电针调控乳酸介导缺氧诱导因子-1α信号通路抑制炎性反应改善血管性痴呆大鼠认知功能障碍

Electroacupuncture improves cognitive function by modulating hippocampal lactatemediated HIF-1αsignaling pathway and inhibiting inflammation response in vascular dementia rats

目的:观察电针“四神聪”“风池”对血管性痴呆(VD)大鼠海马组织中乳酸(Lac)、脯氨酸羟化酶2(PHD2)、缺氧诱导因子-1α(HIF-1α)/核转录因子-κB(NF-κB)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路和炎性因子的影响,探讨电针治疗VD的可能作用机制。方法:SD大鼠随机分为空白组12只、假手术组12只、手术组40只。采用4血管阻断法复制VD大鼠模型,造模成功的24只大鼠随机分为模型组、电针组各12只。电针组电针“四神聪”“风池”,每次30 min,每日1次,连续治疗21 d。采用Morris水迷宫实验检测造模前后及干预后大鼠的学习记忆能力;HE染色法观察海马组织病理变化;比色法检测海马组织Lac含量;ELISA法检测海马组织及血清中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-18含量;免疫组织化学法检测海马组织中PHD2、HIF-1α和磷酸化(p)-NF-κB p65阳性表达;Western blot法检测海马组织中PHD2、HIF-1α、NF-κB p65、p-NF-κB p65和NLRP3蛋白表达。结果:与空白组、假手术组比较,模型组大鼠逃避潜伏期延长及穿越原平台次数减少(P<0.01),海马神经元排列紊乱,细胞结构不同程度受损,海马组织Lac含量及海马和血清中TNF-α、IL-1β、IL-18含量升高(P<0.01);海马组织PHD2阳性表达和蛋白表达降低(P<0.05,P<0.01),HIF-1α、p-NF-κB p65阳性表达和蛋白表达升高(P<0.01),NF-κB p65、NLRP3蛋白表达升高(P<0.01)。与模型组相比,电针组大鼠以上指标均逆转(P<0.01,P<0.05)。结论:电针“四神聪”“风池”可改善VD大鼠认知功能障碍,其作用机制可能与调控大鼠海马组织中乳酸含量,介导HIF-1α信号通路相关蛋白表达,进而抑制炎性反应有关。

Objective To observe the effects of electroacupuncture(EA)stimulation of“Sishencong”(EXHN1)and“Fengchi”(GB20)on lactate(Lac)content,expression of proline hydroxylase 2(PHD2),hypoxia-inducible factor-1α(HIF-1α)/nuclear transcription factor-κB(NF-κB)/NOD-like receptor thermoprotein structural domainassociated protein 3(NLRP3)signaling pathway,and inflammatory factors in hippocampal tissue of vascular dementia(VD)rats,so as to explore its mechanisms underlying improvement of VD.Methods Male SD rats screened by Morris water maze tests were randomly divided into blank control,sham-operation,VD model and EA groups(12 rats in each group).The VD model was replicated using the 4-vessel occlusion(VO)method.EA(2 Hz,1 mA)was applied to EX-HN1 and bilateral GB20 for 30 min,once daily for consecutive 21 days.Morris water maze test was employed to test the rat’s memory learning ability before and after modeling and after the intervention.The hippocampal tissue was sampled for observing histopathologic changes with H.E.staining;and detecting Lac content with colorimetric method,and the contents of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-18(also in serum)by using ELISA,respectively.The immunoactivity levels of PHD2,HIF-1α,and p-NF-κB p65 in hippocampal tissue were detected by immunohistochemistry,and the expression levels of PHD2,HIF-1α,NF-κB p65,p-NF-κB p65 and NLRP3 proteins in hippocampal tissue were detected by Western blot.Results Compared with the blank control and sham-operation groups,the escaping latency,Lac content in hippocampus,the TNF-α,IL-1β,and IL-18 contents in both hippocampus and serum,the immunoactivity of HIF-1αand p-NF-κB p65 and expression levels of HIF-1α,NF-κB p65,p-NF-κB p65,and NLRP3 proteins were significantly increased(P<0.01),while the number of original platform crossing,and PHD2 immunoactivity and protein expression level were significantly decreased(P<0.05,P<0.01)in the model group.Following EA intervention,modeling induced increase and decrease of the indexes mentioned above were all reversed in the EA group(P<0.05,P<0.01).H.E.staining showed disordered arrangement of neurons,uneven cytoplasm stain,blurred nucleolus or disappearance of nucleolus,dilated capillaries,many apoptotic bodies and increased inflammatory cells in the hippocampus tissue of the model group,which was improved to a certain extent in the EA group,including relatively regular arrangement of neurons,reduced apoptotic bodies and inflammatory cells,etc.in the hippocampus.Conclusion EA stimulation of EX-HN1 and GB20 can improve the cognitive function in VD rats,which may be related to its functions in reducing Lac content,regulating the expression of HIF-1αpathway related proteins,and inhibiting inflammatory responses in the hippocampus tissue.