【作者】 范为民；

【导师】 王小琴；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：探讨左归丸对庆大霉素诱导耳肾损伤大鼠模型JJNK通路的影响及机制。方法：(1)将12只SPF级雄性Wistar大鼠适应性喂养1周后,随机分为空白对照组、庆大霉素组。每组6只。空白对照组腹腔注射生理盐水2.5ml·kg-1·d-1,庆大霉素组腹腔注射硫酸庆大霉素100mg·kg-1·d-1。每日根据体重调整给药剂量。2组大鼠连续给药10d。分别在给药前和给药后检测大鼠听力ABR阈值、尿NAG酶值,在实验结束取耳蜗和肾脏标本进行HE染色,在光镜下观察耳蜗和肾脏病理变化。(2)将18只SPF级Wistar大鼠适应性喂养1周后随机分为3组：空白对照组,模型组和左归丸组。每组6只。空白对照组腹腔注射生理盐水2.5ml·kg-1·d-1,模型组腹腔注射硫酸庆大霉素100mg·kg-1·d-1,左归丸组腹腔注射硫酸庆大霉素100mg·kg-1·d-1和灌胃左归丸水溶液10g·kg-1·d-1。每日根据体重调整给药剂量。3组大鼠连续给药10d。分别在给药前和给药后检测大鼠听力ABR阈值、尿NAG酶值、大鼠体重变化。实验结束后,测量大鼠肾脏指数,留取血液检测Scr、BUN。留取耳蜗和肾脏标本光镜和电镜下观察病理变化。(3)将72只SPF级Wistar大鼠适应性喂养1周后随机分为4组：空白对照组,模型组、SP600125组和左归丸组。每组18只。空白对照组腹腔注射生理盐水2.5ml·kg-1·d-1;模型组腹腔注射硫酸庆大霉素100mg·kg-1·d-1; SP600125组左侧腹腔注射硫酸庆大霉素100mg·kg-1·d-1,2h后右侧腹腔注射SP60012515mg·kg-1·d-1;左归丸组腹腔注射硫酸庆大霉素100mg·kg-1·d-1和灌胃左归丸水溶液10g·kg-1·d-1。每日根据体重调整给药剂量。4组大鼠连续给药10d。实验结束后,留取耳蜗和肾脏标本,以免疫组化方法、免疫印迹杂交方法、实时定量PCR方法检测JJNK、p-JNK、c-Jun的表达和蛋白、基因的含量。结果：(1)庆大霉素组大鼠腹腔注射100mg·kg-1·d-1×10d后,听力ABR阈值和尿NAG酶值明显升高,与空白对照组相比,具有统计学差异(P<0.01)。大鼠耳蜗、肾脏病理形态比较：空白对照组螺旋器毛细胞完整无缺失,排列整齐；肾脏小管、间质无特殊改变。而庆大霉素组螺旋器毛细胞出现丢失、变性、排列不规则；肾小管出现炎性细胞浸润,并出现细胞肿胀、变性。(2)3组大鼠ABR阈值、尿NAG酶、血Scr与BUN、肾脏指数结果：与空白对照组相比,模型组ABR阈值、尿NAG酶、血Scr与BUN、肾脏指数值明显升高,具有统计学差异(P<0.01)；与模型组相比,左归丸组ABR阈值、尿NAG酶、血Scr与BUN、肾脏指数值明显下降,具有统计学差异(P<0.05或P<0.01)。大鼠体重结果：与空白组相比,模型组大鼠体重数值明显降低,具有统计学差异(P<0.01)；与模型组相比,左归丸组大鼠体重数值得到升高,具有统计学差异(P<0.05)。耳蜗、肾脏HE染色病理结果：空白对照组耳蜗螺旋器毛细胞无缺失,排列整齐；肾脏小管、间质无特殊改变。而模型组耳蜗螺旋器毛细胞出现核固缩、核破裂,毛细胞丢失严重；肾脏小管上皮细胞出现肿胀、变性和少量坏死。左归丸组耳蜗毛细胞有少量丢失,排列尚可；肾脏小管上皮细胞出现轻微肿胀、变性。透射电镜超微病理结果：空白对照组耳蜗线粒体、粗面内质网结构未发生明显改变；近曲肾小管上皮细胞整体结构正常,基底面有排列整齐的质膜内褶,胞浆及质膜内褶间分布众多线粒体。模型组耳蜗胞浆溶酶体增多,部分出现空泡化；近曲肾小管上皮细胞游离面局部微绒毛脱落,胞浆内多处巨大囊泡扩张。左归丸组耳蜗毛细胞形态结构基本正常,部分胞浆空泡化；近曲肾小管微绒毛排列整齐,胞浆内少量囊泡病变,管腔内有少量絮状物。(3) TUNEL细胞凋亡结果：空白对照组耳蜗和肾脏有少量凋亡细胞,模型组耳蜗和肾脏TUNEL阳性细胞数目明显增多具有统计学差异(P<0.01)；与模型组比较,左归丸组和SP600125组耳蜗和肾脏TUNEL阳性细胞数目明显减少,具有显著统计学差异(P<0.01)。免疫组化结果：空白对照组耳蜗和肾脏JNK、p-JNK、c-Jun蛋白表达较少。与空白对照组相比,模型组JNK、p-JNK、c-Jun蛋白有大量表达,具有显著统计学差异(P<0.01)。耳蜗主要集中在螺旋器、血管纹、螺旋神经节部位表达；肾脏主要集中在肾小管部位。与模型组相比,左归丸组和SP600125组表达减少,具有显著统计学差异(P<0.01)。免疫印迹杂交结果：与空白组相比,模型组耳蜗和肾脏JNK、p-JNK、c-Jun蛋白表达显著升高,具有显著统计学差异(P<0.01)；与模型组相比,左归丸组SP600125组耳蜗和肾脏JNK、p-JNK、c-Jun蛋白表达明显下降,具有显著统计学差异(P<0.01)。实时定量PCR结果：与空白组相比,模型组内耳和肾脏JNK mRNA和c-Jun mRNA表达显著升高(P<0.01)；与模型组相比,SP600125组JNK mRNA和c-Jun mRNA表达显著下调(P<0.01)结论：(1)大鼠腹腔注射庆大霉素100mg·kg-1·d-1×10d可以成功建立耳肾损伤模型。(2)左归丸能够保护耳肾损伤大鼠的听力和肾功能,能够改善耳肾损伤大鼠耳蜗和肾脏病理状况,能够减轻病理性细胞凋亡状态。(3) JNK/c-Jun通路对庆大霉素诱导的耳肾损伤模型中细胞凋亡起重要调控作用,激活的JNK/c-Jun通路促使病理性细胞凋亡的发生,并参与庆大霉素诱导的耳肾损伤。(4)左归丸能够抑制JNK通路在耳肾损伤大鼠模型的激活。左归丸对耳肾损伤大鼠的保护作用可能与抑制JNK通路的激活有关。更多还原

【Abstract】 Objective:To discussion the Intervention of Zuogui pill on JNK/c-Jun pathway of the gentamicin-induced Oto-renal injury in rat model and the Mechanism.Methods:(1)12male Wistar rats were divided randomly into control group, and Gentamic in sulfate (GM) model group. The control group was given intraperitoneal injection of0.9%saline2.5ml·kg-1·d-1, GM group was given intraperitoneal injection of gentamicin sulfate100mg·kg-1·d-1. Two groups of rats administered for10consecutive days. Detection of ABR threshold and NAG enzyme values of rats administered before and after administration, at the end of the experiment to take cochlea and kidney specimens were stained with HE, cochlea and kidney pathological changes in the light microscope.(2)18male Wistar rats were divided randomly into control group, model group and Zuogui pill group. The control group was given intraperitoneal injection of0.9%saline2.5ml·kg-1·d-1, model group was given intraperitoneal injection of gentamicin sulfate100mg·kg-1·d-1, Zuogui pill group was given intraperitoneal injection of gentamicin sulfate100mg·kg-1·d-1and gavage Zuogui pill aqueous solution10g·kg-1·d-1. Three groups of rats administered for10consecutive days. Detected before drug administration and after ABR threshold, urinary NAG enzyme values, changes in body weight of rats. After the end of the experiment, the measurement of the index of the rat kidney specimens from blood tests Scr, BUN. Specimens of pathological changes in light and electron microscopy of the cochlea and kidney specimens.(3)72male Wistar rats were divided randomly into control group, model group, Sp600125group and Zuogui pill group. The control group was given intraperitoneal injection of0.9%saline2.5ml·kg-1·d-1, model group was given intraperitoneal injection of gentamicin sulfate100mg·kg-1·d-1, SP600125group was given intraperitoneal injection of gentamicin sulfate100mg·kg-1·d-1and intraperitoneal injection of SP60012515mg·kg-1·d-1, Zuogui pill group was given intraperitoneal injection of gentamicin sulfate100mg·kg-1·d-1and gavage Zuogui pill aqueous solution10g·kg-1·d-1. Four groups of rats administered for10consecutive days. After the end of the experiment,the specimens from the cochlea and kidney specimens, TUNEL, immunohistochemistry, Western blot hybridization, and real-time PCR method to detect JNK, p-JNK, c-Jun expression and protein, gene content.Results:(1) GM group by intraperitoneal injection of gentamicin100mg·kg-1·d-×10d, the hearing threshold of ABR and urinary NAG enzyme values significantly higher, compared with the control group,with a statistically significant difference (P<0.01).Rat cochlea and kidney pathological comparison:Control group of corti full without missing hair cells, neat rows; Renal tubules and interstitial no special change. GM group of organ of corti appear hair loss, degeneration, irregular arrangement; Renal tubule in inflammatory cell infiltration, and cell swelling and degeneration.(2) Three groups ABR threshold, urinary NAG enzyme, blood Scr and BUN, kidney index results:compared with the control group, the model group ABR threshold, urinary NAG enzyme, blood Scr and BUN, kidney index was significantly higher, with a significant difference (P<0.01); compared with the model group, the Zuogui pill groups is decreased significantly, with a significant difference (P<0.05and P<0.01). Body weight of rats results:After administration, compared with the control group rats, the weight value is significantly lower, with a significant difference (P<0.01); Compared with the model group, Zuogui pill group rats weight values increased, with a statistically significant difference (P<0.05). Cochlea, kidney HE staining pathological findings:Control group in the cochlea corti hair cell loss, neat rows; And the model group the cochlea corti hair a nuclear pyknosis, karyorrhexis, hair loss is serious; Zuogui pill group of cochlear hair cells loss, a small amount of an acceptable arrangement. Control group kidney tubules and interstitial no special change; And model groups renal tubular epithelial cell swelling, degeneration and small amounts of necrosis; Zuogui pill group of renal tubular epithelial cells slight swelling and degeneration. Transmission electron microscopy results:Control group cochlea mitochondria, rough endoplasmic reticulum structure did not change significantly; Proximal tubular epithelial cells in the overall structure of the normal, basal surface of the plasma membrane are arranged neatly tuck, tuck between the cytoplasm and plasma membrane distribution of numerous mitochondria. Model group cytoplasmic lysosomes cochlea, part of the vacuoles appear; Proximal tubular epithelial cells localized free surface microvilli, intracytoplasmic vesicles many huge expansion. Zuogui pill group cochlear hair cell morphology was normal, and some cytoplasmic vacuoles; Proximal tubular microvilli neat, small cytoplasmic vesicle lesions lumen of a small amount of floe.(3) Results of TUNEL Cell apoptosis:Control group cochlea and kidney have few apoptotic cells, the model group cochlea and kidney of TUNEL-positive cells increased significantly in the number with a statistically significant difference (P<0.01); The the Zuogui pill group and SP600125group cochlea and kidney of TUNEL-positive cells significantly reduced the number compared with the model group, with statistically significant differences (P<0.01). Immunohisto-chemical results:Compared with the control group, model group, JNK, c-Jun protein expression of p-JNK, a statistically significant difference (P<0.01); Compared with the model group, The Zuoguipill group and SP600125group JNK, p-JNK, c-Jun protein expression was significantly decreased, with a statistically significant difference (P<0.01). Western blot hresults:Compared with the control group, The model group cochlea and kidney of JNK, p-JNK c-Jun protein expression was significantly increased, with a statistically significant difference (P<0.01); Compared with the model group, the the SP600125group cochlea and kidney of Zuogui pill group of JNK, c-Jun protein expression was significantly decreased p-the JHK, a statistically significant difference (P<0.01). Real-time PCR results:Compared with the control group, the model group, the inner ear and kidney JNK mRNA and c-Jun mRNA expression was significantly higher (P<0.01); Compared with the model group, Zuogui pill group and SP600125JNK mRNA and c-Jun mRNA expression was significantly lower (P<0.01).Conclusions:(1) The intraperitoneal injection100mg·kg-1·d-1gentamicin oto-renal injury model can be successfully established.(2) Zuogui pill able to protect oto-renal injury in rats hearing and kidney function, to improve oto-renal injury in rat cochlea and kidney pathological conditions, can reduce the pathological apoptosis state.(3) JNK/c-Jun pathway play an important regulatory role in apoptosis in the ear gentamicin-induced renal injury model. Activation of JNK/c-Jun pathway to promote the pathological cell apoptosis, and participate in gentamicin-induced oto-renal injury.(4) The protective effect of Zuogui pill oto-renal injury in rats may be related to the inhibition of the activation of the JNK pathway. Zuogui pill able to inhibit the activation of the JNK pathway in oto-renal injury in rats model.更多还原