噪声引起的豚鼠血-迷路屏障损伤及基质金属蛋白酶抑制剂的保护作用

目的通过研究基质金属蛋白酶(matrix metalloproteinase,MMP)2(MMP-2)和基质金属蛋白酶9(MMP-9)在噪声暴露前、后豚鼠耳蜗血管纹上表达和分布的差异,探索MMP在噪声破坏血-迷路屏障完整性中的作用以及广谱MMP抑制剂多西环素对血-迷路屏障噪声性损伤的保护作用,为进一步研究和防治噪声性聋提供参考。方法45只健康成年豚鼠采用随机数表法随机分为对照组(15只,腹腔注射生理盐水,连续4 d)、单纯噪声暴露组(15只,120 dB SPL白噪声每天暴露4 h,连续2 d;腹腔注射生理盐水,连续4 d)及噪声暴露+多西环素组(15只,120 dB SPL白噪声每天暴露4 h,连续2 d;腹腔注射多西环素50 mg/kg/d,连续4 d)。应用免疫荧光染色、蛋白质印迹(western blot)、荧光实时定量PCR等方法分析对比对照组、单纯噪声暴露组及噪声暴露+多西环素组豚鼠耳蜗血管纹上MMP-2和MMP-9的分布和表达差异。观察三组豚鼠血管纹上紧密连接蛋白ZO-1的变化,探索噪声损伤对血管纹的影响。应用听性脑干反应(ABR)测试分析三组豚鼠听力学差异。静脉注射伊文思蓝,观察血管纹毛细血管渗漏情况在三组间的差异,了解噪声导致血-迷路屏障通透性改变的情况。应用SPSS 22.0软件进行统计学分析。结果单纯噪声暴露组与噪声暴露+多西环素组在噪声暴露后2 h听力改变无明显差异;噪声暴露后7 d、14 d和28 d,噪声暴露+多西环素组听力恢复明显好于单纯噪声暴露组(P值均<0.05)。免疫荧光染色发现对照组MMP-2和MMP-9在血管纹上仅有少量表达,ZO-1呈致密线性表达;单纯噪声暴露组可见血管纹上MMP-2和MMP-9表达显著增加,ZO-1结构松散、不连续;噪声暴露+多西环素组血管纹上MMP-2和MMP-9的表达与对照组无明显差异(P值均>0.05),比单纯噪声暴露组明显减少(P值均<0.05),ZO-1仅出现少量破口,但整体仍保持致密的线性结构。血管纹毛细血管中伊文思蓝染料在单纯噪声暴露组的渗漏显著增加,噪声暴露+多西环素组与对照组相比,差异无统计学意义(P>0.05)。结论MMP-2、MMP-9引起的紧密连接蛋白结构的损伤可能是噪声暴露后豚鼠血-迷路屏障破坏的重要机制,多西环素可抑制MMP的分泌,在一定程度上保护血-迷路屏障的完整性,减轻噪声暴露导致的听力损伤。

Noise-induced blood-labyrinth-barrier trauma of guinea pig and the protective effect of matrix metalloproteinase inhibitors

Objective The research is to study the expression and distribution of matrix metalloproteinase(MMPs)-2 and-9 in the guinea pig cochlea after noise exposure,and to explore the role of MMPs in the blood-labyrinth-barrier(BLB).In addition,the role of MMPs inhibitor doxycycline in noise-induced BLB trauma was studied as well,which provides the basis for further studies and prophylaxis of noise-induced hearing loss.Methods A total of 45 healthy adult guinea pigs were randomly divided into the control group(15 received intraperitoneal injection of 0.9%saline for 4 consecutive days),the noise-exposure group(15 exposed by 120 dB SPL white noise for 4 h per day for continuous 2 d,intraperitoneal injection of normal saline for 4 consecutive days)and the noise-exposure+doxycycline group(15 exposed by 120 dB SPL white noise exposure for 4 h per day for 2 consecutive days,and intraperitoneal injection of doxycycline 50 mg/kg/d for 4 consecutive days),respectively.Immunofluorescence staining,western blot,and real-time quantitative PCR were used to analyze the distribution and differential expression of MMP-2 and-9 in the stria vascularis of guinea pigs in comparison with the normal control group,noise only group,and noise&doxycycline treatment group.Immunofluorescence staining was used to observe the changes in tight junction(TJ)protein ZO-1 in stria vascularis in three groups and to investigate the effect of acoustic injury on TJs.And ABR tests were utilized to detect the hearing function of guinea pigs in the three groups.Intravenous Evans blue was administrated intravenously as an indicator of vascular leakage among three groups to study the changes in BLB permeability in context of acoustic injury.SPSS 22.0 was used for statistical analysis.Results There was no significant difference in hearing function between the noise-exposure group and the noise&doxycycline group two hours after noise exposure.After seven,14 and 28 days noise exposure,the hearing recovery of the noise&doxycycline treatment group was significantly greater than that of the noise-exposure group(P<0.05).Immunofluorescence staining showed that there was only a small amount of MMP-2 and-9 in the stria vascular in the normal control group,and ZO-1 showed dense linear expression.While,in the noise-explore group,MMP-2 and-9 in the stria vascular was significantly elevated(P<0.05),and the configuration of ZO-1 became loose and discontinuous.However,the MMP-2 and-9 in the noise&doxycycline treatment group were not significantly different from the normal control group(P>0.05),which were significantly less than that in the noise-exposure group,and just a little break of ZO-1 was observed,however,the overall structure remained dense.The leakage of Evans blue from stria vascular capillary in the noise-exposure group was significantly increased,and the difference between the other two groups did not show any statistical significance(P>0.05).Conclusions The damage of tight junction structure induced by MMP-2 and-9 may play an important role in BLB destruction.In addition,doxycycline can inhibit MMPs secretion,thereby,to some extent,protecting the integrity of BLB from acoustic injury,and contributing to the long-term hearing recovery.