸附法分析大鼠小胶质细胞HAPI中炎性细胞因子水平;Western blot法检测大鼠小胶质细胞HAPI中AURKB及Wnt/β-catenin通路标志蛋白的表达水平;双荧光素酶报告基因法验证miR-204-5p与AURKB的靶定关系。结果与结论:①与假手术组比较,坐骨神经慢性缩窄性损伤大鼠背根神经节中miR-204-5p表达显著降低;②与模型组比较,过表达miR-204-5p可显著提高缩足反应阈值和缩足潜伏期;③在坐骨神经慢性缩窄性损伤大鼠和脂多糖激活的HAPI细胞中,过表达miR-204-5p可以抑制炎性细胞因子的分泌;④双荧光素酶报告基因证实了AURKB是miR-204-5p的下游靶基因;⑤在坐骨神经慢性缩窄性损伤大鼠背根神经节中,AURKB mRNA表达显著升高,沉默AURKB可抑制神经病理性疼痛和神经炎症;⑥在脂多糖激活的HAPI细胞中,miR-204-5p过表达通过抑制AURKB表达降低Wnt/β-catenin信号通路蛋白表达水平,减轻神经炎症反应;⑦以上研究结果表明,miR-204-5p通过AURKB/Wnt/β-catenin通路缓解坐骨神经慢性缩窄性损伤大鼠神经病理性疼痛。

BACKGROUND:MiR-204-5p is a potential biomarker of neuropathic pain,but its role and mechanism in neuropathic pain have not been clarified.OBJECTIVE:To investigate the effect and molecular mechanism of miR-204-5p on neuropathic pain caused by chronic constriction injury in rats.METHODS:(1)A total of 90 male Sprague-Dawley rats were randomized into sham group,model group,empty vector group,miR-204-5p overexpression group,scramble group,and aurora kinase B(AURKB)interference group,with 15 rats in each group.The rat neuropathic pain model was established based on chronic constriction injury of the sciatic nerve.After modeling,rats in each group were injected with the following recombinant lentiviruses(empty vector,miR-204-5p overexpression vector,scrambled shRNA,AURKB shRNA),and the injection volume was 1μg.The expression levels of miR-204-5p and AURKB mRNAs in the dorsal root ganglia of rats were measured by RT-qPCR before surgery and on the 7^(th) and 14^(th) days after surgery.Mechanical allodynia and thermal hyperalgesia were assessed by paw withdrawal threshold and paw withdrawal latency before surgery and on the 3^(rd),7^(th),14^(th),and 21^(st) days after surgery.The levels of inflammatory cytokines in dorsal root ganglion tissues of rats were determined by ELISA on the 7th day after surgery.(2)100 nmol/L lipopolysaccharide was used to induce inflammation in rat microglia cell line(HAPI).The cells were divided into control group,lipopolysaccharide group,lipopolysaccharide+NC mimic group,lipopolysaccharide+miR-204-5p mimic group,lipopolysaccharide+miR-204-5p mimic+empty vector group,lipopolysaccharide+miR-204-5p mimic+AURKB overexpression group.The levels of inflammatory cytokines in HAPI cells were detected using enzyme-linked immunosorbent assay.The expression levels of AURKB and Wnt/β-catenin pathway proteins in HAPI cells were detected by western blot assay.The targeting relationship between miR-204-5p and AURKB was verified by dual luciferase reporter gene assay.RESULTS AND CONCLUSION:Compared with the sham operation group,the expression of miR-204-5p in the dorsal root ganglion of rats with chronic constriction injury was significantly reduced.Compared with the model group,overexpression of miR-204-5p significantly increased the paw withdrawal threshold value and paw withdrawal latency.In rats with chronic constriction injury of the sciatic nerve and HAPI cells activated by lipopolysaccharide,overexpression of miR-204-5p could inhibit the secretion of inflammatory cytokines.The dual luciferase reporter gene confirmed AURKB as the downstream target gene of miR-204-5p.The expression level of AURKB mRNA was significantly increased in the dorsal root ganglion of rats with chronic constriction injury.Whereas silencing of AURKB could inhibit neuropathic pain and neuroinflammation.In lipopolysaccharide-induced HAPI cells,the overexpression of miR-204-5p reduced the expression of Wnt/β-catenin signaling pathway proteins by inhibiting the expression of AURKB,thereby reducing neuroinflammation.All the findings indicate that miR-204-5p relieves neuropathic pain in rats with chronic constriction injury of the sciatic nerve through the AURKB/Wnt/β-catenin pathway.