糖原合酶激酶-3β在大鼠重症急性胰腺炎肾损伤中的作用及机制研究
发布时间：2018-04-10 10:53
本文选题：重症急性胰腺炎 + 肾损伤 ；参考：《武汉大学》2014年博士论文
【摘要】：第一部分：糖原合酶激酶-3p在重症急性胰腺炎大鼠肾损伤中的表达及作用研究 目的：探讨重症急性胰腺炎(SAP)大鼠模型中肾脏的组织形态、超微结构和功能变化,研究GSK-3β在SAP大鼠肾脏组织的变化规律及其可能作用。 方法：采用SPF级雄性Wistar大鼠,随机分为5组(N=12)。假手术组(SO组),重症急性胰腺炎组(SAP3h、6h、12h、24h四个亚组),通过胆胰管逆行注射5%牛磺胆酸钠溶液制备大鼠SAP模型。分别测定各组大鼠血清淀粉酶(AMY)、脂肪酶(LIPA)、肌酐(Cr)和尿素氮(BUN)水平,应用HE染色法观察各组大鼠胰腺和肾脏组织病理学改变,透射电镜观察大鼠肾脏细胞超微结构变化,采用Western-blot法检测肾脏组织中GSK-3β及其磷酸化形式p-GSK-3p ser9在各组中的表达变化。 结果：SAP大鼠血清AMY.LIPA.胰腺病理评分随着时间进展水平逐渐升高,而Cr、BUN及肾脏病理评分在SAP3h、6h、12h呈现逐渐增高的趋势,而SAP12h、24h组基本呈现一个水平表达状态,差异无统计学意义(P0.05),SAP各组大鼠肾脏GSK-3β蛋白表达较SO组表达明显增高(P0.05),但SAP各组之间比较差异无统计学意义(P0.05)；β-GSK-3β ser9在SO组表达不高,而在SAP3h时组,表达较SO组增高,但从SAP6h组开始,随着病程的进展,p-GSK-3β ser9的表达却呈现出逐渐减弱的趋势(P0.05)。结论：随SAP病程进展,大鼠胰腺、肾脏病理损伤呈进行性加重,肾功能检测指标Cr、BUN亦出现相应变化。大鼠肾脏组织中GSK-3β并未随SAP的病程进展出现明显表达变化,但其磷酸化形式的p-GSK-3β ser9表达却呈现出先增强,后逐渐减弱的趋势,表明GSK-3β的磷酸化调控可能在SAP并发肾损伤的发病过程中发挥重要作用。第二部分：不同类型GSK-3p抑制剂对重症急性胰腺炎大鼠肾损伤的作用比较及量效关系探讨 目的：观察TDZD-8干预大鼠SAP肾损伤的量效关系,并将三种常用GSK-3β抑制剂TDZD-8、氯化锂(LiCL)、SB216763对该模型的作用效果进行对比,以探讨针对SAP并发肾损伤大鼠模型最有效的GSK-3β抑制剂类别及其有效、安全的最佳剂量。方法：96只SPF级雄性Vistar大鼠,随机分为8组(N=12)：假手术组(SO组)、重症急性胰腺炎组(SAP组)、TDZD-8预处理组0.25、0.5、1、2mg/kg(TD组,分别标记为TD1、TD2、TD3、TD4组),LiCL预处理组(L组)和SB216763预处理组(SB组),胰胆管逆行注射5%牛磺胆酸钠制作SAP模型。SO组、SAP组均于术前30min经股静脉注射溶剂10%DMSO (O.lml/100g), TDZD-8各剂量预处理组SAP造模前30min经股静脉注射等量10%DMSO溶解的不同剂量的TDZD-8；L组和SB组分别经股静脉注射等体积10%DMSO溶解的LiCL(60mg/kg)和SB216763(1mg/kg)。术后12h剖杀各组大鼠,测定各组大鼠死亡率、腹水量、血清AMY、Cr、BUN和ALT水平,并观察胰腺、肾脏组织病理学变化。 结果：SO、SAP、TD3、L和SB组大鼠死亡率分别为0%、33.3%、0%、8.3%和16.6%；SAP组腹水量、AMY、Cr、BUN、ALT值以及胰腺、肾脏病理评分均较SO组显著升高(P0.05)；TD1组几乎对SAP无缓解作用,TD2、TD3、TD4、L组和SB组均能不同程度地减少SAP大鼠的腹水量,降低血清AMY、Cr、BUN值,并显著降低胰腺组织病理评分,差异有统计学意义P0.05)；TD2、TD3均能不同程度地减少ALT值(P0.05),而TD4组ALT值较高,与SAP组相似；TD2与TD3组比较,对各个指标均有效果,但效果不如TD3组作用显著,两者比较各项指标均有显著差异(P0.05)。TD组中最佳剂量组TD3组在腹水量、ALT值之间与L组和SB组比较,无显著性差异(P0.05)；而TD3组的AMY、Cr、BUN值以及胰腺、肾脏病理评分均较L组和SB降低更显著,差异有统计学意义(P0.05)；L组Cr、BUN值和胰腺、肾脏病理学评分与SB组比较,均较低,差异有统计学意义(P0.05)； 结论：通过对TDZD-8、LiCL和SB216763对大鼠SAP肾损伤模型的作用比较可知TDZD-8是针对该模型最有效的GSK-3β抑制剂。对于牛磺胆酸钠诱导的SAP并发肾损伤大鼠模型,静脉给予TDZD-81mg/kg预处理对该模型是安全有效的最佳剂量。第三部分：GSK-3p抑制剂TDZD-8对大鼠重症急性胰腺炎肾损伤的保护作用机制探讨目的：观察抑制GSK-3β活性对SAP大鼠肾脏组织病理和超微结构的影响,检测肾脏组织NF-κB激活及其依赖性基因的蛋白表达变化,探讨GSK-3β抑制剂TDZD-8对SAP肾损伤保护作用的机制。方法：SPF级雄性Wistar大鼠,随机分为4组(N=20)。SO组(sham+vehicle组)、SAP组(SAP+vehicle组)、TDZD-8治疗组(SAP+TDZD-8组)和TDZD-8药物对照组(sham+TDZD-8组),以12h为观察点。SO组、SAP组均于术前30min经股静脉注射TDZD-8溶剂10%DMSO(0.1ml/100g),TDZD-8治疗组在SAP造模前30min经大鼠股静脉注射GSK-3β抑制剂TDZD-8,TDZD-8药物对照组在操作前30min经大鼠股静脉注射与TDZD-8治疗组等体积的GSK-3β抑制剂TDZD-8,余操作同SO组。术后12h剖杀各组大鼠,测定各组大鼠血清AMY、Cr、BUN水平并观察胰腺、肾脏组织病理学变化,电镜观察肾脏细胞超微形态结构变化,比色法检测肾脏组织髓过氧化物酶(MPO)活性,ELISA法检测IL-1β、IL-6在各组大鼠血清中含量,免疫组化法检测NF-κB p65在大鼠肾脏中的定位表达,Western-blot法检测肾脏组织GSK-3β、p-GSK-3β ser9、NF-κB p65、TNF-α、iNOS、ICAM-1和IL-10的表达水平。 结果：与SAP组比较,TDZD-8可显著降低SAP大鼠血清中AMY、LIPA、Cr、BUN含量,减轻胰腺、肾脏病理损伤,并减轻大鼠肾脏细胞超微结构损伤(P0.05)。TDZD-8治疗组肾脏组织MPO活性、以及血清IL-1p、IL-6活性与SAP组比较,均有明显下降(P0.05)。免疫组化结果示：SAP组中NF-κB p65的表达较SO组增强,且主要在细胞核内表达,而TDZD-8治疗组则较SAP组表达明显减少。Western-blot结果显示：SAP组和TDZD-8治疗组GSK-3p表达较SO组和TDZD-8药物对照组明显增强(P0.05),而SAP组和TDZD-8治疗组表达无显著差异。SAP组肾脏组织中p-GSK-3β ser9的表达较SO组明显减弱(P0.05),而TDZD-8治疗组大鼠肾脏组织中p-GSK-3β ser9的表达较SAP组表达则明显增多(P0.05)；SAP组肾脏组织中NF-kB p65、TNF-α、ICAM-1、iNOS的表达较SO组明显增强,IL-10表达明显减弱(P0.05),阻断GSK-3β活性能够抑制NF-κB p65、TNF-α、ICAM-1、iNOS的表达,提高IL-10的表达(P0.05)。各项指标检测结果显示TDZD-8药物对照组均与SO组无显著差异(P0.05)。 结论：GSK-3β抑制剂TDZD-8可有效导致SAP大鼠肾脏GSK-3β磷酸化而使其活性受到抑制,从而抑制肾脏NF-κB炎症通路激活以及中性粒细胞的募集,进一步抑制其下游炎症介质(TNF-α、IL-1β、IL-6、ICAM-1、iNOS)的释放,提高保护因子IL-10的释放,从而减轻肾脏炎症及病理损伤,缓解SAP病情进展。
[Abstract]:Part one: expression and role of glycogen synthase kinase -3p in renal injury of severe acute pancreatitis in rats
Objective: To investigate the changes of renal morphology, ultrastructure and function in rats with severe acute pancreatitis (SAP), and to explore the possible role of GSK-3 beta in renal tissue of SAP rats.
Methods: male Wistar SPF rats were randomly divided into 5 groups (N=12). Sham operation group (SO group), severe acute pancreatitis group (SAP3h, 6h, 12h, 24h four groups), SAP rat model by retrograde injection of 5% sodium taurocholate solution prepared by serum bile duct. The rats were measured by amylase, lipase (AMY) (LIPA), creatinine (Cr) and urea nitrogen (BUN) level, observe changes of pancreas and kidney tissue of rats with HE pathological staining, observe the ultrastructure changes of rat kidney cells by transmission electron microscope, the expression of GSK-3 in renal tissue and form beta phosphate Western-blot method was used to detect p-GSK-3p ser9 in each group.
Results: SAP rats serum AMY.LIPA. pancreatic pathological score as time progresses levels increased gradually, while Cr, BUN and renal pathological score in SAP3h, 6h, 12h increased markedly, while SAP12h, 24h group showed a level of expression, the difference was not statistically significant (P0.05), compared with the SO group increased significantly. Beta protein GSK-3 in rat kidney SAP expression in each group (P0.05), but there was no significant difference between the groups of SAP (P0.05); beta -GSK-3 beta ser9 expression is higher in the SO group, in SAP3h group, the expression was higher than the SO group, but in SAP6h group, along with the progress of the disease, the expression of p-GSK-3 beta ser9 has gradually weakened (P0.05). Conclusion: with the progression of SAP, rat pancreas, kidney pathological damage is progressive, Cr detection index of renal function, BUN also changed. The renal tissues of rats in GSK-3 beta with SAP progression did not appear Clear expression, but the p-GSK-3 beta ser9 phosphorylated forms of expression are enhanced first, after gradually weakened, showed that phosphorylation of GSK-3 beta may play an important role in the pathogenesis of SAP with renal injury. The second part: comparative study on renal injury in rats with severe acute pancreatitis and the effect of the amount of effect of different types of GSK-3p inhibitors
Objective: To observe the dose effect relationship of TDZD-8 intervention in SAP rats with kidney injury, and three kinds of commonly used GSK-3 beta TDZD-8 inhibitors, lithium chloride (LiCL), SB216763 compares the effect of the model, to explore for SAP patients with renal injury in rats model of GSK-3 beta inhibitors most effective and effective optimal dose not. Safety. Methods: 96 male Vistar SPF rats were randomly divided into 8 groups (N=12): sham operation group (SO group), severe acute pancreatitis group (SAP group), TDZD-8 pretreatment group (group TD, 0.25,0.5,1,2mg/kg were labeled as TD1, TD2, TD3, TD4 group), LiCL treatment group (L group) and SB216763 pretreatment group (SB group), pancreatic duct retrograde injection of 5% sodium taurocholate SAP model.SO group, SAP group were on preoperative 30min by intravenous injection solvent 10%DMSO (O.lml/100g), different doses of TDZD-8 preconditioning group SAP before modeling 30min by intravenous injection of the same amount of 10%DMSO the dissolution of different agents The amount of TDZD-8; group L and group SB were injected with equal volume 10%DMSO dissolved LiCL (60mg/kg) and SB216763 (1mg/kg) respectively via femoral vein. After operation, 12h was used to kill rats in each group. The mortality, ascites volume, serum AMY, Cr, level of 12h and level of rats in each group were measured, and pathological changes in pancreas and kidney were observed.
缁撴灉锛歋O,SAP,TD3,L鍜孲B缁勫ぇ榧犳浜＄巼鍒嗗埆涓，
本文编号：1730896
本文链接：https://www.wllwen.com/yixuelunwen/jjyx/1730896.html
最近更新
教材专著
