【作者】 曹伟；

【导师】 赵彩彦；

【作者基本信息】 河北医科大学 ， 内科学， 2014， 博士

【摘要】 背景：肝衰竭是多种因素（病毒、药物、肝毒性物质、酒精、遗传代谢性疾病等）引起的严重肝脏损害，导致其合成、解毒、排泄和生物转化等功能发生严重障碍或失代偿，出现以凝血功能障碍、黄疸、肝性脑病、腹水等为主要表现的一组临床症候群。急性肝衰竭（acute liverfailure, ALF）是其中一种亚型，起病急、死亡率高，发病2周内出现Ⅱ度以上肝性脑病。其发病机制复杂，目前尚不十分清楚。现已明确，肠源性内毒素血症在ALF的疾病进展中发挥着重要作用。内毒素的主要成分为脂多糖（lipopolysaccharide, LPS），其主要效应细胞是单核巨噬细胞。有学者发现LPS可激活巨噬细胞内Notch信号通路发挥多种生物学功能。目前关于Notch信号通路与炎症关系的研究尚存在争议，尤其是对白介素-10（interleukin-10, IL-10）的影响，有研究显示Notch信号通路可促进IL-10的分泌，但也有研究显示其可抑制IL-10的分泌。此外，最近研究发现LPS激活巨噬细胞Notch信号通路与晚期炎症介质高迁移率族蛋白B1（high mobility group protein B1, HMGB1）的分泌有关。HMGB1是内毒素血症中重要的晚期炎症介质，推测HMGB1在ALF的进展中也发挥着举足轻重的作用。目前，临床上应用益生菌辅助治疗肝病患者已取得很好的疗效，其可以通过调节肠道菌群失调，降低肠道内毒素的产生和吸收，减少血液循环中内毒素的含量，从而改善内毒素血症，对受损的肝脏起保护作用。但对其具体的作用机制尚不明确，且缺乏相关研究。肝衰竭死亡率高，虽然肝移植是最有效的治疗方法，但肝源奇缺、费用昂贵的矛盾仍非常突出。因此肝衰竭的预防显得尤为重要。非酒精性脂肪性肝病（nonalcoholic fatty liver disease, NAFLD）是目前仅次于病毒性肝炎的第二大肝病病因，NAFLD患者比健康人群更易进展为肝衰竭。早期诊断和治疗NAFLD，可在一定程度上预防肝衰竭的发生。非酒精性脂肪性肝炎（nonalcoholic steatohepatitis, NASH）是NAFLD疾病进展的重要限速步骤，因此NASH的早期诊断已成为肝衰竭的主要防治工作之一。目的：本研究通过腹腔注射D-氨基半乳糖建立小鼠ALF模型，并应用益生菌干预模型小鼠，通过检测血清丙氨酸氨基转移酶（alanineaminotransferase, ALT），天冬氨酸氨基转移酶（aspartate aminotransferase,AST）、IL-10、HMGB1及血浆LPS水平，肝组织内Jagged1、Notch1、Hes5mRNA和蛋白、NICD蛋白以及巨噬细胞活化标记物CD68的表达，同时行肝组织病理染色，观察益生菌干预后肝组织病理改变，以及LPS、IL-10、HMGB1与Notch信号通路相关指标的表达变化及意义；体外培养小鼠巨噬细胞株RAW264.7细胞，应用LPS和Notch信号通路的特异性阻断剂氮–[氮–（3，5-二氟苯乙酰）–L–丙氨酰]–S–苯基甘氨酸丁酯（N-[N-(3,5-difluorophenacetyl-L–alanyl)]-S–phenylglycinet-butyl ester, DAPT）进行干预，观察细胞上清液IL-10、HMGB1水平和细胞内Notch信号通路相关指标的表达变化，明确Notch信号通路在外源性LPS刺激巨噬细胞分泌细胞因子中的调控作用；同时应用正常小鼠血浆、ALF小鼠血浆和益生菌干预小鼠血浆分别刺激小鼠巨噬细胞株RAW264.7细胞，观察细胞上清液LPS、IL-10、HMGB1水平和细胞内Notch信号通路相关指标的表达变化，从整体、组织、细胞及分子水平探讨益生菌对于ALF的可能作用机制，为临床应用益生菌预防ALF提供新的理论基础和实验依据。此外，还通过检测NAFLD患者血清中与NAFLD发病密切相关的指标变化，应用统计学方法进行分析处理，建立NASH的无创诊断模型，从而为NASH的早期诊断，也为肝衰竭的预防提供新思路。方法：1益生菌对急性肝衰竭小鼠Notch信号通路的影响健康清洁级6-8周龄雄性BALB/c小鼠30只，体重（18-20）g，由河北医科大学动物实验中心提供。用标准饲料适应性喂养1周，随机分为以下三组：a正常对照组10只：标准饲料喂养；b ALF模型组10只：标准饲料喂养，给予生理盐水（与益生菌干预组剂量相同）灌胃2周，于2周末腹腔注射3.0g/kg的D-氨基半乳糖（以生理盐水溶解，浓度为60mg/ml）；c益生菌干预组10只：标准饲料喂养，给予益生菌（金双歧）900mg/kg/d（生理盐水配成100mg/ml混悬液）灌胃2周，于2周末腹腔注射3.0g/kg的D-氨基半乳糖（以生理盐水溶解，浓度为60mg/ml）。于腹腔注射3.0g/kg D-氨基半乳糖（造模）36小时处死全部小鼠，留取血清、血浆及肝组织。应用全自动生化分析仪测定血清ALT、AST水平；应用酶联免疫吸附（Enzyme-linked immunosorbent assay, ELISA）法测定血清IL-10、HMGB1水平；鲎试剂盒检查血浆LPS水平；取部分肝组织以10%中性甲醛溶液固定，用于常规苏木素-伊红（hematoxylin-eosin,HE）染色；实时荧光定量聚合酶链反应（real-time quantitative polymerasechain reaction, real time-PCR）法检测小鼠肝组织Jagged1、Notch1和Hes5mRNA的表达；Western Blot法检测小鼠肝组织Jagged1、Notch1、NICD和Hes5蛋白的表达变化；免疫组织化学染色方法检测小鼠肝组织CD68的表达。2Notch信号通路在脂多糖刺激巨噬细胞分泌细胞因子中的调控作用小鼠巨噬细胞株RAW264.7细胞购自中国医学科学院基础医学研究所基础医学细胞中心。将处于对数生长期的RAW264.7细胞接种在六孔板中，每孔5×105个细胞，用含10%胎牛血清和90%高糖DMEM培养至70%融合时进行分组干预。正常对照组：常规培养加入DMSO（剂量与LPS+DAPT组相同）培养24小时后，再加入1×PBS缓冲液（剂量与LPS组相同）继续培养30小时；LPS组：常规培养加入DMSO（剂量与LPS+DAPT组相同）培养24小时后，再加入LPS（100ng/ml）继续培养30小时；LPS+DAPT组：常规培养加入DAPT（10μM）培养24小时后[15]，再加入LPS（100ng/ml）继续培养30小时；收取细胞及上清液。ELISA法测定细胞上清液IL-10、HMGB1水平；Real time-PCR法检测RAW264.7细胞Notch1、Hes5mRNA的表达；Western Blot法检测RAW264.7细胞NICD、Hes5蛋白的表达变化。3益生菌干预小鼠血浆调控巨噬细胞分泌细胞因子的分子机制将处于对数生长期的RAW264.7细胞接种在六孔板中，每孔5×105个细胞，用含10%胎牛血清和90%高糖DMEM培养至70%融合时进行分组干预。正常小鼠血浆干预组：80%细胞完全培养基（10%胎牛血清+90%高糖DMEM）+20%正常小鼠血浆培养48小时；ALF小鼠血浆干预组：80%细胞完全培养基（10%胎牛血清+90%高糖DMEM）+20%ALF小鼠血浆培养48小时；益生菌干预小鼠血浆干预组：80%细胞完全培养基（10%胎牛血清+90%高糖DMEM）+20%益生菌干预小鼠血浆培养48小时；收取细胞及上清液。ELISA法测定细胞上清液IL-10、HMGB1和LPS水平；Real time-PCR法检测血浆干预RAW264.7细胞Jagged1、Notch1、Hes5mRNA的表达；Western Blot法检测血浆干预RAW264.7细胞Jagged1、Notch1、NICD、Hes5蛋白的表达。4细胞角蛋白18、天冬氨酸氨基转移酶、血小板、甘油三酯联合预测非酒精性脂肪性肝炎的发生入选患者均需行肝活检，诊断符合中华医学会肝病学分会脂肪肝和酒精性肝病学组制定的非酒精性脂肪性肝病诊疗指南（2010年修订版），同时排除酒精性脂肪性肝病（5年内乙醇摄入量男性≥40克/天或女性≥20克/天）或过量饮酒（男性乙醇摄入量≥140克/周或女性≥70克/周）；病毒性肝炎；自身免疫性肝病；药物或毒物诱导的肝损害；遗传代谢性疾病（Wilson’s病、血色素沉着症、α1抗胰蛋白酶缺乏症等）；胆道梗阻。根据肝组织病理学检查结果，将所有纳入的研究对象分为两组，即非-非酒精性脂肪性肝炎组（non-nonalcoholic steatohepatitis, non-NASH）和NASH组。计算患者体重指数、腰臀比；询问是否合并糖尿病、高血压、血脂异常及吸烟习惯；检测血清ALT、AST、总胆红素（total bilirubin,TB）、白蛋白（albumin, ALB）、碱性磷酸酶（alkaline phosphatase, ALP）、γ-谷氨酰转肽酶（γ-glutamyl transpeptidase, γ-GT）、国际标准化比率（international normalized ratio, INR）、血小板、白细胞（white blood cell,WBC）、肌酐、尿酸（Uric acid, UA）、空腹血糖（fasting glucose, FG）、甘油三酯（triglycerides, TG），胆固醇（total cholesterol, TC）、血红蛋白、超敏反应蛋白（hs-Creactive protein, hs-CRP）和铁蛋白水平；ELISA法测定血清细胞角蛋白18（cytokeratin18, CK18）凋亡片段M30的水平。实验数据以x±ｓ表示，单因素方差分析前进行正态性及方差齐性检验，Least-Significant-Difference法进行组间比较，双侧P<0.05为差异有统计学意义，P<0.01为有显著统计学意义。相关性分析采用直线相关分析法。应用SPSS13.0统计软件分析实验数据。结果：1益生菌对急性肝衰竭小鼠Notch信号通路的影响1.1小鼠的一般情况：正常对照组小鼠精神状态良好，食欲旺盛，对外界刺激或痛觉反应正常；ALF模型组小鼠精神萎靡，进食减少，动作迟缓，对外界刺激反应减弱；益生菌干预组小鼠精神、食欲及对界外刺激反应介于正常对照组与ALF模型组之间。1.2小鼠生化指标、血清HMGB1、IL-10及血浆LPS水平变化：ALF模型组小鼠血清ALT（848.404±94.828U/L）、AST（911.490±67.652U/L）、HMGB1（101.909±12.428μg/L）、IL-10（4627.884±842.453pg/ml）和血浆LPS（11.801±0.887EU/ml）水平均高于正常对照组（38.994±9.628U/L、55.279±7.499U/L、20.733±5.369μg/L、1064.924±238.455pg/ml、0.578±0.119EU/ml）（P值均<0.01）；而益生菌干预组血清ALT（689.891±84.649U/L）、 AST （776.026±61.892U/L）、 HMGB1（82.556±9.719μg/L）、IL-10（3182.596±769.235pg/ml）和血浆LPS（7.396±0.919EU/ml）水平均较ALF模型组明显降低（P值均<0.01）。1.3肝组织病理学变化：光镜下，HE染色可见正常对照组小鼠肝组织内大小一致的肝细胞围绕肝小叶中央静脉呈放射状分布，肝细胞排列整齐，肝索结构完整；ALF模型组小鼠肝组织正常结构被破坏，肝索结构紊乱，可见大面积肝细胞坏死及大量炎性细胞浸润，残存肝细胞肿胀，呈气球样变；益生菌干预组小鼠肝细胞坏死、变性及炎性细胞浸润程度均较ALF模型组改善。1.4肝组织Jagged1、Notch1、Hes5mRNA和蛋白、NICD蛋白的表达：ALF模型组小鼠肝组织内Jagged1、Notch1、Hes5mRNA和蛋白、NICD蛋白的表达量均较正常对照组明显增加（P值均<0.01）；而益生菌干预组上述指标的表达水平均较ALF模型组明显减少，差异有统计学意义（P值均<0.01或<0.05）。1.5肝组织CD68蛋白的表达：ALF模型组小鼠肝组织CD68蛋白（0.631±0.067μm2）较正常对照组（0.339±0.071μm2）明显增加（P<0.01）；与ALF模型组比较，益生菌干预组CD68蛋白表达量（0.460±0.094μm2）明显减少（P<0.01）。1.6相关性分析小鼠血浆LPS水平与ALT、AST、Jagged1mRNA、Notch1mRNA、Hes5mRNA、Jagged1蛋白、Notch1蛋白、NICD蛋白、Hes5蛋白、血清HMGB1、IL-10水平及肝组织CD68蛋白的表达水平均呈正相关，相关系数分别为r=0.936、0.946、0.947、0.945、0.948、0.894、0.829、0.926、0.907、0.942、0.900、0.973（P值均<0.01）。2Notch信号通路在脂多糖刺激巨噬细胞分泌细胞因子中的调控作用2.1RAW264.7细胞上清液中HMGB1和IL-10的含量变化：正常对照组细胞上清液中可检测出低水平的HMGB1（0.213±0.046μg/L）和IL-10（59.192±23.304pg/ml）。经LPS刺激后细胞上清液HMGB（17.441±0.634μg/L）和IL-10（315.188±79.133pg/ml）水平均有不同程度的升高（P值均<0.01），其中以HMGB1升高为主；应用DAPT进行特异性阻断后，细胞上清液HMGB1（6.218±0.711μg/L）和IL-10（252.060±57.633pg/ml）水平均较LPS刺激组下降明显，以HMGB1下降最为显著，差异有统计学意义（P值<0.01或<0.05）。2.2DAPT特异性阻断剂对LPS诱导的RAW264.7细胞Notch1mRNA、Hes5mRNA和蛋白、NICD蛋白的影响：RAW264.7细胞经LPS刺激后，细胞内Notch1mRNA、Hes5mRNA和蛋白、NICD蛋白水平均较正常对照组明显升高（P值均<0.01）；加入DAPT特异性阻断剂进行干预后，与LPS刺激组比较，上述指标的表达水平均显著降低，差异有统计学意义（P值均<0.01）。2.3相关性分析小鼠巨噬细胞株RAW264.7细胞上清液中HMGB1水平与Notch1mRNA、Hes5mRNA、NICD蛋白和Hes5蛋白均呈正相关，相关系数分别为r=0.849、0.933、0.833、0.866（P值均<0.01）；细胞上清液中IL-10水平也与Notch1mRNA、Hes5mRNA、NICD蛋白和Hes5蛋白呈正相关，相关系数分别为r=0.798、0.810、0.821、0.834（P值均<0.01）。3益生菌干预小鼠血浆调控巨噬细胞分泌细胞因子的分子机制3.1小鼠血浆干预RAW264.7细胞上清液中HMGB1、IL-10和LPS的含量变化：正常小鼠血浆干预组细胞上清液中HMGB1（3.433±0.674μg/L）、IL-10（286.524±33.507pg/ml）和LPS（0.090±0.014EU/ml）水平较低；ALF小鼠血浆干预组细胞上清液中HMGB1（34.691±9.058μg/L）、IL-10（1812.736±185.574pg/ml）和LPS（2.102±0.121EU/ml）水平则较正常小鼠血浆干预组明显升高，差异有统计学意义（P值均<0.01）；与ALF小鼠血浆干预组比较，益生菌干预小鼠血浆干预组细胞上清液HMGB1（20.058±2.278μg/L）、IL-10（1338.322±151.339pg/ml）和LPS（1.220±0.069EU/ml）水平均明显下降（P值均<0.01）。3.2小鼠血浆干预RAW264.7细胞内Jagged1、Notch1、Hes5mRNA和蛋白、NICD蛋白的表达变化：RAW264.7细胞经ALF小鼠血浆干预后，细胞内Jagged1、Notch1、Hes5mRNA和蛋白、NICD蛋白水平均较正常小鼠血浆干预组明显升高（P值均<0.01）；加入益生菌干预小鼠血浆后，与ALF小鼠血浆干预组比较，上述指标表达水平显著降低，差异有统计学意义（P值均<0.01）。3.3相关性分析小鼠血浆干预RAW264.7细胞上清液LPS水平与Jagged1mRNA、Notch1mRNA、Hes5mRNA、Jagged1蛋白、Notch1蛋白、NICD蛋白、Hes5蛋白、细胞上清液HMGB1和IL-10水平均呈正相关，相关系数分别为r=0.969、0.911、0.955、0.931、0.889、0.937、0.940、0.913、0.965（P值均<0.01）。4细胞角蛋白18、天冬氨酸氨基转移酶、血小板、甘油三酯联合预测非酒精性脂肪性肝炎的发生4.1人口学及实验室指标的变化：Non-NASH组和NASH组患者年龄、性别构成比、吸烟习惯、糖尿病、高血压、血脂异常、收缩压、舒张压、血清TB、WBC、血红蛋白、肌酐、INR、FG、TC和铁蛋白水平差异均无统计学意义（均P>0.05），而NASH患者BMI、WHR、血清AST、ALT、ALP、γ-GT、UA、hs-CRP、TG和血小板水平均较non-NASH组明显升高，血清ALB水平则明显降低（均P<0.05）。4.2血清CK18片段M30水平及其与肝组织病理学特征的相关性分析：NASH组患者血清CK18片段M30水平（372.9U/L（319.6,431.4））较non-NASH组（248.1U/L（237.5,266.6））明显升高（P<0.001）。其与肝组织脂变（r=0.492）、气球样变（r=0.211）、汇管区炎症（r=0.346）和纤维化分级（r=0.407）均成正相关（P<0.05或P<0.01）。4.3多变量分析：体重指数、腰臀比、血清AST、ALT、ALP、ALB、γ-GT、UA、hs-CRP、TG、CK-18片段M30及血小板均纳入多变量模型分析。其中ALT、血小板、CK-18片段M30和TG是NASH的预测因素。四个指标的AUROC分别为0.811（95%CI：0.722-0.899）、0.631（95%CI：0.515-0.746）、0.892（95%CI：0.824-0.960）和0.714（95%CI：0.611-0.818）。其中血清CK-18片段M30水平与ALT（r=0.639）和TG（r=0.390）水平均成正相关（均P<0.05），而与血小板无相关性（P>0.05）。4.4NASH预测模型：Logistic回归分析得出NASH预测模型，具体公式为-12.764+0.075×ALT（U/L）+0.013×血小板（×109/L）+0.012×CK-18片段M30（U/L）+0.006×TG（mg/dL）。该模型的AUROC为0.920（95%CI：0.866-0.974），临界值为0.361，敏感性、阳性预测值和阴性预测值均为89%，特异性为86%。结论：1应用D-氨基半乳糖腹腔注射小鼠可以成功复制ALF模型，其中血浆LPS水平的升高在ALF的发生发展中起着重要作用。2ALF小鼠血浆中升高的LPS可使肝组织内巨噬细胞活化，激活细胞内Notch信号通路，促进晚期重要炎症介质HMGB1和抗炎细胞因子IL-10的分泌，其中以升高HMGB1为主，参与肝脏的炎症损伤过程，加速ALF的疾病进展。3益生菌可通过降低ALF小鼠血浆LPS水平，减少肝组织中巨噬细胞的活化，抑制Notch信号转导，降低HMGB1和IL-10的水平，从而发挥保护肝脏的作用，为临床预防ALF提供了新的思路和方向。4由CK-18、ALT、血小板和甘油三酯组成的无创诊断模型可准确预测NASH的发生，该模型可能在延缓NAFLD病情进展，改善患者预后及预防肝衰竭中发挥一定作用。更多还原

【Abstract】 Background: Liver failure is a group of clinical syndromecharacterised by coagulopathy, jaundice, hepatic encephalopathy and ascites,with varous precipitating factors containing virus, drugs, alcohol, toxin, andmetabolic disorders. Acute liver failure (ALF) is a subtype known as promptonset, rapid aggravation which presenting hepatic encephalopaghy in nomore than two weeks, and high mortality. Intestinal endotoxemia has beenconsidered to play a key role in the complicating mechanisms of ALF.Lipopolysaccharide (LPS), served as a main component of endotoxin, hasbeen found to activate Notch signaling passway which is closely related tohepatic inflammation, whereas the effects is still controversal. Recent studieshave reported that Notch signaling passway could promote the secretion ofIL-10, while others have inverse results. It was reported, in macrophages,that Notch signaling pathway activated by LPS was associated with thesecretion of high mobility group protein B1(HMGB1), which is animportant mediator in the late phases of inflammation in endotoxemia and issupposed to play a pivotal role in the progression of ALF. Microecologicshave been used to treat liver disease for the therapeutic effects of maintainingthe balance of intestinal flora, decreasing the production and absorption ofthe intestinal endotoxin, and reducing the serum level of endotoxin. However,the definite mechanisms are uncertain. In addition, liver transplantation, themost effective therapy to liver failure, is still limited by the donor shortageand expensive cost. Therefore, the prevention of liver failure is seemedparticularly important. Currently, nonalcoholic fatty liver disease (NAFLD)is an important cause of liver disease second to viral hepatitis, the NAFLDpatients are more likely to progress to liver failure than healthy individuals.To some extent, early diagnosis and early treatment of NAFLD is needed to prevent liver failure. Therefore, nonalcoholic steatohepatitis (NASH), knownas the limited step of NAFLD, should be diagnosed as soon as possible toinhibit the progression of liver failure.Objection: In this study, the mice model of ALF was establishedthrough intraperitoneal injection with D-galactosamine, and probiotics wereadministrated to treat the model mice. To investigate changes andsignificance differences after probiotics, we detected the levels of serumalanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-10,HMGB1, plasma LPS, liver tissue Jagged1, Notch1, Hes5, NICD, and CD68(a specific marker for macrophage activation). Liver tissue pathologicalforms were observed via HE staining. We cultured murine macrophageRAW264.7in vitro with LPS in the absence and presence of DAPT, which isa specific inhibitor of the Notch signaling pathway. To understand theregulatory role of the Notch signaling pathway induced by LPS inmacrophages, the changes in the expressions of Jagged1, Notch1, Hes5, andNICD in RAW264.7, as well as the levels of cells supernatant IL-10andHMGB1were observed. In addition, we cultured RAW264.7with the miceplasma of the normal control, ALF model and probiotic groups, respectively.To study the possible mechanism of ALF treatment with probiotic, theorganization, cellular and molecular levels, levels of LPS, IL-10, andHMGB1in the cell supernatant, and expressions of Jagged1, Notch1, Hes5,and NICD in cells were measured. The results provided a novel andexperimental basis for the prevention of ALF with probiotics. We alsodetected the serum indexes that were in close relationship with thepathogenesis of NAFLD, and then established the diagnostic model ofNASH using statistical methods. This study afforded new ideas for earlydiagnosis of NASH and prevention of liver failure.Methods:1The effects of probiotics on Notch signaling pathway in mice model ofALFThirty BALB/c male mice of clean grade, with6-8weeks old and 18-20g, were purchased from the Animal Experimental Center of HebeiMedical University. All mice were fed adaptively for1week, and thendivided into three groups randomly. All mice were continued to be fed withstandard diet. In addition, mice in the probiotics group (n=10) were perfusedwith probiotics (900mg/kg/d) for2weeks, whereas mice in normal control(n=10) and ALF model groups (n=10) were perfused with the same volumeof normal saline to meet the criteria of body mass for2weeks. At the end of2weeks, D–galactosamine (3.0g/kg) was administered throughintraperitoneal injection in ALF model and probiotics groups. The samevolume of normal saline was administered through intraperitoneal injectionin normal control group. At36h after the intraperitoneal injection, all micewere killed for the experiment. Liver tissue, serum, and plasma samples werecollected. Serum ALT and AST were measured by automatic biochemicalanalyzer, IL-10and HMGB1were examined by enzyme-linkedimmunosorbent assay (ELISA), and plasma LPS was checked by limuliodreagent inspection method. Some liver tissues were fixed in10%formaldehyde, embedded in paraffin, and then stained withHematoxylin-eosin (HE) The expressions of Jagged1, Notch1, NICD andHes5in the liver were detected via real-time quantitative polymerase chainreaction (real time-PCR) and western blot, respectively. The expression ofCD68in mice liver was measured via immunohistochemical stainingmethod.2The regulatory role of Notch signaling pathway in the macrophageswith LPSMurine macrophage RAW264.7were purchased from the Cell Centerof the Institute of Basic Medical Sciences, Chinese Academy of MedicalSciences. RAW264.7in logarithmic phase were inoculated in sixwell plates(5×105cells per well), and cultured in DMEM medium with high glucosecontaining10%fetal bovine serum. After they were grown to70%confluence, the cells were divided into three groups randomly. Normalcontrol group: RAW264.7cells were cultured in DMSO (the same volume of DAPT) for24h, then with1×PBS buffer solution (the same volume ofLPS) for30h. LPS group: RAW264.7cells were cultured in DMSO (thesame volume of DAPT) for24h, then with LPS (100ng/ml) for30h.LPS+DAPT group: RAW264.7cells were cultured in DAPT (10μM) for24h, then with LPS (100ng/ml) for30h. All culture cells and supernatantswere collected. The levels of cell supernatants IL-10and HMGB1wereexamined via ELISA. Moreover, the expressions of Notch1, Hes5and NICDwere measured by real time-PCR and western blot, respectively.3Molecular mechanism of cytokine secretion regulation bymacrophages with probiotics mice plasmaRAW264.7cells in logarithmic phase were inoculated in sixwell plates(5×105cells per well), and cultured in DMEM medium with high glucosecontaining10%fetal bovine serum. After they were grown to70%confluence, the cells were divided into three groups randomly. Normal miceplasma group: RAW264.7cells were cultured in20%normal mice plasmafor48h. ALF mice plasma group: RAW264.7cells were cultured in20%ALF mice plasma for48h. Probiotics mice plasma group: RAW264.7cellswere cultured in20%plasma of probiotics mice for48h. All cells andsupernatants were collected. The levels of cell supernatants IL-10andHMGB1were examined by ELISA, the levels of supernatant LPS waschecked by limuliod reagent inspection method. Moreover, the expressionsof Jagged1, Notch1, NICD and Hes5in RAW264.7cells were detected byreal time-PCR and western blot, respectively.4Cytokeratin18, alanine aminotransferase, platelets and triglyceridespredict the presence of nonalcoholic steatohepatitisLiver biopsy has been done in all enrolled patients, and they werediagnosed with NAFLD based on the guideline for diagnosis and treatmentof NAFLD (new revised edition2010) by the Chinese national workshop onfatty liver and alcoholic liver disease for the Chinese liver diseaseassociation. The exclusion criteria are listed as follows:(a) alcoholic fattyliver disease (alcohol consumption≥40g/d for male or≥20g/d for female during past five years) or excessive alcohol consumption (≥140g/wk formale or≥70g/wk for female);(b) viral hepatitis;(c) autoimmune liverdiseases;(d) drug-or toxin-induced liver steatosis (with no drugs or toxinswhich can induce hepatotoxicity);(e) genetic or metabolic liver diseases,such as Wilson’s disease, hemochromatosis, and alfa-1antitrypsin deficiency;(f) biliary obstruction (by ultrasonography). The patients were divided intotwo groups, namely, non-nonalcoholic steatohepatitis (non-NASH) andNASH (by liver histology). Body mass index (BMI) and waist-on-hip ratio(WHR) were calculated in all patients, and the history of diabetes mellitus(DM), hypertension, dyslipidemia and smoking habits were obtained byregular doctor visit. The laboratory evaluation in all patients included: ALT,AST, total bilirubin (TB), albumin (ALB), alkaline phosphatase (ALP),γ-glutamyl transpeptidase (γ-GT), international normalized ratio (INR),platelets, white blood cell (WBC), creatinine, Uric acid (UA), fasting glucose(FG), triglycerides (TG), total cholesterol (TC), hemoglobin andhs-C-reactive protein (hs-CRP). The level of serum CK-18-Asp396wasdetected by ELISA.The results were presented as means±standard deviation. Normalityand homogeneity of variance were tested before single-factor analysis ofvariance. All data were compared between the two groups throughLeast-Significant-Difference method. Two-tailed P <0.05was consideredstatistically significant, whereas P <0.01was considered to exhibit verysignificant difference. Linear correlation analysis tested the relationshipsamong the variables. SPSS13.0version was used to compute the collecteddata.Results:1The effects of probiotics on Notch signaling pathway in mice model ofALF1.1General condition of mice:Normal mice in good spirits and with good appetite, as well as normalresponse to external stimuli and pain were used. ALF mice in low spirits and with poor appetite, bradykinesia and diminished response to external stimuli.The mental condition, appetite and response to external stimuli in probioticsmice had been ranged between normal mice and model mice.1.2Changes in levels of serum ALT, AST, HMGB1, IL-10, and plasma LPSin mice:The levels of serum ALT (848.404±94.828U/L), AST (911.490±67.652U/L), HMGB1(101.909±12.428μg/L), IL-10(4627.884±842.453pg/ml)and plasma LPS (11.801±0.887EU/ml) in the ALF model group were higherthan those in normal control group (38.994±9.628U/L,55.279±7.499U/L,20.733±5.369μg/L,1064.924±238.455pg/ml,0.578±0.119EU/ml)(all P <0.01). Compared with the ALF model group, the levels of ALT(689.891±84.649U/L), AST (776.026±61.892U/L), HMGB1(82.556±9.719μg/L), IL-10(3182.596±769.235pg/ml), and LPS (7.396±0.919EU/ml) inthe probiotics group were significantly lower (all P <0.01).1.3Changes in liver histopathology:Under a light microscope, normal hepatocytes were observed to bearranged radially from the central veins based on HE staining in normalcontrol group. Histopathological examinations of the liver biopsies in theALF model showed that the structure of liver tissues was destroyed, thehepatic cord in the hepar was disarranged, and large necrosis area andsignificant inflammatory cellular infiltration were present. In addition, theremaining hepatocytes exhibited swelling and ballooning. The degrees ofnecrosis, degeneration, and infiltration of inflammation cells in the liver werelesser in the probiotics group than those in the ALF group.1.4Expressions of Jagged1, Notch1, Hes5and NICD in mice hepatic tissues:Compared with the normal control group, the expressions of Jagged1,Notch1, Hes5and NICD were increased in the liver of ALF model mice (allP <0.01). The expressions of the above indexes were decreased significantlyin the probiotics group compared with those in the ALF model group (P <0.05or P <0.01). 1.5Expression of CD68protein in mice hepatic tissues:The expression of CD68protein in ALF model mice (0.631±0.067μm2)was higher than that in normal control mice (0.339±0.071μm2)(P <0.01).Compared with the ALF model group, the expression of CD68protein in thehepatic tissues of the probiotics group (0.460±0.094μm2) was decreased, thedifference was significant statistically (P <0.01).1.6Correlation analysis:The level of plasma LPS in mice was positively correlated with thelevels of ALT (r=0.936), AST (r=0.946), Jagged1mRNA (r=0.947), Notch1mRNA (r=0.945), Hes5mRNA (r=0.948), Jagged1protein (r=0.894),Notch1protein (r=0.829), NICD protein (r=0.926), Hes5protein (r=0.907),HMGB1(r=0.942), IL-10(r=0.900) and CD68protein (r=0.973), thedifference were significant statistically (all P <0.01).2The regulatory role of Notch signaling pathway in the macrophageswith LPS2.1Changes in levels of HMGB1and IL-10in RAW264.7cell supernatant:Low levels of HMGB1(0.213±0.046μg/L) and IL-10(59.192±23.304pg/ml) were observed in the culture supernatant of normal RAW264.7cells.The levels of HMGB1(7.441±0.634μg/L) and IL-10(315.188±79.133pg/ml) in the supernatant of RAW264.7cells with LPS increased, thedifference were significant statistically (all P <0.01). Compared with theLPS group, the levels of HMGB1(6.218±0.711μg/L), IL-10(252.060±57.633pg/ml) in the LPS+DAPT group were significantlydecreased (P <0.05or P <0.01).2.2Effects of DAPT on the expression of Notch1mRNA, NICD protein,Hes5mRNA and protein in RAW264.7cells through LPS stimulation:Compared with the normal RAW264.7cells group, the expressions ofNotch1mRNA (6.493±0.727), NICD protein (0.646±0.100), Hes5mRNA(7.301±0.851) and protein (0.957±0.097) in the LPS group were increased,the difference were significant statistically (all P <0.01). The expressions ofNotch1mRNA (3.203±0.682), NICD protein (0.424±0.045), Hes5mRNA(4.722±0.665) and protein (0.838±0.087) in the LPS+DAPT group were higher compared with those in the LPS group (all P <0.01).2.3Correlation analysis:The level of HMGB1in culture supernatant was positively correlatedwith Notch1mRNA (r=0.849), NICD protein (r=0.933), Hes5mRNA(r=0.833) and protein (r=0.866) in RAW264.7cells, the difference weresignificant statistically (all P <0.01). The level of IL-10in cells supernatantwas also positively correlated with the levels of cell Notch1mRNA(r=0.798), NICD protein (r=0.810), Hes5mRNA (r=0.821) and protein(r=0.834), the difference were significant statistically (all P <0.01).3Molecular mechanism of cytokine secretion regulation bymacrophages with probiotics mice plasma3.1Changes in levels of HMGB1, IL-10, and LPS in supernatant of RAW264.7cells with mice plasma:Low levels of HMGB1(3.433±0.674μg/L), IL-10(286.524±33.507pg/ml), and LPS (0.090±0.014EU/ml) were observed in the supernatant ofRAW264.7cells with normal mice plasma. The levels of HMGB1(34.691±9.058μg/L)， IL-10(1812.736±185.574pg/ml) and LPS(2.102±0.121EU/ml) in the supernatant of RAW264.7cells with ALF miceplasma were increased, the difference were significant statistically (all P <0.01). Compared with the ALF mice plasma group, the levels of HMGB1(20.058±2.278μg/L), IL-10(1338.322±151.339pg/ml), and LPS(1.220±0.069EU/ml) in the probiotics mice plasma group were significantlydecreased (all P <0.01).3.2Changes in expressions of Jagged1, Notch1, Hes5and NICD in RAW264.7cells with mice plasma:Compared with the normal mice plasma group, the expressions ofJagged1mRNA (6.671±0.790), Notch1mRNA (5.491±0.657), Hes5mRNA(6.301±0.930), Jagged1protein (0.631±0.067), Notch1protein(0.533±0.091), NICD protein (0.600±0.086), and Hes5protein (0.803±0.087)in the ALF mice plasma group were increased, the difference weresignificant statistically (all P <0.01). The expressions of Jagged1mRNA (4.120±0.623), Notch1mRNA (2.375±0.739), Hes5mRNA (3.657±0.755),Jagged1protein (0.460±0.094), Notch1protein (0.326±0.069), NICD protein(0.332±0.064), and Hes5protein (0.605±0.097) were decreased in theprobiotics mice plasma group compared with those in the ALF mice plasmagroup (all P <0.01).3.3Correlation analysis:The level of LPS in supernatant of RAW264.7cells with mice plasmawas positively correlated with the levels of Jagged1mRNA (r=0.969),Notch1mRNA (r=0.911), Hes5mRNA (r=0.955), Jagged1protein (r=0.931),Notch1protein (r=0.889), NICD protein (r=0.937), Hes5protein (r=0.940),HMGB1(r=0.913) and IL-10(r=0.965), the difference were significantstatistically (all P <0.01).4Cytokeratin18, alanine aminotransferase, platelets and triglyceridespredict the presence of nonalcoholic steatohepatitis4.1Characteristics of the study indexes:Compared with the non-NASH group, the levels of BMI, WHR, AST,ALT, ALP, γ-GT, platelets, UA, hs-CRP, and TG were significantly higherin the NASH group (all P <0.05), whereas ALB was lower (P <0.05).Numerous indexes showed no significant differences between two groups.These indexes include the age of patient, gender, smoking habits, systolicblood pressure (SBP), diastolic blood pressure (DBP), serum bilirubin, WBC,hemoglobin, creatinine, INR, Ferritin, FG, TC, and the medical history ofDM, hypertension, and dyslipidemia.4.2Changes of levels of CK-18fragments M30and the correlation analysisbetween levels of CK-18fragments M30and liver histology:CK-18fragments levels were significantly higher in patients withNASH (372.9U/L (319.6,431.4)) than that of the non-NASH group (248.1U/L (237.5,266.6))(P <0.001). The CK-18fragment levels showed asignificant positive correlation with steatosis severity (r=0.492), ballooning(r=0.211), lobular inflammation (r=0.346), and fibrosis stage (r=0.407)(P <0.05or P <0.01). 4.3Multivariate analysis:Multivariate analysis included BMI, WHR, serum AST, ALT, ALB,ALP, γ-GT, platelets, UA, hs-CRP, TG, and CK-18fragments. ALT,platelets, CK-18fragments, and TG were the predictive factors for NASH.The AUROC curves were0.811(95%CI:0.722-0.899),0.631(95%CI:0.515-0.746),0.892(95%CI:0.824-0.960) and0.714(95%CI:0.611-0.818)for the ALT, platelets, CK-18fragments, and TG, respectively (P <0.05or P<0.01). CK-18fragment levels showed a significant positive correlationwith ALT (r=0.639) and TG (r=0.390)(all P <0.01), while no correlationwith platelets (P>0.05).4.4The model to predict NASH in NAFLD patients:A new model that combines ALT, platelets, CK-18fragments, and TGwas established through logistic regression in the NAFLD patients. Theequation of this model was:-12.764+0.075×ALT (U/L)+0.013×platelets(×109/L)+0.012×CK-18fragment levels (U/L)+0.006×TG (mg/dL). TheAUROC curve for the prediction of NASH was0.920(95%CI:0.866-0.974).A cutoff value of0.361, with a sensitivity of89%and a specificity of86%,has a positive predictive value of89%and a negative predictive value of89%.Conclusions:1Mice models of ALF were established through intraperitonealinjection of D-galactosamine, the high level of plasma LPS has an importantfunction in the development of ALF.2Elevated levels of plasma LPS could activate the Notch signalingpathway in macrophages to promote the secretion of HMGB1, a potentiallate inflammatory mediator, and IL-10, an anti-inflammatory cytokine,especially for increasing HMGB1, which is involved in liver injury causedby inflammation and accelerating progression of ALF.3Probiotics protected the liver by reducing the levels of LPS,decreasing the activation of macrophages in the liver tissue, inhibiting theNotch signaling pathway, and lowering the secretion of HMGB1and IL-10, thereby presenting a new idea and direction for clinical prevention of ALF.4The novel scoring system, which combines ALT, platelets, CK-18fragments, and TG, can accurately predict the occurrence of NASH. It mayplay a certain role in improving the progression of NAFLD, the prognosis ofNAFLD patients and the prevention of ALF.更多还原