中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2014年
9期
1537-1545
,共9页
林晓燕%林秋平%许昌声%宁若冰%祝江%林金秀%柴大军
林曉燕%林鞦平%許昌聲%寧若冰%祝江%林金秀%柴大軍
림효연%림추평%허창성%저약빙%축강%림금수%시대군
阿托伐他汀%糖尿病%氧化性应激%动脉粥样硬化%类视黄醇X受体
阿託伐他汀%糖尿病%氧化性應激%動脈粥樣硬化%類視黃醇X受體
아탁벌타정%당뇨병%양화성응격%동맥죽양경화%류시황순X수체
Atorvastatin%Diabetes mellitus%Oxidative stress%Atherogenesis%Retinoid X receptor
目的：观察阿托伐他汀（ Atorv）对链脲佐菌素（ STZ）诱导的糖尿病高脂喂养载脂蛋白E敲除（ apolipoprotein E knockout ， ApoE-/-）小鼠动脉粥样硬化的影响，探讨阿托伐他汀在糖尿病合并高脂饮食条件下对抗动脉粥样硬化的机制。方法：C57小鼠8只作为对照，34只高脂喂养的ApoE-/-小鼠随机分为3组：ApoE-/-组、STZ-ApoE-/-组和STZ-ApoE-/-＋Atorv组。 STZ腹腔注射建立糖尿病动物模型，测定小鼠空腹血糖、血脂水平， HE染色图像分析测定胸主动脉斑块面积；免疫杂交检测主动脉及细胞内NADPH氧化酶亚基gp91phox蛋白水平；Fenton反应Griess显色法测定血清及胸主动脉匀浆上清液活性氧（ ROS）水平。 I型胶原酶消化法培养人脐静脉内皮细胞（HUVECs），流式细胞术检测内皮细胞内ROS的水平，光泽精分析法测定NADPH氧化酶活性。采用干扰RNA和质粒转染的方法评价类视黄醇X受体α（ RXRα）在Atorv抑制氧化应激中的作用。结果：（1）与C57组相比， ApoE-/-组小鼠胸主动脉斑块面积显著增加[（215．88±34．19）μm2 vs 0μm2，P＜0．01]，2组间空腹血糖水平无显著差异，血清甘油三酯（ TG ）、总胆固醇（ TC ）、低密度脂蛋白胆固醇（ LDL-C ）、血清及胸主动脉ROS、胸主动脉gp91phox表达水平显著缩小（P＜0．05）；（2）与ApoE-/-组相比，STZ-ApoE-/-组胸主动脉斑块面积进一步增加[（314．13±35．72）μm2 vs （215．88±34．19）μm2，P＜0．05]，血糖水平升高，血清TC、LDL-C、血清及胸主动脉ROS、胸主动脉gp91phox水平进一步增加（P＜0．05）；（3）与STZ-ApoE-/-组相比，STZ-ApoE-/-＋Atorv组胸主动脉粥样斑块面积显著降低[（217．47±24．56）μm2 vs （314．13±35．72）μm2，P＜0．05]，血糖、血清TG、HDL、TC、和LDL-C无显著变化，血清及胸主动脉ROS、胸主动脉gp91phox水平亦显著降低（P＜0．05）；（4）高糖（25 mmol/L）干预后，HUVECs内ROS含量、gp91phox蛋白水平及NADPH氧化酶活性明显增加（P＜0．05），阿托伐他汀（10-8～10-6 mol/L）显著降低高糖环境下HUVECs胞内ROS含量、gp91phox表达及NADPH氧化酶活性，且具有浓度依赖性；（5）将RXRαsiRNA转染至HUVECs之后，阿托伐他汀（10-6 mol/L）对高糖环境下ROS生成及NADPH氧化酶活性的抑制效应显著减弱，RXRα质粒转染使RXRα过表达后，阿托伐他汀（10-6 mol/L）抑制ROS生成及NADPH氧化酶活性的作用明显增强（ P＜0．05）。结论：阿托伐他汀通过抑制高糖环境下机体的氧化应激反应对抗动脉粥样硬化；核受体RXRα介导阿托伐他汀的抗氧化应激效应。
目的：觀察阿託伐他汀（ Atorv）對鏈脲佐菌素（ STZ）誘導的糖尿病高脂餵養載脂蛋白E敲除（ apolipoprotein E knockout ， ApoE-/-）小鼠動脈粥樣硬化的影響，探討阿託伐他汀在糖尿病閤併高脂飲食條件下對抗動脈粥樣硬化的機製。方法：C57小鼠8隻作為對照，34隻高脂餵養的ApoE-/-小鼠隨機分為3組：ApoE-/-組、STZ-ApoE-/-組和STZ-ApoE-/-＋Atorv組。 STZ腹腔註射建立糖尿病動物模型，測定小鼠空腹血糖、血脂水平， HE染色圖像分析測定胸主動脈斑塊麵積；免疫雜交檢測主動脈及細胞內NADPH氧化酶亞基gp91phox蛋白水平；Fenton反應Griess顯色法測定血清及胸主動脈勻漿上清液活性氧（ ROS）水平。 I型膠原酶消化法培養人臍靜脈內皮細胞（HUVECs），流式細胞術檢測內皮細胞內ROS的水平，光澤精分析法測定NADPH氧化酶活性。採用榦擾RNA和質粒轉染的方法評價類視黃醇X受體α（ RXRα）在Atorv抑製氧化應激中的作用。結果：（1）與C57組相比， ApoE-/-組小鼠胸主動脈斑塊麵積顯著增加[（215．88±34．19）μm2 vs 0μm2，P＜0．01]，2組間空腹血糖水平無顯著差異，血清甘油三酯（ TG ）、總膽固醇（ TC ）、低密度脂蛋白膽固醇（ LDL-C ）、血清及胸主動脈ROS、胸主動脈gp91phox錶達水平顯著縮小（P＜0．05）；（2）與ApoE-/-組相比，STZ-ApoE-/-組胸主動脈斑塊麵積進一步增加[（314．13±35．72）μm2 vs （215．88±34．19）μm2，P＜0．05]，血糖水平升高，血清TC、LDL-C、血清及胸主動脈ROS、胸主動脈gp91phox水平進一步增加（P＜0．05）；（3）與STZ-ApoE-/-組相比，STZ-ApoE-/-＋Atorv組胸主動脈粥樣斑塊麵積顯著降低[（217．47±24．56）μm2 vs （314．13±35．72）μm2，P＜0．05]，血糖、血清TG、HDL、TC、和LDL-C無顯著變化，血清及胸主動脈ROS、胸主動脈gp91phox水平亦顯著降低（P＜0．05）；（4）高糖（25 mmol/L）榦預後，HUVECs內ROS含量、gp91phox蛋白水平及NADPH氧化酶活性明顯增加（P＜0．05），阿託伐他汀（10-8～10-6 mol/L）顯著降低高糖環境下HUVECs胞內ROS含量、gp91phox錶達及NADPH氧化酶活性，且具有濃度依賴性；（5）將RXRαsiRNA轉染至HUVECs之後，阿託伐他汀（10-6 mol/L）對高糖環境下ROS生成及NADPH氧化酶活性的抑製效應顯著減弱，RXRα質粒轉染使RXRα過錶達後，阿託伐他汀（10-6 mol/L）抑製ROS生成及NADPH氧化酶活性的作用明顯增彊（ P＜0．05）。結論：阿託伐他汀通過抑製高糖環境下機體的氧化應激反應對抗動脈粥樣硬化；覈受體RXRα介導阿託伐他汀的抗氧化應激效應。
목적：관찰아탁벌타정（ Atorv）대련뇨좌균소（ STZ）유도적당뇨병고지위양재지단백E고제（ apolipoprotein E knockout ， ApoE-/-）소서동맥죽양경화적영향，탐토아탁벌타정재당뇨병합병고지음식조건하대항동맥죽양경화적궤제。방법：C57소서8지작위대조，34지고지위양적ApoE-/-소서수궤분위3조：ApoE-/-조、STZ-ApoE-/-조화STZ-ApoE-/-＋Atorv조。 STZ복강주사건립당뇨병동물모형，측정소서공복혈당、혈지수평， HE염색도상분석측정흉주동맥반괴면적；면역잡교검측주동맥급세포내NADPH양화매아기gp91phox단백수평；Fenton반응Griess현색법측정혈청급흉주동맥균장상청액활성양（ ROS）수평。 I형효원매소화법배양인제정맥내피세포（HUVECs），류식세포술검측내피세포내ROS적수평，광택정분석법측정NADPH양화매활성。채용간우RNA화질립전염적방법평개류시황순X수체α（ RXRα）재Atorv억제양화응격중적작용。결과：（1）여C57조상비， ApoE-/-조소서흉주동맥반괴면적현저증가[（215．88±34．19）μm2 vs 0μm2，P＜0．01]，2조간공복혈당수평무현저차이，혈청감유삼지（ TG ）、총담고순（ TC ）、저밀도지단백담고순（ LDL-C ）、혈청급흉주동맥ROS、흉주동맥gp91phox표체수평현저축소（P＜0．05）；（2）여ApoE-/-조상비，STZ-ApoE-/-조흉주동맥반괴면적진일보증가[（314．13±35．72）μm2 vs （215．88±34．19）μm2，P＜0．05]，혈당수평승고，혈청TC、LDL-C、혈청급흉주동맥ROS、흉주동맥gp91phox수평진일보증가（P＜0．05）；（3）여STZ-ApoE-/-조상비，STZ-ApoE-/-＋Atorv조흉주동맥죽양반괴면적현저강저[（217．47±24．56）μm2 vs （314．13±35．72）μm2，P＜0．05]，혈당、혈청TG、HDL、TC、화LDL-C무현저변화，혈청급흉주동맥ROS、흉주동맥gp91phox수평역현저강저（P＜0．05）；（4）고당（25 mmol/L）간예후，HUVECs내ROS함량、gp91phox단백수평급NADPH양화매활성명현증가（P＜0．05），아탁벌타정（10-8～10-6 mol/L）현저강저고당배경하HUVECs포내ROS함량、gp91phox표체급NADPH양화매활성，차구유농도의뢰성；（5）장RXRαsiRNA전염지HUVECs지후，아탁벌타정（10-6 mol/L）대고당배경하ROS생성급NADPH양화매활성적억제효응현저감약，RXRα질립전염사RXRα과표체후，아탁벌타정（10-6 mol/L）억제ROS생성급NADPH양화매활성적작용명현증강（ P＜0．05）。결론：아탁벌타정통과억제고당배경하궤체적양화응격반응대항동맥죽양경화；핵수체RXRα개도아탁벌타정적항양화응격효응。
AIM:To explore the effects of atorvastatin (Atorv) on atherosclerosis in streptozotocin (STZ)-in-duced diabetic apolipoprotein E knockout ( ApoE-/-) mice with fat-rich diet and the possible mechanism .METHODS:C57 mice served as control.ApoE-/-mice (n=34) fed with high-fat diet were randomly divided into ApoE-/-group, STZ-ApoE-/-group and STZ-ApoE-/-+Atorv group.Intraperitoneal injection of streptozotocin was performed to create di-abetic animal model .Blood glucose was determined by glucose oxidase method .Blood lipid levels were detected by enzymic method or selective homogeneous method .The plaque area in the thoracic aorta was measured by HE staining .The protein level of nicotinamide-adenine dinucleotide phosphate ( NADPH) oxidase subunit gp91phox in the thoracic aorta was deter-mined by Western blotting .The levels of reactive oxygen species ( ROS) in blood and thoracic aorta homogenates were de-tected by Fenton reaction and Griess reagent .Human umbilical vein endothelial cells ( HUVECs ) were isolated from healthy umbilical cords by collagenase I and cultured .ROS production was detected by flow cytometry .NADPH oxidase ac-tivity was measured using lucigenin assay .Effects of retinoid X receptor α( RXRα) on inhibition of oxidative stress by ator-vastatin were evaluated by RNA interference and plasmid transfection .RESULTS: (1) Compared with C57 group, the plaque areas of the thoracic aorta in ApoE-/-group were increased .No difference of the fasting glucose between the 2 groups was observed.The levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in ApoE-/-group than those in C57 group.(2) Compared with ApoE-/-group, the plaque areas of the thoracic aorta in STZ-ApoE-/-group were further enlarged [(314.13 ±35.72) μm2 vs (215.88 ±34.19) μm2, P<0.05].The levels of blood glucose, TG, TC and LDL-C, thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in STZ-ApoE-/-group than those in ApoE-/-group (P<0.05).(3) Compared with STZ-ApoE-/-group, the plaque areas of the thoracic aorta in STZ-ApoE-/-+Atorv group were reduced [(217.47 ±24.56) μm2 vs (314.13 ±35.72) μm2, P<0.05].The levels of blood glucose , LDL-C, TC, HDL-C and TG showed no significant difference between the 2 groups.Thoracic aorta gp91phox protein level and ROS production in blood and thoracic aorta homogenates were lower in STZ -ApoE-/-+Atorv group than those in STZ-ApoE-/-group (P<0.05).(4) High glucose-induced increases in NADPH oxidase activity and gp91phox expression were significantly inhibited by atorvastatin (10-6 mol/L) in HUVECs.The inhibitory effects of atorvasta-tin on high glucose-induced ROS production and NADPH oxidase activation were largely impaired when the cells were trans -fected with RXRαsiRNA.However , the effect of atorvastatin was significantly strengthened when RXRαwas over-expressed in the HUVECs transfected with RXRαplasmid.CONCLUSION: Atorvastatin inhibits atherogenesis by depressing high glucose-induced oxidative stress in diabetic ApoE-/-mice with fat-rich diet.The anti-oxidative stress effect of atorvastatin is mediated by RXRα.
