活性维生素D3调控NF-?资B信号通路对糖尿病肾病大鼠肾脏保护作用机制研究

AstudyonthemechanismofactionofactivevitaminD3throughregulatingtheNF-?资Bsignalingpathwayinrenalprotectionofdiabeticnephropathyrats

摘要:

目的：探讨活性维生素D3调控NF-?资B炎症信号通路对2型糖尿病肾病大鼠肾脏的保护作用及机制；寻找维生素D在治疗糖尿病肾病中的最佳浓度。方法：将SD大鼠70只随机分成正常对照组（10只）、DN模型组（60只），且DN模型组进一步分为花生油组、厄贝沙坦组、维生素D低剂量组、维生素D中剂量组、维生素D高剂量组（每组各10只）。正常组以普通饲料喂养，模型组分别以上述处理方式进行灌胃。6周后处死大鼠称取体质量，采血检测空腹血糖（fastingbloodglucose，FBG），收集24h尿液计算尿量、测24h尿蛋白定量；留取肾脏组织，HE染色观察肾组织病理学变化；实时荧光定量PCR检测肾脏组织中NF-?资B及其下游单核细胞趋化因子-1（monocytechemotacticprotein1，MCP-1）的mRNA表达；Westernblot检测炎症因子NF-?资B及MCP-1蛋白表达。结果：①在血糖、24h尿蛋白，维生素D低剂量组较花生油组明显下降[分别为18.20（16.25，21.25）、32.24（26.09，37.68）与23.55（22.75，24.60）、72.52（50.38，84.93），均P<0.05]。②正常组大鼠HE染色可见肾小球形态完整，系膜区无明显增生，毛细血管腔规则，毛细血管袢形态规则；花生油组肾小球体积缩小，系膜区明显增宽，肾小球系膜细胞增生，毛细血管壁增厚；厄贝沙坦组较花生油组增生减轻；维生素D高剂量组、中剂量组、低剂量组肾小球系膜区均有增宽，但维生素D低剂量组增宽程度最轻。③维生素D低剂量组的NF-?资B及MCP-1的mRNA水平较正常组花生油组明显下降[分别为1.72±0.002、0.67±0.01；3.57±0.40、3.57±0.37，均P<0.05]；但厄贝沙坦组与维生素D低剂量组比较无统计学差异。④NF-?资B的蛋白表达水平在各组间均无统计学差异，维生素D低剂量组的MCP-1蛋白表达水平较花生油组、维生素D中剂量组下降最为明显[分别为0.47±0.17与0.91±0.30、1.22±0.18、0.93±0.43，均P<0.05）。结论：维生素D能通过下调NF-кB炎症信号通路，有效地缓解糖尿病肾病的肾脏损伤；维生素D低剂量[0.03μg/（kg·d）]在糖尿病肾病的保护作用中为最佳浓度。

Abstract:

Objective：①ToinvestigatethemechanismofactionofactivevitaminD3throughregulatingtheNF-κBsignalingpathwayinrenalprotectionofratswithtype2diabeticnephropathy.②TodeterminetheoptimalconcentrationofvitaminDinthetreatmentofdiabeticnephropathy.Methods：SeventySprague-Dawleyratswererandomlydividedintonormalcontrolgroup（10rats）andDNmodelgroup（60rats），andtheDNmodelgroupwasfurhterdividedintopeanutoilgroup，irbesartangroup，low-dosevitaminDgroup，medium-dosevitaminDgroup，andhigh-dosevitaminDgroup，with10ratsineachgroup.Thenormalcontrolgroupwasfedwithroutinediet，whiletheratsinthemodelgroupweread－ministeredabove-mentioneddrugsbygavage.Theratswerethensacrificedandweighedafter6weeks.Andfastingbloodglucose（FBG）wasmeasuredafterbloodsampling，and24-hoururinewasalsocollectedtocalculateurinevolumeandquantify24-hoururineprotein.RenaltissuewasretainedandHEstainingwasusedtoobservethepathologicalchangesofrenaltissue.Meanwhile，real-timefluorescentquantitativePCRwasusedtodeterminetheexpressionofnuclearfactor-kappaB（NF-κB）mRNAanditsdownstreammonocytechemotacticprotein-1（MCP-1）mRNAinrenaltissue，andtheproteinexpressionofinflammatoryfactors，NF-κBandMCP-1，wasmeasuredbyWesternblot.Results：①ThelevelsofFBGand24-hoururineproteininthelow-dosevitaminDgroupweresignificantlylowerthanthoseinthepeanutoilgroup[18.20（16.25，21.25），32.24（26.09，37.68）vs.23.55（22.75，24.60），72.52（50.38，84.93），P<0.05].②Inthenormalcontrolgroup，HEstainingshowedthattheglomerularmorphologywasintact，themesangialareahadnoobvioushyperplasia，andthecapillarylumenandloopswerebothregularlyshaped；inthepeanutoilgroup，theglomerularvolumewasreduced，themesangialareasignificantlybroadened，theglomerularmesangialcellsproliferated，andcapillarywallbecamethickenedaswell.Theirbesartangroupshowedlesshyperplasiacomparedwiththepeanutoilgroup.Thehigh-，medium-，andlow-dosevitaminDgroupsallhadabroad－enedglomerularmesangialarea，butthelow-dosegroupexhibitedthelowestdegreeofbroadening.③ThemRNAlevelsofNF-κBandMCP-1inthelow-dosevitaminDgroupweresignificantlylowerthanthoseinthenormalcontrolgroupandthepeanutoilgroup（1.72±0.002，0.67±0.01vs.3.57±0.40，3.57±0.37，allP<0.05）.However，therewasnosignificantdifferencebetweentheirbesartangroupandthelow-dosevitaminDgroup.④TherewerenosignificantdifferencesbetweenthesegroupsregardingtheexpressionofNF-κBprotein.Thelow-dosevitaminDgrouphadasignificantreductionintheexpressionofMCP-1proteincomparedwiththepeanutoilgroupandthemedium-dosevitaminDgroup（0.47±0.17vs.0.90±0.30，1.22±0.18，0.93±0.43，allP<0.05）.Conclusion：①VitaminDcaneffectivelyalleviaterenalinjuryindiabeticnephropathybydown-regulationoftheNF-κBinflammatorysignalingpathway.②Asindicatedinthestudy，0.03μg/（kg·d）inthelow-dosevitaminDgroupistheoptimalconcentrationforre－nalprotectionindiabeticnephropathy.