Effects And Mechanisms Of Zuogui Pill On JNK/c-Jun Pathway Of The Gentamicin-induced Oto-renal Injury In Rats Model

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.