miR-143-3p通过靶向KRAS/RalA通路抑制星形胶质细胞活化并缓解坐骨神经分支选择性损伤小鼠神经病理性疼痛OA
miR-143-3p alleviates neuropathic pain in mice with spared nerve injury by targeting the KRAS/RalA pathway and inhibiting astrocyte activation
目的 探讨微小RNA(miR-143-3p)在神经病理性疼痛中的治疗作用,阐明其靶基因及相关分子机制.方法 对8周龄雄性C57BL/6J小鼠建立坐骨神经分支选择性损伤(SNI)模型,假手术组作为对照,治疗组于SNI后第7天单次鞘内注射miR-143-3p agomir,KRAS激动剂组在miR-143-3p agomir的基础上,SNI后第8天单次鞘内注射KRA-533,6只/组.采用vonFrey纤维丝检测术侧足底机械痛阈.miRNA靶基因预测和生物信息学分析用于筛选潜在靶点及相关信号通路.建立脂多糖诱导的C8-D1A星形胶质细胞炎症模型,用或不用miR-143-3p mimic和KRA-533进行干预.qRT-PCR、Western blotting检测基因和蛋白表达.免疫荧光用于脊髓形态学及细胞增殖活性观察.酶联免疫吸附试验(ELISA)检测促炎因子水平.结果 单次鞘内注射miR-143-3p agomir可显著提高SNI小鼠术侧后足的机械痛阈,效果持续近3周(P=0.048).生物信息学确定KRAS是miR-143-3p的直接下游靶点.体内过表达miR-143-3p可显著抑制KRAS表达(P<0.0001),并抑制下游RalA/TBK1/NF-κB信号通路的激活,进而抑制星形胶质细胞活化和炎症因子白细胞介素-6(P<0.0001)、白细胞介素-1β(P<0.0001)、α肿瘤坏死因子上调(P=0.007).KRAS激动剂KRA-533可显著逆转miR-143-3p的镇痛作用(P=0.01),以及对下游通路激活、细胞活化和神经炎症的抑制效应.体外过表达miR-143-3p可显著抑制LPS诱导的非经典Ras信号通路激活、细胞增殖活性及炎症水平,这些均可被KRA-533逆转.结论 miR-143-3p通过靶向KRAS调控非经典Ras信号通路,抑制星形胶质细胞活化及神经炎症,从而缓解神经病理性疼痛,为该疾病的治疗提供了新的理论依据.
Objective To investigate the therapeutic effect of microRNA(miR-143-3p)on neuropathic pain in mice and clarify its target gene and molecular mechanism.Methods Spared nerve injury(SNI)models were established in 8-week-old male C57BL/6J mice,with sham-operated mice as the control group(n=6).The treatment group received a single intrathecal injection of miR-143-3p agomir on day 7 after SNI modeling,and in KRAS agonist group,KRA-533 was intrathecally injected on day 8 following miR-143-3p agomir injection.Mechanical withdrawal thresholds of the ipsilateral paw of the mice were measured using von Frey filaments.Bioinformatics analyses were used to explore the potential targets and signaling pathways.In a C8-D1A astrocyte model of lipopolysaccharide(LPS)-induced inflammation,the effect of miR-143-3p mimic and KRA-533 on the expressions of the identified targets were detected using qRT-PCR and Western blotting.Spinal morphology in the mouse models and proliferative activity of C8-D1A cells were observed with immunofluorescence staining,and the levels of pro-inflammatory factors were determined with enzyme-linked immunosorbent assay(ELISA).Results miR-143-3p agomir significantly increased mechanical withdrawal thresholds of SNI mice,and the effect lasted nearly 3 weeks.KRAS was identified as a direct target of miR-143-3p.In SNI mouse models,miR-143-3p significantly inhibited KRAS expression,RalA/TBK1/NF-κB signaling pathway activation,and astrocyte activation,and upregulated the levels of IL-6,IL-1β,and TNF-α.The KRAS agonist KRA-533 significantly reversed the analgesic effect of miR-143-3p and its inhibitory effects on downstream pathway activation,astrocyte activation,and neuroinflammation.In C8-D1A cells,overexpression of miR-143-3p effectively suppressed LPS-induced noncanonical Ras pathway activation and inhibited cell proliferation and inflammatory response,which were reversed by treatment with KRA-533.Conclusion miR-143-3p overexpression alleviates neuropathic pain in mice by targeting KRAS to regulate the noncanonical Ras pathway and inhibiting astrocyte activation and neuroinflammation,suggesting a new strategy for clinical treatment of SNI.
林奕歆;姚锦忠;陈晔明;秦再生
南方医科大学南方医院麻醉科,广东 广州 510515南方科技大学第一附属医院麻醉科,广东 深圳 518055南方医科大学第三附属医院麻醉科,广东 广州 510630南方医科大学南方医院麻醉科,广东 广州 510515
神经病理性疼痛miR-143-3pKRAS星形胶质细胞神经炎症
neuropathic painmiR-143-3pKRASastrocytesneuroinflammation
《南方医科大学学报》 2026 (5)
1018-1027,10
国家自然科学基金(81973305)Supported by National Natural Science Foundation of China(81973305).
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