GSK-3β抑制剂LiCl对重症急性胰腺炎大鼠肝脏损伤的保护作用及其机制的研究

发布时间：2018-03-07 07:27

  本文选题：重症急性胰腺炎　切入点：氯化锂　出处：《武汉大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分：静脉注射GSK-3β的抑制剂LiCl干预重症急性胰腺炎大鼠量效关系的探讨 目的：探讨静脉注射糖原合成酶激酶-3β (Glycogen synthase kinase-3β, GSK-3β)抑制剂氯化锂(Lithium Chloride, LiCl)干预重症急性胰腺炎(severe acute pancreatitis, SAP)大鼠模型的量效关系,从而为LiCl干预重症急性胰腺炎肝脏损伤(acute pancreatitis associated liver injury)选择适合的用药剂量提供理论依据。 方法：雄性、SPF级、Wistar大鼠50只,体重200-250g,随机将大鼠分为5组(N=10)即为：假手术组(sham operation group, SO组),重症急性胰腺炎模型组(severe acute pancreatitis, SAP组),LiCl-40mg/kg预处理组、LiCl-60mg/kg预处理组以及LiCl-80mg/kg预处理组(LiCl组)。所有大鼠操作之前均需禁食12小时,但可以自由饮水。10%水合氯醛腹腔注射(0.3ml/100g)麻醉大鼠后,取上腹部正中切口入腹,采用微量泵、经胆胰管逆行匀速注射新鲜配制的5%牛磺胆酸钠溶液(sodium taurocholate, STC)(0.1ml/100g)制备重症急性胰腺炎大鼠模型；SO组操作如上说述,但是在胆胰管内注射等量生理盐水替代牛磺胆酸钠。LiCl干预各剂量组均于造模前30min给予股静脉注射对应浓度的LiCl溶液,剂量分别为40、60、80mg/kg。各组大鼠于模型制作后12h剖杀,棉球吸收法检测腹水量,下腔静脉穿刺取血,离心分装冻存待测,使用大鼠右下肺叶检测肺组织湿干比,使用大鼠胰头部组织,固定包埋。切片染色。分别测定各组大鼠腹水量和肺湿干比(反应肺脏含水量)、全自动生化仪测定血清淀粉酶(amylase, AMY)和肝肾功能指标如谷丙转氨酶(alanine aminotransferase, ALT)、肌酐(creatinine, Cr)水平。光镜下观察胰腺组织病理学变化并进行病理学评分。 结果：SAP组大鼠的腹水量、血清淀粉酶、谷丙转氨酶、肌酐水平、肺湿干比以及胰腺病理评分均较SO组显著升高,其差异有统计学意义(P0.05)。LiCl-40mg/kg的干预组上述指标(即腹水量、淀粉酶、肺脏组织湿干比以及胰腺病理学评分)与SAP组比较无明显差异(P0.05)。LiCl-60mg/kg的干预组的全部指标(包括腹水量、淀粉酶、谷丙转氨酶、肌酐、肺组织湿干比以及胰腺病理评分)与SAP组相比较均有明显下降,差异有统计学意义(P0.05)。LiCl-80mg/kg预处理组中上述部分指标(如腹水量、血清AMY、肺湿干比、胰腺病理评分)较SAP组显著降低,差异有统计学意义(P0.05)；但是其ALT水平升高大于SAP组,其差异有统计学意义(P0.05)、Cr值与SAP组比较差异无统计学意义(P0.05)。 结论：60mg/kgLiCl能有效缓解胰腺炎病情,表现为减少腹水量、降低血清淀粉酶水平和缓解胰腺损伤的病理学评分,同时可缓解肝肾功能的损伤。而80mg/kgLiCl虽能缓解胰腺炎病情,但使用此浓度干预存在潜在的肝毒性与肾毒性。因此通过综合分析,我们认为60mg/kg的LiCl剂量是干预重症急性胰腺炎大鼠模型的相对最佳有效剂量。 第二部分：GSK-3β抑制剂LiCl对重症急性胰腺炎大鼠肝损伤的作用 目的：探讨GSK-3β抑制剂LiCl对重症急性胰腺炎(severe acute pancreatitis, SAP)大鼠肝损伤的作用。 方法：雄性、SPF级、Wistar大鼠70只,体重200～250g,随机分为4组。假手术组(SO组)(N=10)；重症急性胰腺炎模型组(SAP组)(N=40),按照时间点分为1h,3h,6h,12h四个亚组,每组大鼠10只；GSK-3β抑制剂LiCl预处理组(LiCl组)(N=10), LiCl药物对照组(Drug-CON组)(N=10)。重症急性胰腺炎模型组及LiCl预处理组大鼠需使用逆行胆胰管注射牛磺胆酸钠法制作SAP模型。需要使用LiCl的LiCl预处理组及LiCl药物对照组需要在制作相关模型之前使用静脉穿刺注射法给予60mg/kg的LiCl溶液。假手术组、GSK-3β抑制剂LiCl预处理组、GSK-3β抑制剂LiCl药物对照组于均选择在12h处死大鼠,SAP各亚组大鼠需根据分组情况,在相应的时间点剖杀。棉球吸收法检测腹水量,下腔静脉穿刺取血,离心分装冻存待测,使用大鼠右下肺叶检测肺组织湿干比,使用大鼠胰头部组织,固定包埋。分别记录和汇总各组大鼠的死亡率、腹水量,全自动生化仪血清淀粉酶(AMY)和磷脂酶水平(PLA2),肝功能通过测定谷丙转氨酶(ALT)与谷草转氨酶(AST)来评价,各组大鼠均行常规胰腺与肝脏病理学HE染色检查,并评分分级。 结果：药物对照组与SO组在死亡率、腹水量、AMY、ALT、AST、PLA2以及胰腺和肝脏的病理评分与分级无明显差异(P0.05)。 SAP各组,随着造模时间的延长,上述指标均有所增加,各组与SO组对比均升高,差异有统计学意义(P0.05)。对比LiCl干预组与SAP组相应时间点亚组发现,使用LiCl干预后,上述指标均有所改善,差异具有统计学意义(P0.05),但这种改变并不能达到治愈效果,相比于SO组水平依旧有所升高(P0.05)。 结论：在使用LiCl干预后,大鼠的胰腺与肝脏酶学指标均有所下降,胰腺组织与肝脏病理学评分分级有所下降,提示LiCl对于SAP大鼠的胰腺炎以及由胰腺炎导致的肝损伤具有一定的保护作用。 第三部分：GSK-3β抑制剂LiCl对重症急性胰腺炎大鼠肝损伤保护作用的机制探讨 目的：通过实验进一步探讨静脉给药对大鼠重症急性胰腺炎肝损伤保护作用的机制。 方法：雄性、SPF级、Wistar大鼠60只,体重200-250g,随机分为3组。假手术组(SO组)(N=10)：重症急性胰腺炎模型组(SAP组)(N=40),根据造模后的时间段分为1h,3h,6h,12h四个亚组,每组大鼠10只；GSK-3β抑制剂LiCl预处理组(LiCl组)(N=10)。重症急性胰腺炎模型组及LiCl预处理组大鼠需使用逆行胆胰管注射牛磺胆酸钠法制作SAP模型。需要使用LiCl的LiCl预处理组需要在制作相关模型之前使用静脉穿刺注射法给予60mg/kg的LiCl溶液。假手术组、GSK-3β抑制剂LiCl预处理组均于12h处死大鼠,SAP各亚组大鼠需根据分组情况,在相应的时间点剖杀。取大鼠胰头部位组织和肝脏右叶部分只固定包埋,切片并行免疫组织化学染色,检测GSK-3β和NF-κB。使用胰尾部分与剩余的右叶肝脏,以Western-Blot法检测NF-κB与ICAM-1水平。 结果：Western-Blot的结果提示：SAP各组大鼠肝脏与胰腺组织NF-κB水平随着造模时间的延长而升高；各组大鼠NF-κB水平相比于SO组均增高,差异具有统计学意义(P0.05)；使用LiCl干预组的大鼠NF-κB水平相比于SAP组明显下降,差异具有统计学意义(P0.05)。ICAM-1的变化与NF-κB基本一致,使用LiCl干预组的ICAM-1表达与同时间点的SAP组对比明显下降,差异具有统计学意义(P0.05)。免疫组化检测结果显示,SAP各组大鼠GSK-3β的水平随着造模时间的延长而不断增加,至12h时达到峰值,在给予LiCl干预后GSK-3β的表达量下降,差异具有统计学意义(P0.05)。NF-ΚB的改变除了进一步证实了Western的结果之外,还提示在SAP过程中NF-ΚB的定位发生了改变,出现由胞浆到核内的移动。 结论：GSK-3p抑制剂Lic1通过抑制GSK-3β的活化下调了肝脏组织NF-κB的活化,通过抑制NF-κB的活化又抑制了ICAM-1的表达,从而通过抑制炎症瀑布效应从而减轻急性胰腺炎肝损伤的程度
[Abstract]:Part one: A Study on the dose effect relationship of GSK-3 beta inhibitor LiCl in rats with severe acute pancreatitis
Objective: To investigate the effect of intravenous injection of glycogen synthase kinase -3 beta (Glycogen synthase kinase-3 GSK-3 beta, beta) inhibitor lithium chloride (Lithium Chloride, LiCl) intervention for severe acute pancreatitis (severe acute, pancreatitis, SAP) dose effect relationship of rat model, and intervention of hepatic injury in severe acute pancreatitis (LiCl acute pancreatitis associated liver injury) selection the ideal dose and provide a theoretical basis.
Methods: male, SPF, 50 Wistar rats, weight 200-250g, the rats were randomly divided into 5 groups (N=10) namely: sham operation group (sham operation, group, SO group), severe acute pancreatitis group (severe acute, pancreatitis, SAP group), LiCl-40mg/kg pretreatment group, LiCl-60mg/kg pretreatment group and LiCl-80mg/kg pretreatment group (LiCl group). All rats before operation are required 12 hours of fasting, but free drinking water.10% intraperitoneal injection of chloral hydrate (0.3ml/100g) in anesthetized rats after taking median incision into the abdomen, the micro pump, 5% sodium taurocholate solution after bile duct retrograde infusion freshly prepared (sodium taurocholate, STC) (0.1ml/100g) prepared in a rat model of severe acute pancreatitis; SO group said the operation above, but instead of sodium taurocholate.LiCl intervention groups were given 30min before modeling in intraductal injection of saline Stock solution of LiCl intravenous injection of the corresponding concentration, dose rate of 40,60,80mg/kg. rats in model 12h after slaughter, cotton absorption method was used to detect the amount of ascites, inferior vena cava puncture blood centrifugal freezing test using packaging, detection of lung tissue of rats with right lobe under wet and dry, the head of the pancreas tissue of rats fixed, embedded. Staining of rats were measured. The ascites volume and lung wet dry ratio (reaction lung moisture), serum amylase automatic biochemical analyzer (amylase, AMY) and liver function indexes such as alanine aminotransferase (alanine aminotransferase ALT), creatinine (creatinine, Cr) level of light. Observe the pathologic changes of the pancreas and the pathological score.
Results: serum amylase, ascites volume, the rats in group SAP, alanine aminotransferase, creatinine level, wet / dry ratio of the lung and pancreas pathological score were significantly increased compared with the SO group, the difference was statistically significant (P0.05.LiCl-40mg/kg) of the intervention group the index (i.e. abdominal content, amylase, lung wet to dry ratio and pancreatic pathology no significant difference was found between the scores) and group SAP (P0.05).LiCl-60mg/kg in the intervention group all indexes (including the amount of ascites, amylase, alanine aminotransferase, creatinine, lung wet / dry ratio and pancreas pathological score) compared with the SAP group were significantly decreased, the difference was statistically significant (P0.05) above indicators.LiCl-80mg/kg treatment group (such as abdominal water, serum AMY, lung wet to dry ratio, pancreatic pathological score) decreased significantly compared with SAP group, the difference was statistically significant (P0.05); but the elevated levels of ALT than in the SAP group, the difference was statistically significant (P0.05 There was no significant difference between the Cr value and the SAP group (P0.05).
Conclusion: 60mg/kgLiCl can effectively alleviate pancreatitis and to reduce the amount of ascites, reduce the level of serum amylase and alleviate the pathological score of pancreatic injury, also can relieve liver and kidney function damage. 80mg/kgLiCl can alleviate pancreatitis, but with this concentration of intervention has the potential for hepatotoxicity and renal toxicity. Therefore, through comprehensive analysis, we that LiCl dose 60mg/kg intervention model of rats with severe acute pancreatitis is the best effective dose.
The second part: the effect of GSK-3 beta inhibitor LiCl on liver injury in rats with severe acute pancreatitis
Objective: To investigate the effect of GSK-3 beta inhibitor LiCl on liver injury in severe acute pancreatitis (SAP) rats.
鏂规硶锛氶泟鎬，

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