猴头菇多糖对胃肠黏膜保护作用的实验研究

发布时间：2018-04-29 07:16

  本文选题：猴头爰舌多裙 + 胃黏膜损伤　； 参考：《广州中医药大学》2014年硕士论文

【摘要】：目的： 通过观察猴头菇多糖对无水乙醇、吲哚美辛、醋酸、水浸拘缚所致大鼠胃黏膜损伤的作用,研究猴头菇多糖对胃黏膜的保护作用及其作用机制,并指导配方中的有效添加剂量；通过观察猴头菇多糖对LPS诱导Caco-2细胞及Caco-2/RAW264.7共培养体系应激炎症反应的保护作用,在细胞水平研究猴头菇多糖对肠黏膜屏障的保护作用。 方法： 1、猴头菇多糖对胃黏膜保护作用的实验研究 采用无水乙醇致大鼠胃黏膜损伤模型观察猴头菇多糖对急性胃黏膜损伤的保护作用,动物随机分成空白对照组、模型组对照组、阳性对照组、猴头菇多糖各剂量组。以猴头菇多糖的人体最小参考剂量(0.2g/d)的5倍作为大鼠的最小给药剂量(HEP1),人体最大参考剂量(0.4g/d)的20倍作为最大给药剂量(HEP4),再在最大、最小剂量间再设置2个梯度(HEP2、HEP3)。剂量设置分别为：HEP117mg/kg, HEP268mg/kg, HEP3102mg/kg, HEP4136mg/kg。实验以无水乙醇模型大鼠的胃黏膜溃疡指数为根据筛选出有效剂量。 在筛选出有效剂量的基础上,分别采用吲哚美辛、醋酸、水浸拘缚致大鼠胃黏膜损伤模型以进一步观察猴头菇多糖对胃黏膜的保护作用并探讨其保护机理。SD大鼠,雄性,180-220g,随机分空白对照组、模型组对照组、阳性对照组、HEP剂量组。灌胃给药1次/d,共10天,对照组给予等量蒸馏水。 (1)末次给药1h后,除空白组外,动物灌胃吲哚美辛80mg/kg,空白组给予等体积蒸馏水。7h后处死动物,取出全胃,测量胃黏膜溃疡面积,酶联免疫法测定胃黏膜组织匀浆上清中的前列腺素(PGE2)、表皮生长因子(EGF)含量。 (2)给药前,以微量注射器平刺入浆膜注射30%醋酸制备大鼠慢性胃黏膜损伤模型,末次给药后禁食24h处死动物,取出全胃,测量胃黏膜溃疡面积,酶联免疫法测定胃黏膜组织匀浆上清中的碱性成纤维生长因子(bFGF)含量,实时荧光定量RT-PCR法测定转化生长因子(TGF-α) mRMA表达的影响。 (3)末次给药0.5h后固定于金属网笼内,直立浸于水温20±1℃的水槽中6h制备大鼠应激性胃黏膜损伤模型,10%水合氯醛麻醉动物,于剑突下正中切开腹前壁,于胃体及胃窦处各作一个长度0.5cm的横向切口,运用激光多普勒灌注监视仪测定胃体及胃窦处血流量(GMBF)。以激光多普勒信号表示血流量的相对数值,以胃体及胃窦处血流量均值表示胃黏膜血流量；处死动物,取出全胃,测量胃黏膜溃疡面积。 2、猴头菇多糖对肠黏膜保护作用的实验研究： 将Caco-2细胞以105浓度接种在Transwell转运培养槽的微孔滤膜上进行培养,培养基为DMEM高糖(pH7.4,含10%胎牛血清,2mmol·L-1L-谷氨酰胺,10mmol-L-1Hepes、NEAA、青链霉素),于37℃,5%CO2培养箱中培养24h换液,以后每隔48h换液,约4-6d细胞融合达90%进行传代。采用形态学观察、跨膜电阻测定、表观渗透系数测定和碱性磷酸酶活性检测对细胞模型进行评估。以脂多糖(LPS)刺激1h引起Caco-2细胞及Caco-2/RAW264.7共培养体系应激炎症反应,加入猴头菇多糖干预24h,收集下侧转运液检测相关炎症因子的分泌情况。 (1)细胞培养至15d,光镜下观察细胞分布均匀,边界清晰,以单层致密的方式排列时,即可进行实验。在Transwell板上部加LPS50pg/ml,1h后在上部加入猴头菇多糖500μg/ml,24h后取下侧培养液酶联免疫法测定细胞因子IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ的含量。 (2)细胞培养至15d,光镜下观察细胞分布均匀,边界清晰,以单层致密的方式排列时,在Transwell板下部以105接种小鼠单核巨噬细胞(RAW264.7),共培养24h。在Transwell板上部加LPS50pg/ml,1h后再加入猴头菇多糖500μg/ml,24h后取下侧培养液酶联免疫法测定细胞因子IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ的含量。 结果： 1、猴头菇多糖对胃黏膜保护作用的实验研究 (1)剂量筛选实验结果表明,猴头菇多糖的17mg/kg、68mg/kg和136mg/kg剂量均能对无水乙醇造成的胃黏膜急性损伤有抑制作用。后续实验研究选择17mg/kg、68mg/kg剂量作为猴头菇多糖的低、高剂量。 (2)猴头菇多糖对吲哚美辛模型作用的实验表明,猴头菇多糖的低、高剂量均能降低模型大鼠胃黏膜溃疡面积,增加模型大鼠胃黏膜组织中前列腺素(PGE2)、表皮生长因子(EGF)含量的表达； (3)猴头菇多糖对醋酸模型作用的实验表明,猴头菇多糖低、高剂量均能降低该模型大鼠胃黏膜埙疡面积,增加模型大鼠胃黏膜组织中碱性成纤维生长因子(bFGF)的含量,并提高其转化生长因子(TGF-α) mRMA的表达； (4)猴头菇多糖对水浸拘缚模型作用的实验表明,猴头菇多糖低、高剂量均能降低模型大鼠胃黏膜溃疡面积,升高大鼠胃黏膜血流量(GMBF);2、猴头菇多糖对肠黏膜保护作用的实验研究： (1)LPS刺激下,Caco-2细胞以及Caco-2/RAW264.7细胞共培养体系IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ分泌量均显著增加； (2)猴头菇多糖可以显著抑制Caco-2细胞以及Caco-2/RAW264.7细胞共培养体系促炎细胞因子IL-6、IL-8, IL-12的分泌,而促进抗炎细胞因子IL-10的分泌,对以上细胞模型的应激炎症反应发挥免疫调节作用。 结论： 胃黏膜保护实验研究表明猴头菇多糖对急慢性、药物诱发型、应激性胃黏膜损伤模型均具有明显的预防和改善作用。进一步机制研究发现,猴头菇多糖可通过增加胃膜黏膜血流量,促进黏膜组织中PGE2、EGF、bFGF及TGF-a mRMA的分泌,从而增强胃黏膜自身的防御功能,达到保护胃黏膜、抗溃疡的目的。 肠黏膜保护实验研究表明猴头菇多糖通过抑制促炎细胞因子分泌,促进抗炎细胞因子分泌来缓解模拟肠黏膜屏障环境的Caco-2细胞及Caco-2/RAW264.7共培养体系的应激炎症反应,其保护作用机制与其免疫调节的生理活性相关。
[Abstract]:Purpose :

The protective effect and mechanism of polysaccharide on gastric mucosa were studied by observing the effect of the polysaccharide on the gastric mucosa of rats induced by absolute ethanol , indometacin , acetic acid and water , and the effective additive amount in the formulation was instructed .
The protective effect of the polysaccharide on LPS - induced inflammatory reaction of Caco - 2 cells and Caco - 2 / RAW264.7 co - culture system was observed .

Method :

An Experimental Study on the Protective Effect of Polysaccharide on Gastric Mucosa

The rats were randomly divided into two groups : blank control group , model group control group , positive control group and monkey head mushroom polysaccharide . The minimum reference dose ( 0 . 2g / d ) of the human body was taken as the minimum dose ( HEP1 ) of rats , the maximum reference dose ( 0.4 g / d ) was 20 times as the maximum dose ( HEP4 ) , then two gradients ( HEP2 , HEP3 ) were set up between the maximum and minimum doses . The dose setting was HEP117mg / kg , HEP26mg / kg , HEP3102mg / kg and HEP4 136 mg / kg , respectively .

On the basis of screening the effective dose , the rat gastric mucosa injury model was induced by indometacin , acetic acid and water . The protective mechanism of the polysaccharide on gastric mucosa was further investigated . The rats were divided into two groups : SD rats , male rats , 180 - 220g , randomly divided control group , model group control group , positive control group and HEP dose group .

( 1 ) After the last dose of 1h , in addition to the blank group , the animals were treated with indometacin 80 mg / kg and the blank group was given equal volume of distilled water . After 7 hours , the animals were sacrificed , the whole stomach was taken out , the area of gastric mucosa ulcer was measured , and the content of prostaglandin ( PGE2 ) and epidermal growth factor ( EGF ) in the supernatant of gastric mucosa tissue homogenate was measured by ELISA .

( 2 ) To prepare rat chronic gastric mucosa injury model by injecting 30 % acetic acid into plasma membrane by a micro - injector . After the last administration , the rats were sacrificed for 24h , the whole stomach was taken out , the area of gastric mucosa ulcer was measured , the alkaline fibroblast growth factor ( bFGF ) content in the supernatant of gastric mucosa tissue homogenate was measured by enzyme - linked immunosorbent assay , and the influence of the expression of transforming growth factor ( TGF - 伪 ) mRMA was measured by real - time fluorescence quantitative RT - PCR .

( 3 ) After the last dose of 0.5 h , the rats were fixed in a metal mesh cage , and the rats were immersed in a water tank at a temperature of 20 卤 1 鈩，

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