丁苯酞联合TLR4抗体鼓室给药小鼠梅尼埃病模型的实验研究  

Intratympanic Administration of Butylphthalide Combined with TLR4 Antibody for A Model of Ménière’s Disease in Mice

作　　者：张静婧 李姝睿 王琼 刘乃嘉 文新 ZHANG Jingjing;LI Shurui;WANG Qiong;LIU Naijia;WEN Xin(Affiliated Hospital of Gansu University of Traditional Chinese medicine,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学附属医院,兰州730000

出　　处：《中华耳科学杂志》2022年第1期15-20,共6页Chinese Journal of Otology

基　　金：甘肃省卫生行业科研计划项目(GSWSKY-2019-33)资助。

摘　　要：目的研究丁苯酞联合TLR4抗体通过调控Hsp70通道蛋白表达对内淋巴积水(Endolymphatic hydrops,ELH)小鼠模型的诊疗价值。方法选取50只BALB/c雌性小鼠,并随机分为对照组(A组)、模型组(B组)、和实验组(C组)。对照组不予任何处理;模型组予内淋巴积水造模;实验组在模型组基础上2天后:A组予以鼓室注射TLR4抗体50μg+丁苯酞25mg/kg灌胃干预;B组予以鼓室注射TLR4抗体100μg+丁苯酞100mg/kg灌胃干预;C组予以鼓室注射TLR4抗体200μg+丁苯酞100mg/kg灌胃干预。连续干预4周。随后观察小鼠行为学改变,耳蜗切片Hsp70表达,测试耳蜗电图SP/AP值。检测MD小鼠在丁苯酞联合TLR4抗体干预前后血清Hsp70水平及TNF-α、IL-6炎性细胞因子表达。结果与对照组比较,模型组小鼠诱发出明显外周前庭症状,符合MD(Ménière’s Disease)动物造模改变;与模型组比较,实验组3个亚组经4w鼓室注射TLR4抗体50、100、200μg+丁苯酞25、100、100mg/kg灌胃干预后,小鼠前庭反应都明显减轻,模型组与实验组耳蜗电图SP/AP值分别为0.4169±0.13,0.3271±0.12,两者差异无统计学意义(P>0.05);耳蜗切片Hsp70表达可见耳蜗螺旋神经节和血管纹细胞在实验组呈较强阳性染色,模型组呈强阳性染色,两者对比有统计学意义(P<0.05)。小鼠血清中Hsp70蛋白、TNF-α及IL-6水平较模型组显著减低,两者比较有统计学意义(P<0.05);实验A组与B组相比,随TLR4抗体鼓室注射及丁苯酞灌胃剂量增加,上述指标水平下降。而实验B组与C组相比,当丁苯酞灌胃剂量不变,继续增加TLR4鼓室注射浓度至200μg时,上述指标水平继续下降。结论丁苯酞联合TLR4抗体鼓室给药可明显干预TLR4/Hsp70信号传导通路在MD小鼠内耳的表达。当TLR4抗体鼓室注射浓度增为200μg,对血清Hsp70表达及减轻炎性因子水平的干预作用最大。Objective To study the effects of butylphthalide combined with TLR4 antibody on a mouse model of endolymphatic hydrops(ELH)by regulating the expression of the HSP70 channel protein.Methods Fifty female BALB/c mice were randomly divided into a control group,a modeling group and a treatment group.The control group received no treatment.Endolymphatic hydrops was induced in the modeling group.Two days after modeling,mice in the treatment group received either intratympanic injection of TLR4 antibody at 50μg and butylphthalide by gavage at 25 mg/kg(group A),or intratympanum injection of TLR4 antibody at 100μg and intragastric butylphthalide at 100 mg/kg(group B),or intratympanic injection of TLR4 antibody at 200μg and intragastric butylphthalide 100 mg/kg(group C)for 4 weeks.Subsequently,behavioral changes were observed and the expression of Hsp70 on cochlear slices was measured by immunohistochemistry,western blot and ELISA.Serum Hsp70 levels in modeled mice were measured before and after butylphthalide/TLR4 antibody treatments,as well as expression of TNF-αand IL-6 inflammatory cytokines.Results Compared with the control group,modeled mice exhibited behaviors consistent with peripheral vestibular symptoms in Ménière’s Disease(MD),which improved after 4 weeks of TLR4 antibody and butylphthalide treatments regardless of doses.The SP/AP ratio was 0.4169±0.13 in the modeling only group and 0.3271±0.12 in the treatment group,respectively(P>0.05).Strong staining of Hsp70 was seen in the spiral ganglion and vascular striatum cells in modeled mice with or without treatment(P<0.05).The serum levels of Hsp70,TNF-αand IL-6 were significantly lower in the control mice as compared with modeled mince(P<0.05),which also decreased with TLR4 antibody/butylphthalide treatment in a dose-dependent manner(Group A vs Group B)and continued to decrease when intratympanic TLR4 dosage was increased to 200μg while the dose of butylphthalide was maintained the same(group B vs group C).Conclusion Butylthalide combined with intratymp

关 键 词：丁苯酞 热休克蛋白70(Hsp70) TLR4抗体 鼓室给药 梅尼埃病 

分 类 号：R764[医药卫生—耳鼻咽喉科]