五味子乙素对链脲佐菌素诱导的糖尿病大鼠视网膜病变的保护作用及机制研究

doi:10.3969/j.issn.1000⁃484X.2019.02.010五味子乙素对链脲佐菌素诱导的糖尿病大鼠视网膜病变的保护作用及机制研究①刘　青　(西京学院医学院,西安710000) 中图分类号　R774 文献标志码　A 文章编号　1000⁃484X (2019)02⁃0176⁃05①本文为陕西省教育厅自然科学研究项目(2013JK1145)㊂作者简介:刘　青,女,硕士,讲师,主要从事中医五官科学眼科方向的研究,E⁃mail:lqqqyynn@㊂[摘　要]　目的:探究五味子乙素(SB)对链脲佐菌素(STZ)诱导的糖尿病视网膜病变(DR)的作用及作用机制㊂方法:腹腔注射STZ 复制大鼠DR 模型,并连续给予SB 9个月,TUNEL 检测细胞凋亡,试剂盒检测氧化应激标记分子的含量,ELISA 法检测炎症因子的表达,Western blot 检测血管新生因子和Cdc42/p38通路蛋白的表达㊂结果:STZ 可明显升高大鼠血糖浓度,表明造模成功;SB 可明显减弱STZ 对凋亡小体形成的促进作用;SB 还能显著促进DR 大鼠抗氧化分子SOD 的表达,降低氧化产物MDA 和LDH 的含量;同时,SB 可明显缓解STZ 对炎症因子(IL⁃6㊁TNF⁃α和IL⁃1β)分泌的促进作用;此外,SB 还能明显减弱STZ 对促血管新生因子VEGF 和通路蛋白Cdc42和p⁃p38表达的促进作用㊂结论:SB 可通过Cdc42/p38通路缓解STZ 诱导的大鼠DR㊂[关键词]　五味子乙素;糖尿病视网膜病变;凋亡;炎症;氧化应激;血管新生Study on protective effect and mechanism of schisandrin B on streptozotocin induced⁃diabetic reinopathyLIU Qing .Xijing University School of Medicine ,Xi′an 710000,China[Abstract ]　Objective :To evaluate the protective effect and underlying mechanism of schisandrin B (SB)on streptozotocin(STZ)induced⁃diabetic reinopathy(DR)in rats.Methods :Following STZ injection,rats received gavage of SB for 9months subse⁃quently.TUNEL assay was performed for apoptosis.The levels of SOD,MDA and LDH were determined by kits.To calculate the levels of inflammatory cytokines,ELISA assay was carried out.And western blot was performed for protein levels.Results :In STZ induced⁃DR rats,glucose level was makedly increased.SB inhibited STZ⁃induced apoptosis as demonstrated by increased apoptotic body.SB significantly increased the level of SOD and decreased the levels of MDA and LDH.Meanwhile,SB notably inhibited STZ induced⁃in⁃flammation by decreasing IL⁃6,TNF⁃αand IL⁃1β.Furthermore,SB partly abolished STZ′s effects on the expressions of VEGF,Cdc42and p⁃p38.Conclusion :SB attenuates STZ induced⁃DR via Cdc42/p38signaling pathway.[Key words ]　Schisandrin B;Diabetic reinopathy;Apoptosis;Inflammation;Oxidative stress;Angiogenesis 糖尿病视网膜病变(Diabetic reinopathy,DR)是糖尿病最常见的微血管并发症[1,2]㊂目前,DR 发病的增长速度虽然已得到了一定的控制,但其仍是导致成年人失明的主要原因之一[3]㊂研究表明,视网膜炎症㊁细胞凋亡㊁脂质过氧化及异常血管增生都是导致DR 恶化的主要原因[4⁃7]㊂因此,若能有效调控DR 的多个病理环节将有利于DR 的治疗㊂五味子乙素(Schisandrin B,SB)是中药五味子的主要有效成分之一,其具有抗炎㊁抗癌㊁抗氧化等药理学功效[8⁃10]㊂研究表明,五味子可抑制糖尿病大鼠钠葡萄糖共转运载体的活性[11]㊂五味子素可对抗烟酰胺腺嘌呤二核苷酸磷酸(Nicotinamide adeninedinucleotide phosphate,NAPDH)诱导的糖尿病肾病氧化损伤,提高糖尿病大鼠对胰岛素的敏感性[12,13]㊂也有研究表明,SB 可减轻四氯化碳(Carbon tetrachloride,CCl 4)诱导的糖尿病大鼠肝损伤[14]㊂但其对DR 的作用还未见报道㊂因此,本文将采用链脲佐菌素(Streptozotocin,STZ)复制大鼠DR 模型,探讨SB 对DR 的作用及作用机制㊂1　材料与方法1.1　试剂与仪器　超氧化物歧化酶(Superoxidedismtase,SOD)㊁丙二醛(Malondiadehyde,MDA)和乳酸盐脱氢酶(Lactate dehydrogenase,LDH)试剂盒购自南京建成生物工程研究所㊂白介素⁃6(Lnterleukin⁃6,IL⁃6)㊁IL⁃1β和肿瘤坏死因子(Tumornecrosis factor,TNF⁃α)ELISA试剂盒购自美国Miliipore公司㊂SB购自北京索莱宝公司㊂内皮细胞生长因子(Vascular endothelial growth factor, VEGF)抗体购自英国Abcam公司,细胞分裂周期蛋白42(Cell division cycle,Cdc42)和p⁃p38一抗购自美国CST公司㊂1.2　方法1.2.1　动物模型的建立　Wistar大鼠购自上海实验动物研究中心㊂将大鼠适应性喂养3d后,随机分为对照组㊁SB组㊁STZ组和STZ+SB组㊂实验前12h禁食禁水㊂STZ组和STZ+SB组大鼠一次性腹腔注射STZ(58mg/kg)诱导高血糖,对照组和SB组注射等量生理盐水,3d后尾静脉取空腹血,用血糖仪检测血糖浓度,血糖浓度高于400mg/dl(22mmol/L)视为造模成功㊂剔除造模未成功的大鼠,SB组和STZ+SB 组大鼠连续灌胃给予SB(50mg/kg)9个月㊂9个月后摘取眼球并收集血液进行后续实验㊂1.2.2　TUNEL　摘取大鼠眼球,用4%多聚甲醛固定过夜后,去除晶状体,使余下组织包含全部视网膜,制作视网膜石蜡切片㊂根据TUNEL试剂盒说明书检测凋亡小体㊂切片脱蜡后,用3%的H2O2处理10min㊂滴加0.5%的Proteinase K37℃孵育10min,随后加入TdT和生物素标记的dUTP混合液37℃处理30min,弃去混合液,待玻片干后,滴加TUNEL混合液,最后滴加DAB显色,苏木精复染㊁脱水㊁封片㊂于显微镜下每片随机选取6个视野进行计数统计㊂1.2.3　SOD㊁MDA和LDH含量的检测　将实验所得全血以3000r/min的转速4℃离心15min,取上清液,根据试剂盒说明书检测上清液中SOD㊁MDA 和LDH的含量㊂1.2.4　ELISA　根据ELISA试剂盒说明书检测全血上清液中IL⁃6㊁TNF⁃α和IL⁃1β的含量㊂1.2.5　Western blot　用蛋白裂解液和蛋白酶抑制剂混合液提取视网膜组织蛋白㊂用BCA试剂盒检测总蛋白质浓度并调平㊂10%SDS⁃PAGE分离各组蛋白质并转移至PVDF膜㊂用5%脱脂牛奶室温封闭PVDF膜2h,随后加入一抗(VEGF,1∶1000; Cdc42,1∶1000;p⁃p38,1∶1000),4℃封闭过夜㊂第2天用TBST洗膜3次后,加入HRP标记的二抗于室温孵育1h,最后加入ECL显色液曝光显影㊂1.3　统计学分析　所有实验数据以x±s表示,采用Graphpad prism6统计软件进行统计分析,用Student′s t⁃test对组间差异进行统计分析㊂以P< 0.05认为差异均有统计学意义㊂2　结果2.1　SB对DR大鼠血糖的影响　为了检测DR大鼠模型是否建立成功,我们检测了STZ注射3d后大鼠的血糖浓度㊂实验结果表明,与对照组比较,SB组大鼠血糖无明显变化(P>0.05,图1),STZ组血糖浓度明显升高(P<0.001,图1),且高于22mmol/L,表明造模成功;同时,SB能明显减弱STZ诱导血糖升高的作用(P<0.05,图1)㊂2.2　SB对视网膜细胞凋亡的影响　为了探究SB 对DR大鼠视网膜细胞凋亡的影响,我们检测了凋亡小体的形成情况㊂STZ可显著促进视网膜凋亡小体的形成(P<0.001,图2),SB则能明显减弱STZ 对凋亡小体形成的诱导作用(P<0.05,图2)㊂2.3　SB对DR大鼠氧化应激的影响　为了探究SB 对STZ诱导的DR大鼠氧化应激的影响,我们检测了外周血中SOD㊁MDA和LDH的含量㊂实验结果表明,STZ可明显降低血清中SOD的含量,促进MDA和LDH的分泌(P<0.01,图3)㊂SD能显著减弱STZ对SOD分泌的抑制作用,降低MDA和LDH 的含量(P<0.05,图3)㊂2.4　SB对DR大鼠炎症反应的影响　ELISA实验结果表明,与对照组比较,SB组炎症因子IL⁃6㊁TNF⁃α和IL⁃1β的含量无显著变化(P>0.05,图4),STZ组IL⁃6㊁TNF⁃α和IL⁃1β的含量明显增多(P<0.01,P< 0.001,图4);与STZ组比较,STZ+SB组血清中IL⁃6㊁TNF⁃α和IL⁃1β含量明显增多(P<0.05,图4)㊂2.5　SB对Cdc42/P38通路的影响　为了探究SB 对STZ诱导的DR的作用机制,我们检测了血管新生标记分子VEGF及其下游信号通路蛋白的表达情况㊂实验结果表明,STZ能显著促进VEGF㊁Cdc42图1　SB对DR大鼠血糖的影响Fig.1　Effect of SB on blood glucose level of rats Note:A.The chemical structural formula of SB;B.The quantification of blood glucose of rats.***.P<0.001vs control group;#.P<0.05vs STZ group.图2　SB 对视网膜细胞凋亡的影响Fig.2　Effect of SB on apoptosis of retina cellsNote:A.The formation of apoptotic body was measured by Tunnel assay;B.The quantification of apoptotic body.Each experiment wasrepeated at least three times.***.P <0.001vs control group;#.P <0.05vs STZgroup.图3　SB 对DR 大鼠氧化应激的影响Fig.3　Effect of SB on oxidative stress of DR ratsNote:The concentrations of SOD,MDA and LDH were measured bykits.**.P <0.01vs control group;#.P <0.05vs STZgroup.图4　SB 对DR 大鼠炎症反应的影响Fig.4　Effect of SB on inflammation of DR ratsNote:The concentrations of IL⁃6,TNF⁃αand IL⁃1βwere determined byELISA assay.**.P <0.01,***.P <0.001vs control group;#.P <0.05vs STZ group.和p⁃p38的表达(P <0.01,P <0.001,图5),SB 能显著下调DR 大鼠视网膜VEGF㊁Cdc42和p⁃p38的蛋白表达水平(P <0.05,图5)㊂图5　SB 对Cdc42/p38通路的影响Fig.5　Effect of SB on Cdc42/p38signaling pathwayNote:A.The protein levels of VEGF,Cdc2and p⁃p38were measured byWestern blot;B.The quantification of protein levels of VEGF,Cdc2and p⁃p38.GAPDH was used as loading control.**.P <0.01,***.P <0.001vs control group;#.P <0.05,##.P <0.001vs STZ group.3　讨论细胞凋亡是导致DR 的重要机制之一[15]㊂在DR 中,新陈代谢紊乱会导致视网膜细胞凋亡㊁糖基化终产物的形成㊁白细胞聚集及氧化应激的发生[6]㊂研究表明,视网膜神经细胞的凋亡会导致视网膜功能减退,周细胞及血管内皮细胞的凋亡是引起血管渗透性增高的主要原因[4,16,17]㊂在本研究中,我们采用腹腔注射STZ 的方法复制大鼠DR 模型并给予大鼠SB 9个月㊂实验结果显示,SB 对正常大鼠的凋亡小体形成没有明显影响,表明该剂量下的SB 对视网膜细胞没有明显的毒副作用㊂但SB 能显著抑制DR 大鼠视网膜凋亡小体形成,表明SB 具有抗DR 大鼠视网膜细胞凋亡的作用㊂研究表明,DR 的高糖和高脂肪酸环境能通过激活线粒体电子传递链诱导过氧化物的生成,从而导致脂质过氧化,脂质过氧化能进一步降低细胞膜的流动性㊁影响Na +⁃K +平衡㊁使细胞膜通透性升高,同时还能改变线粒体膜电位,诱导细胞凋亡[18,19]㊂大量研究表明,中药有效成分具有抗DR 氧化应激的作用㊂如柚苷㊁橙皮素和生草酚等均可通过调控SOD㊁MDA 及NO 等的表达减缓DR 的发展[20⁃22]㊂也有研究表明,SB 可通过调控ROS 通路减轻NAPDH 氧化酶对糖尿病肾病系膜细胞的损伤[12]㊂本文研究发现,SB 可明显促进SOD 的分泌,降低DR 大鼠血清MDA 和LDH 的含量㊂其中,SOD 是超氧阴离子自由基清除剂,可减少过氧化氢的生成[23]㊂MDA 是脂质过氧化反应链的终产物,因此,其含量的变化反映了脂质过氧化程度[24]㊂LDH 是参与糖酵解和糖异生的重要酶㊂在正常情况下,血清中LDH 的含量较少,但当细胞膜通透性增加时,LDH可大量的进入血液[25]㊂结合实验结果表明, SB可减轻STZ诱导的DR大鼠视网膜的脂质过氧化,增强DR大鼠抗自由基损伤的能力,从而降低细胞膜通透性㊂研究表明,TNF⁃α㊁IL⁃1β㊁IL⁃6等炎症因子表达增多可诱导DR模型鼠视网膜结构和功能的改变[26,27]㊂下调IL⁃1β的表达可抑制视网膜毛细血管的退化[26]㊂玻璃体内注射TNF⁃α可抑制糖尿病大鼠视网膜周细胞凋亡和毛细血管变性[27]㊂敲除TNF⁃α基因可降低糖尿病小鼠视网膜血管通透性㊁减少白细胞黏附[28]㊂本文研究发现,TNF⁃α㊁IL⁃1β和IL⁃6在STZ诱导的DR大鼠外周血中均呈高表达状态,SB能显著降低血清中TNF⁃α㊁IL⁃1β和IL⁃6的含量,表明SB可抑制STZ诱导的炎症反应㊂VEGF是调控血管新生的重要细胞因子,能够促进内皮细胞的增殖㊁迁移及基底膜的降解,在促进视网膜血管新生过程中发挥重要作用[29]㊂视网膜血管新生是指视网膜血管的异常生长,也是增殖性DR的重要特征[30]㊂同时,VEGF还可调控DR炎症反应,可促进黏附因子及炎症的表达,诱导白细胞激活[31]㊂因此,下调VEGF表达被认为是目前治疗DR最可行的方法㊂并且哌加他尼㊁兰尼单抗及阿伐斯汀等VEGF抑制剂已进入治疗DR的临床试验阶段[30]㊂我们的研究发现,SB可明显抑制VEGF 的表达㊂此外,SB还能明显抑制DR大鼠Cdc42和p⁃p38的表达㊂Cdc42和p38是VEGF下游Cdc42/ p38信号通路的标记蛋白㊂研究表明,VEGFR2是主要参与调控内皮细胞增殖和迁移的VEGF受体[32]㊂VEGF与VEGFR2结合后,VEGFR2首先进行自身磷酸化进而激活Cdc42,活化的Cdc42和VEGFR2又可进一步激活p38,诱导内皮细胞迁移[33]㊂也有研究表明,IL⁃4可通过调控Cdc42⁃p38 MAPK通路促进IL⁃6的表达,提示Cdc42/p38信号通路可能也参与了炎症反应的调控[34]㊂结合实验结果表明,SB可下调VEGF表达并可通过Cdc42/ p38信号通路抑制STZ诱导的DR大鼠视网膜炎症反应和血管新生㊂综上所述,SB可降低DR大鼠血糖水平㊁抑制视网膜细胞凋亡及氧化应激,可通过抑制炎症因子及VEGF的表达抑制视网膜炎症反应及血管新生,并且其机制与抑制Cdc42/p38信号通路激活有关㊂参考文献:[1]　Duh EJ,Sun JK,Stitt AW.Diabetic retinopathy:currentunderstanding,mechanisms,and treatment strategies[J].JCIInsight,2017,2(14):pii:93751.[2]　Willard AL,Herman IM.Vascular complications and diabetes:current therapies and future challenges[J].J Ophthalmol,2012, 2012:209538.[3]　Leasher JL,Bourne RR,Flaxman SR,et al.Erratum.Globalestimates on the number of people blind or visually impaired by diabetic retinopathy:a meta⁃analysis from1990⁃2010[J].Diabetes Care,2016,39(11):1643⁃1649.[4]　Wu Y,Chen Y,Wu Q,et al.Minocycline inhibits PARP1expression and decreases apoptosis in diabetic retinopathy[J].Mol Med Rep,2015,12:4887⁃4894.[5]　Adamis AP.Is diabetic retinopathy an inflammatory disease?[J].Bri J Ophthalmol,2002,86:363⁃365.[6]　Elahy M,Baindur⁃Hudson S,Cruzat VF,et al.Mechanisms ofPEDF⁃mediated protection against reactive oxygen species damage in diabetic retinopathy and neuropathy[J].J Endocrinol,2014, 222:R129⁃R139.[7]　Wu W,Tang L.Research progress of anti⁃angiogenesis drug targetsin diabetic retinopathy[J].Chin J Ophthalmol,2014,50:871⁃876.[8]　Jiang Y,Zhang Q,Bao J,et al.Schisandrin B suppresses gliomacell metastasis mediated by inhibition of mTOR/MMP⁃9signal pathway[J].Biomed Pharmacother,2015,74:77⁃82. [9]　Thandavarayan RA,Giridharan VV,Arumugam S,et al.Schisandrin B prevents doxorubicin induced cardiac dysfunction by modulation of DNA damage,oxidative stress and inflammation through inhibition of MAPK/p53signaling[J].PLoS One,2015, 10:e0119214.[10]　Mak DH,Ip SP,Li PC,et al.Effects of Schisandrin B and alpha⁃tocopherol on lipid peroxidation,in vitro and in vivo[J].Mol CellBiochem,1996,165:161⁃165.[11]　Qu Y,Chan JY,Wong CW,et al.Antidiabetic effect ofschisandrae chinensis fructus involves inhibition of the sodiumglucose cotransporter[J].Drug Dev Res,2015,76:1⁃8. [12]　Jeong SI,Kim SJ,Kwon TH,et al.Schizandrin prevents damage ofmurine mesangial cells via blocking NADPH oxidase⁃inducedROS signaling in high glucose[J].Food Chem Toxicol,2012,50:1045⁃1053.[13]　Kwon DY,Kim DS,Yang HJ,et al.The lignan⁃rich fractions ofFructus Schisandrae improve insulin sensitivity via the PPAR⁃gamma pathways in in vitro and in vivo studies[J].JEthnopharmacol,2011,135:455⁃462.[14]　Mak DH,Ko KM.Alterations in susceptibility to carbontetrachloride toxicity and hepatic antioxidant/detoxification systemin streptozotocin⁃induced short⁃term diabetic rats:effects ofinsulin and Schisandrin B treatment[J].Mol Cell Biochem,1997,175:225⁃232.[15]　Barber AJ,Gardner TW,Abcouwer SF.The significance ofvascular and neural apoptosis to the pathology of diabeticretinopathy[J].Invest Ophthalmol Vis Sci,2011,52:1156⁃1163.[16]　Ejaz S,Chekarova I,Ejaz A,et al.Importance of pericytes andmechanisms of pericyte loss during diabetes retinopathy[J].Diabetes Obes Metab,2008,10:53⁃63.[17]　Mizutani M,Kern TS,Lorenzi M.Accelerated death of retinal mi⁃crovascular cells in human and experimental diabetic retinopathy[J].J Clin Invest,1996,97:2883⁃2890.[18]　Dias IH,Griffiths HR.Oxidative stress in diabetes⁃circulatingadvanced glycation end products,lipid oxidation and vasculardisease[J].Ann Clin Biochem,2014,51:125⁃127. [19]　Duarte DA,Silva KC,Rosales MA,et al.The concomitance of hy⁃pertension and diabetes exacerbating retinopathy:the role of in⁃flammation and oxidative stress[J].Curr Clin Pharmacol,2013,8:266⁃277.[20]　Liu L,Zuo Z,Lu S,et al.Naringin attenuates diabetic retinopathyby inhibiting inflammation,oxidative stress and NF⁃kappaBactivation in vivo and in vitro[J].Iran J Basic Med Sci,2017,20:813⁃821.[21]　Kumar B,Gupta S,Srinivasan B,et al.Hesperetin rescues retinaloxidative stress,neuroinflammation and apoptosis in diabetic rats[J].Microvasc Res,2013,87:65⁃74.[22]　Bucolo C,Leggio GM,Drago F,et al.Eriodictyol prevents earlyretinal and plasma abnormalities in streptozotocin⁃induceddiabetic rats[J].Biochem Pharmacol,2012,84:88⁃92. [23]　Zhang F,Wu SZ,Zhang L,et al.Effects of simvastatin on plasmaSOD,MDA and8⁃iso⁃PGF2alpha in patients with stable angina[J].Nan Fang Yi Ke Da Xue Xue Bao,2010,30(12):2646⁃2648.[24]　Kumawat M,Kharb S,Singh V,et al.Plasma malondialdehyde(MDA)and anti⁃oxidant status in diabetic retinopathy[J].JIndian Med Assoc,2014,112:29⁃32.[25]　Jurisic V,Radenkovic S,Konjevic G.The actual role of LDH astumor marker,biochemical and clinical aspects[J].Adv Exp MedBiol,2015,867:115⁃124.[26]　Vincent JA,Mohr S.Inhibition of caspase⁃1/interleukin⁃1betasignaling prevents degeneration of retinal capillaries in diabetesand galactosemia[J].Diabetes,2007,56:224⁃230.[27]　Behl Y,Krothapalli P,Desta T,et al.Diabetes⁃enhanced tumornecrosis factor⁃alpha production promotes apoptosis and the lossof retinal microvascular cells in type1and type2models ofdiabetic retinopathy[J].Am J Pathol,2008,172:1411⁃1418.[28]　Huang H,Gandhi JK,Zhong X,et al.TNFalpha is required forlate BRB breakdown in diabetic retinopathy,and its inhibitionprevents leukostasis and protects vessels and neurons fromapoptosis[J].Invest Ophthalmol Vis Sci,2011,52:1336⁃1344.[29]　Gupta N,Mansoor S,Sharma A,et al.Diabetic retinopathy andVEGF[J].Open Ophthalmol J,2013,7:4⁃10. [30]　Tremolada G,Del Turco C,Lattanzio R,et al.The role ofangiogenesis in the development of proliferative diabeticretinopathy:impact of intravitreal anti⁃VEGF treatment[J].ExpDiabetes Res,2012,2012:728325.[31]　Tang J,Kern TS.Inflammation in diabetic retinopathy[J].Progress in Retinal Eye Res,2011,30:343⁃358. [32]　Basagiannis D,Zografou S,Murphy C,et al.VEGF inducessignalling and angiogenesis by directing VEGFR2internalisationthrough macropinocytosis[J].J Cell Sci,2016,129:4091⁃4104.[33]　Lamalice L,Houle F,Huot J.Phosphorylation of Tyr1214withinVEGFR⁃2triggers the recruitment of Nck and activation of Fynleading to SAPK2/p38activation and endothelial cell migrationin response to VEGF[J].J Biolog Chem,2006,281:34009⁃34020.[34]　Wery⁃Zennaro S,Zugaza JL,Letourneur M.IL⁃4regulation of IL⁃6production involves Rac/Cdc42⁃and p38MAPK⁃dependentpathways in keratinocytes[J].Oncogene,2000,19(12):1596⁃1604.[收稿2017⁃12⁃19　修回2018⁃01⁃29](编辑　张晓舟)(上接第175页)[20]　Liao Y,Dong S,Kiyama R,et al.Flos lonicerae extracts andchlorogenic acid protect human umbilical vein endothelial cellsfrom the toxic damage of perfluorooctane sulphonate[J].Inflammation,2013,36(3):767⁃779.[21]　Ren W,Wang Z,Hua F,et al.Plasminogen activator inhibitor⁃1regulates lps⁃induced tlr4/md⁃2pathway activation andinflammation in alveolar macrophages[J].Inflammation,2015,38(1):384⁃393.[22]　Grunz⁃Borgmann E,Mossine V,Fritsche K,et al.Ashwagandhaattenuates tnf⁃alpha⁃and lps⁃induced nf⁃kappab activation andccl2and ccl5gene expression in nrk⁃52e cells[J].BMCComplement Alterna Med,2015,15:434.[23]　Mei XY,Zhou LY,Zhang TY,et al.Scutellaria barbata attenuatesdiabetic retinopathy by preventing retinal inflammation and thedecreased expression of tight junction protein[J].Int JOphthalmol,2017,10(6):870⁃877.[24]　Hong TY,Tzeng TF,Liou SS,et al.The ethanol extract of zingiberzerumbet rhizomes mitigates vascular lesions in the diabetic retina[J].Vasc Pharmacol,2016,76:18⁃27.[25]　Ye HY,Jin J,Jin LW,et al.Chlorogenic acid attenuates lipopo⁃lysaccharide⁃induced acute kidney injury by inhibiting tlr4/nf⁃kappab signal pathway[J].Inflammation,2017,40(2):523⁃529.[26]　Ruifeng G,Yunhe F,Zhengkai W,et al.Chlorogenic acidattenuates lipopolysaccharide⁃induced mice mastitis bysuppressing TLR4⁃mediated NF⁃kappa B signaling pathway[J].Eur J Pharmacol,2014,729:54⁃58.[收稿2017⁃12⁃18　修回2018⁃01⁃12](编辑　张晓舟)。