【作者】 许建慧；

【导师】 邹大进；

【作者基本信息】 第二军医大学 ， 内科学， 2013， 博士

【摘要】 [研究目的]随着生活水平的提高,肥胖已成为威胁人类健康的重要疾病。肥胖产生一系列健康问题,如高血压病,2型糖尿病,心血管疾病,非酒精性脂肪肝等。生活方式干预往往难以坚持,药物治疗的疗效与安全性受到很大的质疑,手术治疗的适应证也比较狭窄,这些都增加了肥胖的治疗难度。小檗碱是存在于黄连、黄柏等药材中的主要生物碱,临床上长期用于抗肠道细菌感染,近年来动物实验及临床研究证实,小檗碱能安全、有效地降脂、降糖、减轻体重,但其作用机制还并不十分明确。药理学研究发现,小檗碱生物利用度很低,基本不被吸收入血。因此,我们认为小檗碱可能主要针对靶器官-肠道发挥作用。研究已经证实,肠道内革兰阴性杆菌溶解释放的脂多糖入血会引起代谢性内毒素血症,通过激活全身性慢性低度炎症系统,从而导致肥胖和胰岛素抵抗等。肠黏膜屏障损伤,也会造成肠道细菌的移位,形成内毒素血症。此外,胃肠道激素对人体的能量代谢平衡也发挥重要作用,研究发现小檗碱能促进糖尿病大鼠肠道GLP-1分泌增加。本研究的目的是检测大鼠门静脉内毒素水平和系统性炎症水平,观察紧密连接蛋白分布和表达的变化,检测胃肠道激素分泌水平的变化,探讨小檗碱治疗对代谢性内毒素血症的保护作用,同时探讨其相关的肠道菌群结构、肠屏障功能、肠道激素分泌等改变对改善内毒素血症的影响,为临床治疗肥胖和胰岛素抵抗等相关疾病提供新的治疗策略和依据。[研究方法]SD大鼠30只,随机分成两组：高脂喂养组(HF, n=20)和低脂喂养组(LF, n=10),14周喂养结束时,以HF组大鼠的体重显著高于LF组为大鼠肥胖模型建造成功。再将高脂喂养的20只大鼠按体重的随机区组设计分为2组：高脂组(HF)和小檗碱治疗组(HB)。LF组继续低脂喂养,HF组继续高脂喂养,HB组在高脂喂养的基础上每天给予小檗碱150mg/kg体重灌胃。实验过程中每隔一天监测1次各组动物的进食、进水量,每周监测1次体重变化情况,小檗碱共干预6周。小檗碱治疗6周后,大鼠禁食12小时,用3%戊巴比妥钠(30mg/Kg)腹腔注射麻醉,肝门静脉取血,用动态比浊法检测内毒素,用Luminex液相芯片技术检测大鼠门静脉总GIP、Glucagon、PP、PYY的水平,ELISA技术检测总GLP-1和GLP-2的水平；眼球后内眦静脉取血,用全自动生化仪检测血糖、血脂、肝功,ELISA技术检测胰岛素。血取净后,取内脏以及腹部皮下脂肪,滤纸吸干,称重,部分用RT-PCR法检测炎性因子、氧化应激因子和巨噬细胞浸润标志物的表达,部分于4%多聚甲醛中固定用于HE染色。取部分肝脏,部分用RT-PCR法检测肝脏炎性因子、氧化应激因子和巨噬细胞浸润标志物的表达,部分于4%多聚甲醛中固定用于油红O染色。取近端结肠组织,RT-PCR法检测结肠紧密连接蛋白和胰高血糖素原的表达,免疫荧光技术检测结肠黏膜表面claudin-1和claudin-2的表达、分布以及结肠L细胞数量。收集大鼠盲肠内新鲜粪便,用焦磷酸测序分析肠道菌群的变化。采用SPSS13.0统计软件进行处理,测量结果以均数±标准差(x±s)表示。两组间比较采用t检验,多样本均数的比较采用单因素方差分析(one way ANOVA),组间比较,方差经正态分布检验,齐性检验采用Test of Homogeneity of Variances,如果方差齐用LSD检验,如果方差不齐用非参数检验的Tamhane’s T2法。p<0.05为差别有统计学意义。[结果]1.高脂饲料喂养14周成功建立肥胖大鼠模型14周结束时,高脂喂养组大鼠的体重显著大于低脂喂养组。2.小檗碱对肥胖大鼠体重和代谢的影响经过小檗碱6周的干预,肥胖组大鼠体重增加显著减少,内脏和皮下脂肪的重量均显著减小,进食量有下降趋势。小檗碱治疗使高脂喂养大鼠的空腹血糖和胰岛素显著降低,胆固醇、低密度脂蛋白胆固醇、游离脂肪酸均显著降低,高密度脂蛋白显著升高,肝谷丙转氨酶也显著降低,甘油三酯有下降趋势但无显著差异。小檗碱治疗使肥胖大鼠肝细胞脂肪变性程度显著减轻,肝脏胆固醇含量显著下降,甘油三酯含量有下降趋势但无显著差异,使肥胖大鼠单个脂肪细胞的面积显著减小。3.小檗碱改善代谢性内毒素血症小檗碱干预6周后,肥胖大鼠门静脉中内毒素水平显著降低。小檗碱治疗使肥胖大鼠肝脏中IL-1β、NADPHox、CD68、TLR4、LBP mRNA的表达均显著降低,使MIF mRNA的表达有增加趋势但无显著差异。小檗碱治疗使肥胖大鼠内脏脂肪中IL-1β、PAI-1、STAMP2、NADPHox、MCP-1、F4/80mRNA的表达均显著降低,使TNF-a mRNA有降低趋势但无显著差异。4.小檗碱改善结肠肠道屏障功能,降低肠道通透性小檗碱干预6周后,肥胖大鼠尾静脉血浆的FITC-dextran曲线下面积显著减小,近端结肠中紧密连接蛋白claudin-1、ZO-1mRNA的表达水平均显著增加,MLCK mRNA的表达显著减少,而occludin mRNA的表达有增加趋势,claudin-2mRNA的表达基本没有变化。免疫荧光显示,小檗碱能抑制肥胖大鼠肠道claudin-1蛋白表达的下降,对claudin-2蛋白的表达没什么影响,但小檗碱能抑制claudin-2蛋白从黏膜表面向隐窝移位。5.小檗碱改善胃肠激素的分泌,促进L细胞增殖小檗碱干预6周使肥胖大鼠门静脉中胃肠激素总GLP-1、GLP-2、PYY的水平均显著增加,总GIP和PP的水平均显著下降,glucagon有下降趋势。小檗碱还能显著增加肥胖大鼠近端结肠中的L细胞数量,使结肠proglucagon mRNA的表达显著增加。6.小檗碱调节肠道菌群结构小檗碱治疗使肥胖大鼠肠道菌群的多样性和菌群丰度显著减少,共有55种菌属受到小檗碱显著影响。其中硬壁菌门中Coprococcus、Lachnospiraceae、Roseburia、 Ruminococcus、Faecalibacterium属的丰度均显著减少,变形菌门中的脱硫弧菌属(Desulfovibrio)丰度也显著降低。[结论]1.小檗碱灌胃能对肥胖大鼠起到减轻体重,改善糖耐量,调节血脂的作用,还能改善大鼠的肝功。2.小檗碱能通过改善代谢性内毒素血症水平,改善肝脏和内脏脂肪组织的炎症和氧化应激水平,减轻系统性炎症,从而达到减轻体重和改善胰岛素抵抗的作用。3.小檗碱能改善紧密连接蛋白的表达和分布,从而改善肠道屏障功能,降低肠道通透性,减少肠源性内毒素血症。4.小檗碱能改善胃肠激素的分泌,促进结肠L细胞增殖,促进结肠胰高血糖素原的表达。5.小檗碱使球形梭菌和柔嫩梭菌亚群中产丁酸盐细菌属的丰度减少,产丁酸盐菌属能够降解植物多糖为宿主提供额外能量。变形菌门中Desulfovibrio属在肠道中能够将硫酸盐还原为硫化物,硫化物对肠道上皮细胞具有毒性作用。小檗碱对肥胖大鼠的体重和胰岛素抵抗的改善功能至少部分是肠道菌群介导的。更多还原

【Abstract】 [Background]With the development of social economy and the improvement of people’s living, obesity has become a threat to the health of mankind. Obesity has brought about many health problems, such as hypertension, type2diabetes, cardiovascular disease, non-alcoholic fatty liver and so on. Lifestyle interventions are difficult to be continued, efficacy and safety of drug treatment are questioned, and the indications for surgical treatment is narrow, which all make the treatment of obesity more difficult.Berberine is the main alkaloid present in the herbs Coptis and Phellodendron and it has been usedfor intestinal bacteria infection for a long time. In recent years animal experiments and clinical studies have proved berberine can decrease blood glucose and lipid and also reduce weight safely and effectively although the mechanism is not very clear. pharmacology study proves the berberine has a very low bioavailability and it is hardly absorbed into the blood. Therefore, we believe the organ-gut may be the target of berberine.It has been proved the release of lipopolysaccharide from lysisof Gram-negative bacilli in the intestinal into the blood can cause metabolic endotoxemia, which will lead to obesity and insulin resistance by activating the chronic low-grade systemic inflammation. Intestinal mucosal barrier damage can also cause a shift of the intestinal bacteria and lead to endotoxemia. In addition, gastrointestinal hormones play an important rolein the balance of the body’s energy balance. It is proved berberine can promote the secretion of intestinal GLP-1in diabetic rats.We aimed to determine the levels of endotoxin in portal vein of rats and the degree of systemic inflammation, to observe changes in expression and distribution of tight junction protein, and to detect the level of gastrointestinal hormones. We want to investigate the protection of berberine on metabolic endotoxemia, at the same time we also want to investigate the effect of change in the intestinal flora structure, intestinal barrier function and gut hormones involved on endotoxemia. We hope the findings can provide a new treatment strategies and basis for the treatment of obesity and insulin resistance and other related diseases.[Methods]30SD rats were randomly divided into two groups:high-fat diet group (HF, n=20) and low-fat diet group (LF, n=10). In the end of the14weeks feeding, if the weight of the HF group is significantly higher than the the LF group obesity rat model is built successfully.Then according to the weight high-fat fed20rats were randomly divided into2groups of10animals per group:high-fat group (HF) and berberine treatment group (HB). The LF group of animals were conventionally raised with low-fat diet, the other two were fed high fat diet. HB group of rats were orally administered150mg/kg body weight BBR once daily whereas the other two groups were used as controls and treated with an equal volume of water. Animal treatments lasted for6weeks, during which the body weight of each animal were measured once a week and food intake three times a week.After six weeks of berberine treatment, the rats were fasted for12hours and were anesthetized by intraperitoneal injection of3%sodium pentobarbital (30mg/Kg). The LPS concentration in hepatic portal vein was determined using Dynamic turbidimetric method; The Plasma total GIP, Glucagon, PP and PYY concentration in hepatic portal vein were determined using Milliplex Map kit; The Plasma total GLP-1and GLP-2concentration were determined using an ELISA kit and following the manufacturer’s instructions. Blood sugar, blood lipids, liver ALT and liver AST in peripheral vein were determined using an automatic biochemical analyzer; The Plasma insulin concentration in peripheral vein was determined using an ELISA kit.Visceral and abdominal subcutaneous fat were removed, Dried with filter paper and weighted. Some fat was used to determine inflammatory cytokines, oxidative stress factors and macrophage infiltration markers gene expression by RT-PCR method, while other fat was fixed in4%poly formaldehyde for HE staining. Some Liver tissue was removed and also used to determine inflammatory cytokines, oxidative stress factors and macrophage infiltration markers gene expression by RT-PCR, while other Liver tissue was fixed in4%poly formaldehyde for oil red O staining. The proximal colon tissue was removed and was used to determine tight junction protein and proglucagon gene expression by RT-PCR. Proximal colon tissue was also used to detect the expression and distribution of claudin-1and claudin-2in colonic mucosal surface as well as colon L cell number by immunofluorescence. Fresh rat cecal feces were collected and were used to analyse changes of intestinal flora by pyrosequencing.Results are presented as means±SE. The statistical significance of differences was analyzed by t test or by one-way ANO VA. After using The normal distribution test and Test of Homogeneity of Variances, Comparison between two groups using LSD test if homogeneity of variance and using nonparametric test Tamhane’s T2if heterogeneity of variance. p<0.05for the difference was statistically significant.[Results]1.Rat model of obesity was successfully established after14weeks of high fat diet feedingIn the end of14weeks feeding, the weight of high-fat fed rats was significantly greater than those low-fat diet fed rats.2.The effect of berberine on body weight and metabolism of obese ratsAfter6weeks of berberine treatment, obese rats had a significantly reduced weight gain and a significantly reduced visceral and subcutaneous fat mass. Food intake of rats had a downward trend. After berberine treatment of obese rats, the level of fasting plasma glucose and insulin significantly decreased, cholesterol, low-density lipoprotein cholesterol and free fatty acids also significantly decreased, high-density lipoprotein significantly increased, and liver alanine aminotransferase significantly decreased, while triglyceride had a downward trend.Berberine could alleviate hepatocytes steatosis significantly. In the same time liver cholesterol content decreased significantly and triglyceride content had a downward trend in obese rats. The size of individual fat cells significantly reduced by berberine.3.Berberine improve metabolic endotoxemiaBerberine could significantly reduce the endotoxin levels in the portal vein of obese rats. Berberine could significantly reduce the gene expression of IL-1β, NADPHox, CD68, TLR4and LBP in the liver of high fat diets fed rats, while MIF gene expression had an increase trend. Besides, Berberine could also significantly reduce the gene expression of IL-1β、PAI-1、STAMP2、NADPHox. MCP-1and F4/80in the visceral fat of rats.4. Berberine improve the colon intestinal barrier function and reduce intestinal permeabilityCompared with rats of HF group, the area under the plasma FITC-dextran curve in rats of HB group significantly reduced. After berberine treatment in the proximal colon claudin-1and ZO-1mRNA expression significantly increased and MLCK mRNA expression significantly decreased. Occludin mRNA expression had an increase trend and claudin-2mRNA expression was essentially unchanged. Immunofluorescence results showed berberine could relieve the decrease of expression of claudin-2protein and inhibited the shift of claudin-2protein from the mucosal surface to the crypt. 5. Berberine improved gastrointestinal hormone secretion and promoted L cell proliferationBerberine increased the gastrointestinal hormone GLP-1, GLP-2and PYY concentration significantly in the portal vein of high fat diet fed rats. Total GIP and PP concentration significantly decreased while glucagon had a downward trend. Besides, berberine also increase the number of L-cells in the proximal colon of HFD fed rats and proglucagon mRNA expression significantly.6. Berberine regulated intestinal floraBerberine treatment significantly reduced the intestinal flora diversity and flora abundance in HFD fed rats and a total of55kinds of the genus was changed significantly. Of Firmacutes the abundance of Coprococcus, Lachnospiraceae, Roseburia, Ruminococcus and Faecalibacterium genus all significantly reduced and of Proteobacteria the abundance of Desulfovibrio genus also significantly reduced.[Conclusions]1.Berberine orally can reduce weight, improve glucose tolerance, regulate blood lipid, and improve liver function in HFD fed rats.2.Berberine can improve metabolic endotoxemia, improve inflammation and oxidative stress levels in liver and visceral adipose tissue, reduce systemic inflammation, so as to lose weight and improve insulin resistance in HFD fed rats.3.Berberine can regulate the expression and distribution of tight junction protein in gene and protein level, so improves intestinal barrier function, reduces intestinal permeability, and ameliorate the enterogenic endotoxemia.4. Berberine can improve gastrointestinal hormone secretion, promot the L cells of colon proliferation, and promote the expression of the proglucagon in colon.5. Berberine can reduce the abundance of butyrate-producing bacterial genus of Spherical Clostridium and Clostridium leptum subgroup. Butyrate-producing bacterial genus is capable of degrading plant polysaccharides so as to provide additional energy for the host. Of Proteobacteria the Desulfovibrio genus in the intestine can restore the sulfate to sulfide,which has a toxic effect on intestinal epithelial cells. The effect of berberine on weight loss and insulin resistance improvement of obese rats at least in part is mediated by intestinal flora.更多还原