基于网络药理学和分子对接法研究六堡茶干预COVID-19的活性成分
Potential active compounds of Liupao tea for prevention and treatment of COVID-19 based on network pharmacology and molecular docking
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摘要: 采用网络药理学和分子对接法探究六堡茶干预COVID-19的物质基础和作用机制。通过文献与中药系统药理数据库和分析平台(TCMSP)等数据库,检索得到六堡茶的化学成分并对其进行筛选得到六堡茶活性成分及对应靶标,并将对应靶标导入Uniprot数据库中进行基因名的校正,运用Cytoscape 3.7.1软件构建“六堡茶-成分-靶标”网络。通过GeneCards数据库对COVID-19疾病靶点进行预测。将六堡茶与疾病的靶标进行映射,采用STRING数据库分析靶标蛋白的互作关系,从中筛选出核心靶标并运用DAVID等数据库对核心靶标进行GO(gene ontology)富集分析和KEGG (kyoto encyclopedia of genes and genomes)通路富集分析。最后将筛选出来的活性成分与受体蛋白SARS-CoV-2 3CL水解酶(Mpro)进行分子对接。筛选得到6种六堡茶活性成分,分别是(+)-儿茶素、(-)-表没食子儿茶素没食子酸酯(EGCG)、α-菠甾醇、(-)-儿茶素没食子酸酯、角鲨烯和氯化天竺葵素,对应作用靶标156个。其中与COVID-19的共同靶标112个,核心靶标38个。GO富集分析 (P < 0.01)主要涉及脂多糖、细胞对缺氧的反应等。KEGG通路富集分析得到六堡茶干预COVID-19相关的HIF-1、IL-17、T细胞受体等信号通路。分子对接结果显示六堡茶中的6种主要化学成分可分别与受体蛋白Mpro结合较好,且结合能与现有报道推荐的临床使用化学药相近。六堡茶中的核心化学成分(+)-儿茶素、(-)-表没食子儿茶素没食子酸酯、α-菠甾醇、(-)-儿茶素没食子酸酯、角鲨烯、氯化天竺葵素与Mpro结合并通过MAPK1、TNF等炎症和免疫相关的靶标介导HIF-1、IL-17、T细胞受体等信号通路发挥干预COVID-19的功效。实验进一步对结合能较低的EGCG进行了Mpro的活力测定,发现IC50为3.4 μmol/L,证实EGCG对Mpro有一定的抑制效果。
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关键词:
- 六堡茶 /
- COVID-19 /
- 网络药理学 /
- 分子对接 /
- SARS-CoV-2 3CL水解酶
Abstract: To investigate the material basis and mechanism of Liupao tea on preventing COVID-19 by network pharmacology and molecular docking.The active ingredients and targets of Liupao tea were searched through the literature and the TCMSP databases and the network between the two was built by Cytoscape 3.7.1.Then using GenCards platform to predict the disease targets,mapping the common targets between Liupao tea and disease.The common targets were imported into the STRING database for exploring the protein-protein interaction.Core targets were enriched by gene ontology (GO) enrichment analysis and KEGG (kyoto encyclopedia of genes and genomes) pathway enrichment analysis using DAVID database etc..Finally,the screened active components were docked with the receptor protein SARS-CoV-2 3CL hydrolase (Mpro).Six active ingredients of Liupao tea were screened,such as (-)-epigallocatechin gallate (EGCG),(+)-catechin,(-)-catechin gallate,α-spinasterol,pelargonidin chloride and squalene,and 156 targets were identified.Among them,there were 112 common targets and 38 core targets with COVID-19.GO enrichment analysis (P<0.01) involved lipopolysaccharide,cell response to hypoxia,etc..And the KEGG pathway enrichment analysis (P<0.01)was conducted to obtain the HIF-1,IL-17,T cell receptor and other signaling pathways associated with COVID-19.The results of molecular docking showed that the active ingredients of Liupao tea were well bound to the receptor protein Mpro.The active ingredients of Liupao tea may control HIF-1,IL-17,T cell receptors signaling pathways by binding Mpro hydrolase and acting on inflammation and immune related targets such as MAPK1,TNF to prevent COVID-19.The EGCG of Mpro activity was determined ,and the IC50 was 3.4 μmol/L,which confirmed that EGCG was a certain inhibition effect on Mpro.-
Keywords:
- Liupao tea /
- COVID-19 /
- network pharmacology /
- molecular docking /
- SARS-CoV-2 3CL hydrolase
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中枢神经系统由神经元和神经胶质细胞两大类组成,神经胶质细胞主要包括星形胶质细胞、小胶质细胞、少突胶质细胞等。小胶质细胞占神经胶质细胞总数的10%~15%,是存在于中枢神经系统内的单核巨噬细胞[1]。正常生理状态下小胶质细胞呈现分枝状并发挥免疫监视作用以维持中枢神经系统的稳态;病理状态下,小胶质细胞能够被β-淀粉样蛋白(amyloid β, Aβ)、α-突触核蛋白(α-synuclein, α-Syn)等错误折叠的蛋白质、细胞碎片以及细菌细胞壁脂多糖(lipopolysaccharide, LPS)等异常激活[2−3]。受损或异常激活的小胶质细胞引发中枢神经系统内的系列炎症反应即神经炎症,已有研究证明神经炎症始终伴随在阿尔茨海默病(Alzheimer’s disease,AD)、帕金森病(Parkinson’s disease,PD)等神经退行性疾病发生发展的整个过程中,并造成神经元丢失和Aβ病理及微管相关蛋白Tau病理加重[4−5]。
激活的小胶质细胞会产生不同的表型,在瞬息万变的脑环境中,精确区分并调节小胶质细胞的表型对治疗AD至关重要[6]。神经炎症初期,小胶质细胞由静息状态激活至M1表型,此时的小胶质细胞分枝缩短变粗,胞体增大并释放大量肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素6(interleukin 6, IL-6)和白细胞介素1β(IL-1β)等促炎细胞因子,这些毒性物质可以消灭入侵的病原体[7]。然而,随着时间推移急性炎症转化为慢性炎症,小胶质细胞的慢性活化以及过度积累的毒性促炎细胞因子会导致神经元损伤[8]。小胶质细胞还可以通过替代激活途径被激活至M2表型,此时的小胶质细胞分泌白细胞介素10(IL-10)、精氨酸酶1(arginase 1, ARG1)等抗炎细胞因子,起到神经保护和组织修复的作用[9],提示逆转小胶质细胞过度激活、调节小胶质细胞极化表型对治疗胶质增多症及AD有着重要作用。
IL-27是IL-12家族中的一种异二聚体细胞因子,由称为EB病毒诱导蛋白3(Epstein-Barr virus-induced protein 3,EBI3)和称为p28的p35相关蛋白组成[10]。IL-27与由gp130和白细胞介素-27受体α(IL-27Rα)组成的异二聚体受体复合物结合,IL-27Rα主要存在于巨噬细胞、小胶质细胞、B细胞等炎症细胞表面[11]。IL-27的功能最初被认为参与Th1细胞的早期发育[12],最近的研究表明IL-27在小鼠缺血性脑卒中模型、小鼠脑出血模型中增加神经元存活率,降低了神经功能缺损和脑部病理[11,13],而其对小胶质细胞作用还有待探究。本研究旨在观察IL-27对LPS或Aβ1-42诱导小胶质细胞过度激活的影响。
1. 材 料
1.1 试 剂
DMEM高糖培养基(美国Gibco公司);胎牛血清(以色列Biological Industries公司);LPS(美国Sigma公司);IL-27(美国MCE公司);Aβ1-42(上海吉尔生化有限公司);BCA蛋白浓度检测试剂盒、RIPA裂解液、SDS-PAGE蛋白上样缓冲液(5×,上海碧云天生物技术有限公司);一步法凝胶制备试剂盒、ECL发光液(杭州福德生物科技有限公司);蛋白Marker(美国Thermo公司);PVDF膜、蛋白酶抑制剂、磷酸酶抑制剂(美国Millipore公司);总RNA提取试剂盒EasyPure RNA Kit(北京全式金生物技术有限公司);HIScriptⅢ RT SuperMix for qPCR(+gDNA wiper)逆转录试剂盒、Taq Pro Universal SYBR qPCR Master Mix RT-qPCR试剂盒(南京诺唯赞生物技术股份有限公司);TNF-α、IL-6、IL-1β ELISA检测试剂盒、TNF-α抗体、IL-1β抗体、β-actin抗体(爱博泰克生物公司);Iba1抗体、NF-кB抗体、p- NFкB抗体、IкBα抗体、p-кBα抗体、山羊抗兔IgG、山羊抗鼠IgG(美国Cell Signaling Technology公司);其余试剂均为国产市售分析纯。
1.2 仪 器
全波长酶标仪、PCR仪、RT-qPCR仪、Nanodrop微量分光光度计(美国Thermo公司);电泳仪、转膜仪(美国Bio-Rad公司);化学发光成像系统(中国Tanon公司);氮吹仪(上海安谱实验科技股份有限公司)。
1.3 细 胞
小鼠小胶质细胞BV-2购自上海iCELL公司。
1.4 动 物
SPF级C57BL/6小鼠,5~6周龄,雄性,购自江苏集萃药康生物科技股份有限公司,实验动物使用许可证号:SCXK(苏)2023-0009。小鼠饲养于独立通风笼盒,实验室温度24~26 ℃,相对湿度60%~80%,保持12 h昼夜交替,给予标准饲料及饮用水。所有动物实验均符合动物伦理委员会标准。
2. 方 法
2.1 Aβ1-42制备
准确称量Aβ1-42 2.0 mg并加入六氟异丙醇500 mL,涡旋后超声10 min使其充分溶解,放置于4 ℃过夜。次日用氮吹仪进行氮吹以充分去除管内六氟异丙醇,−80 ℃放置2 h后放入冻干机冻干。将冻干的Aβ1-42复溶于DMSO中,进行涡旋并超声10 min使其充分溶解,加入已过滤的PBS稀释后置4 ℃冰箱寡聚24 h,BCA法测定蛋白浓度。
2.2 BV-2细胞培养
2.2.1 细胞复苏
将实验室保藏的BV-2细胞从液氮罐中取出后放入37~42 ℃温水中迅速摇动使其快速融化,室温
1000 r/min离心5 min后用含有10%胎牛血清的DMEM高糖培养基重悬并接种至T25细胞瓶中,置含5% CO2的细胞培养箱中37 ℃培养。2.2.2 细胞传代
细胞汇合度约80%时即可传代。弃去细胞瓶中培养基,用PBS润洗细胞后加入含有10%胎牛血清的DMEM高糖培养基3 mL,用移液枪轻柔地将细胞从壁上吹下,按照1∶3的比例传代至T25细胞瓶中,置于含5% CO2的细胞培养箱中37 ℃培养。
2.2.3 细胞冻存
细胞汇合度约80%时即可冻存。按“2.2.2”项下方法获得细胞悬液后,室温
1000 r/min离心5 min,弃去上清液,向离心管中加入细胞冻存液后轻轻吹打混匀,按每管1.5 mL加入冻存管中后封口膜封口,在管上标记细胞名称、代数、日期等信息后于−80 ℃或液氮中保存。2.3 BV-2细胞铺板及分组
将BV-2细胞按每孔5×105个的密度接种于六孔板中,并设置分组如下:空白对照组、LPS(100 ng/mL)组、LPS(100 ng/mL)+IL-27(5 ng/mL)组,造模及药物干预12 h;空白对照组、Aβ1-42(5 μmol/L)组、Aβ1-42(5 μmol/L)+IL-27(5 ng/mL)组,造模及药物干预24 h。
2.4 动物分组、造模及给药
小鼠适应性饲养1周后随机分为空白对照组、模型组(LPS组)、给药组(LPS +IL-27组),每组各12只。IL-27组侧脑室注射IL-27(10 ng/μL,每侧2 μL),术前12h禁食不禁水,麻醉小鼠后将其固定于脑立体定位注射仪上,剔除颅顶毛发后用碘伏消毒,剪开头皮暴露出前囟,在前囟后侧0.5 mm,矢状缝两侧1.0 mm处进行钻孔,微量进样器垂直进入侧脑室后缓慢注射IL-27,缝合伤口;空白对照组和LPS组注射等体积生理盐水,其余操作同给药组。
模型组和给药组于术后第1天开始腹腔注射LPS(1 mg/kg),共4 d。空白对照组按照相同方式注射等体积生理盐水。
2.5 Western blot检测
2.5.1 IL-27对LPS或Aβ1-42诱导BV-2细胞内TNF-α、IL-1β、NF-κB、p-NF-κB、IκBα和p-IκBα表达量的影响
细胞铺板、分组同“2.3”项。造模及IL-27干预后,提取细胞总蛋白。弃去六孔板内的培养基并用预冷的PBS润洗细胞3次,每孔内加入含有磷酸酶抑制剂和蛋白酶抑制剂的RIPA裂解液100 μL,用刮刀将细胞刮下后转移至提前预冷的1.5 mL EP管中并放置于冰上裂解30 min。将细胞裂解液于4 ℃,
12000 r/min离心20 min后小心吸取上清液并通过BCA法测定蛋白浓度,加入1/4体积的5×上样缓冲液后金属浴95 ℃煮沸10 min。配制SDS-PAGE胶并以80 V恒压完成电泳,以300 mA恒流湿转法将条带转移至PVDF膜上,取出PVDF膜用5% BSA封闭液封闭2 h,使用新配制的TBST洗膜5次,孵育TNF-α、IL-1β、NF-κB、p-NF-κB、IκBα、p-IκBα一抗,于4 ℃过夜。次日洗膜后加入与一抗对应的山羊抗兔二抗或山羊抗鼠二抗孵育2 h,洗膜5次,滴加ECL发光液进行成像。2.5.2 IL-27对LPS诱导小鼠脑内TNF-α、IL-1β表达量的影响
动物分组、给药同 “2.4”项。造模及给药完成后处死小鼠,迅速取小鼠全脑后置于4 mL EP管内,每管加入含有磷酸酶抑制剂和蛋白酶抑制剂的RIPA裂解液2 mL,使用手术剪将脑组织剪碎后用组织匀浆机充分研磨脑组织,于冰上涡旋裂解,提取并测定组织总蛋白。制样和电泳等操作同“2.5.1”项。
2.6 RT-qPCR检测
2.6.1 M1/M2型小胶质细胞表面标志物mRNA表达
细胞铺板、分组同“2.3”项。造模及IL-27干预后,按照总提取试剂盒说明书提取细胞内总RNA,于微量分光光度计上测定RNA浓度后,按照HIScriptⅢ RT SuperMix for qPCR(+gDNA wiper)试剂盒进行逆转录获得cDNA。按照Taq Pro Universal SYBR qPCR Master Mix试剂盒进行RT-qPCR。首先配制2×Taq Pro Universal SYBR qPCR Master Mix和引物的混合反应体系,混合均匀后在96孔半裙边板中加入混合体系8 μL/孔,再加入模板cDNA (每孔2 μL)。反应程序如下:预变性95 ℃ 30 s;模板变性95 ℃ 10 s,退火60 ℃ 30 s,循环40次;绘制溶解曲线95 ℃ 15 s,60 ℃ 60 s,95 ℃ 15 s。使用2−ΔΔCt法计算目的基因相对表达量。引物序列参见表1。
Table 1. Primer sequences for RT- PCRBiological indicator Forward primer (5'→3') Reverse primer (5'→3') Tnf TTGGTGGTTTGTGAGTGTGAG GACGTGGAACTGGCAGAAGAG Il6 TTGGTCCTTAGCCACTCCTT TAGTCCTCCTACCCCAATT Il1b ATCTTTTGGGGTCCGTCAACT GCAACTGTTCCTGAACTCAACT Nos2 GTGGACGGGTCGATGTCAC GTTCTCAGCCCAACAATACAAA Nlrp3 ACAAGCCTTTGCTCCAGACCCTAT TGCTCTTCACTGCTATCAAGCCCT Fcgr2b GGGAACCAATCTCGTAGTGTCTGT CCAGAAAGGCCAGGATCTAGTG Cd86 GAGCGGGATAGTAACGCTGA GGCTCTCACTGCCTTCACTC Fcgr3 GTCCAGTTTCACCACAGCCTTC GCCAATGGCTACTTCCACCAC Chil3 GGGCATACCTTTATCCTGAG CCACTGAAGTCATCCATGTC Il10 GGTTGCCAAGCCTTATCGGA ACCTGCTCCACTGCCTTGCT Arg1 GTGAAGAACCCACGGTCTGT CTGGTTGTCAGGGGAGTGTT Actb AGCCATGTACCTAGCCATCC TTTGATGTCACGCACGATTT 2.6.2 小鼠脑内炎症因子mRNA表达
动物分组、给药同“2.4”项。造模及给药完成后处死小鼠,迅速取小鼠全脑后置于研钵中,加入液氮后充分研磨使其成为匀浆。提取RNA、逆转录及RT-qPCR同“2.6.1”项。
2.7 ELISA检测小鼠脑内炎症因子表达
动物分组、给药同“2.4”项。造模及给药完成后处死小鼠,迅速取小鼠全脑后置于4 mL EP管内,每管加入PBS2 mL,使用手术剪将脑组织剪碎后用组织匀浆机充分研磨脑组织,4 ℃,
12000 r/min离心20 min后小心吸取上清液,按照ELISA试剂盒说明书测定小鼠脑内TNF-α、IL-1β和IL-6的表达水平。2.8 免疫组化检测小鼠海马中Iba1的表达
动物分组、给药同“2.4”项。造模及给药完成后处死小鼠,迅速取小鼠全脑并放置于4%多聚甲醛中室温固定24 h。用石蜡将组织包埋,并进行切片、封闭、孵育抗体、DAB显色、复染细胞核和封片等操作,使用扫描仪扫描切片。
2.9 IL-27在正常人和AD患者脑内表达量的分析
通过GEO数据库中的GSE48350数据集分析IL-27在正常人和AD患者脑内的差异表达情况。在海马区,正常组AD组各19例;在内嗅皮层区,正常组19例,AD组15例;在额叶皮层区,正常组27例,AD组21例。
2.10 统计学分析
实验数据使用GraphPad Prism 9.0进行统计分析和作图,每组数据(至少3次独立实验)以$\bar{x} $ ± s表示。多组样本间采用单因素方差分析,以P < 0.05认为有统计学意义。
3. 结 果
3.1 IL-27在正常人和AD患者脑内表达量的分析
使用GEO数据库GSE48350数据集分析IL-27在正常人和AD患者脑内的差异表达情况,结果如图1所示,相较于正常人,AD患者海马体、内嗅皮层以及额叶皮层中的IL-27含量降低[14]。
Figure 1. Analysis of IL-27 expression in the brains of normal and Alzheimer’s disease(AD) patients. In Hippocampus region, control group n=19, AD group n=19. In the entorhinal cortex region, control group n=19, AD group n=15. In the frontal cortex region, control group n=27, AD group n=21. (ns: no significance vs control group)3.2 IL-27对LPS诱导的小鼠脑内小胶质细胞激活的影响
为检测IL-27对小胶质细胞异常激活及神经炎症的影响,使用LPS诱导小鼠并给予IL-27。收集小鼠脑组织进行免疫组化,检测小鼠脑内Iba1表达量,结果如图2所示。与空白对照组相比,LPS组小鼠海马中Iba1+小胶质细胞数量增加;与LPS组相比,IL-27组小鼠海马中Iba1+小胶质细胞数量下降。证明IL-27能缓解小鼠脑内由LPS诱导的小胶质细胞异常激活。
Figure 2. Effect of IL-27 on activation of microglia in lipopolysaccharide(LPS)-induced mice brainA: Expression of Iba1 in the brain of normal mice, LPS-induced inflammation mice, and IL-27 treated mice after LPS-induced inflammation was detected by immunohistochemistry (Scale bar=100 μm); B: Quantitative analysis of Iba1+ levels in A ($\bar{x} $ ± s, n=3, ###P < 0.001 vs control group; **P < 0.01 vs LPS group)3.3 IL-27对LPS诱导的小鼠脑内炎症因子表达量的影响
相较于空白对照组,LPS组小鼠脑内促炎因子IL-1β、IL-6、TNF-α表达量显著增多;相较于LPS组,IL-27组小鼠脑内促炎因子IL-1β、IL-6、TNF-α表达量显著减少,结果如图3所示。说明IL-27能逆转LPS诱导的神经炎症。
Figure 3. Effect of IL-27 on the expression of inflammatory factors in the brain of mice induced by LPS ($\bar{x} $ ± s)A: mRNA levels of Il1b, Il6 and Tnf were detected by RT-qPCR; B: Levels of IL-1β, IL-6, TNF-α were detected by ELISA; C: Representative images of the levels of IL-1β, TNF-α were detected by Western blot; D-E: Quantitative analysis of protein levels of TNF-α/actin and IL-1β/actin(n=5 in ELISA, the other n=3) ###P < 0.001, ##P < 0.01 vs control group; ***P < 0.001, **P < 0.01 vs LPS group3.4 IL-27对LPS诱导的小胶质细胞M1/M2型细胞标志物的影响
为检测IL-27对LPS诱导的小胶质细胞表型的影响,实验分为空白对照组、LPS(100 ng/mL)组和LPS(100 ng/mL)+ IL-27(5 ng/mL)组,造模及药物干预12 h。相较于空白对照组,LPS组M1型小胶质细胞标志物Il1b、Tnf、Nos2、Nlrp3、Fcgr2b、Cd86、Fcgr3及M2型小胶质细胞标志物Il10、Chil3、Arg1的表达量显著增加,说明小胶质细胞由未激活的静息态被LPS激活;给予IL-27后,与LPS组相比,IL-27组M1型小胶质细胞标志物Il1b、Tnf、Nos2、Nlrp3、Fcgr2b的表达量显著减少,而M2型小胶质细胞标志物Il10、Chil3、Arg1的表达量进一步显著增加,结果如图4所示。说明IL-27能够将LPS激活的M1型小胶质细胞转变为M2型,逆转LPS诱导的小胶质细胞过度激活。
Figure 4. Effect of IL-27 on mRNA expression of M1/M2 cell markers in LPS-induced microglia ($\bar{x} $ ± s, n=3)A: mRNA levels of M1-specific makers Il1b, Tnf, Nos2, Nlrp3, Fcgr2b, Cd86 and Fcgr3 were detected by RT-qPCR; B: mRNA levels of M2-specific makers Il10, Chil3 and Arg1 were detected by RT-qPCR ###P < 0.001, ##P < 0.01, #P < 0.05 vs control group; ***P < 0.001, **P < 0.01, *P < 0.05 vs LPS group3.5 IL-27对Aβ1-42诱导的小胶质细胞M1/M2型细胞标志物的影响
为检测IL-27对Aβ1-42诱导的小胶质细胞表型的影响,实验分为空白对照组、Aβ1-42(5 μmol/L)组和Aβ1-42(5 μmol/L)+IL-27(5 ng/mL)组,造模及药物干预24 h。相较于空白对照组,Aβ1-42组M1型小胶质细胞标志物Il1b、Tnf、Nlrp3、Nos2及M2型小胶质细胞标志物Il10、Chil3、Arg1的表达量显著增加,说明小胶质细胞由未激活的静息态被Aβ1-42激活;给予IL-27后,与Aβ1-42组相比,IL-27组M1型小胶质细胞标志物Il1b、Tnf、Nlrp3的表达量显著减少,而M2型小胶质细胞标志物Il10、Chil3、Arg1的表达量显著增加,结果如图5所示。说明IL-27能够将Aβ1-42激活的M1型小胶质细胞转变为M2型,逆转Aβ1-42诱导的小胶质细胞过度激活。
Figure 5. Effect of IL-27 on mRNA expression of M1/M2 cell markers in Aβ1-42-induced microglia ($\bar{x} $ ± s, n=3)A: mRNA levels of M1-specific makers Il1b, Tnf, Nlrp3 and Nos2 were detected by RT-qPCR; B: mRNA levels M2-specific makers of Il10, Chil3 and Arg1 were detected by RT-qPCR ###P < 0.001, ##P < 0.01, #P < 0.05 vs control group; ***P < 0.001, **P < 0.01, *P < 0.05 vs Aβ1-42 group3.6 IL-27对Aβ1-42诱导的小胶质细胞内NF-κB、p-NF-κB、IκBα、p-IκBα激活的影响
为探究IL-27改善小胶质细胞过度激活的途径,用Aβ1-42和IL-27孵育小胶质细胞24h。相较于空白对照组,Aβ1-42组小胶质细胞内NF-κB磷酸化、IκBα磷酸化水平显著增加;相较于Aβ1-42组,IL-27组小胶质细胞内NF-κB磷酸化、IκBα磷酸化水平显著下降;证明IL-27能够改善Aβ1-42诱导的NF-κB、IκBα异常激活。
Figure 6. Effect of IL-27 on activation of NF-κB, p-NF-κB, IκBα, and p-IκBα in microglia induced by Aβ1-42A: Levels of NF-κB, p-NF-κB, IκBα and p-IκBα were detected by Western blot; B-C: Quantitative analysis of protein levels of p-NF-κB/NF-κB and p-IκBα/ IκBα in A (`x ± s, n=3, ###P < 0.001, ##P < 0.01, #P < 0.05 vs control group; ***P < 0.001, **P < 0.01, *P < 0.05 vs Aβ1-42 group)4. 讨 论
LPS也称为内毒素,是革兰氏阴性菌外膜的主要成分,目前被认为是最有效的促炎刺激之一[15]。LPS在神经退行性疾病领域研究十分广泛,其主要通过由Toll样受体4(Toll-like receptor 4,TLR4)信号通路介导的小胶质细胞过度激活进而造成神经元死亡[16],同时还会造成海马和皮质内Aβ的积累[17]。而在AD患者脑内,小胶质细胞主要通过Toll样受体2(Toll-like receptor 2,TLR2)和TLR4识别Aβ[18],进而分泌大量神经毒性的促炎因子造成神经元损伤。因此,本研究选取LPS或Aβ1-42诱导的细胞以及LPS诱导的小鼠模型探究IL-27的抗炎作用。
近年来许多科学家致力于IL-27在中枢神经系统中作用的研究,尽管其发挥作用的途径主要与调节T细胞的反应有关,但有研究指出IL-27还有可能作用于神经系统中的其他细胞[19]。同时,IL-27作为一种多效细胞因子,通过氧化应激、细胞凋亡、自噬、表观遗传修饰、调节细胞因子分泌与表达等多种途径影响神经元存活,且根据作用细胞不同,IL-27可能会产生相反的效果[20]。本研究发现IL-27能够促进LPS或Aβ1-42诱导的小胶质细胞从具有神经毒性M1表型转化为神经保护的M2表型,并抑制LPS诱导的小鼠脑内小胶质细胞过度激活及慢性神经炎症。
核因子-κB(NF-κB)参与广泛的炎症反应,在AD中被认为是神经炎症恶性循环的核心[21]。在非激活状态下,NF-κB与其抑制因子IκB结合形成p65-p50-IκB三聚体,定位于胞质中且无法发挥作用;当受到细胞碎片、病原体、毒性细胞因子等的刺激后,由于磷酸化级联反应的作用,IκBα被蛋白酶体降解,使得p65-p50二聚体得以进入细胞核并且促进下游靶基因的转录[22]。大部分NF-kB的靶基因均为促炎细胞因子,由此引发慢性炎症进而导致神经元死亡[23]。本研究通过Western blot检测Aβ1-42诱导后IL-27对小胶质细胞内NF-κB、p-NF-κB、IκBα、p-IκBα的表达,发现IL-27能逆转Aβ1-42诱导的小胶质细胞内NF-κB、IκBα磷酸化水平。上述结果提示,IL-27可能通过IκB/NF- κB途径改善小胶质细胞过度激活。
综上所述,本研究结果提示,IL-27改善LPS的诱导小鼠脑内小胶质细胞过度激活及M1型小胶质细胞介导的神经炎症,同时IL-27通过IκB/NF- κB途径逆转Aβ1-42诱导的小胶质细胞表型变化。
《中国药科大学学报》入选2024年度“中国高校科技期刊
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