基于TLR4/MyD88/NF-κB通路探讨美洲大蠊虫粉对脊髓损伤大鼠的保护作用

Protective effect of Periplaneta americana powder on rats with spinal cord injury via the TLR4/MyD88/NF-κB pathway

目的探讨美洲大蠊虫粉对脊髓损伤大鼠的保护作用和可能的机制。方法将48只SD大鼠随机分为假手术组、盐水组、美洲大蠊虫粉组、TOLL样受体4(toll-like receptors,TLR4)抑制剂组。除假手术组外其余3组均构建大鼠脊髓半横断损伤模型。术后假手术组不做治疗,盐水组与美洲大蠊虫粉组分别给予生理盐水和美洲大蠊虫粉(630 mg/kg)灌胃处理,TLR4抑制剂组给予TLR4抑制剂(3 mg/kg)腹腔注射处理。术后1、3、7、14 d运用BBB评分评估大鼠后肢运动功能,苏木素-伊红(HE)染色观察脊髓组织病理改变,免疫组化观察神经元数目变化,酶联免疫法检测炎性因子IL-1、IL-6、IL-10和TNF-α表达,Western Blot检测TLR4、髓样分化因子(myeloid differentiation factor 88,MyD88)和NF-κB p65表达。结果与假手术组相比,盐水组BBB评分、神经元数目显著下降,而病理损伤程度、IL-1、IL-6、TNF-α、TLR4、MyD88、NF-κB p65水平显著增加(P<0.05),与盐水组相比,美洲大蠊虫粉组和TLR4抑制剂组增加了BBB评分、神经元数目、降低了病理损伤程度、IL-1、IL-6、TNF-α、TLR4、MyD88、NF-κB p65水平(P<0.05),与TLR4抑制剂组相比,美洲大蠊虫粉组更好地增加了BBB评分、神经元数目,降低病理损伤程度、IL-1、TNF-α表达。结论美洲大蠊虫粉可以通过抑制TLR4/MyD88/NF-κB通路减少脊髓损伤后炎性因子的产生,起到保护神经和促进运动恢复的作用。

Objective To explore the protective effect and possible mechanism of Periplaneta americana powder on rats with spinal cord injury.Methods Forty-eight male SD rats were randomly divided into sham operation,saline,Periplaneta americana powder,and Toll-like receptor 4(TLR4)inhibitor groups.Except for the sham operation group,a rat spinal cord hemi-transection injury model was established in the other three groups.The sham operation group received no treatment after the operation,saline and drug groups were subjected to intragastric administration of equal volumes of normal saline and Periplaneta americana powder(630 mg/kg),respectively,and the TLR4 inhibitor group was administered an intraperitoneal injection of TLR4 inhibitor(3 mg/kg).On days 1,3,7,and 14 after the operation,the motor function of rat hind limbs was evaluated by the Basso,Beattie,and Bresnahan(BBB)score.Histopathological changes of the spinal cord were observed by hematoxylin-eosin staining.Changes in the number of neurons were observed by immunohistochemistry.The levels of inflammatory factors IL-1,IL-6,IL-10,and TNF-αwere measured by ELISA,and expression of TLR4,myeloid differentiation factor 88(MyD88),and NF-κB p65 was detected by Western Blot.Results Compared with the sham operation group,the BBB score and number of neurons in the saline group were significantly decreased,while the degree of pathological damage,and IL-1,IL-6,TNF-α,TLR4,MyD88,and NF-κB p65 levels were significantly increased(P<0.05).Compared with the saline group,periplaneta americana powder and TLR4 inhibitor groups showed an increase in BBB scores and the number of neurons,and decreases in the degree of pathological damage and IL-1,IL-6,TNF-α,TLR4,MyD88,and NF-κB p65 levels(P<0.05).Compared with the TLR4 inhibitor group,the periplaneta americana powder group had better increases in the BBB score,number of neurons and decreases in the degree of pathological damage and expression of IL-1 and TNF-α.Conclusions Periplaneta americana powder reduces the production of inflammatory factors after spinal cord injury by inhibiting the TLR4/MyD88/NF-κB pathway,protects nerves,and promotes motor recovery.