摘要
目的观察重组人干扰素(rh IFN)α-2b对手足口病大鼠的治疗作用及其对Toll样受体(TLR)/髓样分化因子(MyD88)相关信号分子的影响。方法取郑州儿童医院感染性疾病科分离的肠道病毒71-BJ(EV71-BJ),测定EV71-BJ毒株对7日龄无特定病原级Sprauge Dawley(SD)大鼠的半数致死量(LD50)。另取7日龄无特定病原级SD大鼠60只,随机分为对照组、模型组及rh IFNα-2b低、中、高剂量组,每组12只。模型组及rh IFNα-2b低、中、高剂量组大鼠一次性腹腔注射15倍稀释的LD50EV71-BJ毒株100μL,对照组一次性腹腔注射生理盐水100μL,根据各组大鼠的表现进行临床评分,染毒第4天临床评分3~4分者即为建模成功。取rh IFNα-2b低、中、高剂量组建模成功的大鼠,分别给予2、4、8万单位rh IFNα-2b溶入0.2 mL生理盐水中肌肉注射,对照组、模型组大鼠肌肉注射0.2 mL生理盐水,每日1次,连续给药3 d。末次干预后24 h脱颈处死各组大鼠,取腹主动脉血,检测各组大鼠全血T淋巴细胞亚群CD4^+、CD8^+、CD4^+/CD8^+及血清γ干扰素(IFN-γ)、白细胞介素(IL)-4、IL-10水平;实时荧光定量聚合酶链反应检测各组大鼠脑组织中TLR2、My D88、核因子-κB(NF-κB)、肿瘤坏死因子相关因子6(TRAF-6)mRNA相对表达量;Western blot法检测各组大鼠脑组织中TLR2、My D88、NF-κB、TRAF-6蛋白相对表达量。结果各组大鼠全血CD4^+、CD8^+、CD4^+/CD8^+、血清IFN-γ、IL-4、IL-10水平及脑组织中TLR2、My D88、NF-κB、TRAF-6 mRNA和蛋白相对表达量比较差异均有统计学意义(F=72.310、57.107、98.928,F=161.175、28.874、22.637,F=108.493、122.121、120.966、154.783,F=151.001、194.357、89.442、210.210,P<0.05)。与对照组比较,模型组及rh IFNα-2b各剂量组大鼠全血CD4^+、CD4^+/CD8^+及血清IFN-γ水平均显著降低(P<0.05),全血CD8^+、血清IL-4、IL-10水平及脑组织中TLR2、My D88、NF-κB、TRAF-6 mRNA和蛋白相对表达量均显著升高(P<0.05)。与模型组比较,rh IFNα-2b各剂量组大鼠全血CD4^+、CD4^+/CD8^+及血清IFN-γ水平均显著升高(P<0.05),全血CD8^+、血清IL-4、IL-10水平及脑组织中TLR2、MyD88、NF-κB、TRAF-6 mRNA和蛋白相对表达量均显著降低(P<0.05)。与rh IFNα-2b低剂量组比较,rh IFNα-2b中、高剂量组大鼠全血CD4^+、CD4^+/CD8^+及血清IFN-γ水平均显著升高(P<0.05),全血CD8^+、血清IL-4、IL-10水平及脑组织中TLR2、MyD88、NF-κB、TRAF-6 mRNA和蛋白相对表达量均显著降低(P<0.05)。与rh IFNα-2b中剂量组比较,rh IFNα-2b高剂量组大鼠全血CD4^+、CD4^+/CD8^+及血清IFN-γ水平均显著升高(P<0.05),全血CD8^+、血清IL-4、IL-10水平及脑组织中TLR2、MyD88、NF-κB、TRAF-6 mRNA和蛋白相对表达量均显著降低(P<0.05)。结论rh IFNα-2b可有效改善手足口病大鼠的症状和免疫状态,其中高剂量rh IFNα-2b效果最佳,其作用可能与抑制TLR/MyD88信号通路中相关基因和蛋白表达有关。
Objective To investigate the therapeutic effect of recombinant human interferonα-2 b(rh IFNα-2 b)on rats with hand-foot-mouth disease,and to explore its effect on Toll-like receptor(TLR)/myeloid differential protein-88(MyD88)related signaling molecules.Methods Enterovirus 71-BJ(EV71-BJ)isolated from the Department of Infectious Diseases of Children’s Hospital Affiliated to Zhengzhou University was used to determine the lethal dose(LD50)of EV71-BJ strain to7-day-old specified-pathogens free Sprauge Dawley rats.In addition,sixty 7-day-old specified-pathogens free Sprauge Dawley rats were randomly divided into control group,model group and low,medium and high dose of rh IFNα-2 b group,12 rats in each group.The rats in the model group and the low,medium and high dose of rh IFNα-2 b groups were injected with 15-fold dilution of LD50 EV71-BJ strain 100μL in one time intraperitoneally,while the rats in the control group were injected with normal saline 100μL in one time intraperitoneally.Clinical score was made according to the performance of rats in each group,and the model was successful when the clinical score was 3-4 on the fourth day of exposure.The rats in the low,middle and high dose of rh IFNα-2 b groups were given 2,40,80 thousand units of rh IFNα-2 b which were dissolved into 0.2 mL normal saline for intramuscular injection,and the rats in the control group and model group were given intramuscular injection of 0.2 mL normal saline,once a day for 3 days.Twenty-four hours after the last intervention,the rats in each group were sacrificed by neck dissection,and the blood of abdominal aorta was taken.The levels of T lymphocyte subsets CD4^+,CD8^+,CD4^+/CD8^+and serum interferon-γ(IFN-γ),interleukin(IL)-4 and IL-10 were measured.The relative expression of TLR2,MyD88,nuclear factorκB(NF-κB),tumor necrosis factor-related factor 6(TRAF-6)mRNA in brain tissue of rats in each group was detected by real-time fluorescence quantitative polymerase chain reaction.The relative expression of TLR2,MyD88,NF-κB,TRAF-6 protein in brain tissue of rats in each group was detected by Western blot.Results There were significant differences in the percentage of CD4^+,CD8^+,CD4^+/CD8^+in the whole blood,levels of serum IFN-γ,IL-4 and IL-10,and TLR2,MyD88,NF-κB,TRAF-6 mRNA and protein expressions in brain tissue among the groups(F=72.310,57.107,98.928;F=161.175,28.874,22.637;F=108.493,122.121,120.966,154.783;F=151.001,194.357,89.442,210.210;P<0.05).Compared with the control group,in the model group and the low,middle and high dose of rh IFNα-2 b groups,the percentage of CD4^+,CD4^+/CD8^+in whole blood,serum IFN-γlevel were lower(P<0.05),the percentage of CD8^+in whole blood,serum IL-4 and IL-10 levels,TLR2,MyD88,NF-κBp65,TRAF-6 mRNA and protein expressions in brain tissue were higher(P<0.05).Compared with the model group,the levels of CD4^+,CD4^+/CD8^+in whole blood and serum IFN-γlevel were higher in the low,middle and high dose of rh IFNα-2 b groups(P<0.05),the levels of CD8^+in whole blood,levels of serum IL-4 and IL-10,and TLR2,MyD88,NF-κB,TRAF-6 mRNA and protein expressions in brain tissue were lower in the low,middle and high dose of rh IFNα-2 b groups(P<0.05).Compared with the low dose of rh IFNα-2 b group,the levels of CD4^+,CD4^+/CD8^+in the whole blood and serum level of IFN-γin the middle and high dose of rh IFNα-2 b groups were higher(P<0.05),while the levels of CD8^+in the whole blood and serum levels of serum IL-4 and IL-10,TLR2,MyD88,NF-κB,TRAF-6 mRNA and protein expressions in brain tissue in the middle and high dose of rh IFNα-2 b groups were lower(P<0.05).Compared with the middle dose of rh IFNα-2 b group,the levels of CD4^+,CD4^+/CD8^+in the whole blood,serum IFN-γlevel in the high dose of rh IFNα-2 b group were higher(P<0.05),the levels of CD8^+in whole blood and serum IL-4 and IL-10 levels,TLR2,My D88,NF-κB,TRAF-6 mRNA and protein expressions in brain tissue in the high dose of rh IFNα-2 b group were lower(P<0.05).Conclusion rh IFNα-2 b can effectively improve the symptoms and the immune status of rats with hand-foot-mouths.Among them,high dose of rh IFNα-2 b has the best effect,which may be related to the inhibition of related genes and protein expression in TLR/MyD88 signaling pathway.
作者
秦新华
王芳
赵保玲
QIN Xinhua;WANG Fang;ZHAO Baoling(Department of Infectious Diseases,Zhengzhou Children′s Hospital,Zhengzhou 450000,Henan Province,China)
出处
《新乡医学院学报》
CAS
2020年第1期15-20,共6页
Journal of Xinxiang Medical University
基金
河南省科技攻关项目(编号:182102311202)。
关键词
重组人干扰素
手足口病
TOLL样受体
髓样分化因子
recombinant human interferon
hand-foot-mouth disease
toll-like receptor
myeloid differential protein-88
