Sonic Hedgehog信号转导通路中关键蛋白在横纹肌溶解致大鼠急性肾损伤的作用及盐酸戊乙奎醚、山莨菪碱干预的影响

发布时间：2018-07-05 18:42

本文选题：横纹肌溶解 + 急性肾损伤　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:横纹肌溶解(rhabdomyolysis,RM)所致急性肾损伤(acute kidney injury,AKI)为肾脏常见病,如不及时处理可发展为不可逆肾脏损害。挤压伤、过度训练、外伤、中毒等理化因素可以引起横纹肌溶解,进而导致急性肾损伤,但其确切机制尚不完全清楚。因此,探究其发病机制,进行早期干预治疗,以减轻肾脏损害,从而改善预后,降低死亡率,已成为军事医学、运动医学及临床医学的重要课题。本研究旨在通过甘油肌注建立大鼠横纹肌溶解致急性肾损伤模型,拟评价RM时大鼠肾组织Sonic Hedgehog(Shh)信号转导通路中关键蛋白的表达变化及其意义,观察抗胆碱药对其表达变化的影响,为临床研究提供参考。方法:1实验分组:将体重在200-220g之间的72只SD雄性大鼠用随机数字表法平均分为4组。对照组(Control,CN,n=18)、急性肾损伤组(acute kidney injury,AKI,n=18)、山莨菪碱干预组(anisodmine,AD,n=18)、盐酸戊乙奎醚干预组(penehyclidine hydrochloride,PHC,n=18),根据观察时间又分为3个亚组,即注射后6h、24h、72h,每个时间点6只大鼠。2模型制备:将大鼠禁食水,CN组18只大鼠于双后腿内侧注射生理盐水,用量10ml/kg。AKI组、AD组、和PHC组双侧后肢肌肉注射50%甘油生理盐水10 ml/kg,建立大鼠急性肾损伤模型。AD组18只大鼠在注射甘油前的20min,于腹腔注射山莨菪碱,用量10 mg/kg,PHC组18只大鼠在甘油注射前20min,于腹腔注射盐酸戊乙奎醚,用量2mg/kg。随后各组大鼠均给予常规饲料自由进食、水、观察大鼠一般情况。3取材处理:各组于相应时间点分别随机取6只大鼠,于腹腔注入戊巴比妥(60 mg/kg),深度麻醉后大鼠腹部正中切开,于上腔静脉抽取静脉血样,生化分析法测定血浆尿素氮(BUN)、肌酐(Cr)、比色法测血浆、肾髓质髓过氧化物酶(MPO)。取双肾,去除表面脂肪筋膜,右肾放置在-80℃冰箱冻存,采用Western blot法检测Shh、Gli2蛋白的表达;左肾纵行剖开,10%中性福尔马林固定,常规脱水,石蜡包埋,切片;应用HE染色光学显微镜下观察肾组织结构变化,并行肾小管损伤评分,评估损伤程度;采用免疫组化染色(PV法)观察肾组织Shh、Gli2的表达情况。4统计分析:采用SPSS 21.0软件进行统计学分析,以均数±标准差(x±s)表示。各组间比较采用单因素方差分析(one-way ANOVA)分析,两两比较采用Student-Newman-Kuels t检验,以P0.05为有统计学意义。结果:1各组血BUN、Cr变化:与对照组各时间点比较,急性肾损伤组大鼠血清Cr和BUN均增高(P0.05),且进行性升高。山莨菪碱干预组、盐酸戊乙奎醚干预组血清Cr、BUN在24h、72h较AKI组同时间点明显降低(P0.05),但仍高于对照组(P0.05),其中AD组24h、72h血清Cr、BUN较同期PHC组高;2各组血、肾组织MPO变化:与对照组比较,AKI组6小时组MPO明显升高(P0.05),24小时达到高峰、72小时组血MPO下降,但仍高于对照组(P0.05)。与AKI组比较,山莨菪碱干预组、盐酸戊乙奎醚干预组各时间点MPO降低(P0.05),但仍高于对照组(P0.05),与AD组同期比较,PHC血MPO6h、24h,肾MPO24h、72h较低;3各组肾组织病理变化:对照组大鼠双肾病理形态学未见明显改变。急性肾损伤组肌注甘油后6小时可见肾组织充血肿胀,肾小管上皮细胞肿胀、变性,少量坏死、脱落。管腔中形成蛋白管型,肾间质少量炎性细胞浸润。24小时组肾小管上皮细胞变性、坏死加重,大量蛋白管型,间质可见大量炎性细胞浸润,72小时组肾小管管腔内管型逐渐溶解消失,偶可见间断性损伤部位的再生上皮细胞,炎症减轻。山莨菪碱干预组、盐酸戊乙奎醚干预组各时间点亚组上皮细胞变性、坏死程度较急性肾损伤组减轻,肾小管再生增加,肾小管损伤评分,与CN组比较,AKI组6h、24h、72h肾小管损伤评分逐渐升高(P0.05),AD、PHC组肾小管损伤评分较同期AKI组低,但高于CN组(P0.05),与AD组比较,PHC6h组评分较低(P0.05);4各组肾组织Gli2及Shh表达的变化:免疫组化显示,Gli2在正常对照组的肾小管上皮细胞中呈弱表达,急性肾损伤组大鼠于6小时组肾组织Gli2表达增强(P0.05),24小时表达量增加(P0.05),72小时进一步增强(P0.05)。山莨菪碱组、盐酸戊乙奎醚组各时间点蛋白表达均高于急性肾损伤组(P0.05)。Shh在对照组大鼠肾小管上皮细胞表达。急性肾损伤组Shh于甘油肌注后6小时表达减弱(P0.05),24小时组Shh组表达增强(P0.05)。72小时组,Shh表达持续升高(P0.05)。山莨菪碱组、盐酸戊乙奎醚组各时间点与急性肾损伤组同期比较表达增强(P0.05),与AD组同期比较,PHC组Shh 6h、24h的表达,Gli2 24h、72h的表达较强(P0.05)。Western blot结果显示与免疫组化结果一致。结论:1横纹肌溶解致急性肾损伤时,肾组织Shh蛋白早期表达减弱,后期表达升高,说明横纹肌溶解致急性肾损伤可再度激活Shh信号通路。2 Shh信号通路中关键蛋白Shh、Gli2在24小时、72小时表达上调,表明激活Shh信号通路可能对急性肾损伤肾组织保护、修复发挥重要作用。3抗胆碱药山莨菪碱、盐酸戊乙奎醚可能部分通过激活Shh信号通路,减轻急性肾损伤,促进损伤组织早期修复,为临床用药提供参考。
[Abstract]:Objective: acute kidney injury (AKI) is a common kidney disease (acute kidney injury, AKI) caused by rhabdomyolysis (acute). If untimely treatment can be developed into an irreversible renal damage, the physical and chemical factors such as extrusion injury, overtraining, trauma and poisoning can cause rhabdomyolysis and lead to acute renal injury, but the exact mechanism is not completely clear. Therefore, it has become an important subject in military medicine, sports medicine and clinical medicine to explore the pathogenesis and early intervention treatment in order to reduce the renal damage, improve the prognosis and reduce the mortality. This study aims to establish a rat model of acute renal injury induced by rhabdomyolysis through the injection of glycerol muscle, and to evaluate the renal tissue Sonic in RM rats. The changes in the expression of key proteins in the Hedgehog (Shh) signal transduction pathway and its significance were observed to provide reference for clinical study. Methods: 1 experimental groups were divided into 4 groups of 72 SD male rats with weight between 200-220g and the control group (Control, CN, n=18), and acute renal injury. Group (acute kidney injury, AKI, n=18), anisodamine intervention group (anisodmine, AD, n=18), amyl hydrochloric ether intervention group (penehyclidine hydrochloride, PHC, n=18), according to the observation time, divided into 3 subgroups, namely, after the injection, 6 rats were prepared at each time point: the rat fasting water, 18 rats in the inside of the double hind legs In group 10ml/kg.AKI, group AD, group AD, and group PHC, the muscles of bilateral hind limbs were injected with 50% glycerol 10 ml/kg, and 18 rats in group.AD were injected with Anisodamine before glycerol injection, and the dosage of anisodamine was injected into the abdominal cavity, and 18 rats in group PHC were injected with glutamine hydrochloride before glycerol injection. The rats were given free feeding of conventional feed after the dosage of 2mg/kg., and water was given to the rats. The rats were treated with.3. 6 rats were randomly selected at the corresponding time points and injected into the abdominal cavity with pentobarbital (60 mg/kg). After deep anesthesia, the abdominal median incision was taken, the venous blood samples were extracted from the superior vena cava and the biochemical analysis was used to determine the blood. Plasma urea nitrogen (BUN), creatinine (Cr), colorimetric method to measure plasma and renal medullary myeloperoxidase (MPO), take two kidneys, remove the surface of the fat fascia, the right kidney is placed at -80 centigrade refrigerator and frozen, the expression of Shh, Gli2 protein is detected by Western blot; the left kidney is opened in a longitudinal way, 10% neutral formalin is fixed, routine dehydration, paraffin embedding and slicing; HE staining light is applied. The changes of renal tissue structure were observed under the microscope, the renal tubule injury score was evaluated and the degree of injury was evaluated. The expression of Shh and Gli2 in renal tissue was observed by immunohistochemical staining (PV method).4 statistical analysis: SPSS 21 software was used for statistical analysis with mean standard deviation (x + s). The single factor variance analysis (one-wa) was used in each group. Y ANOVA) analysis, 22 compared with Student-Newman-Kuels t test, with P0.05 as a statistical significance. Results: 1 groups of blood BUN, Cr changes: compared with the control group, the serum Cr and BUN increased in acute renal injury rats (P0.05), and progressively increased. Compared with the AKI group at the same time point (P0.05), it was still higher than the control group (P0.05), and the serum Cr and BUN in AD group were higher than that of the same group in the same period, and BUN was higher than that of the same group in the same period. 2 groups of blood and renal tissue MPO changes: compared with the control group, the MPO significantly increased in the 6 hour group of AKI group, reached the peak at 24 hours, and decreased in the 72 hour group, but still higher than the control group. In the group of anisodamine intervention, MPO decreased (P0.05) at all time points in the treatment group, but it was still higher than the control group (P0.05). Compared with the AD group, PHC blood MPO6h, 24h, renal MPO24h and 72h were lower. 3 the pathological changes of kidney tissues in the control group were not obviously changed. The kidney was seen in the acute renal injury group after 6 hours of glycerol. Tissue hyperemia and swelling, renal tubular epithelial cells swelling, degeneration, a small amount of necrosis, shedding. The tubule formation in the lumen, a small amount of inflammatory cells in the renal interstitial cells infiltrated.24 hours, the renal tubular epithelial cells degeneration, necrosis aggravated, a large number of protein tube type, mass of inflammatory cells infiltration, 72 hours group of renal tubules gradually dissolved in the group. I can see the regenerative epithelial cells in the discontinuous injury site and the inflammation alleviated. In the anisodamine intervention group, the epithelial cell degeneration in each time point group of the amyl hydrochloric acid group, the necrosis degree was less than the acute renal injury group, the renal tubule regeneration increased and the renal tubule injury score was increased. Compared with the CN group, the 6h, 24h and 72h renal tubule injury scores in group AKI increased gradually. (P0.05), the score of renal tubule injury in group AD and PHC was lower than that of group AKI, but higher than that in group CN (P0.05). Compared with group AD, the score of PHC6h group was lower (P0.05); 4 the changes of Gli2 and Shh expression in renal tissue in each group: immunohistochemistry showed that Gli2 was weakly expressed in the tubule epithelial cells of the normal control group, and the rats in the acute renal injury group were in the 6 hour group of renal tissue. Enhancement (P0.05), increased expression of 24 hours (P0.05) and further enhancement (P0.05). The expression of each time point in the group of anisodamine and amyl hydrochloric acid was higher than that in the acute renal injury group (P0.05).Shh in the renal tubular epithelial cells in the control group. The expression of Shh in the acute renal injury group was weakened at 6 hours after the glycerol injection (P0.05) and 24 hours. Group Shh expression enhanced (P0.05).72 hour group and Shh expression increased continuously (P0.05). The time points of anisodamine group and amyl hydrochloric acid group were compared with those of acute renal injury group at the same time (P0.05). The expression of Shh 6h, 24h expression in group PHC was compared with that of group AD in the same period of the AD group. Conclusion: when 1 rhabdomyolysis induced acute renal injury, the early expression of Shh protein in renal tissue decreased and later expression increased, indicating that rhabdomyolysis induced acute renal injury could reactivate the key protein Shh in the Shh signaling pathway.2 Shh signaling pathway, Gli2 at 24 hours, up regulation up to 72 hours, indicating that activation of Shh signaling pathway may be associated with acute renal damage. Renal tissue protection and repair play an important role in.3 anticholine drug anisodamine. Amyl hydrochloric acid may partially activate Shh signaling pathway to alleviate acute renal injury and promote early repair of damaged tissue, and provide reference for clinical use.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692;R685.5

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