不同剂量桃仁提取物对急性胰腺炎大鼠肠道黏膜屏障功能及免疫功能的作用

Effect of different dose of persicae semen extract to barrier function of intestinal mucous membrane and immunologic function in acute pancreatitis rats

目的:研究不同剂量桃仁提取物对急性胰腺炎大鼠肠道黏膜屏障功能及免疫功能的作用。方法:48只大鼠制备SAP模型后随机分为模型对照组、桃仁提取物低剂量、中剂量和高剂量组,每组12只。另取12只大鼠作为假手术组,造模麻醉苏醒即开始干预,桃仁提取物低剂量组、中剂量组和高剂量组分别灌胃给予桃仁提取物0.12、0.248和0.36 g/kg,假手术组及模型组给予等体积蒸馏水灌胃,各组灌胃均1次/6 h,连续4次。给药后24 h应用10%水合氯醛麻醉各组大鼠,打开胸腔和腹腔,腹主动脉分别抽取5 ml血样于EDTA抗凝管和非抗凝管内,分别应用荧光直接标记法和流式细胞仪进行CD4+、CD8+和Treg细胞测定,采用免疫比浊法测定Ig A、Ig G和Ig M,采用EPS-G7底物法测定血清淀粉酶水平,采用酶学分光光度法检测血清D-乳酸水平,采用活性比色法测定血清二胺氧化酶;取小肠组织HE染色后采用光学显微镜进行病理学检查;取小肠组织采用放射免疫法进行s Ig A测定以及采用RT-PCR法进行TLR4和NF-κBp65 mRNA测定。结果:(1)中剂量和高剂量组大鼠血清淀粉酶、D-乳酸和二胺氧化酶水平均较低剂量组大鼠显著降低(P<0.01),小肠黏膜s Ig A较低剂量组显著升高(P<0.01),并且高剂量组和中剂量组差异具有统计学意义(P<0.01);(2)桃仁提取物中剂量和高剂量组血液CD4+、CD4+/CD8+较低剂量组大鼠显著升高(P<0.01),CD8+、Treg细胞较低剂量组大鼠显著降低(P<0.01),并且高剂量组和中剂量组差异均具有统计学意义(P<0.01);(3)桃仁提取物中剂量和高剂量组血清Ig A、Ig G、Ig M较低剂量组大鼠显著升高(P<0.01),并且高剂量组和中剂量组差异均具有统计学意义(P<0.01);(4)假手术组大鼠小肠黏膜无显著损伤,模型对照组大鼠小肠黏膜显著损伤,低剂量组大鼠小肠黏膜病理情况与模型组基本相似,中剂量和大剂量组大鼠小肠黏膜损伤显著降低;(5)桃仁提取物中剂量和高剂量组小肠组织TLR4和NF-κBp65 mRNA较低剂量组大鼠显著降低(P<0.01),并且高剂量组和中剂量组差异均具有统计学意义(P<0.01)。结论:桃仁提取物对急性胰腺炎大鼠肠道屏障功能具有保护作用,并且显著改善急性胰腺炎大鼠的免疫功能。

Objective： To study the effect of different dose of persicae semen extract extract （PSE） to barrier function of the intestinal mucous membrane and immunologic function in acute pancreatitis rats. Methods： A total of 48 rats were divided into model control group,low dose,medial dose and high dose PSE groups, and there were 12 rats in each group. Another 12 rats were Sham- operation group. After anesthesia recovery,rats in low dose,medial dose and high dose PSE groups respectively received PSE 0. 12 g/ kg,0. 248 g/kg and 0. 36 g/kg,and rats in Sham-operation group and model control group receive isovolumetric distilled water,once per 6 h,4 times in 24 hours. All rats were anesthetized by 10% chloral hydrate after in 24th hour after dosing. Thorax and enterocoelia were opened; 5 ml of blood were respectively drawed to EDTA-anticoagulation＇ tube and un-anticoagulation tube from aorta abdominalis. CD4＋,CD8~ and Treg cells were determined by direct fluorescent-labelded flow cytometry. IgA, IgG and IgM were determined by immunoturbidimetry. Serum amylase was determined by EPS-G7 substrate, D-lactic acid was determined by enzymology, and serum diamine oxidase was determined by active ration of colorimetry method. Pathological examination of small intestine mucous membrane tissue was taken after HE staining, sIgA in small intestine was determined by radioimmunoassay, mRNA of TLR4 and NF- KBp65 in small intestine tissue was determined by RT-PCR. Results： （1） Serum amylase,D-lactic acid and diamine oxidase in medial dose and high dose PSE groups were significantly decreased （P〈0. 01 ）, and sIgA in small intestine was significantly increased（ P〈 0. 01 ）. These indicators were significantly different in medial dose and high dose PSE groups（ P〈0. 01 ）. （2） CD4~ and CD4~/CD8~ in medial dose and high dose PSE groups were significantly increased（ P〈0. 01 ）, and CD8~ ,Treg cells were significantly decreased（ P〈 0. 01 ） compared with those in low dose PSE group. These indicators were significantly different in medial dose and high dose PSE groups（P〈0. 01 ）. （3） IgA,IgG and IgM in medial dose and high dose PSE groups were significantly decreased（P〈0.01 ） compared with those in low dose PSE group. These indicators were significantly different in medial dose and high dose PSE groups （P〈0. 01 ）. （4） Small intestine mucous membrane tissue in Sham-operation group was not damaged significantly, but that in model control group was damaged significantly. Small intestine mucous membrane tissue in low dose PSE group was similar to that in model control group, and damage in medial dose and high dose PSE groups was decreased significantly. （5） mRNA of TLR4 and NF-KBp65 in small intestine tissue in medial dose and high dose PSE groups were significantly increased （P〈 0. 01 ） compared with those in low dose PSE group. These indicators were significantly different in medial dose and high dose PSE groups（P〈0.01 ）. Conclusion： PSE has protective effect to barrier function of the intestinal mucous membrane, and significantly improve the immunologic function.