艾灸对庆大霉素致听力损伤豚鼠模型的干预效应及其作用机制

Effect and mechanism of moxibustion on gentamicin-induced hearing loss in guinea pig model

目的 研究艾灸治疗庆大霉素耳毒性导致的豚鼠耳聋的作用机制,为艾灸治疗突发性耳聋(sudden sensorineural hearing loss, SSHL)提供科学证据。方法 将18只雄性豚鼠随机分为3组,每组6只:空白组、庆大霉素组、庆大霉素+艾灸组,按照庆大霉素组腹腔注射硫酸庆大霉素120mg/(kg·d),连续14天;庆大霉素+艾灸组肌注硫酸庆大霉素120 mg/(kg·d),并予艾灸治疗15分钟1次/天,连续14天;空白组腹腔注射生理盐水,连续14天,进行干预。14天后对豚鼠进行听觉脑干诱发电位(auditory brainstem response, ABR)阈值检测以观测其听力变化,光学显微镜观察耳蜗血管纹形态,并用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)法检测耳蜗超氧化物歧化酶(supper oxide dismutase, SOD),丙二醛(malondial dehyde, MDA)的含量及血清一氧化氮(nitric oxide, NO),内皮素(endothelin, ET)的含量,PCR检测耳蜗表达的B淋巴细胞瘤-2基因(B-cell lymphoma-2, Bcl-2)及Bcl-2相关蛋白X(BCL2-Associated X,Bax)的含量,免疫荧光染色半定量法观察血管内皮生长因子(vascular endothelial growth factor, VEGF)在血管纹的表达及分布。结果 (1)听力阈值:ABR的听力阈值庆大霉素组明显高于空白组;庆大霉素+艾灸组明显低于庆大霉素组而高于空白组(P<0.05)。(2)耳蜗螺旋韧带血管纹形态:空白组螺旋韧带血管纹显示基本正常,粗细均匀顺畅;庆大霉素组可见耳蜗螺旋韧带血管纹屈曲变窄,血管粗细不均;庆大霉素+艾灸组可见耳蜗螺旋韧带血管纹结构稍有不流畅,粗细稍显不均(3)耳蜗组织SOD、MDA含量:庆大霉素组耳蜗组织中SOD含量与空白组比较明显降低,MDA含量与空白组比较明显升高;庆大霉素+艾灸组耳蜗中SOD含量高于庆大霉素组,MDA含量低于庆大霉素组(P<0.05)。(4)血清NO、ET含量:庆大霉素组与空白组比较,血清NO、ET均有所上升(P<0.05)。庆大霉素+艾灸组与庆大霉素组比较,血清NO上升(P<0.05)。(5)耳蜗Bcl-2、Bax、Bcl-2/Bax基因表达:庆大霉素组与空白组比较,庆大霉素组豚鼠耳蜗组织Bcl-2、Bcl-2/Bax均比空白组有所降低(P<0.05),Bax含量两组差异无统计学意义(P>0.05);庆大霉素+艾灸组与庆大霉素组比较,Bax明显下降,Bcl-2/Bax明显上升(P<0.05),Bcl-2两组差异无统计学意义(P>0.05)。(6)血管纹VEGF的分布和含量:庆大霉素组耳蜗血管纹周围组织VEGF蛋白(绿色)荧光强度较空白组稍有增强;庆大霉素+艾灸组VEGF蛋白(绿色)荧光强度比空白组增强,与庆大霉素组相比在管腔结构附近荧光强度增加明显。结论 艾灸能通过提高机体清除自由基的能力,扩张血管促进微循环,保护耳蜗组织细胞和神经等功能,起到改善听力,防治突发性耳聋的作用。

Objective To identify the effect of moxibustion in the intervention of Gentamic in ototoxicity.Methods Eighteen guinea pigs were randomly divided into 3 groups, 6 in each: normal saline group, the gentamicin-made module group and the gentamicin-made+moxibustion group. According to the gentamicin-made module group and gentamicin-made +moxibustion group, gentamicin sulfate were intraperitoneally injected 120 mg/(kg·d) for 14 consecutive days. The guinea pigs in gentamicin-made+moxibustion group were treated with moxibustion 15 min time per day after injection for 14 consecutive days;the guinea pigs in the normal saline group were intraperitoneally injected with normal saline for 14 days. 14 days later, ABR threshold detection was performed on guinea pigs;the distribution of the striated veins were observed by optical microscope;SOD, MDA, serum NO, ET were detected by ELISA. The expression of BAX and BCL2 genes in cochlea tissue was detected by PCR, and the distribution and content of VEGF in stria vascularis were observed by immunofluorescence staining. Results(1)Hearing threshold: The hearing threshold of ABR in the gentamicin group was significantly higher than that in the normal saline group(P<0.05), and the gentamicin+moxibustion group was significantly lower than that in the gentamicin group and higher than that in the normal saline group(P<0.05).(2)Morphology of cochlear spiral ligament striae: the striae of spiral ligament in the normal saline group were basically normal, with uniform and smooth thickness;in the gentamicin group, the stria vascularis of cochlea spiral ligament were flexed and narrowed, and the thickness of vessels was uneven. In the gentamicin+moxibustion group, the vascular stria of cochlea spiral ligament was slightly not smooth, and the thickness was slightly uneven.(3)SOD and MDA content in the cochlea: the SOD content in the cochlear tissue of the gentamicin group was significantly lower than that in the normal saline group, the MDA content was significantly higher than that in the normal saline group(P<0.05). The SOD content in the cochlea of the gentamicin+moxibustion group was higher than that of the gentamicin group, and the MDA content was lower than that in the gentamicin group(P<0.05).(4)Serum NO/ET content: comparing the gentamicin group with the normal saline group, serum NO and ET increased significantly(P<0.05). Comparing the gentamicin+moxibustion group with the gentamicin group, serum NO increased significantly(P<0.05).(5)Bcl-2/Bax: Comparing the gentamicin group with the normal saline group, the Bcl-2 and Bcl-2/Bax of the guinea pig cochlea in the gentamicin group were lower than that in the normal saline group(P<0.05), The comparison of Bax between the two groups were not statistically significant(P>0.05). Comparing the gentamicin+moxibustion group with the gentamicin group, Bax decreased, Bcl-2/Bax increased(P<0.05). There was no statistical difference between the two groups with regard to Bcl-2 and Bax(P>0.05).(6)The distribution and content of VEGF in stria vascularis: the fluorescence intensity of VEGF protein(green) in the tissue around the stria vascularis in the gentamicin group was slightly increased compared with that in the normal saline group, and the fluorescence intensity of VEGF protein(green) in the gentamicin+moxibustion group was significantly increased. The fluorescence intensity increases significantly near the lumen structure.Conclusion Moxibustion can improve the body’s ability to scavenge free radicals, expand blood vessels, promote microcirculation, protect the function of cochlear tissue cells and nerves, and improve hearing.