NALP3炎性体在大鼠重症急性胰腺炎肺损伤中的表达及大黄附子汤干预作用的实验研究

发布时间：2018-07-04 22:14

本文选题：重症胰腺炎 + 急性肺损伤　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:建立重症急性胰腺炎大鼠模型,观察重症急性胰腺炎导致肺损伤过程中NALP3炎性体和水通道蛋白AQP-5的表达以及应用大黄附子汤对SAP-ALI大鼠进行干预后的变化,阐明AQP-5、IL-1β、NALP3在SAP-ALI发病中的作用。方法:SPF级雄性SD大鼠(购自大连医科大学实验动物中心)24只,体重220-280 g,随机分为4组:空白对照组(n=6),SAP组(n=6),大黄附子汤治疗A组(n=6),大黄附子汤治疗B组(n=6)。采用异氟烷麻醉;空白对照组开腹后,找到胰腺,翻动胰腺数次后关腹;SAP组、治疗组经胰胆管缓慢注入4%牛磺胆酸钠(1ml/kg)诱导SAP-ALI模型,造模成功后SAP组用2ml生理盐水于术后12、24小时灌肠;治疗A组用2ml大黄附子汤于术后12小时灌肠,治疗B组用2ml大黄附子汤于术后12、24h灌肠干预治疗。各组大鼠在各自时间点腹主动脉取血测血浆内毒素、血淀粉酶、血钙,在显微镜下观察肺和胰腺组织形态学变化,通过Smith评分法对实验各组肺组织进行病理评分,改良Schmidt评分标准对胰腺组织进行病理评分,用ELISA法检测血清、肺组织IL-1β水平,免疫组化法检测肺组织AQP-5蛋白表达与分布,Western-blot和免疫组化法检测肺组织NALP3蛋白表达及分布。结果:1.血清内毒素含量:与空白对照组比较,SAP组血清内毒素较对照组明显升高(P0.05);与SAP组比较,治疗组血浆内毒素含量较模型组减少(P0.05)。治疗B组内毒素含量较治疗A组降低,差异有统计学意义(P0.05)。2.血清淀粉酶、血钙含量:与空白对照组比较,SAP组淀粉酶明显升高,血钙含量下降(P0.05);与SAP组比较,治疗组血淀粉酶含量下降,血钙含量升高(P0.05)。治疗B组淀粉酶较治疗A组低,血钙升高,差异有统计学意义(P0.05)。3.HE染色观察:空白对照组胰腺腺泡完整;SAP组胰腺坏死明显,腺体结构紊乱甚至消失,大量炎性细胞浸润伴出血;治疗A组较SAP组腺体水肿减轻,组织间隙宽,炎性细胞浸润减少,部分有出血和坏死;治疗B组腺体轻度水肿,组织间隙轻度增宽,少量炎性细胞浸润。空白对照组肺结构组织正常;SAP组肺泡间隔明显增宽,肺泡结构破坏,肺泡腔内有明显的出血渗出,大量中性粒细胞浸润;治疗A组肺泡结构有破坏,肺泡间隔增宽较SAP组减轻,肺泡出血及中性粒细胞浸润减少;治疗B组肺泡结构破坏不明显,肺泡间隔略增宽,中性粒细胞浸润明显减少。损伤评分有统计学差异(P0.05)。4.ELLSA:SAP组、治疗组血清、肺组织IL-1β与空白对照组比较含量升高(P0.05);治疗组与SAP组比较,治疗组IL-1β含量较模型组低(P0.05)。治疗B组较治疗A组比较含量降低(P0.05)。5.免疫组化:与空白对照组比较,SAP组大鼠NALP3高表达,AQP5低表达(P0.05);与SAP组比较,治疗组大鼠NALP3表达降低,AQP5表达升高(P0.05)。6.Western-blot:与空白对照组比较,SAP组大鼠NALP3表达量高(P0.05);与SAP组比较,治疗组大鼠NALP3表达量降低(P0.05)。结论:(1)重症急性胰腺炎肺损伤形成过程中,NALP3表达增加,促使IL-1β的表达增加,此过程降低了AQP-5的表达,使肺内水转运出现障碍,肺含水量增多。因此,重症急性胰腺炎急性肺损伤的发生可能与NALP3炎性体和AQP-5有关。(2)大黄附子汤可能通过下调NALP3炎性小体,促使IL-1β的表达下降,上调AQP-5表达,从而减轻重症急性胰腺炎肺损伤程度。
[Abstract]:Objective: to establish a rat model of severe acute pancreatitis (SAP), to observe the expression of NALP3 and aquaporin AQP-5 in the process of severe acute pancreatitis and the changes in the application of Rhubarb Decoction on SAP-ALI rats, and to clarify the role of AQP-5, IL-1 beta, NALP3 in the pathogenesis of SAP-ALI. Methods: SPF grade male SD rats (purchased from the rats). 24 rats at the experimental animal center of Dalian Medical University, weighing 220-280 g, were randomly divided into 4 groups: blank control group (n=6), group SAP (n=6), A group (n=6) treated with Rhubarb Decoction (n=6), rhubarb decoction for B group (n=6) and isoflurane anesthesia. After laparotomy in the blank control group, the pancreas was found, the pancreas was turned over several times, and the group of SAP, the treatment group was injected slowly through the pancreatic bile duct. 4% sodium taurocholate (1ml/kg) was used to induce the SAP-ALI model. After the model was successful, the SAP group was treated with 2ml saline after 12,24 hour postoperatively, and the A group was treated with 2ml Rhubarb decoction for 12 hours after the operation, and the group B was treated with 2ml Rhubarb decoction for 12,24h enema after operation. The blood plasma endotoxin was measured at the abdominal aorta at each time point in each group. The changes of lung and pancreas histomorphology were observed under the microscope by blood amylase and blood calcium. The pathological scores of lung tissues in the experimental groups were evaluated by Smith scoring method. The pathological score of the pancreas tissue was evaluated by improved Schmidt score standard. The serum level of the lung and the level of IL-1 beta in the lung tissue were detected by ELISA, and the expression and distribution of AQP-5 protein in lung tissue was detected by immunohistochemical method. The expression and distribution of NALP3 protein in lung tissue were detected by Western-blot and immunohistochemistry. Results: 1. serum endotoxin content: compared with the blank control group, the serum endotoxin in SAP group was significantly higher than that in the control group (P0.05). Compared with the SAP group, the plasma endotoxin content was lower than that in the model group (P0.05). The content of endotoxin in the treatment group was lower than that in the treatment group of the treatment group, compared with that of the treatment group of the treatment group A. The difference was statistically significant (P0.05).2. serum amylase, blood calcium content: compared with the blank control group, the amylase in the SAP group increased significantly and the blood calcium content decreased (P0.05). Compared with the SAP group, the blood amylase content decreased and the blood calcium content increased (P0.05). In the B group, the amylase was lower than that in the treatment group and the blood calcium increased, the difference was statistically significant (P0.05). 3.HE staining showed that the pancreatic acinus in the blank control group was complete, and the pancreatic necrosis was obvious in group SAP, the structure of the gland was disorganized and even disappeared, and a large number of inflammatory cells were infiltrated with bleeding. In the group A, the edema of the glands in the group SAP was relieved, the space of the tissue was wide, the infiltration of inflammatory cells decreased, and the necrosis of the inflammatory cells, and the mild edema of the glands in the B group and the light widening of the space of the tissue were treated in the B group. The pulmonary structure and tissue in the blank control group were normal, the alveolar space in the SAP group was obviously widened, the alveolar structure was destroyed, the alveolus had obvious bleeding and exudation, and a large number of neutrophils were infiltrated. The alveolar structure in the A group was destroyed, the alveolar septum widened more than that of the SAP group, and the alveolar hemorrhage and neutrophils infiltration were reduced, and the lung of the B group was treated. The damage of the alveolar structure was not obvious, the alveolar septum increased slightly and the neutrophils infiltration decreased significantly. The damage score was statistically different (P0.05).4.ELLSA:SAP, the serum of the treatment group, the IL-1 beta in the lung tissue and the blank control group increased (P0.05); the treatment group was compared with the SAP group, the IL-1 beta content was lower than that in the model group (P0.05). The treatment group was compared with the treatment A for A. Compared with the blank control group, the NALP3 high expression and low expression of AQP5 (P0.05) were compared with the blank control group. Compared with the SAP group, the expression of NALP3 in the treatment group was lower and the AQP5 expression was higher (P0.05).6.Western-blot: than that in the blank control group. The expression of NALP3 in the SAP group was higher than that in the control group. Compared with the SAP group, the expression of the rats was higher than that in the control group. Compared with the SAP group, the expression of NALP3 in the rats was higher than that in the group of SAP. ALP3 expression decreased (P0.05). Conclusion: (1) during the formation of severe acute pancreatitis, the expression of NALP3 is increased and the expression of IL-1 beta is increased. This process reduces the expression of AQP-5, causes the transport of water in the lungs and increases the water content of the lungs. Therefore, the occurrence of acute lung injury in severe acute pancreatitis may be associated with the NALP3 inflammatory and AQP-5. (2) Rhubarb decoction may reduce the expression of IL-1 beta by down regulation of NALP3 inflammatory corpuscle, and increase the expression of AQP-5, thus reducing the degree of lung injury in severe acute pancreatitis.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R576

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