柴黄清胰活血颗粒对重症急性胰腺炎模型大鼠肠粘膜屏障的影响

发布时间：2018-05-21 01:38

本文选题：重症急性胰腺炎 + 肠粘膜屏障　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:探讨“柴黄清胰活血颗粒”对重症急性胰腺炎(severe acute pancreatitis,SAP)模型大鼠肠粘膜屏障的影响及作用机制。方法:选用雄性健康SD大鼠120只,采用完全随机法分为5组:假手术组、SAP模型组、柴黄清胰活血颗粒低浓度组、柴黄清胰活血颗粒中浓度组、柴黄清胰活血颗粒高浓度组,每组各24只。通过向胰胆管内逆行注射3.5%牛磺胆酸来诱导SAP模型。待造模成功的大鼠麻醉苏醒后立即进行灌胃,每4小时1次,按1ml/100g体重给药,高、中、低浓度组分别予高、中、低浓度柴黄清胰活血颗粒溶液灌胃(中浓度为0.1g/ml,高、中、低浓度比分别为4:2:1),模型组、假手术组予以等量生理盐水灌胃。造模第6h、12h、24h每组各取8只大鼠麻醉后开腹,观察胰腺出血坏死、肠积气情况;取腹主动脉血制备血清,分别检测血淀粉酶、二胺氧化酶(diamine oxidase,DAO)、D-乳酸、内毒素;取胰腺、十二指肠组织予以HE染色后在光镜下观察;并运用免疫组化法检测小肠黏膜紧密连接蛋白Occludin的表达。结果:1、建立模型:大鼠造模6h后可见胰腺组织出现片状出血和紫黑色坏死;HE染色光镜下可见胰腺间质水肿、点片状出血、炎细胞浸润、灶性变性坏死;血淀粉酶有不同程度升高。上述观察结果均符合SAP改变,提示造模成功。2、肉眼观察:SAP模型组大鼠胰腺组织出现片状出血和紫黑色坏死;胃肠道积气明显,肠管管壁变薄,透亮;大网膜、肠系膜上有较多皂化斑。而高、中、低浓度组大鼠胰腺坏死及肠道积气较模型组减轻,大网膜、肠系膜上有较少皂化斑。3、血淀粉酶:与假手术组比较,模型组及高、中、低浓度组的血淀粉酶在第6、12、24小时均升高(p0.05)。高、中、低浓度组第12、24小时血淀粉酶较模型组均降低(p0.05);第12小时中、低浓度组的血淀粉酶比高浓度组高(p0.05)。第24小时血淀粉酶高浓度组低浓度组中浓度组(p值均0.05)。4、通过对大鼠血清dao、d-乳酸、内毒素检测,模型组及高、中、低浓度组的d-乳酸、dao、内毒素均较假手术组升高(p0.05),说明sap造模成功后,血清d-乳酸、dao、内毒素增加。第12小时高、中、低浓度组的内毒素组间比较均有差异(p0.05),第12小时的d-乳酸低浓度组分别与高、中浓度组比较有差异(p0.05),第12小时低浓度组的dao比模型组高(p0.05),第24小时中浓度组的dao比模型组高(p0.05),除第24小时低浓度组的dao比高浓度、中浓度组低(p0.05)外,其它浓度组组间未见明显差异。提示:柴黄清胰活血颗粒可减少sap大鼠血d-乳酸、血浆内毒素的产生,降幅由大到小依次为高浓度组、中浓度组、低浓度组。本次实验未观察到该制剂对sap大鼠血dao明显的量效、时效关系。5、胰腺组织he染色光镜下显示:模型组胰腺组织受损严重,且随着时间递增逐渐加重,第6h即可见胰腺间质水肿、点片状出血,炎细胞浸润,灶性变性、坏死。高、中、低浓度组第6h与模型组比较未见明显改变,而第12h、24h与模型组比较损伤有所减轻。高、中、低浓度组同时间点对比,胰腺组织损伤好转幅度由大到小依次为高浓度组、中浓度组、低浓度组。胰腺病理学评分:第24小时高、中、低浓度组病理学评分较模型组有显著变化,且呈降低趋势(p0.05);第24小时高、中、低浓度组的病理学评分组间比较均有统计学差异(P0.05)。提示:柴黄清胰活血颗粒可降低SAP大鼠胰腺病理学评分,降幅由大到小依次为高浓度组、中浓度组、低浓度组。6、大鼠十二指肠HE染色光镜下显示:模型组肠粘膜受损严重,且随着时间递增逐渐加重,第6h即可见肠绒毛充血水肿,大量中性粒细胞浸润、杯状细胞减少,顶端上皮坏死、脱落,溃疡形成。第12h、24h高、中、低浓度组与模型组比较损伤均有所减轻。高、中、低浓度组同时间点对比,肠粘膜损伤好转幅度由大到小依次为高、中浓度组低浓度组。提示柴黄清胰活血颗粒可减少SAP大鼠肠粘膜损伤程度,且肠粘膜损伤改善程度为高、中浓度组低浓度组。7、大鼠十二指肠肠粘膜紧密连接蛋白Occludin的表达显示:模型组及高、中、低浓度组的细胞浆、细胞膜表达水平均较假手术组显著下降,提示SAP大鼠造模成功后,肠黏膜组织中Occludin的表达减弱。而高、中、低浓度组与同时间点的模型组比较,Occludin的表达逐渐恢复,且随着时间递增表达逐渐增强。提示柴黄清胰活血颗粒可恢复SAP大鼠紧密连接蛋白Occludin表达,且恢复程度为高、中浓度组低浓度组。结论:早期应用柴黄清胰活血颗粒能够改善重症急性胰腺炎模型大鼠胰腺组织及肠道黏膜损伤,其机制可能与降低大鼠血清乳酸、血浆内毒素含量,上调Occludin蛋白的表达有关,从而起到改善肠黏膜屏障的作用。
[Abstract]:Objective: To investigate the effect and mechanism of "Chai Huang Qingyi Huoxue Granule" on intestinal mucosal barrier of severe acute pancreatitis (SAP) model rats. Methods: 120 male healthy SD rats were selected and divided into 5 groups by complete random method: sham operation group, SAP model group, Chai Huang Qing pancreas Huoxue granule low concentration group, Chai Huang Qingyi The concentration group in Huoxue granule, the high concentration group of Chahuang Qingyi Huoxue granule, 24 in each group. The SAP model was induced by retrograde injection of 3.5% taurocholic acid into the pancreatic duct. After the rats were awakened, the rats were irrigated immediately after the anesthesia was awakened, and every 4 hours and 1 times, the high, middle, and low concentration groups were given high, middle, and low concentration of Chai Huang Qing respectively. Pancreas Huoxue granule (0.1g/ml, high, medium and low concentration ratio was 4:2:1). The model group and sham operation group were given the same amount of normal saline. 8 rats in each group were anaesthetized to open the abdomen to observe the hemorrhagic necrosis of the pancreas and the condition of intestinal accumulation. The blood serum was prepared from the abdominal aorta, and the blood amylase and two amine were detected respectively. Diamine oxidase (DAO), D- lactic acid, endotoxin, pancreatic and duodenal tissue were observed under light microscopy after HE staining; and immunohistochemical method was used to detect the expression of tight connexin Occludin in small intestinal mucosa. Results: 1, the model was established: after the rat model 6h, the pancreatic tissue appeared flaky bleeding and purple black necrosis; HE staining light was found. The pancreatic interstitial edema, point bleed, inflammatory cell infiltration, focal degeneration and necrosis were seen under the microscope, and the blood amylase was elevated in different degrees. The results of the above observation all conformed to the changes of SAP, suggesting that the model was successful.2, the naked eye was observed in the SAP model group, the pancreas tissue appeared flaky bleeding and purple black necrosis, gastrointestinal tract accumulation was obvious, the intestinal tube wall became thinner. There were more saponification spots on the greater omentum and mesentery. The pancreas necrosis and intestinal accumulation in the high, middle and low concentration rats were less than that in the model group. The greater omentum and the mesentery had less saponification spots.3 and blood amylase: compared with the sham operation group, the blood amylase in the model group and the high, middle and low concentration groups increased (P0.05) at the 6,12,24 hour. High, medium and low concentration In group 12,24 hours, the blood amylase was lower than that in the model group (P0.05); in the twelfth hour, the blood amylase in the low concentration group was higher than that in the high concentration group (P0.05). The concentration group in the low concentration group of the high concentration group of blood amylase (P value was 0.05).4, through the serum Dao, d- lactic acid, endotoxin test, the model group and the d- lactic acid in the high, middle and low concentration group, Dao, Endotoxin was higher than that of sham operation group (P0.05), indicating that after the success of SAP model, serum d- lactic acid, Dao, endotoxin increased. Twelfth hours high, middle and low concentration of endotoxin group were different (P0.05), Twelfth hours low concentration of d- lactate group were respectively higher than medium concentration group (P0.05), Twelfth hours low concentration group Dao ratio model group High (P0.05), the concentration of Dao in the twenty-fourth hour concentration group was higher than the model group (P0.05), except the high concentration of Dao in the twenty-fourth hour low concentration group and the lower concentration group (P0.05), there was no obvious difference between the other concentration groups. It suggested that the Chai Huang Qing pancreas Huoxue granule could reduce the blood d- lactic acid and the plasma endotoxin production in the SAP rats, and the decrease from large to small was the high concentration group. In the medium concentration group and low concentration group, the dose effect of this preparation on the blood Dao of SAP rats was not observed, the aging relationship was.5, and the pancreas tissue HE staining light microscope showed that the pancreas tissue of the model group was seriously damaged and gradually increased with the time, and the 6h was the pancreatic edema, the dot bleed, the inflammatory cell infiltration, the focal degeneration and necrosis. High, middle, low concentration group 6h and model group no significant change, but 12h, 24h and model group, compared with the model group, compared with the time point, the group of pancreatic tissue damage improved from large to small in order of high concentration group, middle concentration group, low concentration group. The pancreatic pathology score: twenty-fourth hours high, middle, low concentration group disease Compared with the model group, there was a significant change in the score of the model group (P0.05), and there was a statistically significant difference between the twenty-fourth hours, middle and low concentration groups. It was suggested that Chai Huang Qing pancreas Huoxue granule could reduce the pathological score of pancreas in SAP rats, and the decrease from large to small was high concentration group, middle concentration group and low concentration group.6. HE staining in the rat duodenum showed that the intestinal mucosa of the model group was seriously damaged and increased with time. 6h was seen in the intestinal villus congestion and edema, a large number of neutrophils infiltrated, the goblet cells decreased, the epithelia of the apical necrosis, exfoliation, and ulcers formed. The injury of the group 12h, 24h, low concentration and the model group were all lightened. The lower concentration group compared with the time point, the intestinal mucosal injury improved from large to small in order of high, middle concentration group low concentration group. It suggests that Chai Huang Qing pancreas Huoxue granule can reduce the degree of intestinal mucosal injury in SAP rats, and the improvement of intestinal mucosa damage is high, middle concentration group low concentration group.7, rat duodenal intestinal mucosa close connexin Occludi The expression of N showed that the expression level of cell membrane in the model group and the high, middle and low concentration groups was significantly lower than that in the sham operation group. It suggested that the expression of Occludin in the intestinal mucosa was weakened after the successful model of SAP rats. The high, middle and low concentration groups were compared with the model group at the same time point, and the expression of Occludin was gradually restored with the increase of time. It is suggested that Chai Huang Qing yhuoxue granules can restore the expression of tight connexin Occludin in SAP rats, and the degree of recovery is high and the concentration group is low in concentration group. Conclusion: the early application of Chai Huang Qing pancreas Huoxue granule can improve the injury of pancreatic tissue and intestinal mucosa in rats with severe acute pancreatitis, and its mechanism may reduce the blood of rats. Clear lactic acid, plasma endotoxin content, upregulated the expression of Occludin protein, thereby playing a role in improving intestinal mucosal barrier.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332

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