基于烟酰胺磷酸核糖转移酶（NAMPT）探讨红景天苷在非酒精性脂肪性肝病小鼠模型中的保护作用.docx

脂肪性肝病DO1:10.12449/JCH240112基于烟酰胺磷酸核糖转移酶(NAMPT)探讨红景天首在非酒精性脂肪性肝病小鼠模型中的保护作用李荣军L徐春霞I,张庭2,张志红2,王缕21泸州市中医医院脾胃病肝病科，四川泸州6460002西南医科大学附属医院全科医学科，四川泸州646000三三储:例，WangIVo507(ORQD:0000X)002-0876-1029)摘要：目的研究红景天昔对非酒精性!旨肪性肝病(NAFLD)的保护作用，并探讨其作用机制.方法将24只雄性KM小鼠随机分为正常组、匐歌食(HFD)组、HFD+空白对照组、HFD+红景天昔组，锲S6只，正常组小鼠予以普通饲料喂养，其余各组高脂饮食，造模14周开始给予100mgkg"d红景天吉灌胃给药，22周末取材.酶联免疫法测定血清ALT、AST、TG、TC、HDL-CxLDL-C等相关生化指标；HE染色和NAFLD活动评分(NAS)观察小面组织病理.WesternBlot检测肝组织内烟酰胺磷酸核糖转移酶(NAMPT)xSirtl.AMPK,SREBPl的表达变化。多组间比较采用单因素方差分析，步两两比较采用LSDM佥验结果与正常组比较，HFD组小鼠肝组织存在广泛较大的脂肪滴，脂肪变性明显，NAS评分升高(P<0.01),外周血AST、ALT、TG、TC、LDLC的含量明显增高(P值均0.05),HDL-C含量降低(P<。05),肝组织内NAMPT、AMPKa、Sirtlfl氐(鼻酸<。05),SERBPl加(P<0.01)。而HFD较HFD组、HFD+空白就颔肝组织空泡状旨滴分布、小叶内炎症减少，肝细胞气球样变减轻，NAS评分下降(4。01),外周血AST、ALT、TG、LDL-C的明显B制氐(P侬<0.05),HDL-C含量升高(P<0,05),NAMPTxAMPKa.Sirtl(P(li¾<0,05),SERBPl的表达减少(P<0.Ol).结论红景天昔能明显改善高月歌食诱导小鼠NAFLD的病理状态,并通过增加NAMPLSirtLAMPKa的表达，减少SERBPl的表达，发t辕对NAFLD的保护作用。关键词：烟酰胺磷酸核糖基转移酶；红景天昔；非酒精性脂肪性肝病；小鼠基金项目：西南医科大学-泸州市中医医院基地项目(2017-LH012)Roleofsalidrosideinamousemodelofnon-alcoholicfattyliverdisease:Astudybasedonnicotinamidephosphoribosyltransferase1.IRongjun1,XUChunxia',ZHANGTin,ZHANGZhihonlWANGLyif.(1.DepartmentOfSpIeen,StomachandUverDiseases,LuzhouHospitalofTraditionalChineseMedicine,Luzhou,Sichuan646000,China;2.DepartmentofGeneralPractice,TheAffiliatedHospitalOfSouthwestMedicalUniversitylLuzhou,Sichuan646000,China)Correspondingauthor:WANGLv,WangM)507(ORCID:0000-0002-0876-1029)Abstract:ObjectiveToinvestigatetheprotectiveeffectofsalidrosideagainstnonalcoholicfattyliverdisease(NAFLD)anditsmechanismofaction.MethodsAtotalof24maleKMmicewererandomlydividedintonormalgroup,HFDgroup,HFD+blankcontrolgroup,andHFD+salidrosidegroupfwith6miceineachgroup.ThemiceinthermalgroupWeregivennormaldiet,andthoseintheothergroupsWeregivenhigh-fatdietAfter14veeksofmodefing,themiceweregivensalidroside100mgkg<bygavage,andrelatedsampleswerecollectedattheendofweek22.Enzyme-Iinkedimmunosortoentassaywasusedtomeasurethese11jmlevelsofrelatedbiochemicalparametersincludingalanineaminotransferase(ALT),aspartateaminotransferase(AST),triglyceride(TG),totalcholesterol(TC),high-densitylipoproteincholesterol(HDL-C),andlow-densitylipoproteincholesterol(LDL-C);HEstainingandNAFLDactivityscore(NAS)wereusedtoobservetheliverhistopathologyofmice;WesternblotwasusedtomeasurethechangesintheexpressionofNAMPT,Sirtl,AMPKa,andSREBPlinlivertissue.Aone-wayanalysisofvariancewasusedforcomparisonbetweenmultiplegroups,andtheleastsignificantdifferenceMeStwasusedforfurthercomparisonbetweentwogroups.ResultsComparedwiththermalgroup,theHFDgrouphadObVioUSsteatosisandextensivelargelipiddropletsinlivertissue,withsignificantincreasesinNASscore(P<0.01)andthecontentofAST,ALT,TG,TC,andLDL-Cinperipheralblood(allP<0.05)andasignificantreductioninthecontentofHDL-C(P<0,05),aswellassignificantreductionsinteexpressionlevelsofNAMPT,AMPKa,andSirtlinlivertissue(allP<0.05)andasignificantincreaseintheexpressionlevelfSERBPl(P<0,01).ComparedwithteHFDgroupandtheHFD+blankcontrolgrp,theHFD+salidrosidegrouphadreductionsinthedistributionofvacuolarlipiddropletsandintratobdarinflammationinlivertissue,alleviationofteballningdegeneratiofhepatocytes,significantreductionsinNASscore(P<0.01)andthecontentofAST,ALT,TG,andIDL-Cinperipheralblood(allP<0.05),andasignificantincreaseinthectentofHDL-C(P<0,05),aswellassignificantineasesintheexpressilelsfNAMPT,AMPKa,andSirtlinlivertissue(allP<0.05)andasignificantreductionintheexpressionlevelofSERBPl(P<0.01).CondusionSalidrosidecansignificantlyimprovethepathologicalstateofmicewithNAFLDinducedbyhigh-fetdietandexertaprotectiveeffectagainstNAFLDbyincreasingtheexpressionofNAMPT,Sirtl,andAMPKaandreducingtheexpressionofSERBPLKeywords:NicotinamidePhosphoribosyltransferase;Salidroside;Non-alcoholicFattyUverDisease;MiceResearchfunding:SouthwestMedicalUniVerSity-LUZhoUTraditionalChineseMedicineHospitalBaseProject(2017-LH012)不良的生活方式及饮食习惯导致非酒精性脂肪性LL4画OALT、AST、TG.TC.HDLCLDL-C检测试肝病（nonalcoholicfattyliverdisease,NAFLD）患增长，已成为我国第一大慢性肝病和健康体检肝脏生化指标异常的首要原因【】。最新的流行病学调查123j结果显示，包括中国在内的多数亚洲国家NAFLD患病率处于中上水平（25%）红景天昔在NAFLD中可通过改善胰岛素抵抗、抗氧化应激、抗纤维化等途径发挥保护作用a?）。同时，研究表明，细胞内烟酰胺雕核糖转移酶（nicotinamidePhosphoribosyltransferas,NAMPT）具有调节肝脂质代谢的潜在作用。本研究通过建立NAFLD模型，并以NAMPT为切入点，探讨红景天音在NAFLD中的调控作用及其可能机制，以期为红景天昔的临床应用提供理论依据。1材料与方法1.1主要材料1.1.1实验动物SPF级6周龄雄性KM小鼠24只，体质量（30±5）g,由成都达硕实验动物有限公司提供,饲养于西南医科大学实验动物中心，实验动物生产许可证编号:SCXK（JII）2015-030,物使用许可ii三:SYXK（Jll）2014-0001.1.2药物制备红景天首（98%,大连美仑生物技术有限公司）批号：J1206A。用时以蒸储水配置成浓度为10mg/mL的药液。1. 1.3高月胸斗配方：82.5%普通饲料、10%猪油、5%蔗糖、2%胆固醇、0.5%胆酸钠，购自成都达硕实验动物有限公司。剂盒（Mindray公司）；NAMPT、AMPKaSERBPl抗体、Sirtl（Affinity公司）,actin抗体（AbdOnal公司）,Western及IP细胞裂解液、BCA蛋白浓度测定试剂盒（Beyotime公司）预染蛋白Marker（ABelone公司）;ECL发光试剂盒（Affinity公司）,ImmobiIon-PSQPVDF膜（Sigmaaldrich公司）;PAGE凝胶快速制备试剂盒（上海雅酶生物科技有限公司）；GlyCine（BiOfrOXX公司）；TriS缓冲液、SDS（BiOfroXX公司）1.2实验方法1.2.1分组及造模将小鼠适应性喂养1周，随机分为正常组（普通饲料喂养）、翩敬食（HFD）组、HFD+空白对照组、HFD+红景天昔组，每组6只。HFD组通过高月施料喂养14周建立NAFLD动物模型，14周末以HFD组小鼠肝脏病理切片中出现大小不