布拉氏酵母菌对NAFLD大鼠肠黏膜屏障保护作用的研究
发布时间：2018-08-27 08:26
【摘要】：目前,非酒精性脂肪性肝病(Non-alcoholic fatty liver disease,NAFLD)是全球重要的健康问题之一,也是我国越来越受重视的慢性肝病问题。它是一组由单纯肝脏脂肪变性,发展到脂肪性肝炎(Non-alcoholic steatohepatitis,NASH)、肝硬化、肝癌的临床症候群。近年来随着肠道微生态与NAFLD发病关系研究的深入,肠道菌群过度生长、肠黏膜通透性增加所致的内毒素血症刺激机体炎症反应在NAFLD的发生发展中的作用得到越来越多的认可。布拉氏酵母菌是目前唯一上市的真菌微生态制剂,它为肠道过路菌,在停药一周后粪便中就不可检测到该菌,不会影响肠道内菌量。口服后不受胆汁、胃酸等破坏。它可与致病菌在肠上皮的黏附形成竞争,可以抑制炎症信号的传递表达。我们通过饲以高脂饮食成功造模,研究布拉氏酵母菌对NAFLD大鼠肠黏膜屏障的保护作用,将有助于为NAFLD的治疗及预防提供理论基础。目的:观察布拉氏酵母菌对NAFLD大鼠肠黏膜屏障的保护作用,并对其可能机制进行探讨。方法:实验用雄性健康Sprague-Dawley大鼠36只,体重为180±20g,适应性喂养1周后随机分为三组,即正常对照组(Control组,C组)、高脂模型组(Model组,M组)、高脂治疗组(Treatment组,T组),每组12只;C组饲以基础饲料(其热量为碳水化合物65.5%,蛋白质24.2%,脂肪10.3%)喂养12周;M组和T组饲以高脂饲料(碳水化合物和蛋白质各20.1%,脂肪59.8%)喂养12周;每组各随机处死2只,苏丹黑染色观察肝脏脂质沉积程度确定造模成功。随后T组开始给予布拉氏酵母菌灌胃(75*108 CFU/kg?d-1),C、M组给予等容量的生理盐水灌胃;治疗过程中每周随体重变化调整灌胃量。20周时在麻醉状态下腹主动脉取血处死大鼠,收集血液测定丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、甘油三酯(TG)、肠型脂肪酸结合蛋白(IFABP)、肿瘤坏死因子-α(TNF-α)、内毒素水平;观察肝脏的大体情况,称肝脏湿重,采用苏丹黑染色观察肝脏病理变化;肠组织行HE染色观察肠绒毛完整程度,免疫组化染色检测肠黏膜occludin蛋白的表达情况。留取大鼠盲肠内粪便,用于粪便大肠杆菌、拟杆菌定量检测。结果:1大鼠体重(g)、肝重(g)和肝指数(肝重/体重×100%):M组大鼠的体重(548.7±16.1)、肝重(17.516±1.112)、肝指数(3.191±0.170)均明显高于C组的体重(423.2±19.2)、肝重(9.493±0.739)及肝指数(2.241±0.112),差异有统计学意义(P0.05)。T组体重(499.1±18.1)、肝重(13.062±1.307)和肝指数(2.616±0.230)与M组相比均明显降低,差异有统计学意义(P0.05)。2血清TG(mmol/l)、ALT(u/l)、AST(u/l)水平:M组TG(0.585±0.057)、ALT(78.75±10.92)、AST(205.74±10.05)较C组TG(0.373±0.081)、ALT(30.99±8.59)、AST(108.04±11.60)显著增高,差异有统计学意义(P㩳0.05);T组的AST(194.42±13.74)与M组相比明显降低,差异有统计学意义(P0.05);T组TG(0.541±0.072)、ALT(72.36±9.87)与M组相比稍有下降,无统计学差异(P0.05)。3血清内毒素(EU/ml)及TNF-α(ng/ml)水平:M组内毒素(0.306±0.054)、TNF-α(1.160±0.129)较C组内毒素(0.201±0.047)、TNF-α(0.917±0.115)显著升高,差异有统计学意义(P0.05);T组内毒素(0.249±0.026)、TNF-α(1.042±0.078)与M组相比均明显降低,有显著性差异(P0.05)。4血清IFABP(pg/ml)水平:M组IFABP(288.76±59.17)较C组IFABP(137.19±39.12)明显升高,差异有统计学意义(P0.05);T组IFABP(193.02±27.13)与M组相比明显降低,差异有统计学意义(P0.05)。5组织病理结果:(1)在12周时苏丹黑染色显示M、T组肝细胞脂肪变明显增加,证实造模成功。20周时肝组织苏丹黑染色提示M组肝细胞脂肪变(2.90±0.32)较C组(0.00±0.00)明显增加(P0.05);治疗后,T组肝细胞脂肪变(1.80±0.79)较M组明显减少(P0.05)。(2)肠黏膜组织HE染色结果:光镜下,C组肠绒毛排列整齐,结构完整;M组肠绒毛变短、局部有绒毛脱落,排列欠整齐,可见炎性细胞浸润;与M组相比,T组肠绒毛轻度水肿,排列较整齐,炎性细胞浸润较少。6免疫组织化学结果:M组的肠黏膜occludin蛋白(0.042±0.006)较C组(0.177±0.007)明显减少,差异有统计学意义(P0.05);T组(0.049±0.010)较M组表达稍有增加,但无统计学意义(P0.05)。7粪便PCR结果(log10(CFU/g)):(1)M组的大肠杆菌(E.Coli)数量(5.98±0.08)较C组(4.00±0.09)明显增加,差异有统计学意义(P0.05),与M组相比,T组大肠杆菌数量(4.92±0.09)明显下降,差异有统计学意义(P0.05)。(2)M组的拟杆菌数量(8.04±0.17)较C组(9.47±0.09)明显减少,差异有统计学意义(P0.05),与M组相比,T组的拟杆菌数量(8.89±0.10)明显增加,差异有统计学意义(P0.05)。结论:1高脂饮食可成功诱导大鼠NAFLD,经布拉氏酵母菌干预可以起到减轻体重和改善肝细胞脂肪变的作用。2布拉氏酵母菌能通过减轻NAFLD大鼠内毒素血症,减轻机体炎症反应。3布拉氏酵母菌能调节NAFLD大鼠肠道大肠杆菌和拟杆菌的比例。
[Abstract]:Nowadays, non-alcoholic fatty liver disease (NAFLD) is one of the most important health problems in the world, and it is also a chronic liver disease that has been paid more and more attention in China. In recent years, with the deepening of the relationship between intestinal microecology and the pathogenesis of NAFLD, intestinal flora overgrowth and intestinal mucosal permeability increase caused by endotoxemia stimulate the role of inflammation in the occurrence and development of NAFLD has been more and more recognized. Passage-passing bacteria can not be detected in the stool after a week of drug withdrawal and do not affect the amount of bacteria in the intestine.They are not damaged by bile and gastric acid after oral administration.They can compete with pathogenic bacteria in the intestinal epithelium and inhibit the transmission and expression of inflammation signals.We successfully established a model by feeding high-fat diet to study the effect of Saccharomyces brasiliensis on NAF. OBJECTIVE: To observe the protective effect of Saccharomyces brasiliensis on intestinal mucosal barrier in rats with NAFLD and to explore its possible mechanism. Then they were randomly divided into three groups: normal control group (Control group, C group), high-fat model group (Model group, M group), high-fat treatment group (Treatment group, T group), 12 rats in each group; group C was fed with basic diet (65.5% carbohydrate, 24.2% protein, 10.3% fat) for 12 weeks; group M and group T were fed with high-fat diet (20.1% carbohydrate and 20.1% protein, respectively). Fat 59.8% was fed for 12 weeks; 2 rats in each group were sacrificed randomly, and Sudan black staining was used to observe the degree of liver lipid deposition to determine the success of modeling. Rats were sacrificed by taking blood from the lower abdominal aorta and blood samples were collected to determine the levels of ALT, AST, TG, IFABP, TNF - alpha and endotoxin. HE staining was used to observe intestinal villi integrity and immunohistochemical staining was used to detect occludin protein expression in intestinal mucosa. The body weight, liver index (3.191 (+0.170)) and liver index (9.493 (+0.739) of group C were significantly higher than those of group C (423.2 (+19.2)), and liver index (2.241 (+0.112)). The body weight, liver weight (13.062 (+1.307) and liver index (2.616 (+0.230)) of group T were significantly lower than those of group M (P 0.05). Mmol / l, ALT (u / l), ALT (u / l) and AST (u / l) levels: TG (0.585 (+ 0.057), ALT (78.75 (+ 10.92), ALT (78.75 (+ 10.92), AST (205.74 (+ 10.05) (205.74 (+ 10.74 (+ 10.05) TG (0.373 [(0.373 +0.081), ALT (30.373 [(30.99 [8.99 [.59]), ALT (30.99 [(30.99 [8.59]]), AST (108.04 [(108.04 [11.04 [11.60] 0.05), AST A The levels of endotoxin (EU/ml) and TNF-alpha (ng/ml) in serum of group M were significantly higher than those of group C in endotoxin (0.306+0.054), TNF-alpha (1.160+0.129) and TNF-alpha (0.201+0.047) and TNF-alpha (0.917+0.115), respectively (P 0.05). The levels of serum IFABP (pg/ml) in group M (288.76+59.17) were significantly higher than those in group C (137.19+39.12) (P 0.05); IFABP (193.02+27.13) in group T (193.02+27.13) was significantly lower than that in group M (P 0.05). 5 histopathological results were statistically significant: (1) At 12 weeks, IFABP in group M (288.76+59.17) was significantly higher than that in group C (137.19+39.12) (P 0.05). Sudan black staining showed that hepatocyte steatosis in group M and T was significantly increased, which confirmed the success of modeling. At 20 weeks, Sudan black staining showed that hepatocyte steatosis in group M (2.90.32) was significantly increased compared with group C (0.00.00) (P 0.05); after treatment, hepatocyte steatosis in group T (1.80.79) was significantly decreased compared with group M (P 0.05). (2) HE staining of intestinal mucosa The intestinal villi of group C were arranged neatly and structurally intact; the intestinal villi of group M were shortened, some villi were shedding off, and irregular arrangement, and inflammatory cell infiltration was observed; compared with group M, the intestinal villi of group T were slightly edematous, arranged neatly, and inflammatory cell infiltration was less.6 Immunohistochemical results showed that occludin protein (0.042 + 0.006) of intestinal mucosa of group M was higher than that of group C (0.177 + 0.007). The expression of E. coli in group T (0.049.010) was slightly higher than that in group M (P 0.05), but there was no significant difference (P 0.05). 7 Fecal PCR results (log10 (CFU / g): (1) The number of E. coli in group M (5.98.08) was significantly higher than that in group C (4.00.09), and the number of E. coli in group T (4.05) was significantly higher than that in group M (P 0.05). (2) The number of Bacteroides in group M (8.04 + 0.17) was significantly lower than that in group C (9.47 + 0.09), and the difference was statistically significant (P 0.05). Compared with group M, the number of Bacteroides in group T (8.89 + 0.10) was significantly increased, and the difference was statistically significant (P 0.05). The intervention of Brucella yeast can reduce body weight and improve hepatic steatosis. 2 Brucella yeast can alleviate inflammation by reducing endotoxemia in NAFLD rats. 3 Brucella yeast can regulate the proportion of enteric E. coli and Bacteroides in NAFLD rats.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R575.5
本文编号：2206679
[Abstract]:Nowadays, non-alcoholic fatty liver disease (NAFLD) is one of the most important health problems in the world, and it is also a chronic liver disease that has been paid more and more attention in China. In recent years, with the deepening of the relationship between intestinal microecology and the pathogenesis of NAFLD, intestinal flora overgrowth and intestinal mucosal permeability increase caused by endotoxemia stimulate the role of inflammation in the occurrence and development of NAFLD has been more and more recognized. Passage-passing bacteria can not be detected in the stool after a week of drug withdrawal and do not affect the amount of bacteria in the intestine.They are not damaged by bile and gastric acid after oral administration.They can compete with pathogenic bacteria in the intestinal epithelium and inhibit the transmission and expression of inflammation signals.We successfully established a model by feeding high-fat diet to study the effect of Saccharomyces brasiliensis on NAF. OBJECTIVE: To observe the protective effect of Saccharomyces brasiliensis on intestinal mucosal barrier in rats with NAFLD and to explore its possible mechanism. Then they were randomly divided into three groups: normal control group (Control group, C group), high-fat model group (Model group, M group), high-fat treatment group (Treatment group, T group), 12 rats in each group; group C was fed with basic diet (65.5% carbohydrate, 24.2% protein, 10.3% fat) for 12 weeks; group M and group T were fed with high-fat diet (20.1% carbohydrate and 20.1% protein, respectively). Fat 59.8% was fed for 12 weeks; 2 rats in each group were sacrificed randomly, and Sudan black staining was used to observe the degree of liver lipid deposition to determine the success of modeling. Rats were sacrificed by taking blood from the lower abdominal aorta and blood samples were collected to determine the levels of ALT, AST, TG, IFABP, TNF - alpha and endotoxin. HE staining was used to observe intestinal villi integrity and immunohistochemical staining was used to detect occludin protein expression in intestinal mucosa. The body weight, liver index (3.191 (+0.170)) and liver index (9.493 (+0.739) of group C were significantly higher than those of group C (423.2 (+19.2)), and liver index (2.241 (+0.112)). The body weight, liver weight (13.062 (+1.307) and liver index (2.616 (+0.230)) of group T were significantly lower than those of group M (P 0.05). Mmol / l, ALT (u / l), ALT (u / l) and AST (u / l) levels: TG (0.585 (+ 0.057), ALT (78.75 (+ 10.92), ALT (78.75 (+ 10.92), AST (205.74 (+ 10.05) (205.74 (+ 10.74 (+ 10.05) TG (0.373 [(0.373 +0.081), ALT (30.373 [(30.99 [8.99 [.59]), ALT (30.99 [(30.99 [8.59]]), AST (108.04 [(108.04 [11.04 [11.60] 0.05), AST A The levels of endotoxin (EU/ml) and TNF-alpha (ng/ml) in serum of group M were significantly higher than those of group C in endotoxin (0.306+0.054), TNF-alpha (1.160+0.129) and TNF-alpha (0.201+0.047) and TNF-alpha (0.917+0.115), respectively (P 0.05). The levels of serum IFABP (pg/ml) in group M (288.76+59.17) were significantly higher than those in group C (137.19+39.12) (P 0.05); IFABP (193.02+27.13) in group T (193.02+27.13) was significantly lower than that in group M (P 0.05). 5 histopathological results were statistically significant: (1) At 12 weeks, IFABP in group M (288.76+59.17) was significantly higher than that in group C (137.19+39.12) (P 0.05). Sudan black staining showed that hepatocyte steatosis in group M and T was significantly increased, which confirmed the success of modeling. At 20 weeks, Sudan black staining showed that hepatocyte steatosis in group M (2.90.32) was significantly increased compared with group C (0.00.00) (P 0.05); after treatment, hepatocyte steatosis in group T (1.80.79) was significantly decreased compared with group M (P 0.05). (2) HE staining of intestinal mucosa The intestinal villi of group C were arranged neatly and structurally intact; the intestinal villi of group M were shortened, some villi were shedding off, and irregular arrangement, and inflammatory cell infiltration was observed; compared with group M, the intestinal villi of group T were slightly edematous, arranged neatly, and inflammatory cell infiltration was less.6 Immunohistochemical results showed that occludin protein (0.042 + 0.006) of intestinal mucosa of group M was higher than that of group C (0.177 + 0.007). The expression of E. coli in group T (0.049.010) was slightly higher than that in group M (P 0.05), but there was no significant difference (P 0.05). 7 Fecal PCR results (log10 (CFU / g): (1) The number of E. coli in group M (5.98.08) was significantly higher than that in group C (4.00.09), and the number of E. coli in group T (4.05) was significantly higher than that in group M (P 0.05). (2) The number of Bacteroides in group M (8.04 + 0.17) was significantly lower than that in group C (9.47 + 0.09), and the difference was statistically significant (P 0.05). Compared with group M, the number of Bacteroides in group T (8.89 + 0.10) was significantly increased, and the difference was statistically significant (P 0.05). The intervention of Brucella yeast can reduce body weight and improve hepatic steatosis. 2 Brucella yeast can alleviate inflammation by reducing endotoxemia in NAFLD rats. 3 Brucella yeast can regulate the proportion of enteric E. coli and Bacteroides in NAFLD rats.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R575.5
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