TLR4/NF-κB信号通路在阿尔茨海默病炎性反应中的表达及电针干预影响

Role of TLR4/NF-κB Signaling Pathway and Effect of Electroacupuncture on Alzheimer’s Disease Inflammatory Response

目的观察TLR4/NF-κB信号通路在阿尔茨海默病(Alzheimer’s disease,AD)炎性反应中的表达及益肾调督法电针的效应。方法40只雄性SD大鼠,随机等分成对照组、假手术组、模型组和治疗组。采用双侧海马注射Aβ1-42建立AD模型。对照组、假手术组和模型组不予任何治疗;治疗组选取百会、肾俞,电针20 min,1次/d,6 d为1个疗程,2个疗程间休息1日。采用Elisa法检测血清TNF-α、IL-1β、IL-4水平,Western Blot法检测脑内APP、TLR4、NF-κBp65蛋白含量,免疫组织化学染色法检测海马CA1区胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达。结果Elisa结果显示,假手术组与对照组相比,各项指标含量差异均无统计学意义。模型组血清TNF-α、IL-1β水平较对照组显著升高(P<0.01)、IL-4水平显著下降(P<0.01)。治疗组血清TNF-α、IL-1β水平较模型组显著降低(P<0.01),与对照组比较差异无统计学意义(P>0.05),治疗组血清IL-4水平较模型组显著上升(P<0.01),与对照组比较差异无统计学意义(P>0.05);Western Blot结果显示,假手术组与对照组相比,各项指标含量差异均无统计学意义。模型组脑内APP、TLR4、NF-κBp65蛋白含量较对照组显著增高(P<0.01),治疗组含量则显著低于模型组含量(P<0.01),但仍显著高于对照组(P<0.05);免疫组织化学染色法检测结果显示:假手术组与对照组相比,GFAP表达量无统计学意义。模型组GFAP阳性表达量较对照组显著增高(P<0.01),治疗组GFAP阳性表达量与模型组比较差异无统计学意义(P>0.05),但仍显著高于对照组(P<0.01)。结论"益肾调督"法电针明显改善AD大鼠血清中炎性因子水平,减轻脑组织损伤程度,这与抑制TLR4/NF-κB通路密切相关,并可能与电针激活星形胶质细胞保护脑组织功能相关。

Objective To observe the role of TLR4/NF-κB signaling pathway in the inflammatory response of Alzheimer’s disease(AD)and the therapeutic effect of tonifying kidney and regulating Du meridian electroacupuncture therapy.Methods Forty male SD rats were randomly divided into control group,sham operation group,AD model group and treatment group.AD models were established by a bilateral hippocampal injection of Aβ1-42.Control group,sham operation group and AD model group did not receive any treatment."Baihui"(GV20)and bilateral"Shenshu"(BL23)were selected to be needled in treatment group.An individual EA session was administered daily for 20 min,6 days,and 1 day rest for a course of 6 days,totally for 2 courses.The levels of serum TNF-α,IL-1βand IL-4 were detected by ELISA method.The contents of APP,TLR4 and NF-κBp65 protein in brain were detected by Western Blot.The glial fibrillary acidic protein(GFAP)in hippocampal CA1 region was detected by immunohistochemical staining.Results ELISA results showed that there was no significant difference in the content of each index between sham operation group and control group.The levels of serum TNF-αand IL-1βin the model group were significantly higher than those in the control group(P<0.01),and the levels of IL-4 were significantly decreased(P<0.01).The levels of serum TNF-αand IL-1βin the treatment group were significantly lower than those in the model group(P<0.01)but there was no significant difference when compared with those of control group.The serum IL-4 level in the treatment group was significantly higher than that in the model group(P>0.05)but there was no significant difference when compared with those of the control group(P>0.05).Western Blot results showed that there was no significant difference in the content of each index between the sham operation group and the control group.The contents of APP,TLR4 and NF-κBp65 in the model group were significantly higher than those in the control group(P<0.01),and the content in the treatment group was significantly lower than that in the model group(P<0.01),but was still significantly higher than that of the control group(P<0.05).The results of immunohistochemical staining showed that there was no statistically significant difference in the expression of GFAP between the sham operation group and the control group.The positive expression of GFAP in the model group was significantly higher than that in the control group(P<0.01).The positive expression of GFAP in the treatment group was not significantly different from that in the model group(P>0.05),but the expression of the treatment group was still significantly higher than that of the control group(P<0.01).Conclusion The electroacupuncture therapy for tonifying kidney and regulating Du meridian significantly improves the inflammatory index in AD rats,and reduces the degree of brain damage,which is closely related to the inhibition of TLR4/NF-κB signaling pathway,possibly related to activation of astrocytes that mediates an effect of protecting brain tissue.