【作者】 张伟；

【导师】 黄飞骏；

【作者基本信息】 四川大学 ， 法医病理学与法医临床学， 2023， 博士

【摘要】 研究背景与目的:甲基苯丙胺(Methamphetamine,METH)依赖是多基因、多机制共同作用的结果。METH依赖主要与神经炎症、氧化应激、转录因子活化、DNA损伤、线粒体功能紊乱等多种病理生理过程有关。目前对METH依赖神经炎症的机制并不完全清楚,筛选METH依赖神经炎症的关键基因及机制对阐明METH依赖的神经生物学功能具有重要意义。生物信息学技术广泛用于发掘和筛选肿瘤、神经退行性疾病、免疫相关疾病、神经炎症、神经损伤等关键基因及生物学功能。本实验采用生物信息学方法从多个数据库筛选出CD44和PI3K/Akt信号通路是METH依赖的hubgene和主要富集信号通路。CD44和PI3K/Akt信号通路参与调控神经系统的发育、突触可塑性变化(学习和记忆)、中枢神经系统肿瘤的侵袭、神经炎症及神经退行性疾病的发生发展过程。CD44与PI3K/Akt信号通路在METH依赖中的作用机制鲜见研究报道。本实验拟构建METH依赖小鼠模型和BV2细胞模型,采用siRNAs敲低CD44、pc DNA 3.1(+)过表达CD44、Ly294002抑制PI3K/Akt信号通路,初步探讨CD44和PI3K/Akt信号通路在METH依赖神经炎症中的作用机制,可为进一步阐明METH依赖神经炎症的病理生理机制提供理论依据和数据支撑。研究方法:1.生物信息学分析筛选METH依赖的hubgene和主要信号通路通过GEO数据库下载METH依赖相关的基因芯片数据集,采用GEO2R和R语言筛选METH依赖的差异表达基因(differentially expressed genes,DEGs);Venn图筛选共表达DEGs(common DEGs,CDEGs);基因本体论(Gene Ontology,GO)和京都基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)对CDEGs进行功能注释和富集分析;STRING在线数据库、Cytoscape软件进行蛋白互作(protein–protein interaction network,PPI)可视化分析;Cytoscape MCODE和Cyto Hubba插件筛选功能模块及hubgene。2.CD44和PI3K/Akt信号通路在METH依赖小鼠脑功能区的作用2.1 METH依赖小鼠模型构建及行为学指标检测C57BL/6小鼠连续腹腔注射METH(2.0mg/kg×21d)建立METH依赖小鼠模型,观察小鼠一般情况、体温和体重。条件位置偏好(conditioned place preference,CPP)评价METH依赖小鼠模型;高架十字迷宫(Elevated plus maze,EPM)及旷场实验(open filed test,OFT)评价METH依赖小鼠焦虑样行为;Morris水迷宫(Morris Water Maze,MWM)和Y迷宫(Y-maze)评价METH依赖小鼠学习能力和空间记忆力。2.2.METH依赖小鼠脑功能区损伤及神经炎症指标检测HE和尼氏染色检测METH依赖对小鼠脑功能区(海马、前额皮层、纹状体)形态学的影响。免疫组织化学法(Immunohistochemistry,IHC)分别检测NLRP3、CD11b蛋白在METH依赖组和Control组小鼠上述脑功能区的表达。Western Blotting检测METH依赖组和Control组小鼠脑功能区NLRP3炎性小体复合体及其效应器蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleave IL-1β、AIM2和ASC/TSM1)、炎症指标(CD11b、NF-κB、TNF-α、IL-6)、凋亡蛋白和抗凋亡蛋白(Caspase-9、Caspase-3、cleaved Caspase-3、Bax、Bcl2)的蛋白表达水平。ELISA检测METH依赖组和Control组小鼠外周血血浆炎症指标(IL-6、TNF-α、IL-1β)的蛋白表达水平。2.3 METH依赖小鼠脑功能区CD44和PI3K/Akt信号通路关键因子的蛋白检测IHC检测CD44蛋白在METH依赖组和Control组小鼠脑功能区(海马、纹状体、前额皮层)表达;Western Blotting检测上述脑功能区CD44、TLR4、PI3K/Akt信号通路关键因子及磷酸化蛋白(p-PI3K、PI3K、p-Akt、Akt)表达。3.CD44激活PI3K/Akt信号通路在METH依赖神经炎症的机制3.1敲低/过表达CD44对METH诱导神经炎症的影响采用1.0m M METH构建METH BV2细胞模型。siRNA-CD44敲低CD44,pc DNA 3.1(+)-CD44过表达CD44。采用CCK8检测细胞活性;流式细胞术(flow cytometry,FCM)检测细胞凋亡;q PCR检测CD44、TLR4、PI3K、Akt、CD11b、TNF-α、IL-6、Bax和Bcl2 mRNA相对表达水平;Western Blotting检测CD44、TLR4、PI3K/Akt信号通路关键因子及其磷酸化蛋白(p-PI3K、PI3K、p-Akt、Akt)、NLRP3炎性小体复合体及其效应蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleave IL-1β、AIM2、ASC/TSM1)、炎症指标(CD11b、NF-κB、TNF-α、IL-6)、凋亡蛋白和抗凋亡蛋白(Caspase-9、Caspase-3、cleaved Caspase-3、Bax、Bcl2)的蛋白表达水平。ELISA检测细胞培养上清中TNF-α、IL-6和IL-1β的蛋白表达水平。3.2 Ly294002抑制PI3K/Akt信号通路对METH诱导神经炎症的影响。采用Ly294002抑制PI3K/Akt信号通路;CCK8检测上述细胞活性;FCM检测细胞凋亡;Western Blotting检测CD44、TLR4、PI3K/Akt信号通路关键因子及其磷酸化蛋白(p-PI3K、PI3K、p-Akt、Akt)、NLRP3炎性小体复合体及其效应器蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleave IL-1β、AIM2和ASC/TSM1)、炎症指标(CD11b、NF-κB、TNF-α、IL-6)、凋亡蛋白和抗凋亡蛋白(Caspase-9、Caspase-3、cleaved Caspase-3、Bax、Bcl2)的蛋白表达。ELISA检测细胞培养上清中TNF-α、IL-6和IL-1β的蛋白表达水平。研究结果:1.生物信息学筛选METH依赖hubgene和主要信号通路最终GSE111925、GSE12387、GSE15774、GSE17665、GSE30305共5个数据库纳入研究。5个数据库包含56个METH依赖数据集,55个Controls数据集。共筛选出8197个DEGs,其中299个CDEGs。GO和KEGG富集分析发现CDEGs主要富集在DNA和RNA转录调控、细胞凋亡、损伤刺激的反应、药物反应等46个生物学进程(BP),细胞核、细胞质、胞浆、细胞外区等21个细胞学组分(CC),蛋白质、DNA、大分子复合体、细胞因子、蛋白质同分异构体、转录因子、蛋白酶、病原体受体活化等25个分子功能。PI3K/Akt信号通路是CDEGs最主要富集的信号通路之一。采用6种算法筛选出CD44、Syk、Fos、Dusp1、Klf4、Hspa1b、Cdkn1a、Egr1/2、Gata4、Brd4、Gnaq、Crem和Creb1是METH依赖的的hubgene。14个hubgene生物学功能高度相关,主要富集在凋亡(Dusp1,CD44,Cdkn1a,Gnaq,Gata4,Hspa1b),c AMP、视黄醇、光、激素、药物等刺激(Dusp1,Fos,Cdkn1a,Crem,Gata4,Klf4),细胞增殖(Dusp1,Cdkn1a,Klf4),转录调控(Egr1/2,Creb1,Crem,Gata4,Fos,Klf4,Brd4),心脏发育(Cdkn1a,Gnaq,Gagt4),生理周期(Egr1,Creb1,Crem)等生物学进程。MCODE共筛选出11个功能模块,其中14个hubgene主要集中在模块1和模块2,CD44和Dusp1也是模块的核心基因。2.CD44和PI3K/Akt信号通路在METH依赖小鼠脑功能区的作用2.1 METH依赖小鼠模型构建、一般情况及行为学变化C57BL/6小鼠连续腹腔注射METH(2.0mg/kg×21d)成功构建METH依赖小鼠模型。METH依赖组CPP分数、伴药室滞留时间显著高于Control组(P<0.001)。与Control组相比,METH依赖组小鼠注射METH后活动明显增加,出现竖尾,不停转圈,好斗,易激惹,2h-4h出现精神萎靡、进食及饮水减少、卷缩成团。METH依赖组和Control组在注射后30min和1h体温均较注射前显著增加,METH依赖组注射METH 2h体温显著高于Control组、METH依赖组注射前(P<0.01);Control组2h内体温恢复正常(P=0.409)。METH依赖组小鼠体重在实验前后无统计学差异(P=0.054),Control组小鼠体重逐渐增加(P<0.001),METH依赖组和Control组用药后体重存在统计学差异(P=0.004)。EPM发现METH依赖组在开放臂进入次数(open arm entry,OE)百分比、开放臂滞留时间(open arm time,OT)及其百分比、开放臂运动距离及其百分比、探头次数均显著低于Control组(P<0.05)。OFT发现METH依赖组小鼠中心区穿越次数百分比、中心区滞留时间百分比、中心区运动距离百分比均显著低于Control组(P<0.05)。Y-maze发现METH依赖组小鼠自由交替率、新异臂滞留时间及其百分比、新异臂穿梭次数百分比及新异臂运动距离百分比均显著低于Control组(P<0.05)。MWM定位巡航实验显示METH依赖组小鼠主要沿着Morris壁寻找平台,潜伏期未见明显变化;MWM空间探索实验显示METH依赖组小鼠首次到达平台时间显著高于Control组(P<0.001),平台所在象限滞留时间、穿越平台次数、平台象限滞留时间百分比及平台象限运动距离百分比均显著低于Control组(P<0.05)。2.2 METH依赖小鼠脑功能区神经炎症指标变化HE和尼氏染色显示,与Control组比较,METH依赖组海马、前额皮层、纹状体神经细胞数目减少,形态多样,细胞变圆,排列欠规整,神经细胞周围间隙增宽,神经纤维网疏松,胞体肿胀,染色加深,核固缩、碎裂,尼氏颗粒减少。METH依赖组小鼠上述脑功能区NLRP3炎性小体复合体及其效应蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleave IL-1β、AIM2、ASC/TSM1)、炎症指标(CD11b、NF-κB、TNF-α、IL-6)、凋亡蛋白(Caspase-9、Caspase-3、Bax/Bcl2)的蛋白表达水平均显著高于Control组(P<0.05)。cleaved Caspase-3蛋白在METH依赖组和Control组小鼠纹状体表达水平无统计学差异(P>0.05)。IHC发现METH依赖组小鼠NLRP3和CD11b蛋白在海马、前额皮层、纹状体表达水平均明显高于Control组(P<0.05)。ELISA发现METH依赖组小鼠外周血血浆IL-6、TNF-α和IL-1β蛋白表达水平均显著高于Control组(P<0.001)。2.3 METH依赖小鼠脑功能区CD44和PI3K/Akt信号通路蛋白表达Western Blotting发现CD44、TLR4、p-PI3K、PI3K、p-Akt和Akt蛋白在METH依赖组小鼠海马、前额皮层、纹状体表达水平均显著高于Control组(P<0.05);IHC观察CD44蛋白在上述脑功能区表达得出相似的结果(P<0.05)。3.CD44激活PI3K/Akt信号通路在METH依赖神经炎症的机制3.1敲低/过表达CD44对METH依赖神经炎症的影响采用1.0m M METH成功构建METH BV2细胞模型。METH组CD44、TLR4、PI3K和Akt mRNA表达水平均显著高于Control组(P<0.05),Western Blotting检测上述因子蛋白表达得出相似的结果(P<0.05)。siRNA-CD44+METH组TLR4、p-PI3K、PI3K、p-Akt蛋白表达较siRNA-NC+METH组显著降低(P<0.05),Akt蛋白表达水平在两组间无统计学差异(P=0.302)。Western Blotting发现METH组NLRP3炎性小体复合体及其效应蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleave IL-1β、AIM2、ASC/TSM1)、炎症指标(CD11b、TNF-α、IL-6、NF-κB)、凋亡蛋白(Caspase-9、Caspase-3、cleaved Caspase-3、Bax/Bcl2)的蛋白表达水平均显著高于Control组(P<0.05)。FCM检测细胞凋亡、ELISA检测细胞培养上清TNF-α、IL-6、IL-1β蛋白表达亦得出相似结果(P<0.05)。与siRNA-NC+METH组相比,siRNA-CD44+METH组NLRP3炎性小体复合体及其效应蛋白(NLRP3、Caspase-1、cleaved Caspase-1、cleaved-IL-1β、AIM2、ASC/TMS1)、炎症指标(CD11b、NF-κB)、凋亡蛋白(Caspase-9、cleaved Caspase-3、Bax/Bcl2)的表达显著增加(P<0.05);TNF-a、IL-6、IL-1β、Caspase-3蛋白表达在两组间无统计学差异(P>0.05)。ELISA显示siRNA-CD44+METH组细胞培养上清TNF-a、IL-6、IL-1β蛋白表达显著高于siRNA-NC+METH组(P<0.001)。FCM检测细胞凋亡得出相似结果(P=0.001)。pc DNA 3.1(+)-CD44组CD44蛋白表达水平显著高于pc DNA 3.1(+)-NC组(P=0.014)。Western Blotting显示pc DNA 3.1(+)-CD44+METH组TLR4、p-PI3K、Akt、p-Akt蛋白表达水平显著高于pc DNA 3.1(+)-NC+METH组(P<0.05);PI3K蛋白表达水平在两组间无统计学差异(P=0.357)。Western Blotting显示,与pc DNA 3.1(+)-NC+METH组相比,pc DNA 3.1(+)-CD44+METH组NLRP3炎性小体复合体及其效应蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleaved IL-1β、AIM2)、炎症指标(CD11b、NF-κB、TNF-a、IL-6)、凋亡蛋白(Caspase-3、cleaved caspase-3、Bax、Bax/Bcl2)的蛋白表达水平显著降低(P<0.05);ASC/TMS1、Caspase-9的蛋白表达在两组间无统计学差异(P>0.05)。ELISA检测细胞培养上清TNF-a、IL-6、IL-1β的蛋白、FCM检测细胞凋亡均得出相似的结果(P<0.01)。3.2 Ly294002抑制PI3K/Akt信号通路对METH依赖神经炎症的影响。采用Ly294002抑制PI3K/Akt信号通路激活,Ly294002+METH组p-PI3K、PI3K、p-Akt、Akt蛋白表达水平显著低于DMSO+METH组(P<0.01),Ly294002+METH组CD44、TLR4、Akt蛋白表达与DMSO+METH组相比无统计学差异(P>0.05),结合上述结果说明CD44是PI3K/Akt信号通路上游蛋白。Western Blotting发现Ly294002+METH组NLRP3炎性小体复合体及其效应蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleaved IL-1β、AIM2)、炎症指标(CD11b、NF-κB、TNF-a、IL-6)、凋亡蛋白(Caspase-9、cleaved caspase-3、Bax/Bcl2)的蛋白表达水平均显著高于DMSO+METH组(P<0.05)。ELISA发现Ly294002+METH组细胞培养上清IL-6、TNF-α和IL-1β的蛋白表达水平均显著高于DMSO+METH组(P<0.05)。FCM检测细胞凋亡得出相似结果(P=0.007)。siRNA-CD44和Ly294002共同预处理BV2细胞,Western Blotting发现siRNA-CD44+Ly294002+METH组NLRP3炎性小体复合体及其效应器蛋白(NLRP3、Caspase-1、cleaved caspase-1、IL-1β、cleaved IL-1β、AIM2)、炎症指标(CD11b、NF-κB、TNF-a、IL-6)、凋亡蛋白(Caspase-9、caspase-3、cleaved caspase-3、Bax/Bcl2)的蛋白表达均显著高于siRNA-CD44+METH组或Ly294002+METH组(P<0.05)。ELISA发现siRNA-CD44+Ly294002+METH组细胞培养上清IL-6、TNF-α、IL-1β蛋白表达均显著高于siRNA-CD44+METH组或Ly294002+METH组(P<0.001)。FCM检测细胞凋亡得出相似结果(P<0.01)。研究结论:1.生物信息学分析发现CD44、Syk、Fos、Dusp1、Klf4、Hspa1b、Cdkn1a、Egr1/2、Gata4、Brd4、Gnaq、Crem和Creb1是METH依赖的hubgene;PI3K/Akt信号通路是METH依赖的主要富集信号通路。2.CD44和PI3K/Akt信号通路关键因子及其磷酸化蛋白在METH依赖组小鼠脑功能区及BV2细胞株表达升高。CD44可能通过激活PI3K/Akt信号通路调控METH依赖神经炎症,参与METH依赖的病理生理过程。更多还原

【Abstract】 Background: Methamphetamine(METH)was one of the most commonly abused drugs in the whole world.Long-term abuse could impair the structure and function of the nervous system.It also could result the METH dependence.Multiple genes and mechanisms were related to METH dependence,such as neuroinflammation,oxidative stress,transcription factor activation,DNA damage,mitochondrial dysfunction.However,the mechanism of METH dependence induced neuroinflammation was not fully understood.It was necessary to identify more useful genes and mechanisms of METH dependence induced neuroinflammation.Bioinformatics technology was an economical and efficient technology.It had been widely performed to discover and screen the hubgenes and key molecular mechanism of injuries and diseases,such as tumor,neurodegeneration,immune-related diseases,neuroinflammation,traumatic brain injury.CD44 and PI3K/Akt signaling pathway were identified as the hubgene and key signaling pathway of METH-dependence,which was involved in the regulation of nervous system development,synaptic plasticity changes(learning and memory),invasion of nervous system tumor,neuroinflammation,neurodegeneration,and other pathophysiological processes.However,the role of CD44 and PI3K/Akt signaling pathway in METH dependence were rarely reported.In order to clarify the role of CD44 in METH dependence induced neuroinflammation via PI3K/Akt signaling pathway,the siRNA-CD44 were used to knockdown the expression of CD44.The pc DNA 3.1(+)-CD44 were performed to overexpress CD44.The Ly294002 was selected to inhibit the activating of PI3K/Akt signaling pathway.It could provide theoretical basis and data support for further elucidating the pathophysiological mechanism of METH dependence induced neuroinflammation.Material and method:1.Bioinformatics analysis to screen hubgenes and key signaling pathwaysThe expression profiling by array of METH dependence were downloaded from GEO database.The GEO2 R and R languages were performed to screen the differentially expressed genes(DEGs).Venn diagram was used to screen common DEGs(CDEGs).The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were used to explore the biological functional annotation and signaling pathway.STRING online database were used to explore the special and nonspecific interactions of proteins.Cytoscape software was used for visual analysis.The MCODE and Cyto Hubba plug-ins of Cytoscape were used to screen functional modules and hubgenes.2.The role of CD44 and PI3K/Akt signaling pathway in METH dependent mice2.1 To establish the METH-dependent model and detect the behavioristicsThe METH dependent mouse model was established by intraperitoneal injection with 2.0mg/kg×21d of METH.The general state,temperature,and weight were observed and record.The conditioned place preference(CPP) was used to evaluate the METH dependent model.The Elevated plus maze(EPM)and open field test(open field test,OFT)were selected to evaluate the anxiety-like behavior in METH dependent mice.The Morris Water Maze (MWM)and Y-maze were used to evaluate the learning ability and spatial memory in METH dependent mice.2.2 To detect the damage of brain functional areas and neuroinflammatory markers in METH dependent miceThe morphological features of hippocampus,prefrontal cortex,and striatum of METH dependence and controls were detected by HE and Nissl staining.The expression of IL-6,TNF-α,and IL-1β in peripheral blood between METH dependent group and the controls were detected by ELISA.The expression of NLRP3 and CD11 b protein in brain functional areas was detected by Immunohistochemistry(IHC).The expression of NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved caspase-1,IL-1β,cleave IL-1β,AIM2,and ASC/TSM1),inflammatory markers(CD11b,NF-κB,TNF-α,IL-6),apoptotic proteins(Caspase-9,Caspase-3,cleaved caspase-3,Bax/ Bcl2)in hippocampus,prefrontal cortex,and striatum ofMETH dependent group and controls were detected by western blotting.2.3 To detect the expression of CD44 and key factors of PI3K/Akt signaling pathways in brain functional areas in METH dependent miceThe expression of CD4 protein in brain functional areas was detected byImmunohistochemistry(IHC).The relative protein expression of CD44,TLR4,key factors of PI3K/Akt signaling pathway(p-PI3 K,PI3K,p-Akt,Akt) in brain functional areas of METH dependent group and the controls were detected by western blotting.3.CD44 regulate METH dependence induced neuroinflammation through PI3K/Akt signaling pathway3.1 Effect of CD44 knockdown/overexpression on METH dependence induced neuroinflammationThe BV2 cell treated with 1.0m M METH to construct the vitro model of methamphetamine dependence.The siRNA-CD44 was performed to knockdown the expression of CD44.The pc DNA 3.1(+)-CD44 was used to overexpression of CD44.Cell viability and cell apoptosis was detected by CCK8 assay and flow cytometry.q PCR was used to detect the mRNA of CD44,TLR4,PI3 K,Akt,CD11 b,TNF-α,IL-6,Bax and Bcl2.The expression of CD44,TLR4,key factors of PI3K/Akt signaling pathway(p-PI3 K,PI3K,p-Akt,Akt),inflammatory markers(CD11b,NF-κB,TNF-α,IL-6),NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved caspase-1,IL-1β,cleave IL-1β,AIM2,and ASC/TSM1),apoptotic and anti-apoptotic proteins(Caspase-9,Caspase-3,cleaved caspase-3,Bax,and Bcl2)were detected by western blotting in BV2 cell,respectively.ELISA was used to detect the expression of TNF-α,IL-6,and IL-1β in cell culture supernatant as well.3.2 Effect of Ly294002 on METH dependence induced neuroinflammation by inhibiting PI3K/Akt signaling pathway.The Ly294002 was used to inhibit the activation of PI3K/Akt signaling pathway.Cell apoptosis was detected by CCK8 and flow cytometry.The expression of CD44,TLR4,key factors of PI3K/Akt signaling pathway(p-PI3 K,PI3K,p-Akt,Akt),inflammatory markers(CD11b,NF-κB,TNF-α,IL-6),NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved caspase-1,IL-1β,cleave IL-1β,AIM2,and ASC/TSM1),apoptotic proteins(Caspase-9,Caspase-3,cleaved caspase-3,Bax/Bcl2)were detected by western blotting.ELISA was used to detect the expression of TNF-α,IL-6,and IL-1β in cell culture supernatant as well.Result:1.Bioinformatics analysis screening the hubgenes and key signaling pathways of METH dependenceFinally,the GSE111925,GSE12387,GSE15774,GSE17665,and GSE30305 were included in the study.Total of 56 METH dependent microarray and 55 controls were included.299 CDEGs were identified from the 8197 DEGs.The GO analysis showed that the CDEGs were mainly enriched in 46 biological processes(BP),21 cytological components(CC),and25 molecular functions.BP category was mainly including DNA and RNA transcriptional regulation,cell apoptosis,cell response to DNA damage stimulation,and drug response.The CC was mainly including nucleus,cytoplasm,cytoplasm,and extracellular region.And CC mainly included binding of proteins and DNA,macromolecular complexes,enzymes,cytokines,protein isomers,transcription factors,proteases,and pathogen receptor activation.KEGG enrichment analysis showed that the PI3K/Aky signaling pathway was the predominant enrichment pathway of CDEGs.CD44,Syk,Fos,Dusp1,Klf4,Hspa1 b,Cdkn1a,Egr1/2,Gata4,Brd4,Gnaq,Crem,and Creb1 were identified as the hubgene by Cyto Hubba plugin with six algorithms.The 14 hubgenes biological functions were highly correlated,and the mainly enriched in apoptotic process(Dusp1,CD44,Cdkn1 a,Gnaq,Gata4,Hspa1b)c AMP,retinol,light,hormones,drugs and other stimuli(Dusp1,Fos,Cdkn1 a,Crem,Cdkn1a),stimulation of c AMP,retinol,light,hormone,drug(Gata4,Klf4),cell proliferation(Dusp1,Cdkn1 a,Klf4),transcriptional regulation(Egr1/2,Creb1,Crem,Gata4,Fos,Klf4,Brd4),cardiac development(Cdkn1a,Gnaq,Gagt4),physiological cycle(Egr1,Creb1,Crem).In addition,a total of 11 functional modules were screened by MCODE plug-in.The 14 hubgenes were mainly concentrated in module 1 and module 2,and CD44 and Dusp1 were also the key module genes.2.Roles of CD44 and PI3K/Akt signaling pathways in METH dependent mice2.1 To establish the METH dependent mice and detect the behavioristicsC57BL/6 mice were intraperitoneally injected with METH(2.0mg/kg×21d)to establish the METH dependent mouse model.The CPP score and retention time in the METH dependent group were significantly higher than the controls(P<0.001).It indicated that the METH dependent mouse model were successfully established.After METH injection,the activity of the METH dependent mice significantly increased than the controls.The METH dependent mice were also showing vertical tail,non-stop turning around,aggressiveness,irritability.The METH dependent mice were listlessness,decreased food and water intake,and huddled at 2h-4h after injection.The temperature of both METH dependent group and the controls increased at 30 min and 1h after injection.The temperature of the METH dependent group was significantly higher than the controls(P<0.05).The temperature METH dependent mice was higher than before injection at 2hours(P=0.004).The temperature of controls returned to normal at 2 hours after injection(P=0.409).Furthermore,There were no significant difference in weight of METH dependent group before and after the experiment(P=0.054).While the weight of the controls gradually increased(P<0.001).There was significant difference in weight between the METH dependent group and the controls after the experiment(P=0.004).The EPM showed that the percentage of open arm entry(OE),time spent in open arm(OT)and its percentage,the distance in open arm and its percentage,and headdip times of the METH dependent group were significantly lower than the controls(P<0.05).The time spent and distance in the center and its percentage of METH dependent mice were significantly lower than those of controls in the OFT(P <0.05).Y-maze showed that the spontaneous alternation rate,entries times and time spent and its percentage of METH dependent group in new arm were significantly lower than the controls(P<0.05).MWM test showed that the METH dependent mice mainly searched the platform along the Morris wall.There was no significant difference in the latency during 5-day training period in METH dependent group.The time of first arrived at the platform in METH dependent group was significantly longer than the controls in MWM test(P<0.001).The time spent in the platform quadrant,entries times of the platform,the percentage of time spent and distance in the platform quadrant were significantly lower than the controls(P<0.05).2.2 To detect the damage of brain functional areas and neuroinflammatory markers in METH dependent miceCompared with the controls,the METH dependent group had fewer nerve cells,pleomorphism,arranged irregularly,dilated intercellular space.The neurofiber network was loose,the cell body was swollen,the staining was deepened,and the nuclear condensation.In METH dependent group,inflammatory markers(CD11b,NF-κB,TNF-α,IL-6),NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved caspase-1,IL-1β,cleave IL-1β,AIM2,and ASC/TSM1),and apoptotic proteins(Caspase-9,Caspase-3,Bax/Bcl2)in brain functional areas of METH dependent group were higher than the controls(P<0.05).There was no significant difference in the expression of cleaved caspase-3 protein in striatum between METH dependent group and the controls(P>0.05).IHC also showed that the expression of NLRP3 and CD11 b in the brain functional areas of METH dependent group were significantly higher than the controls(P<0.05).The ELISA analysis showed that the protein of IL-6,TNF-α,and IL-1β protein in the plasma of METH dependent group were significantly higher than the controls(P<0.001).2.3 To detect the protein of CD44 and key factors of PI3K/Akt signaling pathways in brain functional areas of METH dependent miceWestern blotting analysis showed that the expressions of CD44,TLR4,p-PI3 K,PI3K,p-Akt and Akt in the hippocampus,prefrontal cortex and striatum of METH dependent group were significantly higher than the controls(P<0.05).IHC showed that the expression of CD44 protein in the brain functional areas was also significantly higher than the controls(P<0.05).3.The role of CD44 in METH dependence induced neuroinflammation via PI3K/Akt signaling pathway3.1The knockdown/overexpression of CD44 on METH dependence induced neuroinflammationThe mRNA of CD44,TLR4,PI3 K,and Akt in METH group treatment with the 1.0 m M METH were significantly higher than the controls(P<0.05).Similar results were obtained in the expression of protein by western blotting(P<0.05).After knockdown the CD44 expression by siRNA-CD44,the protein of TLR4,p-PI3 K,PI3K,and p-Akt in siRNA-CD44+METH group were significantly lower than the siRNA-NC+METH group(P<0.05).There was no statistic difference in Akt protein between the two groups(P=0.302).Compared with the siRNA-NC+METH group,the NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved caspase-1,cleaved IL-1β,AIM2,and ASC/TMS1),inflammatory markers(CD11b and NF-κB),apoptotic proteins(Caspase-9,cleaved caspase-3 and Bax/Bcl2)in the siRNA-CD44+METH group were significantly increased(P<0.05).There were no significantly differences in the expression of TNF-a,IL-6,IL-1β,and Caspase-3 proteins between the two groups(P>0.05).ELISA showed that the protein expression levels of TNF-a,IL-6 and IL-1β in the siRNA-CD44+METH group were significantly higher than the siRNA-NC+METH group(P<0.001).Similar results were observed in cell apoptosis by flow cytometry(P=0.001).The expression of CD44 protein in pc DNA 3.1(+)-CD44 group was significantly higher than the pc DNA 3.1(+)-NC group(P=0.014).The expression of TLR4,p-PI3 K,Akt and p-Akt protein in pc DNA3.1(+)-CD44+METH group were significantly higher than the pc DNA3.1(+)-NC+METH group(P<0.05).We failed to find any difference in PI3 K protein between the two groups(P=0.357).Compared with the pc DNA3.1(+)-NC+METH group,the expression of NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved caspase-1,IL-1β,cleaved IL-1β,and AIM2),inflammatory markers(CD11b,NF-κB,TNF-a,IL-6),apoptotic proteins(Caspase-3,cleaved caspase-3,Bax,Bax/Bcl2)in pc DNA 3.1(+)-CD44+METH group were significantly decreased(P<0.05).The ASC/TMS1 and Caspase-9 protein had no significantly difference between the two groups(P>0.05).Similar results were obtained for TNF-a,IL-6 and IL-1βprotein in the culture supernatant by ELISA and apoptosis by flow cytometry(P<0.01).3.2 Effect of Ly294002 on METH dependence induced neuroinflammation.The expression of p-PI3 K,PI3K,p-Akt,and Akt in the Ly294002+METH group were significantly lower than the METH group(P<0.01).The CD44,TLR4 and Akt protein were not significantly differece between the two groups(P>0.05).The above results indicated that CD44 may be an upstream protein of PI3K/Akt signaling pathway.The NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved)caspase-1,IL-1β,cleaved IL-1β,AIM2),inflammatory markers(CD11b,NF-κB,TNF-a,IL-6),apoptotic proteins(Caspase-9,cleaved caspase-3,and Bax/Bcl2)in Ly294002+METH group were significantly higher than the DMSO+METH group(P<0.05).The IL-6,TNF-α,and IL-1β protein in the cell culture supernatant of Ly294002+METH group were significantly higher than the DMSO+METH group by ELISA(P<0.05).Similar results were obtained for apoptosis by flow cytometry(P=0.007).Furthermore,when the BV2 cells were pretreated with both siRNA-CD44 and Ly294002,the IL-6,TNF-α and IL-1β protein levels in the cell culture supernatant of siRNA-CD44+Ly294002+METH group were significantly higher than the either siRNA-CD44+METH group or Ly294002+METH group(P<0.001).The NLRP3 inflammasome effector protein(NLRP3,Caspase-1,cleaved caspase-1,IL-1β,cleaved IL-1β,AIM2),inflammatory markers(CD11b,NF-κB,TNF-a,IL-6),apoptosis proteins(Caspase-9,caspase-3,cleaved caspase-3,and Bax/Bcl2)expression in the siRNA-CD44+METH group were significantly higher than the siRNA-CD44+METH group and Ly294002+METH group(P<0.05).Flow cytometry also showed that the apoptosis rate in siRNA-CD44+Ly294002+METH group was significantly higher than that of siRNA-CD44+METH group and Ly294002+METH group(P<0.01).Conclusion:1.Bioinformatics analysis showed that CD44,Syk,Fos,Dusp1,Klf4,Hspa1 b,Cdkn1a,Egr1/2,Gata4,Brd4,Gnaq,Crem,and Creb1 were the hubgenes of methamphetamine dependence.The PI3K/Akt signaling pathway was the key pathway of METH dependence.2.CD44 and key factors of PI3K/Akt signaling pathway and their phosphorylated protein increased in the brain functional areas of METH dependent mice and BV2 cell model.The CD44 may regulate the pathophysiological process of METH dependence inducde neuroinflammation via PI3K/Akt signaling pathway.更多还原