【作者】 陈丽华；

【导师】 刘文恩；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景：心血管疾病是世界范围内致病率和致死率主要的原因,动脉粥样硬化是心血管病的病理基础。降脂治疗是防治心血管疾病的一种常规手段,常用的降脂药由于是化学合成的药物,各有其副作用和不良反应,临床上迫切需要疗效更好更安全的降胆固醇药。乳酸杆菌从公元3世纪起即被人们用于发酵食物,悠久的使用历史证明了乳酸杆菌是一种安全的益生菌并具有多种功效。从20世纪六七十年代起人们开始关注乳酸杆菌的降胆固醇作用并迅速成为益生菌研究领域的热点。然而,乳酸杆菌降胆固醇的效果一直存在争议,有关乳酸杆菌对心血管疾病作用的研究非常有限,而直接研究乳酸杆菌对动脉粥样硬化形成影响的报道更加少。目的：研究嗜酸乳酸杆菌ATCC4356对ApoE-/-小鼠血清胆固醇和动脉粥样硬化形成的影响并从乳酸杆菌对炎症、氧化应激和肠道菌群的调节三方面阐明可能的机制。方法：(1)实验对象分组和饲养：野生型C57BL/6J小鼠灌胃生理盐水作为正常对照组(WT组)；选取ApoE-/-小鼠为动脉粥样硬化动物模型,ApoE-/-小鼠灌胃生理盐水作为动脉粥样硬化模型对照组(Vehicle组);ApoE-/-小鼠灌胃5×107CFU嗜酸乳酸杆菌ATCC4356作为菌液低剂量组(La.L组)；ApoE-/-小鼠灌胃5×108CFU嗜酸乳酸杆菌ATCC4356组作为菌液高剂量组(La.H组)。小鼠均为8周龄的雄性小鼠,每天灌胃体积为0.5mL,连续12周。WT组小鼠饲料为普通饲料,ApoE-/-小鼠用高脂高胆固醇饲料(含15%猪油+0.25%胆固醇)喂养以加速动脉粥样硬化模型的形成。(2)实验过程中,定期记录小鼠体重并观察体重变化,实验结束后将小鼠麻醉处死、取材、保存。(3)检测乳酸杆菌对血清胆固醇的影响：利用全自动生化分析仪检测血清总胆固醇(total cholesterol, TC)、甘油三酯(tryglyeride, TG)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol, HDL-C)和低密度脂蛋白胆固醇(low density lipoprotein cholesterol, HDL-C)的水平。(4)检测小鼠主动脉粥样硬化形成情况：对小鼠主动脉大体和主动脉根部冰冻切片行油红O染色,对动脉粥样斑块面积进行定量；对主动脉根部切片进行H&E染色观察粥样斑块的病理变化。(5)检测氧化应激分子的变化：用酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测各组小鼠血清中氧化型低密度脂蛋白(oxidized low density lipoprotein, ox-LDL)的水平,用生化检测试剂盒检测小鼠血清和肝脏中丙二醛(Malondialdehyde, MDA)、超氧化物歧化酶(Superoxide dismutase, SOD)和谷胱甘肽(Glutathione, GSH)的水平。(6)检测炎症因子的变化：采用ELISA方法检测小鼠血清肿瘤坏死因子-α (Tumor necrosis factor alpha, TNF-α)和白介素-10(Interleukin10, IL-10)的蛋白水平；采用实时荧光定量聚合酶链式反应(real-time polymerase chain reaction, real-time PCR)检测各组小鼠主动脉的TNF-α和IL-10的mRNA表达水平；(7)检测核因子P65(nuclear factor kappa B, NF-κB P65)信号通路的激活情况：用western blot检测总蛋白核因子κB抑制蛋白α蛋白(nuclear factor-kappa-B inhibitor alpha, IκB-α)、胞浆蛋白和胞核蛋白NF-κB P65的蛋白表达水平。(8)检测小鼠肠道菌群的变化：对小鼠粪便进行倍比稀释后,利用选择性培养基,Mac用于大肠杆菌、EC用于肠球菌、MRS用于乳酸杆菌和BS用于双岐杆菌的分离培养并进行菌落的鉴定和计数。用MTT方法检测嗜酸乳酸杆菌ATCC4356对肠上皮细胞Caco-2细胞增殖的影响。结果：(1)成功构建动脉粥样硬化小鼠模型。(2)小鼠体重的变化：高脂高胆固醇喂养的ApoE-/-小鼠体重增加要大于普通饮食喂养的野生型C57BL/6J小鼠,但是嗜酸乳酸杆菌ATCC4356对小鼠体重没有影响。(3)乳酸杆菌对胆固醇的影响：ApoE-/-小鼠血清的LDL-C、TC和HDL-C都显著高于WT组,但是乳酸杆菌对ApoE-/-小鼠的血清胆固醇并没有影响。(4)粥样斑块的形成情况：高脂高胆固醇喂养的ApoE-/-小鼠在实验结束时可以形成明显的动脉粥样斑块病变,给予乳酸杆菌处理的小鼠,动脉粥样斑块面积减少,病变减轻,La.H组与Vehicle组比较有显著性差异(P<0.05)。(5)氧化应激分子的检测结果：与WT组相比,、Vehicle组小鼠的血清ox-LDL、MDA显著升高,SOD活力降低,用嗜酸乳酸杆菌ATCC4356干预可以降低ox-LDL、MDA水平,并增加SOD活性,但对GSH无影响。(6)炎症分子的变化结果：乳酸杆菌可以降低血清TNF-α水平,La.H组与Vehicle组比有显著性差异(P<0.05),但是对IL-10的蛋白含量无影响。Real-time PCR结果显示,与WT组小鼠相比,Vehicle组小鼠主动脉TNF-α mRNA表达水平增高,IL-10mRNA表达水平减低,嗜酸乳酸杆菌ATCC4356干预可以逆转这些变化。(8)NF-κB信号通路的激活情况：Western Blot结果显示,Vehicle组小鼠主动脉总蛋白IκB-a蛋白降解显著,胞浆NF-κB P65蛋白表达减少,胞核NF-κB P65蛋白表达增加,应用嗜酸乳酸杆菌ATCC4356处理后,IκB-α蛋白降解减少,胞浆NF-κB P65蛋白表达增加,胞核NF-κB P65蛋白表达减少,提示NF-κB P65核转位减少。(9)肠道菌群的改变结果：高脂高胆固醇喂养的Vehicle组ApoE-/-小鼠与普通饮食喂养的WT组小鼠相比,肠道内容物中双岐杆菌、乳酸杆菌数量减少,大肠杆菌数量增加,肠球菌数量无明显改变。给ApoE-/-小鼠灌胃嗜酸乳酸杆菌ATCC4356菌液12周后,小鼠肠道内容物的双岐杆菌、乳酸杆菌和肠球菌数量增加,大肠杆菌数量减少。MTT结果显示1×108CFU/mL的嗜酸乳酸杆菌ATCC4356可以显著增加Caco-2细胞的增殖。结论：嗜酸乳酸杆菌ATCC4356有减轻动脉粥样硬化形成的作用,该作用不依赖于降胆固醇机制,而与降低炎症、抗氧化应激和调节肠道菌群有关。抗炎和抗氧化应激可能是通过抑制NF-κB P65信号通路的激活来实现的。对肠道菌群的调节可能与促进肠上皮细胞细胞增殖,保护肠上皮屏障有关。图33幅,表1个,参考文献115篇更多还原

【Abstract】 Background:Cardiovascular disease is the major cause leading to mortality and morbidity throughout the world and its basic pathogenesis is atherosclerosis. Lipid-lowering therapy is a kind of conventional treatment. However, as the commonly used lipid-lowering drugs are synthesized by chemical methods and have side-effects and adverse reactions, the drugs having better curative effects and being safer are urgently needed in clinic. Lactobacillus has been used in fermenting foods since the third century, AD. The long term used history proves that Lactobacillus is a kind of safe probiotics and owns variety effects. In1960’s and1970’s, people began to paid attention to its cholesterol-lowering effect, which is also rapidly becoming the hot field of probiotics research area. Yet there has been controversy on the cholesterol-lowering effect of Lactobacillus. In addition, the research about the effect of Lactobacillus on cardiovascular disease is limited and fewer studies have investigated the role of Lactobacillus directly in atherogenesis.Objective (1) To investigate the effect of Lactobacilus (L.) acidophilus ATCC4356on the development of atherosclerosis (As) and serum cholesterol in ApoE-/-mice. The underlying mechanisms were further studied through detecting the modulation of oxidative and inflammatory process, and the regulation of intestinal microflora.Methods (1) Grouping and feeding of mice:8week-old male mice were randomly assigned to4groups:(a)WT group: Wild Type C57BL/6J mice were given normal saline (NS) as normal control group (WT);(b) Vehicle group:ApoE-/-mice were treated with NS as model control group;(c) La. L group:ApoE-/-mice treated with low dose (5×107CFU) of L. acidophilus ATCC4356daily;(d) La. H group:ApoE-/-mice treated with high dose (5×10CFU) of L. acidophilus ATCC4356daily. C57BL/6J mice were fed with normal chow, while all the ApoE-/-mice were fed with high fat and high cholesterol diet (including15%fat and0.25%cholesterol). The volume of NS and L. acidophilus ATCC4356was0.5mL/day per mouse. The experimental time was12weeks.(3) During the course of experiment, mice body weights were monitored once a week. After12weeks of intervention the mice were sacrificed, the needed materials were drawn from responding organ and frozen in liquid nitrogen or-80℃.(4) Detecting the effects of L. acidophilus ATCC4356on mice serum cholesterol:Serum lipid levels, including total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C), were tested by automatic biochemical analyzer.(5) Testing the development of As in mice aorta: Aortic en face analysis and cryosections of aortic root were stained with Oil Red O to examine the aortic atherosclerotic lesions; The morphologic changes in the atherosclerotic lesions were identified by hematoxylin-eosin (H&E) staining.(6) Examining the oxidative status: Eenzyme-linked immunosorbent assay (ELISA) kits was used to test the serum levels of oxidized low density lipoprotein (ox-LDL); Malondialdehyde (MDA) levels, superoxide dismutase (SOD) activities and glutathione (GSH) concentrations were detected by biochemical analyzed kits to show the oxidative status.(7) Detecting the changes of inflammatory factors:The protein levels of tumor necrosis factor alpha (TNF-α) and interleukin10(IL-10) in mice serum were assessed by ELISA kits; the mRNA levels of TNF-a and IL-10in aorta were analyzed with quantitative real-time PCR.(8) Testing the activation of nuclear factor kappa-B P65(NF-κB p65) signaling pathway:The expression levels of nuclear factor-kappa-B inhibitor alpha (IκB-α), cytoplasmic and nuclear NF-κB p65in aorta were determined by western blot.(9) Analyzing the changes of intestinal microflora:The contents of colon were homogenized and diluted with NS, then certain diluted contents were plated on responding selective media to distinguish bacterial and count the number of colony forming units (CFU). The effect of L. acidophilus ATCC4356on proliferation of Caco-2cells was detected by MTT method.Results (1) The atherosclerotic animal models were successfully established.(2) The changes of mice body weights:Body increments in high fat and high cholesterol fed ApoE-/-mice were higher than those in normal chow fed wide type C57BL/6J mice, while L. acidophilus ATCC4356could not affect the body weights.(3) The effect of L. acidophilus ATCC4356on mice serum cholesterol:The serum levels of TC, LDL-C and HDL-C in ApoE-/-mice were significantly higher that those in WT group mice, but there were no differences among ApoE-/-mcie treated with or without L. acidophilus ATCC4356.(4) Atherosclerotic lesion area:At the end of experiment, the Vehicle group mice could develop apparent atherosclerotic lesions in aorta. However, ApoE-/-mice treated with L. acidophilus ATCC4356showed decreased atherosclerotic lesion size in both en face aorta and aorta root. H&E staining also showed attenuated morphologic severity in La.H group.(5) The results of oxidative status examination:Compared to WT mice, ApoE-/-mice in Vehicle group showed higher levels of serum ox-LDL and MDA, but lower levels of SOD. Administration of L. acidophilus ATCC4356could decrease the levels of ox-LDL and MDA while increase the activities of SOD in a dose-dependent manner. However, the levels of GSH showed no changes.(6) The results of inflammatory molecules detection:Serum levels of TNF-α were increased while IL-10levels were reduced in Vehicle group compared with WT group. The aortic mRNA expression of TNF-α and IL-10also showed the same trend. Treatment with L. acidophilus ATCC4356could reverse these trends.(7) The activation of NF-κB p65signal pathway was suppressed:The results of Western blot demonstrated that compared to ApoE-/-mice in Vehicle group, the degradation of aortic IκB-α was suppressed, expression of cytoplasmic NF-κB was upregulated while nuclear NF-κB p65was downregulated in ApoE-/-mice treated with L. acidophilus ATCC4356, suggesting the inhibition of NF-κB p65nuclear translocation.(8) The changes of intestinal microflora:The numbers of both intestinal Lactobacillus and Bifidobacterium were lowered in high fat and high cholesterol fed Vehicle group mice than those in WT mice. Administration of L. acidophilus ATCC4356to ApoE-/-mice showed significant increases in the numbers of intestinal Lactobacillus, Bifidobacterium and Enterococcus spp. colonies as compared to ApoE-/-mice treated with vehicle only.1×108CFU/mL L. acidophilus ATCC4356could augment the proliferation of Caco-2cells.Conclusions Administration of L.acidophilus ATCC4356in ApoE-/-mice can attenuate the development of atherosclerotic lesions through the mechanisms of reducing oxidative stress, anti-inflammatory response and regulating intestinal microflora, rather than lowering cholesterol levels. The effects on preventing oxidative stress and inflammation are possably associated with inhibiting the activation of NF-κB singal pathway. L.acidophilus ATCC4356can promote the proliferation of Caco-2cells to strengthen the intestinal barrier function, which is the possible underlying mechanism of regulating gut flora. Figures:33, Table:1, References:115更多还原