聪耳通窍剂干预豚鼠庆大霉素耳毒性聋作用的机制探讨

Cong'er-Tong'qiao Agent for Gentamycin-Induced Ototoxic Deafness in Guinea Pigs

目的探讨基于中医脾肾理论的中药聪耳通窍剂干预庆大霉素(gentamycin,GM)耳毒性聋作用与可能机制。方法选择杂色成年豚鼠60只,随机分为正常对照组,GM对照组,中药+GM组,每组各20只。正常对照组按常规饲养至第11天期满;GM对照组按200mg/kg·d剂量,每天分两次连续注射9天,第11天终止饲养;中药+GM组按3.994g/kg·d剂量(1/2人工灌服,1/2自动饮用),预先服用聪耳通窍剂10天,然后在继续服用中药同时开始注射每天同等剂量GM,连续9天,第11天终止饲养。其中每组6只动物于第5、6、9天检测ABR评估听力;6只动物利用RT-PCR检测耳蜗SOD1;剩余8只动物用于全耳蜗基底膜铺片,琥珀酸脱氢酶染色,进行耳蜗毛细胞形态学研究,以及检测血清BUN和Cr,肾脏切片经苏木素-伊红染色,了解肾功能和肾组织病理变化,其中每组6只动物一侧肾脏应用western blot检测ASK-1、Trx、caspase-3。结果正常对照组ABR阈值一直保持相对恒定正常范围。与正常对照组比较,GM对照组ABR阈值第5天即出现明显差异(P<0.05),与中药+GM组比较,第7天开始出现明显差异(P<0.05),随着天数增加,差异继续扩大(P<0.01)。中药+GM组ABR阈值递增差异不显著(P>0.05)。全耳蜗基底膜检查显示,正常对照组各回三排内、外毛细胞形态正常,数量完整,排列有序;GM对照组各回三排外毛细胞损毁严重,绝大部分细胞出现严重崩解不全,或凋亡缩小,但内毛细胞仍基本存在;中药+GM组各回三排外毛细胞除少量和散在崩解不全外,大多仍存留可辨,形态如常,内毛细胞仍保持完整,各回外毛细胞损毁状况明显少于GM对照组(P<0.01)。中药+GM组对耳蜗SOD1表达明显高于正常对照组和GM对照组(P<0.01,P<0.05),而血清BUN和Cr含量则明显低于GM对照组(P<0.01)。肾脏切片染色显示,与正常对照组比较,GM对照组肾小球和近曲肾小管等受损严重,病理改变明显,而中药+GM组则受损不明显,甚至基本保持正常。中药+GM组对肾组织ASK-1、caspase-3表达明显低于GM对照组(P<0.05),对Trx表达则明显高于GM对照组(P<0.05)。结论一定剂量GM连续使用可造成严重耳、肾毒性损害,耳蜗损害以外毛细胞为主要损害靶细胞,肾脏损害以肾小球和近曲小管为主。聪耳通窍剂具有显著防护GM耳、肾毒性损害作用,其作用机制与其抑制氧化应激反应,干预ROS介导的细胞死亡径路有关,而通过护肾,促进GM排泄,是其间接护耳的重要机制;中医脾肾理论防治耳聋具有科学基础和应用价值。

Objective To report outcomes and possible mechanisms of Cong’er-Tong’qiao agent (CETQA), based on Chinese herbs (CH) and the traditional Chinese Medicine (TCM)“spleen-kidney”theory, in treating gentamycin (GM)-induced deafness. Methods Variegated adult guinea pigs (n=60) were randomly divided into a normal control, a GM control, and a CETQA treatment (CH+GM) group (20 in each group). Animals in the normal control group received no treatment. Those in the GM control group received GM injection (200 mg/kg/day, bid) for 9 successive days, and were sacrificed on day 11. Animals in the last group received CETQA (3.994g/kg/day, half artificial and half automatic feeding) for 10 days before starting GM treatment, followed by simultaneous GM injection and CETQA treatment for 9 days, and were sacrificed on day 11. From each group, 6 animals were selected for auditory brainstem response (ABR) evaluation on the 5th, 7th and 9th day. A second group of 6 animals from each group were examined for expression of superoxide dismutase-1(SOD1) in the cochlea by real time PCR. For the remaining 8 animals in each group, blood urea nitrogen (BUN) and serum creatinine (Cr) were tested by spectrophotometer, pathological changes in renal tissue were examined under a microscope with hematoxylin and eosin staining, and the whole cochlear basilar membrane was micro- dissected out, trimmed and mounted in glycerin on glass slides as flat surface preparations for morphology examination with succinate dehydrogenase staining. In 6 animals from each group, one kidney was harvested for tests of apoptosis signal regulating kinase-1(ASK-1), Thioredoxin (Trx) and caspase-3 by western blot. Results ABR thresholds remained unchanged in the normal range in animals of the normal control group, while ABR thresholds in the GM control group were significantly elevated compared with the normal control group on the 5th day and the CH+GM group on the 7th day (P<0.05), with the difference continuing to increase along with time(P<0.01). ABR threshold elevation in the CH+GM group was not significant (P>0.05). On the whole cochlear basilar membrane preparation, inner and outer hair cells in each turn showed normal morphology and orderly arrangement with no missing cells in the normal control group; all three rows of outer hair cells showed severe damage in all turns (with most cells showing obvious signs of disintegration, apoptosis or shrinkage) in the GM control group, with inner hair cells mostly spared; and most outer hair cells in all turns remained identifiable with normal morphology in the CH+GM group, except for a few partially disintegrated or damaged cells, with no damage to inner hair cells. Outer hair cell damage in the CH+GM group was significantly less as compared with the GM control group (P<0.01). SOD1 expression in the cochlea was significantly higher in the CH+GM group than in the GM control and normal control group(P<0.05 and P<0.01, respectively), and serum BUN and Cr levels were significantly lower in the CH+GM group than in the GM control group ( P<0.01). Renal tissue section staining showed severe damage to glomeruli and proximal convoluted tubules in the GM control group, but only mild or no damage in the CH+GM group. ASK-1 and caspase-3 expression in renal tissues was significantly lower(P< 0.01 and P<0.05, respectively), but Trx expression was significantly higher(P<0.01), in the CH+GM group than in the GM control group. Conclusion Continuous use of GM can cause severe ototoxic and nephrotoxic damage. Outer hair cells are the main target in the cochlea, while renal damage involved mainly glomeruli and proximal convoluted tubules. CETQA can significantly mitigate GM-induced ototoxic and nephrotoxic damage, probably through inhibiting oxidative stress responses and disrupting the ROS-mediated cell death pathway. Another indirect mechanism of hearing protection may be preservation of kidney function and thus effective GM excretion. The TCM“spleen-kidney”theory provides a theoretical basis applicable to prevention and treatment of deafness.