TLR-7对阿尔茨海默病细胞模型β淀粉样蛋白表达及炎症反应的调控作用及机制

The regulation and mechanism of TLR-7 on the expression of amyloidβprotein and inflammatory response in Alzheimer's disease cell model

目的探究Toll样受体7(TLR-7)在阿尔茨海默病人神经母细胞瘤细胞(SH-SY5Y)模型中对β淀粉样蛋白表达及神经炎症反应的调控作用及机制。方法构建稳定表达β淀粉样前体蛋白瑞典型突变(APPswe)的阿尔茨海默病模型细胞SH-SY5Y/APPswe,将细胞分为SH-SY5Y组、SH-SY5Y/APPswe组、TLR-7敲低SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-TLR-7)组、敲低对照SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-NC)组、TLR-7敲低+miR-15b抑制SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-TLR-7+miR-15b inhibitor)组和TLR-7敲低+微小RNA-15b(miR-15b)抑制对照SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-TLR-7+inhibitor NC)组。蛋白质印迹法检测β淀粉样前体蛋白(APP)的表达;实时荧光定量PCR实验检测TLR-7、miR-15b、β淀粉样前体蛋白裂解酶1(BACE1)、白介素-1β(IL-1β)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)mRNA的表达;酶联免疫吸附实验检测β淀粉样蛋白42(Aβ42)以及IL-1β、IL-6和TNF-α的分泌;荧光素酶报告基因实验检测核因子κB(NF-κB)活性。结果与SH-SY5Y组比较,SH-SY5Y/APPswe组细胞APP表达水平上调,Aβ42分泌水平增加[(11.24±0.76)ng/mL比(2.09±0.17)ng/mL,P<0.01)];与SH-SY5Y/APPswe+sh-NC组比较,SH-SY5Y/APPswe+sh-TLR-7组miR-15b表达水平上调[(2.98±0.21)比(1.10±0.05),P<0.01],BACE1 mRNA表达水平下降[(0.72±0.06)比(1.02±0.03),P<0.01],Aβ42分泌水平下降[(6.09±0.42)ng/mL比(10.52±0.67)ng/mL,P<0.01],IL-1β、IL-6和TNF-αmRNA表达水平下降[IL-1β:(3.34±0.25)比(4.62±0.41),P<0.01;IL-6:(3.75±0.34)比(5.43±0.31),P<0.01;TNF-α:(1.25±0.11)比(2.07±0.18),P<0.01],IL-1β、IL-6和TNF-α分泌水平下降[IL-1β:(61.40±7.76)pg/mL比(101.76±7.95)pg/mL,P<0.01;IL-6:(547.29±39.11)pg/mL比(893.35±101.43)pg/mL,P<0.01;TNF-α:(256.17±24.32)pg/mL比(375.28±33.99)pg/mL,P<0.01],NF-κB活性下降[(3.25±0.31)比(5.01±0.52),P<0.01]。与SH-SY5Y/APPswe+sh-TLR-7+inhibitor NC组比较,SH-SY5Y/APPswe+sh-TLR-7+miR-15b inhibitor组miR-15b表达水平下降[(0.67±0.13)比(2.96±0.35),P<0.01],BACE1 mRNA表达水平升高[(1.39±0.42)比(0.71±0.05),P<0.05],Aβ42分泌水平升高[(19.54±3.01)ng/mL比(7.47±1.58)ng/mL,P<0.01],IL-1β、IL-6和TNF-αmRNA表达水平升高[IL-1β:(6.07±0.43)比(3.55±0.33),P<0.01;IL-6:(5.98±0.28)比(4.02±0.22),P<0.01;TNF-α:(1.88±0.21)比(1.36±0.20),P<0.01],IL-1β、IL-6、TNF-α分泌水平升高[IL-1β:(155.32±13.67)pg/mL比(68.62±5.41)pg/mL,P<0.05;IL-6:(1255.49±119.79)pg/mL比(439.73±60.01)pg/mL,P<0.05;TNF-α:(423.66±34.32)pg/mL比(227.82±26.53)pg/mL,P<0.05],NF-κB活性升高[(5.91±0.66)比(3.58±0.27),P<0.01]。结论TLR-7可能通过miR-15b调控NF-κB信号通路,并影响阿尔茨海默病模型细胞β淀粉样蛋白的分泌及神经炎症反应。

Objective To investigate the regulation and mechanism of TLR-7 on amyloidβprotein expression and neuroinflammatory response in SH-SY5Y cell model of Alzheimer's disease.Methods An Alzheimer's disease cell model SH-SY5Y/APPswe with stable expression of APPswe was constructed.The cells were divided into SH-SY5Y,SH-SY5Y/APPswe,SH-SY5Y/APPswe+sh-TLR-7,SH-SY5Y/APPswe+sh-NC,SH-SY5Y/APPswe+sh-TLR-7+miR-15b inhibitor and SH-SY5Y/APPswe+sh-TLR-7+inhibitor NC groups.The expression of APP protein was detected using western blot,the mRNA expression levels of TLR-7,miR-15b,BACE1,IL-1β,IL-6,and TNF-αwere detected using real-time fluorescent quantitative PCR,and the levels of secreted amyloidβprotein 42(Aβ42),IL-1β,IL-6,and TNF-αwere detected using enzyme-linked immunosorbent assay.NF-κB activity was detected using luciferase reporter gene assay.Results Compared with the SH-SY5Y group,the SH-SY5Y/APPswe group showed upregulated cellular APP expression and increased secretion levels of amyloidβprotein[(11.24±0.76)ng/mL vs.(2.09±0.17)ng/mL,P<0.01)].Compared with the SH-SY5Y/APPswe+sh-NC group,the SH-SY5Y/APPswe+sh-TLR-7 group exhibited upregulated miR-15b expression[(2.98±0.21)vs.(1.10±0.05),P<0.01],decreased BACE1 mRNA expression[(0.72±0.06)vs.(1.02±0.03),P<0.01],reduced secreted Aβ42 level[(6.09±0.42)ng/mL vs.(10.52±0.67)ng/mL,P<0.01]and decreased mRNA expression of IL-1β[IL-1β:(3.34±0.25)vs.(4.62±0.41),P<0.01],IL-6[(3.75±0.34)vs.(5.43±0.31),P<0.01],and TNF-α[(1.25±0.11)vs.(2.07±0.18),P<0.01].Additionally,the SH-SY5Y/APPswe+sh-TLR-7 group also exhibited decreased secreted levels of IL-1β[(61.40±7.76)pg/mL vs.(101.76±7.95)pg/mL,P<0.01],IL-6[(547.29±39.11)pg/mL vs.(893.35±101.43)pg/mL,P<0.01],and TNF-α[(256.17±24.32)pg/mL vs.(375.28±33.99)pg/mL,P<0.01],as well as decreased NF-κB activity[(3.25±0.31)vs.(5.01±0.52),P<0.01].Compared with SH-SY5Y/APPswe+sh-TLR-7+inhibitor NC group,the SH-SY5Y/APPswe+SH-TLR-7+miR-15b inhibitor group showed decreased miR-15b expression[(0.67±0.13)vs.(2.96±0.35),P<0.01],increased BACE1 mRNA expression level[(1.39±0.42)vs.(0.71±0.05),P<0.05],increased secreted Aβ42 level[(19.54±3.01)ng/mL vs.(7.47±1.58)ng/mL,P<0.01],and increased mRNA expression of IL-1β[(6.07±0.43)vs.(3.55±0.33),P<0.01],IL-6[(5.98±0.28)vs.(4.02±0.22),P<0.01],and TNF-α[(1.88±0.21)vs.(1.36±0.20),P<0.01].Additionally,the SH-SY5Y/APPswe+SH-TLR-7+miR-15b inhibitor group also showed increased secreted levels of IL-1β[(155.32±13.67)pg/mL vs.(68.62±5.41)pg/mL,P<0.05],IL-6[(1255.49±119.79)pg/mL vs.(439.73±60.01)pg/mL,P<0.05],and TNF-α[(423.66±34.32)pg/mL vs.(227.82±26.53)pg/mL,P<0.05],as well as increased NF-κB activity[(5.91±0.66)vs.(3.58±0.27),P<0.01].Conclusion TLR-7 may regulate the NF-κB signaling pathway through miR-15b and influence the secretion of amyloid beta protein and neuroinflammatory response in Alzheimer's disease model cells.