【作者】 陈诚；

【导师】 徐萍；

【作者基本信息】 南京医科大学 ， 内科学， 2013， 硕士

【摘要】 背景和目的重症急性胰腺炎发病早期,在各种损伤因子的刺激下单核/巨噬细胞等被激活,释放出大量的促炎细胞因子。多种促炎细胞因子(TNF-α、IL-1β、IL-6等)在SAP的病情演变过程中起着至关重要的作用。研究表明NF-κB、p38MAPK信号通路在SAP发病中起重要作用,促炎细胞因子产生与NF-κB、p38MAPK等细胞内信号转导通路异常激活有关。我们的前期工作发现SAP时NF-κB、p38MAPK信号通路均可被活化,并通过产生大量细胞因子,在SAP发病中起重要作用。近年来,磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路信号转导通路介导炎症、应激等细胞反应在急性胰腺炎发生发展中的作用引人注目。有研究表明SAP时促炎细胞因子的表达和释放与PI3K/Akt信号转导通路异常激活有关,在重症急性胰腺炎的全身炎症反应中发挥作用,而抑制PI3K/Akt信号通路的激活可以明显减轻SAP发病时的炎症反应,但作用机制尚不清楚。本研究观察SAP发病早期PI3K/Akt信号转导通路在大鼠胰腺组织中的表达情况,并通过抑制PI3K/Akt信号转导通路来研究PI3K/Akt通路对细胞因子(TNF-α、IL-1β、IL-6)转录和表达以及胰腺组织病理评分的影响和PI3K/Akt通路在NF-κB、p38MAPK通路活化中的作用,探讨PI3K/Akt信号通路在重症急性胰腺炎大鼠发病早期的作用及其机制。方法第一部分:将SD大鼠60只随机分为重症急性胰腺炎组(S组)、假手术组(C组)、生理盐水组(NS组)、溶剂(DMSO)对照组(R组)、SAP+PI3K/Akt抑制剂wortmannin组(W组)。采用改良的Aho法制作SAP模型,分别在造模3、6h后抽血并处死大鼠,收集胰腺组织。采用酶联免疫吸附试验(ELISA)检测血清细胞因子(TNF-α、IL-1β、IL-6)表达。Real-time PCR方法检测胰腺组织细胞因子(TNF-α、IL-1β、IL-6)m RNA转录水平变化,并常规HE染色进行胰腺组织病理评分,同时观察各组腹水量、血清及腹水淀粉酶的变化。第二部分:72只SD大鼠随机分为:假手术对照组(SO组)、重症急性胰腺炎组(SAP组)、SAP+PI3K抑制剂wortmannin组(W组),每组24只。采用改良的Aho法制作SAP模型。假手术或造模3、6h后分别处死12只大鼠并立即心脏取血,收集大鼠胰腺组织。采用实时定量PCR(Real-time PCR)和Western Blot检测胰腺组织中Akt、p38MAPK、NF-κBp65 m RNA及蛋白磷酸化的表达情况。采用酶联免疫吸附试验(ELISA)检测血清细胞因子(TNF-α、IL-1β、IL-6)表达。同时检测血清淀粉酶水平,HE染色评估胰腺损伤程度。结果第一部分:S组和R组大鼠术后3、6小时的腹水量、血清及腹水淀粉酶水平、胰腺组织病理评分较C组和NS组增高,差异有统计学意义(P<0.05);W组的腹水量、血清及腹水淀粉酶水平、胰腺组织病理评分较同时段的S组和R组有所降低,差异有统计学意义(P<0.05)。与C组和NS组相比,S组和R组大鼠术后3、6小时的血清细胞因子TNF-α、IL-1β、IL-6表达水平和胰腺组织内TNF-α、IL-1β、IL-6 m RNA转录水平增高,差异有统计学意义(P<0.05);W组的上述指标均低于S组和R组,差异有统计学意义(P<0.05)。第二部分:SAP组与SO组比较,大鼠胰腺组织中Akt、p38MAPK、NF-κBp65 m RNA表达及蛋白磷酸化水平、血清细胞因子(TNF-α、IL-1β、IL-6)含量、血清淀粉酶表达水平和胰腺组织病理学评分在3h、6h均升高,差异有统计学意义(P<0.05)。SAP模型建成3h、6h后,W组与同时段的SAP组相比,大鼠胰腺组织中Akt的转录活性和蛋白磷酸化程度降低(P<0.05)的同时,p38MAPK、NF-κBp65 m RNA表达和磷酸化水平也明显降低(P<0.05)。W组的血清细胞因子(TNF-α、IL-1β、IL-6)表达、血清淀粉酶、胰腺组织病理学评分均低于同时段的SAP组,差异有统计学意义(P<0.05)。结论PI3K/Akt信号转导通路的激活介导重症急性胰腺炎时p38MAPK、NF-κB通路的活化,影响SAP大鼠细胞因子的转录和表达水平,与SAP大鼠早期炎症反应密切相关。抑制PI3K/Akt信号转导通路不仅可以抑制Akt活化,还可以下调p38MAPK、NF-κB的活性,减少促炎因子(TNF-α、IL-1β、IL-6)的转录和表达,减轻大鼠胰腺组织的病理损伤,对SAP大鼠具有保护作用。更多还原

【Abstract】 ObjectiveIn the early stage of severe acute pancreatitis, monocytes/macrophages are activated under the stimulation of various injury factors, and release a large number of pro-inflammatory cytokines. Multiple pro-inflammatory cytokines(TNF-α, IL-1β, IL-6, etc.) play an important role in the occurrence and development of severe acute pancreatitis. Studies shows that the activation of NF-κB and p38 MAP kinase signaling pathway is an early and critical event in SAP. Production of pro-inflammatory cytokines are related to the activation of NF-κB and p38 MAPK pathway.We have found that NF-κB and p38 MAPK signaling pathway can be activated and play a significant role in the SAP by the release of massive cytokines.In recent years, the role of cell responses such as inflammation and stress mediated by phosphatidylinositol 3-kinase/ser-ine threonine kinase(PI3K/Akt) signaling pathway in the occurrence and development of acute pancreatitis has aroused widespread interest. Some research has shown that the expression and release of cytokines is associated with abnormal activation of PI3K/Akt signal pathway during SAP and PI3K/Akt pathway plays a role in systemic inflammatory response induced by SAP. While inhibition of PI3K/Akt signaling pathway’s activation can significantly reduce inflammatory response of SAP, but the mechanism was still not clear. In this study, we observe the expression of PI3K/Akt signal transduction pathway in pancreatic tissue at the early stages of SAP. And we inhibit PI3K/Akt signal transduction pathway to research the effects on the transcription and expression of cytokines(TNF- α, IL-1 beta, IL-6) and pathological scores of pancreatic tissue, to investigate the role of PI3K/Akt pathway in the activation of NF-κb and p38 MAPK pathways, and to clarify the effects of PI3K/Akt signaling pathway and its possible mechanisms during the early stages of severe acute pancreatitis.MethodsPart One: Sixty male Sprague-Dawley(SD) rats were randomly divided into five groups, including SAP group, sham operation group, normal saline group, DMSO control group, wortmannin group. The modified Aho’s method was used to reproduce the SAP model. The rats were sacrificed 3 and 6 h after treatment. The levers of inflammatory cytokines TNF-α, IL-1β and IL-6 in serum were examined by ELISA.Transcription levers of these inflammatory cytokines in pancreatic tissue were determined by real-time PCR. In addition, the amount of serum amylase, the activities of serum amylase and ascites amylase, and the pathological scores of pancreatic tissue were also measured.Part Two: Seventy-two male Sprague-Dawley(SD) rats were randomly divided into three groups, including SAP group, sham operation group, wortmannin group. The modified Aho’s method was used to reproduce the SAP model. The rats were sacrificed 3 and 6 h after treatment. The levers of inflammatory cytokines TNF-α, IL-1β and IL-6 in serum were examined by ELISA. Transcription levers of Akt, p38 MAPK and NF-κBp65 in pancreatic tissue were determined by real-time PCR. Protein expression and phosphorylation of Akt, p38 MAPK, NF-κB p65 were detected by Western blot. In addition, the activities of serum amylase and pathological scores of pancreatic tissue were measured.ResultsPart One: After 3 and 6 h after treatment,all parameters tested, including the amount of ascites, the levels of serum and ascites amylase, the pathological scores of pancreatic tissue, serum levels of TNF-α, IL-1β and IL-6, and the transcription levels of TNF-α, IL-1βand IL-6 m RNAs in the pancreatic tissue in SAP group and DMSO group were significantly higher than those in the normal saline group and sham operation group(all P<0.05). Compared to the SAP group and DMSO group, The above parameters decreased significantly in the wortmannin group(all P<0.05).Part Two: After 3 and 6 h after treatment,all parameters tested, including the levels of protein phosphorylation of Akt, p38 MAPK, NF-κB p65, the transcription levels of Akt 、p38MAPK and NF-κBp65 m RNAs in the pancreatic tissue,serum levels of TNF-α, IL-1β and IL-6,the levels of serum amylase, the pathological scores of pancreatic tissue in SAP group were significantly higher than those in sham operation group(all P<0.05). Compared to the SAP group, the above parameters decreased significantly in the wortmannin group(all P<0.05).ConclusionPI3K/Akt signaling transduction pathway induces the activation of p38 MAPK and NF-κB pathway, affects the transcription and expression of cytokines during severe acute pancreatitis(SAP), plays an important role in the early-stage inflammatory response in rats with SAP. By inhabiting PI3K/Akt signaling transduction pathway, the severity of inflammatory response of SAP is attenuated through down-regulateing the activation of Akt、p38MAPK and NF-κB and decreaseing the transcription and expression of cytokines(TNF-α、IL-1β and IL-6).更多还原