CAJ | 학술논문

本研究观察葛根素是否减轻部分由过氧亚硝基阴离子(peroxynitrite,ONOO-)导致的糖尿病大鼠晶状体上皮细胞(lens epithelium cell,LEC)凋亡.采用大鼠腹腔注射链脲佐菌素(streptozotocin,STZ)的方法建立糖尿病动物模型.36只大鼠作为对照组,腹腔注射生理盐水;其他72只大鼠腹腔注射STZ(45 mg/kg)后分为STZ组和STZ+葛根素组,每组36只.STZ注射3 d后,STZ+葛根素组大鼠每天腹腔注射葛根素(140 mg/kg).于实验开始后第20、40和60天用裂隙灯检查晶状体的形态学变化后处死动物.用流式细胞仪检测LEC凋亡,用免疫组化方法检测晶状体中ONOO的标志物--硝基酪氨酸(nitrotyrosine,NT)的表达,用基因芯片分析技术检测LEC凋亡相关基因iNOS等的表达.结果发现,对照组大鼠晶状体均透明,各项指标基本正常;STZ组大鼠第20天时即出现晶状体混浊,40～60 d期间混浊不断加重;STZ+葛根素组大鼠20～40 d时晶状体混浊呈加重趋势,但40～60 d以后明显减轻.对照组LEC轻度凋亡,而STZ组凋亡细胞呈持续性增长,STZ+葛根素组大鼠20～40 d时细胞凋亡呈增长趋势,但40～60 d以后明显下降.对照组大鼠晶状体NT未见明显表达;STZ组大鼠NT表达明显加强;STZ+葛根素组大鼠20～40 d时NT表达呈增长趋势,但40～60 d以后明显下降.对照组凋亡相关基因未见明显变化,STZ组凋亡相关基因iNOS表达明显上调.其他凋亡相关基因如BCL-2、SOD表达明显下调,但NF-кB和TNFR1-FADD-caspase信号转导途径明显上调;STZ+葛根素组凋亡相关基因表达则呈相反改变.上述结果表明,在糖尿病大鼠晶状体中有ONOO- 的标志物NT表达,证明糖尿病大鼠LEC凋亡部分由ONOO诱导,这可能是氧化损伤导致白内障形成的新途径.葛根素能够部分逆转ONOO- 对LEC的致凋亡作用,提示葛根素可能是治疗糖尿病性白内障的有效药物,其治疗机制可能与葛根素直接抑制凋亡和对抗ONOO-对糖尿病大鼠LEC的损伤有关. 本研究觀察葛根素是否減輕部分由過氧亞硝基陰離子(peroxynitrite,ONOO-)導緻的糖尿病大鼠晶狀體上皮細胞(lens epithelium cell,LEC)凋亡.採用大鼠腹腔註射鏈脲佐菌素(streptozotocin,STZ)的方法建立糖尿病動物模型.36隻大鼠作為對照組,腹腔註射生理鹽水;其他72隻大鼠腹腔註射STZ(45 mg/kg)後分為STZ組和STZ+葛根素組,每組36隻.STZ註射3 d後,STZ+葛根素組大鼠每天腹腔註射葛根素(140 mg/kg).于實驗開始後第20、40和60天用裂隙燈檢查晶狀體的形態學變化後處死動物.用流式細胞儀檢測LEC凋亡,用免疫組化方法檢測晶狀體中ONOO的標誌物--硝基酪氨痠(nitrotyrosine,NT)的錶達,用基因芯片分析技術檢測LEC凋亡相關基因iNOS等的錶達.結果髮現,對照組大鼠晶狀體均透明,各項指標基本正常;STZ組大鼠第20天時即齣現晶狀體混濁,40～60 d期間混濁不斷加重;STZ+葛根素組大鼠20～40 d時晶狀體混濁呈加重趨勢,但40～60 d以後明顯減輕.對照組LEC輕度凋亡,而STZ組凋亡細胞呈持續性增長,STZ+葛根素組大鼠20～40 d時細胞凋亡呈增長趨勢,但40～60 d以後明顯下降.對照組大鼠晶狀體NT未見明顯錶達;STZ組大鼠NT錶達明顯加彊;STZ+葛根素組大鼠20～40 d時NT錶達呈增長趨勢,但40～60 d以後明顯下降.對照組凋亡相關基因未見明顯變化,STZ組凋亡相關基因iNOS錶達明顯上調.其他凋亡相關基因如BCL-2、SOD錶達明顯下調,但NF-кB和TNFR1-FADD-caspase信號轉導途徑明顯上調;STZ+葛根素組凋亡相關基因錶達則呈相反改變.上述結果錶明,在糖尿病大鼠晶狀體中有ONOO- 的標誌物NT錶達,證明糖尿病大鼠LEC凋亡部分由ONOO誘導,這可能是氧化損傷導緻白內障形成的新途徑.葛根素能夠部分逆轉ONOO- 對LEC的緻凋亡作用,提示葛根素可能是治療糖尿病性白內障的有效藥物,其治療機製可能與葛根素直接抑製凋亡和對抗ONOO-對糖尿病大鼠LEC的損傷有關.
본연구관찰갈근소시부감경부분유과양아초기음리자(peroxynitrite,ONOO-)도치적당뇨병대서정상체상피세포(lens epithelium cell,LEC)조망.채용대서복강주사련뇨좌균소(streptozotocin,STZ)적방법건립당뇨병동물모형.36지대서작위대조조,복강주사생리염수;기타72지대서복강주사STZ(45 mg/kg)후분위STZ조화STZ+갈근소조,매조36지.STZ주사3 d후,STZ+갈근소조대서매천복강주사갈근소(140 mg/kg).우실험개시후제20、40화60천용렬극등검사정상체적형태학변화후처사동물.용류식세포의검측LEC조망,용면역조화방법검측정상체중ONOO적표지물--초기락안산(nitrotyrosine,NT)적표체,용기인심편분석기술검측LEC조망상관기인iNOS등적표체.결과발현,대조조대서정상체균투명,각항지표기본정상;STZ조대서제20천시즉출현정상체혼탁,40～60 d기간혼탁불단가중;STZ+갈근소조대서20～40 d시정상체혼탁정가중추세,단40～60 d이후명현감경.대조조LEC경도조망,이STZ조조망세포정지속성증장,STZ+갈근소조대서20～40 d시세포조망정증장추세,단40～60 d이후명현하강.대조조대서정상체NT미견명현표체;STZ조대서NT표체명현가강;STZ+갈근소조대서20～40 d시NT표체정증장추세,단40～60 d이후명현하강.대조조조망상관기인미견명현변화,STZ조조망상관기인iNOS표체명현상조.기타조망상관기인여BCL-2、SOD표체명현하조,단NF-кB화TNFR1-FADD-caspase신호전도도경명현상조;STZ+갈근소조조망상관기인표체칙정상반개변.상술결과표명,재당뇨병대서정상체중유ONOO- 적표지물NT표체,증명당뇨병대서LEC조망부분유ONOO유도,저가능시양화손상도치백내장형성적신도경.갈근소능구부분역전ONOO- 대LEC적치조망작용,제시갈근소가능시치료당뇨병성백내장적유효약물,기치료궤제가능여갈근소직접억제조망화대항ONOO-대당뇨병대서LEC적손상유관.
The present study was designed to observe if puerann decreases lens epithelium cell(LEC)apoptosis induced partly by peroxynitrite(ONOO-).One hundred and eight rats were randomly divided into control group(n=36),streptozotocin(STZ)group (n=36)and STZ+puerarin group(n=36).The rats in the control group intraperitoneally(i.p.)received 0.5 ml of saline.The rats in STZ group and STZ+puerarin group received intraperitoneal injection of STZ(45 mg/kg).Three days later,the rats in STZ+puerarin group were given puerarin(140 mg/kg per day,i.p.).On days 20,40 and 60 of the experiment,morphologic changes of lenses were observed with slit lamp.Then the animals were sacrificed for further analysis.The amount and percentage of apoptotic LECs were determined by flow cytometry.Nitrotyrosine(NT, the foot print of ONOO-)was examined by immunohistochemistry.Apoptosis-related genes (iNOS,etc.)were analyzed by gene array.The results showed that in the control group,all the lenses were clear.In STZ group,gradually severe opacity of the lens was observed on days 20,40 and 60.But in STZ+puerarin group,mild opacity of the lens was observed on day 20 and more severe on day 40,but markedly decreased on day 60.In the control group,mild apoptosis of LECs was observed.In STZ group,time-dependent increase in apoptosis of LECs was observed.In STZ+puerarin group,mild apoptosis of LECs was observed on day 20,significantly increased on day 40,but markedly decreased on day 60.There was no expression of NT in the lens in the control group,but an increased expression of NT in STZ group.In STZ+puerarin group,mild expression of NT was observed on day 20,significantly increased on day 40,but markedly decreased on day 60.There was no expression of iNOS in the lens in the control group,but continuous up-regulation of iNOS expression in STZ group.In STZ+ puerarin group,mild expression of iNOS was observed on day 20,significantly increased on day 40,but markedly decreased on day 60.Except the changes of iNOS related to caspase signal transduction way were up-regulated in STZ group.The results were opposite in STZ+puerarin group and the control group.These findings show that NT is expressed in diabetic rat lens,which proves that LEC apoptosis in diabetic lens is partly induced by ONOO- which may be a new oxidative damage way to form cataract.Puerarin partly decreases LEC apoptosis induced by ONOO- and is a potential medicine for therapy of diabetic cataract.The mechanism of puerarin dealing with diabetic cataract may be related to its direct inhibition of LEC apoptosis and antagonism of ONOO- in diabetic rats.