【作者】 范海清；

【导师】 徐萍；

【作者基本信息】 南昌大学 ， 内科学， 2008， 硕士

【摘要】 目的:研究核转录因子-κB在牛磺胆酸钠诱导的大鼠重症急性胰腺炎中的动态变化规律,以及PPARγ激动剂吡格列酮对大鼠重症急性胰腺炎的干预作用。方法:共130只健康雄性Sprague-Dawley大鼠分为二大部分:第一部分:40只健康雄性Sprague-Dawley大鼠,体重160-200g,随机分为假手术组(C组)、重症急性胰腺炎组(A组)和吡格列酮预处理组三个剂量组(P1组、P2组、P3组),每组8只。采用盐酸氯胺酮腹腔注射麻醉下逆行胰胆管内加压注射5%牛磺胆酸钠(0.1ml/100g)建立SAP模型,P组大鼠在术前2h腹腔内注射吡格列酮(20mg/kg、50mg/kg、100mg/kg),造模后大鼠自由摄食和饮水,比较各组大鼠的生存时间和60小时的生存率。第二部分:90只健康雄性Sprague-Dawley大鼠,体重160-200g,随机分为假手术组(C组)、重症急性胰腺炎组(A组)和吡格列酮预处理组三个剂量组(P1组、P2组、P3组),每组18只。采用盐酸氯胺酮腹腔注射麻醉下逆行胰胆管内加压注射5%牛磺胆酸钠(0.1ml/100g)建立SAP模型。P组大鼠在术前2h腹腔内注射吡格列酮(20mg/kg、50mg/kg、100mg/kg)。分别于术后3、6、12h 3个时间点采用腹主动脉放血法将大鼠分批处死(每次每组6只),取腹水进行淀粉酶检测,并取腹主动脉血采用ELISA法测血清炎症细胞因子IL-6的水平;取胰腺组织,分为两部分:第一部分立即放于10%中性甲醛溶液(100ml/L)固定,用于进行常规HE染色;第二部分立即放入液氮罐内冻存用于Western blot法检测。采用Western blot法检测胰腺组织NF-κB蛋白的表达,同时进行胰腺组织病理学评分的测定,并观察吡格列酮预处理后上述各指标的变化。结果:1.五组大鼠的生存时间和60小时的生存率(第一部分):P组大鼠生存时间较A组明显延长,但低于C组(P<0.01),P2组的生存时间与A组的差异有统计学意义(P<0.05),P2组和P3的生存时间与A组的差异无统计学意义。A组大鼠造模后60h的生存率是12.5%,P1、P2、P3组的生存率分别为25%、37.5%、37.5%,A组与P2组比较,差异有统计学意义(P<0.05)。说明P2疗效优于P1和P3组(见表3.1、表3.2)。2.腹水淀粉酶变化:C组无腹水形成,P组腹水淀粉酶水平在各时间点与A组相比,有所降低,但差异无统计学意义(P>0.05)(见表3.3)。3.胰腺组织病理学评分:与C组相比,A组、P组各时间点均明显升高,差异有统计学意义(P<0.01);P组与A组相比,在3h差异无统计学意义(P>0.05),在6h、12h差异有统计学意义(P<0.05)(见表3.4)。4.血清IL-6水平变化:与C组相比,A组、P组各时间点均明显升高,差异有统计学意义(P<0.01),P组与A组相比,在3h差异无统计学意义(P>0.05),在6h、12h相差异有统计学意义(P<0.05)(见表3.5)。5.胰腺组织NF-κB p65蛋白的表达变化:胰腺组织NF-κBp65的活化程度:与C组相比,A组、P组各时间点活化程度均明显升高,差异有显著性(P<0.01); A组与P2组相比,在3h差异无统计学意义,在6h、12h差异有统计学意义( (P<0.05)(见附图17,表3.6)。结论:1.吡格列酮预处理组可延长重症急性胰腺炎大鼠的生存时间,同时降低重症急性胰腺炎大鼠60小时的死亡率,以50mg/kg剂量组(P2组)疗效最为显著。2.IL-6是反应急性胰腺炎严重程度的可靠指标,研究显示IL-6的水平高低与急性胰腺炎的严重程度相平行,当有效的抑制NF-κB活性后,IL-6显著下降。3.急性重症胰腺炎的炎症介质的产生始发于NF-κB的过度活化,应用NF-κB激活的抑制剂阻断NF-κB激活,在转录水平下调炎症介质表达,从而改善病情。因此,选择一个适当时机抑制NF-κB的活化必定可以减轻SAP的进展,降低胰腺炎对机体所造成损害,这将为今后SAP的治疗提供了一条新的途径。4.吡格列酮通过活化PPARγ途径,抑制NF-κB的活性,减轻大鼠SAP炎症程度,因此,其有望成为一种新的临床治疗SAP的方法。更多还原

【Abstract】 Objective: To study the dynamic changes and effect of p65, an important subtype of nuclear transcription factor-κB (NF-κB) in the pancreas of sodium taurocholic acid-induced severe acute pancreatitis (SAP),and to assess the preventive effects of pioglitazone, a ligand of Peroxisome proliferator-activated receptor gamma, on the development of STC-induced severe acute pancreatitis.METHODS: 130 Male Sprague-Dawley(SD) rats (160-200g) were randomly allocated into two parts:(1) Male Sprague-Dawley(SD) rats (160-200g) were randomly allocated into five groups(n=8 for each group): (a) SAP group. Acute pancreatitis(AP) was induced in male SD rats by the retrograde injection of 1ml/kg.m of 50 g/L sodium taurocholate (STC) in the pancreatic duct. 10% dimethyl sulphoxide (DMSO) was injected intraperitoneally two hours prior to STC; (b) Pioglitazone groups (same as SAP group, but 10% DMSO was replaced by pioglitazone administered intraperitoneally, 2mg/100g、5mg/100g、10mg/100g in DMSO); (c)Sham operation group. Sham-operated animals served as control. Operation was executed, STC was not injected, but pancreas was flipped and striked gently three times. After operation, rats were given freedom to drink water, but were fasted.The survival time and survival rate of each group were compared .(2)Male Sprague-Dawley(SD) rats (160-200g) were randomly allocated into five groups(n=18 for each group): (a) SAP group. Acute pancreatitis(AP) was induced in male SD rats by the retrograde injection of 1ml/kg.m of 50 g/L sodium taurocholate (STC) in the pancreatic duct. 10% dimethyl sulphoxide (DMSO) was injected intraperitoneally two hours prior to STC; (b) Pioglitazone groups (same as SAP group, but 10% DMSO was replaced by pioglitazone administered intraperitoneally, 2mg/100g、5mg/100g、10mg/100g in DMSO); (c)Sham operation group. Sham-operated animals served as control. Operation was executed, STC was not injected, but pancreas was flipped and striked gently three times. After operation, rats were given freedom to drink water, but were fasted. Rats were killed by abdominal aorta exsanguination at 3、6 and 12h after the inducetion of pancreatitis. Serum and ascitic activities of amylase were measured .The pancreatic tissue was divided into two parts: I,Samples of pancreatic tissues for histological examination were fixed in 10% formalin and stained with hematoxylin and eosin,and histologic score was performed. II,Samples of pancreatic tissues were rapidly frozen in liquid nitrogen and stored at -80℃for analysis against p65.RESULTS:(1) The survival time of Pioglitazone group(5mg/100g) was longer than that of SAP group(P<0.05), while lower than that of sham group(P<0.05). The 36-hour survival rate in of Pioglitazone group(5mg/100g) was longer than that of SAP group(P<0.05), while lower than that of sham group(P<0.05).(2) The concentration of serum amylased and ascite amylased in SAP group were significantly higher than those in sham group (P<0.001). Pioglitazone administered(2mg/100g、5mg/100g、10mg/100g i.g.) 2 hours prior to STC,attenuated dose-dependently the pancreatic tissue damage in STC-induced pancreatitis as demonstrated by the improvement of pancreatic histology,reduce ascitic activities of amylase, the concentrations of inflammatory cyctokines ( IL-6) of the serum and increased serum levels of IL-6.(P<0.01 or P<0.05).(3) The concentration of the pancreatic histologic score in SAP group were significantly higher than those in sham group (P<0.001).According to Schmidt criteria, the pancreatic histologic score showed that there existed significant difference in the SAP group in the interstitial edema, inflammatory infiltration, parenchyma necrosis and parenchyma hommorrhage in comparison with those of the sham group and pioglitazone groups respectively (P<0.01, P<0.05) .(4) The activity of NF-κB p65: In A group and Pgroup,the activity of NF-κB p65 was markedly upgraded compared with C group at all pionts (P<0.01),which was decreased significantly in P2 group compared with A group (P<0.05) at 6h and 12h.Conclusions:(1) The results of our study demonstrate that pioglitazone could raise the 36-hour survival rate and prolong survival time.(2) IL-6 is the reaction of the severity of acute pancreatitis reliable indicators, studies show that it is in parallel between the level of severity of acute pancreatitis and IL-6, when the effective inhibition of NF-κB activity, IL-6 decreased significantly(3) NF-κB is involved in the inflammation response of SAP. The expression of NF-κB has a positive correlation with pathology damage of pancreatic tissue. Inhibition of the expression of NF-κB in pancreatic tissue can be helpful for treatment of SAP by downregulating the above cytokines.(4) Through activation of PPARγway Pioglitazone, inhibiting NF-κB activity and reducing inflammation in rats SAP, is expected to become a new kind of treatment methods SAP更多还原