【作者】 王雪；

【导师】 李文生；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2022， 博士

【摘要】 研究背景:非诺贝特作为过氧化物酶体增殖物激活受体alpha(Peroxisome proliferator activated receptor alpha,PPARα)激动剂,已被美国食品和药物管理局(Food and Drug Administration,FDA)批准治疗高脂血症。临床研究表明,非诺贝特治疗可以减少糖尿病视网膜病变患者对激光治疗的需求。关于非诺贝特在视网膜方面的有益作用,目前有脂质相关机制和非脂质调节机制两种。研究目的:本文主要为了研究非诺贝特对高脂饮食(High-fat diet,HFD)小鼠肠道-视网膜轴的影响;以及非诺贝特对高脂饮食诱导的高脂血症小鼠模型视网膜色素上皮(Retinal pigment epithelial,RPE)组织细胞病理改变的影响及机制。研究方法:C57BL/6J小鼠随机分为4组:标准脂肪饮食(Standard diet,SD)组、高脂饮食组(HFD)、标准脂肪饮食加非诺贝特(SD＿Fe)组、高脂饮食加非诺贝特(HFD＿Fe)组。造模成功后,检测小鼠粪便中肠道菌群组成及短链脂肪酸(Short-chain fatty acid,SCFAs)水平。评估小鼠肠道屏障功能和肠道紧密连接蛋白的表达。检测结肠和肝脏中的炎症因子水平。检测血清中脂质、炎症因子、脂多糖(Lipopolysaccharide,LPS)、和SCFAs水平。评估小鼠视网膜功能。检测视网膜氧化应激损伤水平,小胶质细胞和Müller细胞的激活状态,炎症信号通路激活、下游炎症因子表达和SCFAs水平。检测小鼠RPE组织的氧化应激、炎症和PI3K/AKT信号通路水平。通过体外培养ARPE-19细胞系,进一步研究棕榈酸和非诺贝特酸对RPE氧化应激和炎症反应的影响及机制。研究结果:非诺贝特可显著抑制高脂饮食小鼠血脂水平。非诺贝特可显著抑制高脂饮食小鼠粪便中LPS相关菌群的上调,包括“门”水平厚壁菌门和变形菌门,“科”水平的脱硫弧菌科以及“属”水平的脱硫弧菌属,产醋菌属,黄杆菌属,颤杆菌克属和厌氧棍状菌属。除此之外,非诺贝特可显著上调高脂饮食小鼠粪便中SCFAs相关菌群的丰度,包括“门”水平的拟杆菌门,在“科”水平的紫单胞菌科,“属”水平的紫单胞菌属,巴恩斯氏菌属、拟普雷沃菌属和双歧杆菌属。非诺贝特可以抑制高脂饮食小鼠粪便中SCFAs下调和肠道屏障的损害。非诺贝特可显著下调高脂小鼠系统性(血清,结肠,肝脏)炎症因子水平和血清中LPS水平,以及上调高脂小鼠血清中SCFAs水平。相关性分析表明血清炎症因子分别与血清LPS水平呈正相关,与血清SCFAs水平呈负相关。非诺贝特可抑制高脂饮食小鼠视网膜功能受损,氧化应激水平,炎症细胞激活,TLR4、NF-k B和JNK信号通路的激活,下游炎症因子的上调和SCFAs下调。视网膜炎症因子分别与血清LPS水平呈正相关,与血清及粪便中SCFAs呈负相关。非诺贝特可以显著抑制高脂饮食诱导的RPE功能受损和RPE组织氧化应激、炎症反应以及PI3K/AKT信号通路的下调。非诺贝特酸(非诺贝特在体内代谢后的活性形式)通过上调PI3K/AKT信号通路抑制棕榈酸诱导的ARPE-19细胞氧化应激反应和炎症反应。研究结论:我们的研究证明了非诺贝特能显著减轻高脂饮食诱导的高脂血症,肠道菌群及代谢物紊乱,肠道屏障功能受损,系统性炎症反应以及视网膜和RPE的功能受损、氧化应激损伤和炎症反应。我们推断非诺贝特对高脂饮食诱导的视网膜损伤的影响可能通过以下两条途径:1)非诺贝特可部分通过抑制“肠道菌群紊乱-SCFA下调/LPS上调-肠道屏障损伤-系统性炎症反应-视网膜炎症/氧化应激”轴,间接抑制高脂饮食小鼠视网膜炎症反应和氧化应激损伤;2)非诺贝特可通过上调PI3K/AKT信号通路直接抑制高脂饮食诱导的高脂血症小鼠视网膜RPE组织的脂质过氧化/氧化应激及炎症反应损伤。图38幅,表30个,参考文献218篇更多还原

【Abstract】 Background: As a peroxisome proliferator activated receptor alpha(Peroxisome proliferator activated receptor alpha,PPAR α)agonist,fenofibrate has been approved by the Food and Drug Administration(FDA)for the treatment of hyperlipidemia.Clinical studies have shown that fenofibrate treatment can reduce the demand for laser treatment in patients with diabetes retinopathy.There are two mechanisms of the beneficial effects of fenofibrate on the retina: lipid and non-lipid related mechanisms.Objectives: This study aimed to investigate the effect of fenofibrate on the gut-retina axis in high-fat diet(HFD)-induced mice.In addition,we also investigated the effect and its mechanism of fenofibrate on the pathological changes of retinal pigment epithelial(RPE)in hyperlipidemia model induced by HFD.Methods: C57BL/6J mice were randomly allocated into four groups:standard diet(SD)group;HFD group;SD plus fenofibrate(SD＿Fe)group;HFD plus fenofibrate(HFD＿Fe)group.After successfully establishing models,gut microbiota composition and short-chain fatty acids(SCFAs)levels in the feces were detected.Gut barrier function and the expression of tight junction protein(ZO-1 and Occludin)were evaluated.Proinflammatory factors levels in colon and liver were measured.The levels of lipid,inflammatory factors,lipopolysaccharide(LPS)and SCFAs in serum were detected.The oxidative stress indicators levels,activation of microglia and Müller cells,activation of inflammatory signaling pathways,inflammatory factors levels and SCFAs levels in neuroretina were evaluated.The levels of oxidative stress,inflammatory response and PI3K/AKT signaling pathway in RPE were measured.The effect and its mechanism of fenofibric acid(an active form of fenofibrate after metabolism)on oxidative stress and inflammatory response of RPE induced by PA were further studied using ARPE-19 cell in vitro.Results: Fenofibrate markedly inhibited blood lipid levels.Fenofibrate supplementation down-regulated the abundances of LPS-associated bacteria in HFD mice,including Firmicutes and Proteobacteria at the phylum level,Desulfovibrionaceae at the family level,as well as unclassified＿Desulfovibrionaceae,Acetatifactor,Flavonifractor,Oscillibacter and Anaerotruncus at the genus level.However,fenofibrate treatment up-regulated the abundances of SCFAs-associated bacteria in HFD mice,including Bacteroidetes at the phylum level,Porphyromonadaceae at the family level,as well as unclassified＿Porphyromonadaceae,Barnesiella,Alloprevotella and Bifidobacterium at the genus level.Fenofibrate alleviated SCFAs reduction and intestinal barrier function damage in HFD-induced mice.Fenofibrate could significantly inhibit HFD-induced up-regulation of inflammatory factors in colon and liver.Fenofibrate down-regulated the levels of inflammatory factors and LPS,and up-regulated SCFAs levels in the serum of HFD-fed mice.Correlation analysis showed that the serum levels of inflammatory factors were positively correlated with serum LPS levels,and were negatively correlated with the serum SCFAs levels,respectively.Fenofibrate inhibited the increased levels of oxidative stress indicators,the activation of inflammatory cells,the activation of TLR4,NF-k B and JNK signaling pathways,the up-regulation of inflammatory factors and the down-regulation of SCFAs in the neuroretina of HFD-fed mice.Correlation analysis showed that the neuroretinal levels of inflammatory factors were positively correlated with serum LPS levels,and were negatively correlated with the SCFAs levels in serum and feces,respectively.Fenofibrate significantly ameliorated function damage,oxidative stress,inflammatory response and an inhibition of PI3K/AKT pathway in the RPE of HFD-induced mice.Fenofibric acid significantly alleviated the increases of oxidative stress levels and inflammatory response in the PA-induced ARPE-19 cell via the upregulation of PI3K/AKT signaling pathway.Conclusions: Our results confirmed fenofibrate attenuated hyperlipidemia,gut microbiota and its metabolites dysbiosis,gut barrier damage,systemic inflammatory response,as well as the retinal function damage,oxidative stress and inflammatory response in HFD-fed mice.We inferred that the effects of fenofibrate on HFD-induced retinal damage might be related with the following two pathways: 1)fenofibrate might indirectly inhibit retinal inflammation and oxidative stress in HFD-fed mice,in part,through inhibiting the “gut microbiota dysbiosis-SCFAs downregulation/LPS upregulation-intestinal barrier damage-systemic inflammation-inflammation/oxidative stress in retina” axis.2)fenofibrate might directly inhibit lipid peroxidation/oxidative stress and inflammatory response of the RPE in hyperlipidemia mice induced by HFD feeding via upregulation of PI3K/AKT signaling pathway.更多还原