目的 分析下调Toll样受体4(TLR4)mRNA对手足口病(HFMD)模型乳鼠树突状细胞的影响及作用机制,为手足口病防治提供依据。方法 选取45只SPF级12日龄BALB/c乳鼠,分离并培养树突状细胞,设计合成TLR4 siRNA序列并做细胞感染。建立手足口病模型,分为模型组、下调组、上调组各15只,下调组乳鼠腹腔注射下调TLR4基因的树突状细胞,上调组腹腔注射上调TLR4基因的树突状细胞,模型组腹腔注射等量灭菌用水。检测三组乳鼠树突状细胞增殖、凋亡,血清白介素-6(IL-6)、白介素-12(IL-12)、肿瘤坏死因子-α(TNF-α)水平及丝裂原活化蛋白激酶(MAPK)通路蛋白表达量,并进行比较。结果 下调组不同时间点树突状细胞增殖率低于上调组(t=20.160、19.050、55.380,P＜0.001);下调组不同时间点树突状细胞凋亡率高于上调组(t=34.740、84.570、48.020,P＜0.001)。下调组血清IL-6、IL-12、TNF-α水平低于上调组(t=39.330、12.660、33.690,P＜0.001)。下调组树突状细胞p-p38MAPK、p-JNK、p-ERK1/2蛋白表达量低于上调组(t=33.690、17.470、27.800,P＜0.001)。结论 下调TLR4 mRNA经抑制MAPK通路活化,调控通路蛋白,下调IL-6、IL-12、TNF-α表达,促进手足口病乳鼠树突状细胞凋亡,可以为手足口病防治提供新思路。
Abstract
Objective To explore the effect and mechanism of down-regulation of Toll like receptor 4(TLR4) mRNA on dendritic cell in hand-foot-mouth disease (HFMD) mice model,so as to provide reference for the prevention and treatment of HFMD. Methods Forty-five 12-day-old BALB/c suckling mice of SPF grade were selected to isolate and culture the dendritic cells.TLR4 siRNA sequence was designed and synthesized for cell infection.The suckling mice model of HFMD was established and divided into model group,down-regulated group and up-regulated group,with 15 animals in each group.Mice in the down-regulated group were intraperitoneally injected with dendritic cells that down-regulated TLR4 gene,and those in the up-regulated group were intraperitoneally injected with dendritic cells that up-regulated TLR4 gene,and the mice in the model group were intraperitoneally injected with the same amount of sterilized water.The proliferation and apoptosis of dendritic cells,the levels of serum interleukin-6 (IL-6),interleukin-12 (IL-12),tumor necrosis factor-α (TNF-α) and the expression of mitogen activated protein kinase (MAPK) pathway protein were detected and were compared among three groups. Results The proliferation rates of dendritic cells in the down-regulated group were lower than those in the up-regulated group at different time points (t=20.160,19.050,55.380,P<0.001).The apoptosis rates of dendritic cells in the down-regulated group at different time points were higher than those in the up-regulated group (t=34.740,84.570,48.020,P<0.001).The serum levels of IL-6,IL-12 and TNF-α in the down-regulated group were lower than those in the up-regulated group(t=39.330,12.660,33.690,P＜0.001).The expression levels of P-P38MAPK,p-JNK and p-ERK1/2 protein in the down-regulated group were lower than those in the up-regulated group (t=33.690,17.470,27.800,P＜0.001). Conclusion Down regulation of TLR4 mRNA can down regulating the expression of IL-6,IL-12 and TNF-α through inhibiting MAPK pathway activation and regulating pathway protein,thereby promoting the apoptosis of dendritic cells in HFMD mice.
关键词
Toll样受体 /
手足口病 /
树突状细胞
Key words
Toll like receptor /
hand foot mouth disease /
dendritic cell
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参考文献
[1] Zhao TS,Du J,Sun DP,et al.A review and meta-analysis of the epidemiology and clinical presentation of coxsackievirus A6 causing hand-foot-mouth disease in China and global implications[J].Rev Med Virol,2019,6(1):e2087.
[2] Wang QW,Su Y.Sheng JT.et a1.Anti-lnfluenza A virus actjvity of rhein through regulating oxjdative stress,TLR4,Akt,MAPK,and NF-KB signaI pathways[J].PLoS One,2018,13(1):e0191793.
[3] 张中馥,常海霞,李清华,等.手足口病患儿外周血中TLR4、NF-κB表达变化及意义[J].河北医科大学学报,2019,40(8):911-914.
[4] 王辉强,蒋建东,李玉环.应用2周龄BALB/c小鼠建立EV71动物模型[J].药学学报,2013,48(3):343-346.
[5] 苏强,程凯,高艳萍,等.肠道病毒71型在树突状细胞上的主要受体[J].中华实验和临床病毒学杂志,2016,30(1):100-103.
[6] Fan S,Liao Y,Lian Y,et al.Role of innate lymphoid cells and dendritic cells in intradermal immunization of the enterovirus antigen[J].NPJ Vaccines,2019,4(1):14.
[7] 许红梅,潘招军.肠道病毒71型感染所致儿童手足口病与天然免疫细胞的研究进展[J].中华实用儿科临床杂志,2018,33(22):1681-1686
[8] Zhang Y,Suo X,Zhang Y.Association of IL-13,S100B,and TLR-7 gene polymorphismswith enterovirus 71 infection in hand,foot,and mouth disease in China[J].Genet Test Mol Biomarkers,2019,23(3):188-196.
[9] Jin Y,Zhang R,Wu W,et al.Innate immunity evasion by enteroviruses linked to epidemic hand-foot-mouth disease[J].Front Microbiol,2018,9(1):2422.
[10] 朱磊,齐共健,席晓莹,等.Toll样受体4和核因子-κB 在危重型手足口病中的表达及艾司洛尔的干预作用[J].中华微生物学和免疫学杂志,2015,35(11):827-831.
[11] 赵楚生,吴令杰,林泽鸿,等.轻重症手足口病外周血单个核细胞TLR4、NF-κB表达联合血清干扰素在病情和预后判断中的价值[J].广东医学,2018,39(3):394-397.
[12] 邵海岩,秦思文,杨雪葳,等.手足口病患儿外周血单个核细胞中TLR4通路检测与病情、炎症反应、免疫功能的关系[J].临床和实验医学杂志,2020,19(3):286-289.
[13] Lin YL,Hu YC,Liang CC,et al.Enterovirus-71 virus-like particles induce the activation and maturation of human monocyte-derived dendritic cells through TLR4 signaling[J].PLoS One,2014,9(10):e111496.
[14] Li Y,Wang Y,Wu Y,et al.Echinacea pupurea extracts promote murine dendritic cell maturation by activation of JNK,p38 MAPK and NF-κB pathways[J].Dev Comp Immunol,2017,73(1):21-26.
[15] Li Y,Li Y,Yang T,et al.Dioscin attenuates oxLDL uptake and the inflammatory reaction of dendritic cells under high glucose conditions by blocking p38 MAPK[J].Mol Med Rep,2020,21(1):304-310.
[16] Guindi C,Cloutier A,Gaudreau S,et al.Role of the p38 MAPK/C/EBPβ pathway in the regulation of phenotype and IL-10 and IL-12 production by tolerogenic bone marrow-derived dendritic cells[J].Cells,2018,7(12),256.
[17] 李冬.EV71感染对手足口病患儿DCs成熟、凋亡及MAPK信号通路和炎性细胞因子表达的影响[J].山东医药,2017,57(9):82-84.
[18] 高勇,徐元宏,陈晓枫,等.EV71型手足口病患儿外周血树突状细胞及细胞因子产生的调节机制研究[J].安徽医科大学学报,2017,52(1):78-82.
[19] Zhu L,Li W,Qi G,et al.The immune mechanism of intestinal tract Toll-like receptor in mediating EV71 virus type severe hand-foot-and-mouth disease and the MAPK pathway[J].Exp Ther Med,2017,13(5):2263-2266.
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