目的:在中国古代,蜂毒被广泛用于治疗各种疾病。虽然使用蜂毒不是目前的主流医疗方法,有些人把它用来治疗某些疾病,包括特发性面神经麻痹(IFP)。最近,melittin(Mel),蜂毒的主要活性成分,已显示出强烈的抗炎和镇痛作用。然而,蜂毒如何改善面瘫患者的神经功能障碍仍不清楚。本研究旨在探讨Mel对雪旺氏细胞(SCs)的抗神经创伤作用,神经元鞘的主要细胞,氧化应激损伤。
方法:建立低氧SCs模型,和CCK-8测定,siRNA转染,酶联免疫吸附测定,定量逆转录聚合酶链反应,westernblot,免疫荧光,和细胞超微结构分析进行了研究,以减轻缺氧诱导的SCs体外损伤,揭示Mel对SCs氧化应激损伤的影响。
结果:CoCl2诱导的SCs中HIF-1α的过表达(p<0.05)表明SCs缺氧模型的建立。通过CCK-8实验(p<0.0001)和S-100mRNA表达检测(p<0.0001),与CoCl2组相比,在Mel处理组中低氧SCs的增殖和再生过程增强。CoCl2诱导的SCs中活性氧(ROS)水平升高(p<0.001)和超氧化物歧化酶(SOD)水平降低(p<0.05)表明Mel可以减轻CoCl2诱导的SCs的氧化应激损伤。Mel通过减少促炎细胞因子IL-1β(p<0.0001)和TNF-α(p<0.0001)减轻低氧SCs的氧化应激和炎症。此外,Mel增强了细胞活力和与氧代谢相关的调节指标,细胞修复,神经代谢,缺氧后血管内皮的形成,如C-JUN(p<0.05),胶质细胞源性神经营养因子(GDNF;p<0.001),血管内皮生长因子(VEGF;p<0.05),缺氧诱导因子1-α(HIF-1α;p<0.05),白细胞介素-1受体1型(IL-1R1;p<0.05),enolase1(ENO1;p<0.05),醛糖还原酶(AR;p<0.01),SOD(p<0.05),神经生长因子(NGF;p<0.05),和诱导型一氧化氮合酶(iNOS;p<0.05)。就其机制而言,Mel抑制与NF-κB途径相关的蛋白如IKK的表达(p<0.01),p65(p<0.05),p60(p<0.001),IRAK1(p<0.05),IKB-α增加(p<0.0001)。此外,与Mel治疗组相比,si-IL-1R1组中的IL-1R1的敲除增强了Mel的治疗效果(所有P<0.05)。
结论:本研究提供了IL-1R1在SCs缺氧引起的氧化应激损伤中的重要作用,证明Mel通过靶向IL-1R1下调NF-κB介导的炎症反应,减轻SCs的氧化应激损伤。Mel可能会成为治疗IFP的创新治疗方法。
OBJECTIVE: In ancient China, bee venom was widely used to treat various diseases. Although using bee venom is not currently a mainstream medical method, some have applied it to treat certain conditions, including idiopathic facial paralysis (IFP). Recently, melittin (Mel), the main active component of bee venom, has been shown strong anti-inflammatory and analgesic effects. However, how bee venom improves neurological dysfunction in facial paralysis remains unknown. This study aimed to investigate the anti-neurotraumatic effect of Mel on Schwann cells (SCs), the main cells of the neuron sheath, injured by oxidative stress.
METHODS: A model of hypoxic SCs was established, and CCK-8 assay, siRNA transfection, enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, western blot, immunofluorescence, and cell ultrastructure analyses were conducted to investigate the mitigation of hypoxia-induced damage to SCs in vitro, revealing the effects of Mel on oxidative stress injury in SCs.
RESULTS: The overexpression of HIF-1α in CoCl2-induced SCs (p < 0.05) indicated the establishment of an SCs hypoxia model. The proliferation and regeneration process of the hypoxic SCs enhanced in the Mel-treated group compared to the CoCl2 group has been proven through the CCK-8 experiment (p < 0.0001) and S-100 mRNA expression detection (p < 0.0001). The increased level of reactive oxygen species (ROS) (p < 0.001) and decreased superoxide dismutase (SOD) levels (p < 0.05) in the CoCl2-induced SCs indicated that Mel can alleviate the oxidative stress damage to SCs induced by CoCl2. Mel alleviated oxidative stress and inflammation in hypoxic SCs by reducing pro-inflammatory cytokines IL-1β (p < 0.0001) and TNF-α (p < 0.0001). In addition, Mel augmented cellular vitality and regulated indicators related to oxygen metabolism, cell repair, neurometabolism, and vascular endothelial formation after hypoxia, such as C-JUN (p < 0.05), glial cell line-derived neurotrophic factor (GDNF; p < 0.001), vascular endothelial growth factor (VEGF; p < 0.05), hypoxia-inducible factor 1-alpha (HIF-1α; p < 0.05), interleukin-1 receptor type 1 (IL-1R1; p < 0.05), enolase1 (ENO1; p < 0.05), aldose reductase (AR; p < 0.01), SOD (p < 0.05), nerve growth factor (NGF; p < 0.05), and inducible nitric oxide synthase (iNOS; p < 0.05). In terms of its mechanism, Mel inhibited the expression of proteins associated with the NF-κB pathway such as IKK (p < 0.01), p65 (p < 0.05), p60 (p < 0.001), IRAK1 (p < 0.05), and increased IKB-α (p < 0.0001). Moreover, knocking out of IL-1R1 in the si-IL-1R1 group enhanced the therapeutic effect of Mel compared to the Mel-treated group (all of which p < 0.05).
CONCLUSIONS: This research provided evidence of the substantial involvement of IL-1R1 in oxidative stress damage caused by hypoxia in SCs and proved that Mel alleviated oxidative stress injury in SCs by targeting IL-1R1 to downregulate the NF-κB-mediated inflammatory response. Mel could potentially serve as an innovative therapeutic approach for the treatment of IFP.