【作者】 宋新玲；

【导师】 贾乐；

【作者基本信息】 山东农业大学 ， 生物学， 2022， 博士

【摘要】 酒精性肝损伤（Alcoholic liver disease,ALD）是由长期或过量饮酒所造成的肝脏损伤性疾病,其发病率呈逐年升高趋势,已成为严重危害人类健康的常见疾病之一。目前临床上主要采用药物疗法改善或缓解ALD的发生与发展,但其作用过程相对缓慢,且易产生不良反应,限制了药物疗法的推广。因此,明确ALD的病理机制以及探索有效的干预策略具有重大意义。秀珍菇（Pleurotus geesteranus）是一种味道鲜美,营养丰富的食用菌,素有“菇中极品”的美誉。秀珍菇多糖具有多种生物学活性,如抗氧化衰老、降血脂血糖及免疫调节等,但其对ALD的保护作用及机理研究较少。本课题对秀珍菇菌丝体多糖（P.geesteranus mycelium polysaccharides,PGPs）进行了结构表征,采用Lieber-De Carli液体饲料构建了ALD小鼠模型,基于“肝-肠轴”分析了ALD的发病机理和PGPs抗ALD的生物活性,旨在为天然保肝药物的筛选提供新思路。主要研究结果如下:（1）液体深层发酵获得秀珍菇菌丝体,其生物量为4.57±0.18g/L,经脱脂、热水浸提、去蛋白、分离纯化及冷冻干燥等处理获得了PGPs,其含量为83.23±0.38%。（2）经红外光谱、甲基化及气质联用（Gas chromatography-mass spectrometer,GC-MS）分析表明PGPs是以→4)-Glcp-(1→和Glcp-(1→为主,兼具α和β构型的吡喃型糖环。结合¹H、¹³C、COSY、HSQC、HMBC、NOESY、TOCSY和DEPT135等多维核磁波谱对其进行碳氢信号归属分析,初步推测PGPs的重复单元结构如下所示:（?）（3）结构表征显示,PGPs在扫描电镜（Scanning electron microscope,SEM）下的形貌为相互交错的片状结构,在原子力电镜（Atomic force electron microscopy,AFM）下为聚集的圆锥形,呈现明显聚集效应。（4）毒性试验表明,在试验周期内,小鼠未出现中毒或死亡现象,行为活动表现正常,血清学指标未出现异常,表明PGPs是一种安全无毒的天然活性成分。（5）通过建立ALD小鼠模型,经干预后发现PGPs能有效保护肝细胞结构、显著抑制氧化应激和炎症应激、调节脂质代谢,进而保护肝脏组织。（1）PGPs能显著抑制酒精引起的肝脏肿胀,降低肝脏指数;同时降低血清中肝损伤标志酶如谷丙转氨酶（Alanine transaminase,ALT）和谷草转氨酶（Aspartate aminotransferase,AST）活力;（2）肝脏病理学及透射电镜超微结构表明,PGPs可有效减轻酒精造成的脂滴堆积、细胞肿胀、细胞边界消失、细胞核皱缩、内质网扩张、核糖体脱落以及炎性细胞浸润等病理变化;（3）PGPs能降低细胞色素P450 2E1（Cytochrome P450 2E1,CYP2E1）的蛋白水平,升高乙醇脱氢酶（Alcohol dehydrogenase,ADH）和乙醛脱氢酶（Aldehyde dehydrogenase,ALDH）的蛋白水平,进而调节酒精代谢酶系统;（4）PGPs能有效减少活性氧（Reactive oxygen species,ROS）的过度积累,通过调节核因子E2相关因子2（Nuclear factor erythroid2-related factor 2,Nrf2）信号通路,促进Nrf2核转位,增强下游抗氧化酶如血红素氧合酶1（Heme oxygenase-1,HO-1）、超氧化物歧化酶（Superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（Glutathione peroxidase,GSH-Px）和过氧化氢酶（Catalase,CAT）的活性,抑制丙二醛（Malondialdehyde,MDA）的生成,减轻氧化应激损伤;（5）PGPs可激活腺苷酸活化蛋白激酶（Adenosine monophosphate activated protein kinase,AMPK）信号通路,抑制固醇调节元件结合蛋白（Sterol regulatory element binding proteins-1c,SREBP-1c）和乙酰辅酶A羧化酶（Acetyl Co A carboxylase,ACC）的表达,同时增加过氧化物酶体增殖物激活受体-α（Peroxisome proliferator-activated receptor-α,PPARα）和沉默信息转录调控因子1（Sirtuin type 1,SIRT-1）的蛋白水平,减少脂肪酸合成,加速脂肪酸氧化分解,降低胆固醇（Total cholesterol,TC）和甘油三酯（Triacyl glycerols,TG）在肝脏和血清中的水平,有效改善脂代谢紊乱,减少脂质堆积;（6）PGPs能显著抑制髓样分化因子（Myeloid differentiation factor 88,My D88）依赖的Toll样受体/核转录因子κB（Toll-like receptor 4（TLR4）/nuclear factorκB（NF-?B））信号通路的激活,降低肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）、白介素-1β（Interleukin,IL-1β）、白介素-6（IL-6）和白介素-8（IL-8）的释放,调节机体免疫平衡,减轻肝脏炎症。（6）PGPs可通过“肝-肠轴”发挥对ALD小鼠结肠的保护作用。（1）PGPs能显著改善酒精引起的结肠黏膜层变薄、肠绒毛脱落严重、内质网空泡化及紧密连接消失等病理学变化;（2）Western blot结果表明PGPs能显著升高紧密连接蛋白（Zona occludens-1,ZO-1;Occludin;Claudin-1;Claudin-4）和黏蛋白（Mucin,MUC1、MUC2、MUC4）的表达水平,有效保护肠屏障完整性;（3）16S r RNA基因测序表明PGPs能在一定程度上影响小鼠肠道菌群的Alpha和Beta多样性,在门和属水平改变肠道菌群的群落组成结构,抑制肠道LPS（Lipopolysaccharide,LPS）产生菌的扩增,增加短链脂肪酸（Short chain fatty acids,SCFAs）产生菌的丰度,维持肠道菌群平衡,进而抑制LPS的肠渗漏,维持肠道稳态,通过“肝-肠轴”维持机体的健康状态。综上所述,PGPs能通过介导Keap1/Nrf2、AMPK/SREBP和TLR4/NF-κB等多信号通路抑制氧化应激损伤、改善脂质代谢紊乱和抑制肝脏炎症等发挥保肝作用。此外,基于“肝-肠轴”理论,PGPs可通过降低肠道通透性,维持肠道屏障的完整性,正向调节肠道菌群等显著改善肝/肠内环境,证实了PGPs对ALD具有较好的预防和治疗功效。更多还原

【Abstract】 As a form of liver injury caused by long-term/excessive alcohol consumption,the incidence rate of alcoholic liver disease（ALD）is increasing year by year,becoming one of the common diseases that seriously threaten human health.Currently and clinically,the main therapy against ALD was pharmacotherapy,but it was limited in application and promotion owing to the easily accompanied by side-effects.Therefore,it was of great significance to clarify the pathological mechanisms of ALD and explore effective intervention strategies.Pleurotus geesteranus,an edible mushroom with delicious taste and higher nutrition,showed potential effects of antioxidant,anti-aging,hypolipidemic and immunomodulatory.However,scarce literature about the protective effects and mechanism of polysaccharides on ALD had been published.In the present work,the characterizations of P.geesteranus mycelium polysaccharides（PGPs）were conducted firstly.And the ALD mice models using Lieber-De Carli liquid diet were established to explore the hepatoprotective effects against ALD of PGPs based on the liver-intestinal axis,aiming to provide new ideas for the exploitation of natural hepatoprotective drugs.The main results were listed as follows:（1）P.geesteranus mycelium was obtained by the liquid submerged fermentation,and the biomass was 4.57±0.18g/L.After defatting,extraction,deproteinization,separation,purification and freeze-drying,the PGPs with the content of 83.23±0.38%was obtained.（2）Fourier transform infrared spectroscopy（FTIR）,methylation and gas chromatography-mass spectrometry（GC-MS）analysis presented that PGPs were pyranose rings linked byα-andβ-glycosidic bonds,and predominantly bonded with→4)-Glcp-(1→and Glcp-(1→.The attribution of PGPs was analyzed by multi-dimensional nuclear magnetic resonance（NMR）spectra including ¹H,¹³C,COSY,HSQC,HMBC,NOESY,TOCSY and DEPT135,and the repeat units of PGPs were preliminarily deduced and shown as follows:（?）（3）The morphological characteristics exhibited PGPs were shown obvious aggregation effects with interlocked lamellar structures under the scanning electron microscope（SEM）and aggregated conical shapes under atomic force electron microscopy（AFM）.（4）The toxicity tests showed no poisoning or death,as well as normal behavioral activities and the serological parameters were observed during the test period,indicating PGPs was the safe and non-toxic natural active ingredient.（5）The ALD models in mice were established successfully,and the results demonstrated that PGPs showed potential hepatoprotective effects by effectively protecting hepatocyte structure,significantly inhibiting oxidative stress and inflammatory stress,and regulating lipid metabolism disorder.（1）PGPs could significantly inhibit alcohol-induced liver swelling,reduce liver index,and significantly reduce the serum activities of alanine transaminase（ALT）and aspartate aminotransferase（AST）.（2）The liver histopathology and transmission electron microscopy（TEM）ultrastructure showed that PGPs could effectively reduce alcohol-induced hepatic lesions including lipid droplet accumulation,cell swelling,cell boundary disappearance,nuclear shrinkage,endoplasmic reticulum expansion,ribosome abscission and inflammatory cell infiltration.（3）PGPs could inhibit the disorder of alcohol metabolic enzyme system by down-regulating cytochrome P450 2E1（CYP2E1）and up-regulate alcohol dehydrogenase（ADH）and aldehyde dehydrogenase（ALDH）.（4）PGPs could effectively reduce over-accumulations of reactive oxygen species（ROS）and reduce oxidative stress damage through the nuclear factor erythroid 2-related factor 2（Nrf2）signaling pathway by promoting the nuclear translocation of Nrf2,up-regulating the antioxidant activities of enzymes such as heme oxygenase-1（HO-1）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）and catalase（CAT）,and inhibiting the production of malondialdehyde（MDA）.（5）PGPs could improve the disorder of lipid metabolism and reduce lipid accumulation.It could activate the signaling pathway of AMP-activated protein kinase（AMPK）,inhibit expressions of sterol regulatory element binding proteins-1c（SREBP-1c）and acetyl Co A carboxylase（ACC）,and up-regulate peroxisome proliferator-activated receptor-α（PPARα）and sirtuin type 1（SIRT-1）,with the results of reducing fatty acid synthesis,accelerating fatty acid oxidative decomposition,and lowing the levels of liver lipid and blood lipid including triglyceride（TG）and total cholesterol（TC）.（6）PGPs showed effects on reducing liver inflammation by inhibiting the activation of myeloid differentiation factor 88（My D88）-dependent TLR4/NF-?B（Toll-like receptor 4（TLR4）/nuclear factor kappa B（NF-?B））signaling pathway,reducing the release of inflammatory factors including tumor necrosis factor-α（TNF-α）、interleukin（IL-1β、IL-6,and IL-8）.（6）According to the“liver-gut axis”theory,we further evaluated the protective effects of PGPs on colon in ALD mice.（1）PGPs could significantly improve the pathological changes caused by alcohol,such as thinning of colonic mucosa,severe shedding of intestinal villi,vacuolation of mitochondria and disappearance of tight junctions.（2）The western blot results showed that PGPs significantly protected intestinal barrier by up-regulating the expressions of tight junction proteins（zona occludens-1（ZO-1）,Occludins,Claudin-1 and Claudin-4）,and mucins（MUC1,MUC2 and MUC4）.（3）The high-throughput sequencing results presented that PGPs could affect the Alpha and Beta diversity of gut microbiota,alter the gut microbial community at the Phylum and Genus levels,inhibit the expansion of intestinal LPS（lipopolysaccharide）-producing bacteria,increase the abundance of short chain fatty acids（SCFAs）-producing bacteria,and maintain the balance of intestinal flora,thereby inhibiting the intestinal leakage of LPS,and maintaining the body homeostasis.In conclusion,PGPs showed potential hepatoprotective effects against ALD by reducing oxidative stress,regulating lipid metabolism,and inhibiting the hepatic-inflammations through the Keap1/Nrf2,AMPK/SREBP and TLR4/NF-κB signaling pathways.Besides,PGPs could significantly improve the internal environment of the liver and gut by reducing the intestinal permeability,protecting the integrity of the intestinal barrier,reducing the LPS intestinal leakage,and regulating the gut microbial.All the conclusions confirmed that PGPs showed obvious hepatoprotection against ALD.更多还原