布拉氏酵母菌对NAFLD小鼠肝脏Egr-1表达及肠黏膜机械屏障作用的初步研究

发布时间：2018-04-16 01:29

  本文选题：非酒精性脂肪性肝病 + 脂多糖　； 参考：《河北医科大学》2017年硕士论文

【摘要】：目的:西方饮食建立非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)小鼠模型,观察血清内毒素水平,肝脏早期生长反应因子-1(early growth response-1,Egr-1)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达及肠黏膜机械屏障变化情况,进而探讨布拉氏酵母菌干预后的影响,初步探讨该药物对NAFLD的疗效以及相关作用机制。方法:将30只6-8周龄的清洁级雄性C57BL/6小鼠适应性喂养1周后应用随机数字表将其随机分为正常对照组(CG,n=12)和模型组(MG,n=18)。正常对照组给予正常对照饮食(AIN93M对照饲料+正常饮用水)喂养,模型组给予西方饮食(高脂饲料+高糖饮用水)喂养。于喂养的24周末随机处死各组6只小鼠,留取相应标本,并进行肝脏HE染色,病理改变证实NAFLD小鼠模型成功。自第25周起,将模型组剩余12只小鼠随机分为布拉氏酵母菌灌胃组(TB,n=6)和模型灌胃对照组(TD,n=6),继续西方饮食喂养。正常对照组剩余6只小鼠为正常灌胃对照组(NG,n=6),继续给予正常对照饮食。TB组给予布拉氏酵母菌(5.98×108 CFU/kg/day)灌胃,TD组及NG组同时开始给予布拉氏酵母菌相应辅料果糖(284mg/kg/day)+乳糖(19mg/kg/day)溶于蒸馏水中灌胃,每日灌胃1次,共6周,6周后处死全部小鼠,留取标本进行相应检测。观察小鼠的毛色、行为、精神状态等情况,同时每周测定小鼠体重。处死小鼠后,观察肝脏及回肠的大体情况,并完整切除肝脏,留取末端回肠组织,称肝脏湿重;采用全自动生化分析仪检测血清丙氨酸氨基转氨酶(alanine aminotransferase,ALT)、门冬氨酸氨基转氨酶(aspartate aminotransferase,AST)、甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)水平;采用鲎试剂法测定血清脂多糖(lipopolysaccharide,LPS)水平;HE染色观察肝脏及末端回肠黏膜病理变化;透射电镜观察回肠组织超微结构的改变;采用Real-time Q-PCR(Real-time quantitation PCR)方法检测各组小鼠肝脏Egr-1、TNF-α及末端回肠组织中TNF-α、Occludin m RNA的表达情况;蛋白免疫印迹(Western Blot)法检测肝脏Egr-1、TNF-α及末端回肠组织中Occludin、TNF-α的蛋白表达情况。结果:1实验动物的一般情况正常对照组小鼠皮毛顺滑、浓密、有光泽,精神状态良好,活动灵活,反应灵敏,体重逐渐增加;模型组小鼠自造模第4周开始皮毛欠光滑,稍显油腻,精神差,体重增长迅速,逐渐呈腹型肥胖,至24周,小鼠精神萎靡,喜卧懒动,反应迟缓,毛发无光泽,造模完成。给予布拉氏酵母菌灌胃干预后,TB组小鼠体重较前减轻,活动增多,情况好转。实验期间,TB组及TD组小鼠各死亡1只,剩余5只,其余组小鼠无死伤发生。2实验动物体重、肝重指数的比较MG组较CG组小鼠体重及肝重指数显著增高(P0.05);灌胃干预后,TB组较TD组体重及肝重指数显著降低(P0.05),但仍明显高于NG组(P0.05),差异有统计学意义。3实验动物肝脏及末端回肠组织病理肝脏病理:CG组小鼠肝小叶结构完整,肝细胞排列整齐,大小正常,可见围绕中央静脉呈放射状排列的肝细胞索,细胞核大而饱满,未见脂肪滴及炎性细胞浸润;MG组肝细胞排列疏松、紊乱,脂肪滴呈弥漫性堆积,肝细胞呈气球样变,可见不同程度的炎性细胞浸润,点状坏死。经灌胃后,TB组较TD组肝脏脂肪变性程度减轻,炎症细胞浸润减少,但与NG组相比其脂肪变及炎症程度仍较明显。回肠病理:CG组小鼠末端回肠黏膜结构完整,绒毛及腺体排列整齐,上皮细胞形态正常,呈高柱状;MG组小鼠回肠绒毛变疏松,上皮细胞出现坏死和脱落,间隙增宽,可见上皮层与固有层分离。灌胃干预后,TB组较TD组黏膜损伤减轻,但仍比NG组损伤严重。4透射电镜观察回肠组织超微结构改变CG组肠黏膜上皮细胞排列紧密,表面微绒毛丰富,排列规则,紧密连接比较长,连接缝隙小;MG组肠黏膜上皮排列较规则,部分上皮细胞表面微绒毛倒伏、稀疏,紧密连接结构破坏。布拉氏酵母菌灌胃后,TB组较TD组上皮细胞表面微绒毛逐渐丰富,紧密连接结构恢复,连接缝隙减小,但仍比NG组损伤严重。5血清生化指标比较MG组较CG组血清ALT、AST、TC及TG显著升高(P0.05);灌胃干预后,与TD组相比,TB组血清ALT、AST、TC及TG显著降低(P0.05),但仍高于NG组(P0.05),差异有统计学意义。6血清LPS水平比较MG组较CG组血清LPS显著升高(P0.05);灌胃干预后,TB组血清LPS较TD组降低(P0.05),但仍高于NG组(P0.05),差异有统计学意义。7 Egr-1、TNF-α在动物肝脏组织的表达情况应用PCR及Western Blot技术从基因及蛋白水平检测肝组织中Egr-1、TNF-α表达,结果均显示,MG组Egr-1、TNF-α表达明显高于CG组(P0.05);灌胃干预后,TB组表达量较TD组显著降低(P0.05),但仍明显高于NG组(P0.05),差异有统计学意义。8 Occludin、TNF-α在动物末端回肠的表达情况PCR及Western Blot结果均显示:MG组回肠组织Occludin蛋白较CG组表达降低(P0.05);灌胃干预后,TB较TD组表达增加(P0.05),但仍低于NG组(P0.05),差异有统计学意义。MG组回肠组织中TNF-α较CG组表达增加(P0.05);灌胃干预后,TB较TD组表达降低(P0.05),但仍高于NG组(P0.05),差异有统计学意义。结论:1西方饮食可诱导NAFLD,NAFLD存在内毒素血症。2 NAFLD小鼠肠粘膜机械屏障受损,肠道通透性增加,可能加重了肠源性内毒素血症。3 NAFLD小鼠肝脏Egr-1表达增加,可能参与了NAFLD的发病过程。4布拉氏酵母菌可能通过改善肠粘膜机械屏障,减轻肠源性内毒素血症,降低了肝脏Egr-1的表达,从而改善了肝脏炎症及脂肪变程度。
[Abstract]:Objective: to establish nonalcoholic fatty liver disease (non-alcoholic fatty liver disease in the Western diet, NAFLD) mouse model, observe the level of serum endotoxin and liver of early growth response factor -1 (early growth response-1, Egr-1), tumor necrosis factor alpha (tumor alpha factor- alpha necrosis, TNF-) expression and intestinal mucosal changes. And to explore the intervention effect of Saccharomyces boulardii, preliminary discuss the curative effect of drugs on NAFLD and related mechanism. Methods: 30 6-8 week old male C57BL/6 mice were fed for 1 weeks after application were randomly divided randomly into normal control group (CG, n=12) and model group (MG. N=18). Normal control group was given normal diet (AIN93M control diet + normal drinking water feeding), model group were given the Western diet (high fat diet + high glucose in drinking water) feeding. Feeding 24 weekends were killed Each group of 6 mice, take corresponding specimens, the liver and the HE staining, the pathological changes of NAFLD mouse model confirmed successful. Since the twenty-fifth week, the model group, the remaining 12 mice were randomly divided into Saccharomyces boulardii gavage group (TB, n=6) model and gavage control group (TD, n=6), to the Western diet the normal control group feeding. The remaining 6 mice into normal gavage control group (NG, n=6), continue to give normal diet.TB group was given boulardii (5.98 x 108 CFU/kg/day) by gavage, TD group and NG group also began to give the corresponding materials of Saccharomyces boulardii fructose (284mg/kg/day) + lactose (19mg/kg/day) solution by intragastric administration in distilled water gavage 1 times, a total of 6 weeks, all mice were killed after 6 weeks, the specimen of the corresponding detection. Observe the hair color, behavior, mental state, and body weight was recorded weekly. After the death of small rats, the general situation of the observation of liver and ileum, And complete resection of the liver, specimens from the distal ileum, called wet liver weight; the serum alanine aminotransferase automatic biochemical analyzer (alanine aminotransferase, ALT), aspartate aminotransferase (aspartate, aminotransferase, AST), triglycerides (triglyceride, TG), total cholesterol (total, cholesterol, TC) level of serum; lipopolysaccharide using Limulus amebocyte lysate (lipopolysaccharide, LPS) level; observe the pathological changes of liver and terminal ileum mucosa HE staining; TEM ileal tissue changes; using Real-time Q-PCR (Real-time quantitation PCR) method to detect the mouse liver Egr-1, TNF- alpha TNF- alpha and terminal ileum tissue, the expression of Occludin m RNA; Western blot (Western Blot) method for the detection of liver Egr-1, TNF- alpha and terminal ileum tissue of Occludin, expression of TNF- protein. Results: 1 The general situation of the experimental animal control mice skin smooth, dense, shiny, good mental state, flexible, responsive, body weight increased gradually; the model mice model was made fourth weeks fur less smooth, slightly greasy, poor spirit, weight increase rapidly, gradually abdominal obesity, to 24 week, apathetic, lie lazy, sluggish, hair dull, modeling is completed. Given boulardii gavage intervention, group TB mice weight decreased, the activity increased, the situation improved. During the experiment, TB group and TD group were 1 rats died, the remaining 5, the remaining group no casualties occurred in mice.2 experimental animal body weight, liver weight and liver weight index in MG group compared with CG group of mice weight index increased significantly (P0.05); gavage intervention, TB group than in TD group significantly decreased body weight and liver weight index (P0.05), but still higher than that in group NG (P0.05), there are differences statistical significance The meaning of liver and.3 terminal ileum tissue pathology experimental animal liver pathology: in CG group the structure of hepatic lobule integrity, liver cells arranged in neat rows, normal size, visible around the central vein radially cords of liver cells, the nucleus is big and full, no fat droplets and inflammatory cell infiltration; MG group of liver cells arranged in loose and disorder that fat droplets diffuse accumulation, the liver cells showed ballooning, showed different degrees of inflammatory cell infiltration, spotty necrosis. After intragastric administration, TB group than in TD group reduced the degree of liver steatosis, inflammatory cells infiltration, but compared with the NG and the degree of fatty degeneration and inflammation of the ileum was still obvious. Pathology: CG group were terminal ileum mucosa structure integrity, villi and glands lined epithelial cells, normal, columnlike; MG group of mouse ileum villi became loose, epithelial cell necrosis and shedding, gap widened, visible epithelial layer and solid A layer of separation. Intragastric administration intervention, reduce the TB group than in TD group mucosal injury, but still higher than the NG group was seriously damaged.4 transmission electron microscope to observe the ultrastructural changes of ileal tissue in CG group intestinal epithelial cells arranged closely, microvilli on the surface of rich, arranged and closely connected to long connection gap of small intestinal mucosa in MG group; epithelial cells were regular, part of epithelial cell microvilli on the surface of lodging, sparse, tight junction damage. Boulardii after intragastric administration, TB group and TD group of epithelial cell surface microvilli gradually, tight junction recovery, gap junction decreased, but still higher than the NG group was seriously damaged.5 serum biochemical indexes in MG group than in CG group serum ALT, AST, TC and TG were significantly increased (P0.05); gavage intervention, compared with the TD group, TB group, serum ALT, AST, TC and TG were significantly reduced (P0.05), but still higher than that of NG group (P0.05), the difference had statistical significance between the serum level of LPS in.6 MG group than in CG 缁勮��娓匧PS鏄捐憲鍗囬珮(P0.05);鐏岃儍骞查�勫悾�

本文编号：1756715