海兔素对慢性酒精性肝损伤大鼠TLR4信号转导通路和肠道菌群的影响

发布时间：2018-06-03 13:25

本文选题：海兔素 + 酒精性肝病　；参考：《青岛大学》2016年博士论文

【摘要】：目的:海兔素(Aplysin)是从海洋三列凹顶藻中提取的脂溶性化合物,属于溴代倍半萜。本实验室研究证实海兔素具有多样生物学活性,主要包括抗氧化、抗炎、抗肿瘤、免疫增强等。并且初步发现其对酒精诱导肝损伤大鼠具有保护效果,机制与调控线粒体介导的肝细胞凋亡有关。本课题在此研究基础之上,从海兔素调控Kupffer细胞中TLR4信号转导通路与调节肠道菌群两个方面,继续探讨海兔素保肝功能与作用机制。方法:1.海兔素改善慢性酒精性肝损伤的效果评价(1)动物分组及模型建立:本实验选取两月龄健康雄性Wistar大鼠,共60只,体重大约180-220g,根据体重进行随机数字表法分组,随机分为3组,每组各20只。酒精模型组给予56度白酒(北京红星二锅头)8 ml/(kg·bw·d),灌胃2周后,剂量提升到12 ml/(kg·bw·d),继续灌胃6周;海兔素组酒精剂量同模型组,同时每日给予海兔素150 mg·kg-1·d-1灌胃,持续8周;正常组以等体积生理盐水灌胃,持续8周。末次灌胃12小时后,使用代谢笼留取粪便,大鼠称重后给予7%水合氯醛麻醉,腹主动脉取血,剥取肝组织及小肠组织。(2)肝脏病理学检查:HE染色观察大鼠肝脏组织病理学改变及透射电镜观察大鼠肝细胞超微结构变化。(3)肝功能评价:采用赖氏法检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)活性;PNP比色法检测血清碱性磷酸酶(ALP)活性;采用羟胺三氯化铁比色法检测血清胆碱酯酶(CHE)活性;采用IFCC速率法检测血清谷氨酰转移酶(GGT)活性。2.海兔素对慢性酒精性肝损伤大鼠Kupffer细胞吞噬能力和TLR4信号转导通路影响(1)原代Kupffer细胞获取:采用大鼠门静脉胶原酶Ⅳ原位灌注法和Percoll密度梯度离心法分离大鼠肝脏Kupffer细胞,所得Kupffer细胞在37℃和5%CO2条件下进行培养。(2)ED2免疫化学染色鉴定Kupffer细胞纯度,Giemsa染色观察Kupffer细胞形态,墨汁吞噬实验检测Kupffer细胞吞噬能力,MTT实验检测Kupffer细胞活力水平。(3)采用显色鲎试剂检测血浆内毒素水平。(4)逆转录PCR法检测Kupffer细胞中TLR4信号转导通路关键蛋白CD14,TLR4和NF-κB m RNA水平的表达。(5)Western Blot法检测CD14,TLR4蛋白表达水平。(6)ELISA法检测Kupffer细胞中TLR4通路下游炎性细胞因子TNF-α和IL-1β含量。3.海兔素对慢性酒精性肝损伤大鼠肠道菌群的影响(1)小肠病理学检查:HE染色观察小肠上皮组织病理学改变,透射电镜对大鼠小肠上皮细胞超微结构进行观察。(2)大鼠粪便肠道菌群基因组DNA提取:采用特异性结合DNA的离心吸附柱和抑制剂吸附片Inhibit EX提取粪便肠道菌群基因组DNA。(3)肠道菌群代表菌株定量分析:采用实时荧光定量PCR技术对粪便大肠杆菌、粪肠球菌、双歧杆菌和乳酸杆菌16S r DNA V3可变区进行定量分析。(4)肠道菌群菌株数量与肝功酶学指标相关性分析:使用SPSS软件,对肠道菌群的菌株数量与肝功酶学指标作Pearson相关性分析。结果:1.海兔素改善慢性酒精性肝损伤大鼠的效果评价HE染色结果显示与正常对照组相比,酒精模型组中央静脉出现大量炎性细胞浸润,脂肪变性明显且发现Mallory小体。正常对照组和海兔素干预组肝小叶结构正常,肝细胞索以中央静脉为中心呈现辐射状,没有发现典型脂肪变性和炎性细胞浸润。透射电镜观察发现相比正常对照组,酒精模型组内质网扩张明显,出现大量溶酶体,正常对照组与海兔素干预组核膜光滑,核孔清晰,线粒体结构完整,但海兔素组出现少量脂滴。通过对血清中五种肝功能酶学指标检测发现,模型对照组血清中ALT,AST,ALP,CHE和GGT活性水平相对正常对照组分别显著增高(P0.05);而海兔素干预组血清中ALT,AST,ALP,CHE和GGT活性水平相对酒精模型组分别显著降低(P0.05)。2.海兔素对慢性酒精性肝损伤大鼠Kupffer细胞吞噬能力和TLR4信号转导通路影响原代培养Kupffer细胞呈现典型梭子形状或者不规则三角形形状。ED2免疫化学染色发现Kupffer细胞纯度达到97%以上。Giemsa染色结果发现Kupffer细胞表现出典型“荷包蛋”形状。墨迹吞噬实验发现各组Kupffer细胞胞浆中均可见吞噬的碳素颗粒,正常对照组细胞多维持细胞原有形状,呈多角形、菱形等不规则形状,酒精模型组细胞胞浆中可见大量碳素颗粒积聚,大部分细胞胀大呈圆形或椭圆形,Kupffer细胞吞噬能力明显下降;海兔素干预组多数细胞维持原有形状,部分细胞胀大呈圆形,吞噬能力相对酒精模型组明显回升。MTT实验结果显示酒精模型组Kupffer细胞活力较正常对照组明显增强,而海兔素组Kupffer细胞活力较酒精模型组显著下降。酒精模型对照组血浆内毒素水平相对正常对照组显著升高20%(P0.05),海兔素组血浆内毒素水平相对酒精模型组显著下降7.1%(P0.05)。逆转录PCR结果显示Kupffer细胞中酒精模型组CD14,TLR4和NF-κB m RNA表达相对正常对照组分别上升(1.1倍,1.7倍和2.0倍,P0.05)。海兔素组中CD14,TLR4和NF-κB m RNA表达水平相对酒精模型组显著降低38%,20%和23%。Western Blot结果显示,酒精模型组大鼠肝脏CD14,TLR4蛋白表达水平相较正常对照组显著性上升1.25倍和1.13倍,海兔素干预组CD14,TLR4蛋白表达水平相较酒精模型组显著性下降23%和20%。ELISA结果显示酒精模型组TNF-α和IL-1β含量相较正常对照组分别显著提高2.1倍和1.7倍,海兔素组TNF-α和IL-1β含量相较酒精模型组分别下降39%和31%(P0.05)。3.海兔素对慢性酒精性肝损伤大鼠肠道菌群的影响HE染色结果显示酒精模型组中小肠绒毛结构发生明显萎缩。透射电镜观察到酒精模型组细胞连接变宽,微绒毛结构稀疏,海兔素组出现大量溶酶体。菌株定量分析显示相对正常对照组,酒精模型组大肠杆菌(P0.05)数量显著升高,粪肠球菌数量呈上升趋势(P0.05)。相对酒精模型组,海兔素组大肠杆菌数量显著下降(P0.05),粪肠球菌数量呈下降趋势(P0.05)。另外相对正常对照组,酒精模型组乳酸杆菌与双歧杆菌数量都显著下降(P0.05),海兔素干预后,乳酸杆菌数量呈上升趋势(P0.05),双歧杆菌数量显著升高(P0.05)。通过4个代表菌株数量与肝功能酶学指标作相关性分析结果发现大肠杆菌数量与血清谷丙转氨酶活性呈正相关的高度相关关系(P0.05)。结论:1.根据肝脏病理学观察和肝功能酶活性检测,海兔素对慢性酒精性肝损伤大鼠具有改善作用。2.海兔素通过增强慢性酒精性肝损伤大鼠肝脏Kupffer细胞吞噬能力,加大对内毒素清除,抑制Kupffer细胞中TLR4信号转导通路的激活,降低CD14,TLR4和NF-κB表达和下游炎性因子TNF-α和IL-1β含量达到保肝效果。3.海兔素对慢性酒精性肝损伤大鼠小肠同样起保护效果;海兔素保肝效果可能与其调节肠道菌群有关;大肠杆菌可能是海兔素调节肠道菌群改善慢性酒精性肝损伤大鼠的相关菌株。
[Abstract]:Objective: hahnin (Aplysin) is a fat soluble compound extracted from three marinas in the sea and belongs to brominated sesquiterpene. This laboratory study confirmed that hahnin has a variety of biological activities, mainly including antioxidant, anti-inflammatory, anti-tumor, immune enhancement and so on. And it has been found to have protective effect on alcohol induced liver injury in rats. It is related to the regulation of mitochondria mediated hepatocyte apoptosis. On the basis of this study, we continue to explore the function and mechanism of Rabbit Hare liver preservation from the two aspects of the TLR4 signal transduction pathway and the regulation of intestinal microflora in Kupffer cells. Methods: the evaluation of the effect of 1. hare on the improvement of slow alcoholic liver injury (1) animal score. Group and model establishment: a total of 2 month old healthy male Wistar rats were selected, with a total of 60 rats with a weight of about 180-220g. According to the weight, random numbers were divided into 3 groups and 20 were randomly divided into 3 groups. The alcohol model group was given 56 degree liquor (Beijing red Star Erguotou) 8 ml/ (kg. BW. D). After gavage for 2 weeks, the dose was promoted to 12 ml/ (kg. BW d) and continued. Continue After 6 weeks gavage, the alcohol dose in the hare group was the same as that in the model group. At the same time, the rabbits were given the hare 150 mg. Kg-1. D-1 for 8 weeks. The normal group was given the stomach with equal volume of normal saline for 8 weeks. After the last perfusion of the stomach for 12 hours, the excrement was taken by the metabolic cage, the rats were weighed with 7% chloral anaesthesia, the abdominal aorta was taken blood, and the liver tissue and small intestine were stripped. Organization. (2) liver pathological examination: HE staining observed the pathological changes of liver tissue and ultrastructural changes in rat liver. (3) liver function evaluation: serum glutamic prop aminotransferase (ALT) and cereal transaminase (AST) activity were detected by Ryan's method; PNP colorimetry was used to detect the activity of serum alkaline phosphatase (ALP); hydroxylamine three chloride was used. Serum cholinesterase (CHE) activity was detected by ferric colorimetry, and IFCC rate method was used to detect the effect of serum glutamyl transferase (GGT) activity.2. hare on the phagocytosis of Kupffer cells and TLR4 signal transduction pathway of Kupffer cells in rats with chronic alcoholic liver injury (1) primary Kupffer cells: using rat portal vein collagenase IV in situ perfusion and Percoll Kupffer cells of rat liver were separated by density gradient centrifugation. The obtained Kupffer cells were cultured at 37 and 5%CO2. (2) ED2 immunochemistry staining was used to identify the purity of Kupffer cells, Giemsa staining was used to observe the morphology of Kupffer cells, the phagocytic energy of Kupffer cells was detected by the ink phagocytosis test, and the MTT test was used to detect the activity level of Kupffer cells. (3) The level of plasma endotoxin was detected by chromogenic limulus reagent. (4) the expression of TLR4 signal transduction pathway key protein CD14, TLR4 and NF- kappa B m RNA level in Kupffer cells was detected by reverse transcriptase PCR. (6) Western Blot method was used to detect the expression level of CD14 and protein. The effect of hare on intestinal flora of rats with chronic alcoholic liver injury (1) pathological examination of small intestine: Observation of pathological changes of intestinal epithelium by HE staining, ultrastructure of small intestinal epithelial cells in rats by transmission electron microscopy. (2) extraction of genomic DNA from fecal intestinal flora of rats: centrifuge column and inhibition by specific combination of DNA Inhibit EX extraction of fecal intestinal microflora DNA. (3) intestinal microflora representative strains quantitative analysis: the quantitative analysis of fecal Escherichia coli, Enterococcus faecalis, bifidobacteria and Lactobacillus 16S R DNA V3 variable region by real-time fluorescence quantitative PCR. (4) the correlation analysis of the number of intestinal microflora and liver function enzymology index: SPSS software was used to analyze the Pearson correlation between the number of bacteria in the intestinal flora and the liver function enzymology index. Results: 1. the effect of hare on the improvement of chronic alcoholic liver injury in rats. The results of HE staining showed that there was a lot of inflammatory cell infiltration in the central vein of the alcohol model group compared with the normal control group, and the fatty degeneration was obvious and the Mallor was found. The hepatic lobules in the normal control group and the hare intervention group were normal, the hepatic cord was radiated with the central vein as the center, and no typical fatty degeneration and inflammatory cell infiltration were found. The transmission electron microscope observed that the endoplasmic reticulum of the alcohol model group showed a large number of lysosomes, the normal control group and the hare, compared with the normal control group. In the vegetarian intervention group, the nuclear membrane was smooth, the nuclear pore was clear and the structure of mitochondria was complete, but a small amount of lipid droplets appeared in the hare group. The serum levels of ALT, AST, ALP, CHE and GGT were found to be Zhuo Zenggao (P0.05) in the serum of the model control group compared with the normal control group, and the serum ALT, AST, ALP, C in the hare intervention group. The activity level of HE and GGT decreased significantly compared with the alcohol model group (P0.05) the phagocytosis of Kupffer cells and the TLR4 signal transduction pathway in the Kupffer cells of the rats with chronic alcoholic liver injury were influenced by the TLR4 signal transduction pathway, and the original culture Kupffer cells presented the typical shuttle shape or the irregular triangle shape.ED2 immunochemical staining found that the purity of Kupffer cells was reached. More than 97%.Giemsa staining results showed that Kupffer cells showed a typical "Lotus egg" shape. The phagocytic carbon particles were found in the cytoplasm of Kupffer cells in each group. The cells in the normal control group maintained the original shape of the cells, were polygonal, rhombus and other irregular shapes, and a large amount of carbon was found in the cytoplasm of the alcohol model group. The accumulation of prime particles, most of the cells swelled round or oval, the phagocytosis of Kupffer cells decreased obviously, most of the cells in the hare intervention group maintained the original shape, some cells swelled and round, and the phagocytic ability compared with the alcohol model group.MTT experimental results showed that the activity of Kupffer cells in the alcohol sperm model group was more obvious than that of the normal control group. The activity of Kupffer cells in the hare group was significantly lower than that in the alcohol model group. The plasma endotoxin level in the alcohol model control group was significantly increased by 20% (P0.05), and the plasma endotoxin level in the hare group decreased by 7.1% (P0.05) significantly (P0.05). The reverse transcriptase PCR results showed that the alcohol model group in Kupffer cells was CD14, TLR The expression of 4 and NF- kappa B m RNA increased (1.1 times, 1.7 times and 2 times, P0.05). The RNA expression level of CD14, TLR4 and NF- kappa B m in the hare group was significantly lower than that of the alcohol model group by 38%, 20% and 23%.Western results showed that the level of protein expression in the alcohol model group was significantly higher than that of the normal control group. 1.25 times and 1.13 times the CD14, the expression level of TLR4 protein in the hare intervention group was significantly lower than that of the alcohol model group by 23% and 20%.ELISA results showed that the content of TNF- A and IL-1 beta in the alcohol model group was significantly increased by 2.1 and 1.7 times compared with the normal control group. The TNF- alpha and IL-1 beta content in the hare group decreased by 39% and 31% (P0.05), respectively. The effect of.3. hare on the intestinal flora of rats with chronic alcoholic liver injury HE staining showed that the structure of small intestinal villi in the alcohol model group was obviously atrophied. The transmission electron microscope showed that the cell connections in the alcohol model group became wider, the microvilli structure was sparse, and the hare group appeared a large number of lysosomes. The number of Escherichia coli (P0.05) in the alcohol model group increased significantly and the number of Enterococcus faecalis increased (P0.05). Compared with the alcohol model group, the number of Escherichia coli in the hare group decreased significantly (P0.05) and the number of Enterococcus faecalis declined (P0.05). In addition, the number of lactobacillus and Bifidobacterium in the alcohol model group decreased significantly (P0.05). P0.05), the number of Lactobacillus increased significantly (P0.05) and the number of bifidobacteria increased significantly (P0.05). The correlation analysis between the number of 4 representative strains and the hepatic functional enzymology indicators found that the number of Escherichia coli was highly correlated with the activity of serum glutamic pyruvic aminotransferase (P0.05). Conclusion: 1. according to the liver Histopathological observation and detection of liver function enzyme activity, hahnin has an improved effect on chronic alcoholic liver injury in rats..2. hahnin can enhance the phagocytosis of Kupffer cells in liver of rats with chronic alcoholic liver injury, increase the clearance of endotoxin, inhibit the activation of TLR4 signal transduction pathway in Kupffer cells and reduce the expression of CD14, TLR4 and NF- kappa B The TNF- alpha and IL-1 beta content of downstream inflammatory factors can protect the liver from the effect of.3. hahnin on the small intestine of rats with chronic alcoholic liver injury, and the effect of hhnin protecting liver may be related to the regulation of intestinal flora, and E. coli may be the related strain of the rabbit rabbit with the regulation of intestinal microflora to improve the chronic alcoholic liver injury in rats.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5

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