过表达miR-202-5p通过抑制PCSK9减轻阿尔茨海默病神经损伤

Overexpression of miR-202-5p attenuates nerve damage in Alzheimer’s disease by inhibiting PCSK9

目的探讨miR-202-5p对前蛋白转化酶枯草溶菌素9(PCSK9)的调控作用,及对阿尔茨海默症(AD)神经元损伤的影响。方法SD大鼠用侧脑室注射β-淀粉样蛋白(Aβ1-42)法建立AD模型,随机分正常对照组、模型组、ago-miR-202-5p组、ago-NC组、PCSK9抑制剂组。给药结束后进行空间记忆及学习能力检测;尼氏染色检测脑皮层组织神经元变化;神经元核抗原(NeuN)免疫荧光染色法检测脑皮层组织神经元数目;ELISA法检测脑脊液中PCSK9、β-淀粉样蛋白42(Aβ42)、β-淀粉样蛋白40(Aβ40)、总胆固醇(TC)及脑皮层组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、磷酸化Tau蛋白(P-tau)水平;qRT-PCR法检测脑皮层组织miR-202-5p表达水平;Western blot法检测脑皮层组织PCSK9、LDL受体相关蛋白1(LRP-1)、载脂蛋白E(ApoE)及淀粉前体蛋白(APP)、β位点APP剪切酶1(BACE1)蛋白表达。双荧光素酶验证miR-202-5p对PCSK9的靶向调控作用。建立体外AD细胞模型,共转染miR-202-5p mimic及PCSK9过表达载体(pcDNA-PCSK9),探究PCSK9过表达对miR-202-5p作用的逆转。结果与正常对照组相比,模型组大鼠空间记忆及学习能力降低,脑皮质神经元损伤及数目减少,脑脊液中PCSK9、Aβ42、Aβ40、胆固醇水平升高,脑皮质组织中P-tau及炎症因子水平升高,miR-202-5p表达降低,PCSK9活化介导的BACE1-APP-Aβ生成途径活性升高,LRP-1-ApoE介导的促胆固醇摄取及Aβ清除活性降低(P<0.05)。miR-202-5p过表达或PCSK9抑制剂干预治疗,均可抑制Aβ42、Aβ40沉积引起的神经元损伤,减少炎症因子分泌,并抑制PCSK9活化介导的BACE1、APP、Aβ生成,提高LRP-1-ApoE介导的促胆固醇摄取及Aβ清除(P<0.05)。miR-202-5p与PCSK9之间存在靶向调控作用。PCSK9上调,可部分减弱miR-202-5p过表达发挥的抗AD作用(P<0.05)。结论过表达miR-202-5p可通过抑制PCSK9活化抑制Aβ生成并促进LRP-1-ApoE介导的促胆固醇摄取及Aβ清除,进而发挥抗AD神经损伤作用。

Objective To investigate the regulatory effect of miR-202-5 p on proprotein convertase subtilisin/kexin type 9(PCSK9)and its effect on neuronal damage in Alzheimer’s disease(AD).Methods SD rats were injected with amyloid β(Aβ)1-42 into the lateral ventricle to establish an AD model and were randomly divided into normal control,model,ago-miR-202-5 p,ago-NC and PCSK9 inhibitor groups.After the administration,spatial memory and learning ability tests were performed.Nissl staining was used to detect neuronal changes in cerebral cortex tissue.Neuron nuclear antigen(NeuN)immunofluorescence was used to detect the number of neurons in cerebral cortex tissue.ELISAs were used to measure the levels of PCSK9,β-amyloid 42(Aβ42),β-amyloid 40(Aβ40),and total cholesterol(TC)in cerebrospinal fluid and tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and phosphorylated Tau protein(P-tau)in cerebral cortex tissues.qRT-PCR was used to measure miR-202-5 p expression in cerebral cortex tissue.Western blot was used to detect PCSK9,LDL receptor-related protein 1(LRP-1),apolipoprotein E(ApoE),amyloid precursor protein(APP),andβsite APP shearing enzyme 1(BACE1)protein expression in cerebral cortex tissue.Dual luciferase assays were used to verify targeted regulation of PCSK9 by miR-202-5 p.An in vitro AD cell model was established and cotransfected with miR-202-5 p mimic and an PCSK9 overexpression vector(pcDNA-PCSK9),and then reversal of the effect of PCSK9 overexpression on miR-202-5 p was explored.Results Compared with the normal control group,the spatial memory and learning ability of rats in the model group were decreased,cerebral cortex neuron damage and number were decreased,PCSK9,Aβ42,Aβ40 and cholesterol levels in the cerebrospinal fluid were increased,P-tau and inflammatory factors levels in the cerebral cortex were increased,miR-202-5 p expression was decreased,activity of the PCSK9 activation-mediated BACE1-APP-Aβproduction pathway was increased,and LRP-1-ApoE-mediated promotion of cholesterol uptake and Aβclearance activity were decreased(P<0.05).miR-202-5 p overexpression or PCSK9 inhibitor treatment suppressed neuronal damage caused by Aβ42 and Aβ40 deposition,reduced inflammatory factor secretion,inhibited BACE1,APP and Aβproduction mediated by PCSK9 activation,and increased LRP-1-ApoE-mediated promotion of cholesterol uptake and Aβclearance(P<0.05).There was a targeted regulatory effect between miR-202-5 p and PCSK9.Upregulation of PCSK9 partially attenuated the anti-AD effect of miR-202-5 p overexpression(P<0.05).Conclusions miR-202-5 p overexpression inhibits Aβproduction by suppressing PCSK9 activation and promoting LRP-1-ApoE-mediated cholesterol uptake and Aβclearance,thereby exerting anti-AD nerve injury effect.