【作者】 张楠；

【导师】 杜洪印；

【作者基本信息】 天津医科大学 ， 麻醉学， 2019， 硕士

【摘要】 目的:肝移植手术已成为治疗终末期肝病的最佳有效方法。然而在其手术过程中,缺血再灌注损伤对移植肝是一次巨大的打击。目前,由于供体来源有限,扩大供体来源、增加边缘供肝利用率如脂肪变性供肝等变得极其重要。值得注意的是,脂肪供肝由于其本身病理学特点,对围术期肝缺血/再灌注损伤的敏感性明显增高。小檗碱是一种传统中药,可通过抑制氧化应激和炎症反应,减轻肝缺血再灌注损伤。因此,本实验旨在探讨小檗碱对脂肪肝形成的影响,研究小檗碱对脂肪肝缺血再灌注损伤的保护作用及其相关机制。方法:18只健康雄性wistar大鼠,6周龄,随机分为3组(n=6):正常组(Sham组,正常饮食12周);脂肪肝组(HFD组,高脂饮食12周);小檗碱组(HFD+BBR组,高脂饮食12周,第9周开始小檗碱灌胃200mg/kg,持续4周)。分别在喂养4、8、12周后眼球取血,测定血清甘油三酯浓度;12周后取血测定肝功能指标,取肝组织进行病理学检测。进一步实验,30只大鼠随机分为5组(n=6):假手术组(SD组,正常饮食12周,仅行开关腹操作);脂肪肝组(HFD组,高脂饮食12周,仅行开关腹操作);缺血再灌注组(HFD+IR组,高脂饮食12周,建立大鼠缺血再灌注模型);小檗碱组(HFD+IR+BBR组,高脂饮食12周,于11周进行小檗碱灌胃200 mg/kg,持续7天,建立大鼠缺血再灌注模型);毒胡萝卜素组(HFD+IR+BBR+TG组,高脂饮食12周,于11周进行小檗碱灌胃200mg/kg,持续7天,术前1天腹腔注射TG 20mg/kg,建立大鼠缺血再灌注模型)。再灌注6h后,采集血液检测肝功能指标,取肝组织进行HE病理学检测,硫代巴比妥酸法检测MDA含量,黄嘌呤氧化酶法检测SOD水平;使用ELISA法检测炎症因子释放情况;蛋白印迹法检测Bip、CHOP、p-PERK、PERK、LC3、Beclin-1和p62的表达情况,反映内质网应激反应和自噬彼此之间的变化;透射电镜检测内质网形态和自噬小体数量在各组中的变化情况。KDEL是内质网标记蛋白,LC3B是膜型自噬体的标记物,通过KDEL和LC3B双标免疫荧光法观察内质网和自噬小体的共定位情况。FAM134B是内质网自噬标记蛋白,采用免疫组化法检测肝组织内质网自噬的表达情况。结果:与Sham组相比,HFD组与HFD+BBR组在各时点甘油三酯浓度明显升高;肝功能指标损伤严重;肝细胞内出现脂肪空泡。与HFD组相比,HFD+BBR组12周末甘油三酯浓度下降;肝功能指标数值下调;肝细胞内脂肪空泡减少。相较于SD组,其余各组ALT和AST水平明显升高,肝功能损伤加重,氧化应激指标显著增加,炎症因子释放增多,HE病理学检测显示肝细胞结构破坏,出现脂肪变性,炎症因子在脉管处聚集;内质网应激相关蛋白和自噬相关蛋白表达增强,透射电镜下观察显示内质网扩张成囊泡状,呈脱颗粒状态,自噬小体数量增加;双标免疫荧光显示KDEL与LC3的共定位增强,免疫组化显示FAM134B的表达明显增加。给予BBR预处理后,肝功能指标、氧化应激指标和炎症因子减轻,HE病理学检测显示肝细胞脂肪空泡减少,炎性因子减少;内质网应激相关蛋白和自噬相关蛋白表达下调,透射电镜观察可见内质网形态得到改善,自噬活动减轻;双标免疫荧光法发现KDEL与LC3共定位减轻,免疫组化显示FAM134B表达水平降低。然而TG预处理使BBR保护作用消失。结论:小檗碱预处理可减轻肝组织脂肪化程度。同时,小檗碱可通过减轻内质网应激反应介导的内质网自噬,缓解大鼠脂肪肝缺血再灌注损伤,从而发挥保护作用。更多还原

【Abstract】 Objective Liver transplantation has been an effective treatment for end-stage liver disease.However,ischemia reperfusion（IR）injury causes the huge strike for liver.Owing to the lack of organ donation,expanding the liver donor pool and increasing the use ratio of marginal donor such as steatotic liver are of vital importance.It’s worth noting that steatotic livers are more susceptible to IR injury,and enhance a higher risk of primary graft non-function.Berberine（BBR）is a traditional Chinese medicine.A large body of experimental data has supported the notion that BBR preconditioning attenuates IR injury via inhibiting oxidative stress and inflammation parameters.Therefore,this research aimed to investigate the role of BBR on steatitoc liver,determine the protective effect of BBR on steatotic liver ischemia reperfusion and explore the mechanisms.Methods Eighteen wistar rats were randomly assigned to the three groups（n=6）:Sham group（fed with normal fat diet for 12 weeks）,HFD group（fed with high-fat diet for 12 weeks）,HFD+BBR group(fed with high-fat diet for 12 weeks,BBR200mg·kg^-1 was intragastrically for last 4 week).The blood lipid level was detected at4 weeks,8 weeks and 12 weeks,the serum liver enzymes were analyzed at 12 weeks,histopathologic changes was observed by HE staining.Thirty wistar rats were randomly assigned to the five groups（n=6）:SD group（fed with normal fat diet for 12weeks,and then underwent abdominal abdominal operation）,HFD group（fed with high-fat diet for 12 weeks,and then underwent abdominal abdominal operation）,HFD+IR group（established cold I/R model）,HFD+IR+BBR group(BBR200mg·kg^-1 was intragastrically for 1 week before performing cold I/R treatment),HFD+IR+BBR+TG group(BBR 200mg·kg^-1 was intragastrically for 1 week before performing cold I/R treatment,six days later TG 0.2mg/kg was intravenously).All liver tissues and blood were collected at 6 hours after reperfusion.The liver function was detected,histopathologic changes were observed by HE staining,the oxidative stress and inflammation were determined by ELISA kit,and expression of Bip,CHOP,p-PERK,PERK,LC3,Beclin-1 and p62 were detected by western blot.The morphology of endoplasmic reticulum and the number of autophagy in liver tissue were observed by transmission electron microscope.We detected the co-localization of ER marker（KDEL）and autophagic protein（LC3B）by immunofluorescence microscopy.The level of reticulophagy hallmark（FAM134B）was determined by immunohistochemistry.Results Compared with Sham group,the level of serum lipid was increased in HFD and HFD+BBR group.At the same time,liver function became severe,and fat vacuoles appeared in hepatocytes.Compared with HFD group,the level of serum lipid,liver function and fat vacuoles were decreased in HFD+BBR group.Compared with SD group,the level of ALT and AST,oxidative stress index and inflammatory factor were up-regulated.Histological examination revealed that micro-and macro-vesicular steatosis scattered and severe sinusoidal congestion,necrosis,inflammatory infiltration.The expression of endoplasmic reticulum stress-related proteins and autophagy related proteins were increased.Under Transmission electron microscopy（TEM）,the endoplasmic reticulum was dilated into cystic bubble shape,the number of autophagy was increased.The co-localization of KDEL and LC3B was enhanced by double-labeled immunofluorescence.The expression of FAM134B was increased obviously.After BBR pretreatment,liver function,oxidative stress and inflammatory factor were reduced.HE staining showed that hepatocyte fat cavitation and inflammatory factor decreased.The expression of endoplasmic reticulum stress related proteins and autophagy related proteins were reduced.The morphology of endoplasmic reticulum was improved,and the autophagy activity was reduced under TEM.The co-localization of KDEL and LC3B was down-regulated,and immunohistochemical showed that the expression of FAM134B decreased.However,TG reversed the beneficial effects of BBR.Conclusions Berberine pretreatment can reduce the adipose degree of liver tissue.At the same time,Berberine can play a protective role by reducing the endoplasmic reticulum stress-mediated reticulophagy,and attenuating ischemia reperfusion injury of steatotic liver in rats.更多还原