CAJ | 학술논문

目的观察κ阿片受体激活对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的心肌细胞肥大的影响,并探讨其作用机制.方法 SD大鼠,雌雄不拘,2～3天龄.体外培养大鼠乳鼠的心肌细胞.实验分为对照组、CSA(1μmol/L)组、AngⅡ(1μmol/L)组、AngⅡ(1μmol/L)+U50488H(1 μmol/L)组、AngⅡ(1μmol/L)+ CSA(1μmol/L)组、AngⅡ(1μmol/L)+Rp-cAMPS(1μmol/L)、AngⅡ(1μmol/L)+ CSA(1μmol/L)+U50488H(1μmol/L)组和AngⅡ(1μmol/L) +PTX(5 mg/L)+U50488H(1 μmol/L)组,共8组.以AngⅡ1 μmol/L诱导心肌细胞肥大,观察κ阿片受体激动剂U50488H 1 μmol/L对其的作用,并进一步采用钙调神经磷酸酶(calcineurin,CaN)特异性抑制剂环孢菌素A(cyclosporin A,CSA)l μmol/L、PKA抑制剂cAMP三乙胺盐(RP-cAMPS)1μmol/L、百日咳毒素(pertussis toxin,PTX)5 mg/L分别进行干预,观察上述干预因素对κ阿片受体激活对心肌细胞肥大的影响.通过Lowry法测心肌细胞蛋白含量.Fluo-3/AM为荧光探针,共聚焦显微镜下测量心肌细胞内游离钙离子浓度([Ca2+]i)瞬变.蛋白免疫印迹法测心肌细胞CaN的相对表达水平.结果 (1)各组大鼠乳鼠心肌细胞总蛋白含量的检测结果:AngⅡ组乳鼠心肌细胞的总蛋白含量明显高于对照组(P＜0.01).U50488H组、CSA组和Rp-cAMPS组乳鼠心肌细胞的总蛋白含量均明显低于AngⅡ组(P均＜0.01),且三组间差异无统计学意义.AngⅡ+PTX+ U50488H组乳鼠心肌细胞的总蛋白含量与AngⅡ组比较差异则无统计学意义.(2)各组大鼠乳鼠心肌细胞[Ca2+]i的检测结果:AngⅡ组乳鼠心肌细胞[Ca2+]i明显高于对照组(P＜0.01).U50488H组、CSA组和Rp-cAMPS组乳鼠心肌细胞[Ca2+]i均明显低于AngⅡ组(P＜0.01),且三组间差异无统计学意义.AngⅡ+PTX+ U50488H组乳鼠心肌细胞内[Ca2+]i与AngⅡ组比较差异无统计学意义.(3)各组大鼠乳鼠心肌细胞CaN表达的检测结果:AngⅡ组乳鼠心肌细胞CaN的表达水平明显高于对照组(P＜0.01).U50488H组、CSA组和Rp-cAMPS组乳鼠心肌细胞CaN的表达水平均明显低于AngⅡ组(P＜0.01),且三组间比较差异无统计学意义.AngⅡ+PTX+ U50488H组与AngⅡ组比较差异无统计学意义.结论 κ阿片受体可通过CaN通路抑制AngⅡ诱导的心肌肥大,其机制与降低细胞内[Ca2+]i及CaN表达有关. 目的觀察κ阿片受體激活對血管緊張素Ⅱ(angiotensinⅡ,AngⅡ)誘導的心肌細胞肥大的影響,併探討其作用機製.方法 SD大鼠,雌雄不拘,2～3天齡.體外培養大鼠乳鼠的心肌細胞.實驗分為對照組、CSA(1μmol/L)組、AngⅡ(1μmol/L)組、AngⅡ(1μmol/L)+U50488H(1 μmol/L)組、AngⅡ(1μmol/L)+ CSA(1μmol/L)組、AngⅡ(1μmol/L)+Rp-cAMPS(1μmol/L)、AngⅡ(1μmol/L)+ CSA(1μmol/L)+U50488H(1μmol/L)組和AngⅡ(1μmol/L) +PTX(5 mg/L)+U50488H(1 μmol/L)組,共8組.以AngⅡ1 μmol/L誘導心肌細胞肥大,觀察κ阿片受體激動劑U50488H 1 μmol/L對其的作用,併進一步採用鈣調神經燐痠酶(calcineurin,CaN)特異性抑製劑環孢菌素A(cyclosporin A,CSA)l μmol/L、PKA抑製劑cAMP三乙胺鹽(RP-cAMPS)1μmol/L、百日咳毒素(pertussis toxin,PTX)5 mg/L分彆進行榦預,觀察上述榦預因素對κ阿片受體激活對心肌細胞肥大的影響.通過Lowry法測心肌細胞蛋白含量.Fluo-3/AM為熒光探針,共聚焦顯微鏡下測量心肌細胞內遊離鈣離子濃度([Ca2+]i)瞬變.蛋白免疫印跡法測心肌細胞CaN的相對錶達水平.結果 (1)各組大鼠乳鼠心肌細胞總蛋白含量的檢測結果:AngⅡ組乳鼠心肌細胞的總蛋白含量明顯高于對照組(P＜0.01).U50488H組、CSA組和Rp-cAMPS組乳鼠心肌細胞的總蛋白含量均明顯低于AngⅡ組(P均＜0.01),且三組間差異無統計學意義.AngⅡ+PTX+ U50488H組乳鼠心肌細胞的總蛋白含量與AngⅡ組比較差異則無統計學意義.(2)各組大鼠乳鼠心肌細胞[Ca2+]i的檢測結果:AngⅡ組乳鼠心肌細胞[Ca2+]i明顯高于對照組(P＜0.01).U50488H組、CSA組和Rp-cAMPS組乳鼠心肌細胞[Ca2+]i均明顯低于AngⅡ組(P＜0.01),且三組間差異無統計學意義.AngⅡ+PTX+ U50488H組乳鼠心肌細胞內[Ca2+]i與AngⅡ組比較差異無統計學意義.(3)各組大鼠乳鼠心肌細胞CaN錶達的檢測結果:AngⅡ組乳鼠心肌細胞CaN的錶達水平明顯高于對照組(P＜0.01).U50488H組、CSA組和Rp-cAMPS組乳鼠心肌細胞CaN的錶達水平均明顯低于AngⅡ組(P＜0.01),且三組間比較差異無統計學意義.AngⅡ+PTX+ U50488H組與AngⅡ組比較差異無統計學意義.結論 κ阿片受體可通過CaN通路抑製AngⅡ誘導的心肌肥大,其機製與降低細胞內[Ca2+]i及CaN錶達有關.
목적 관찰κ아편수체격활대혈관긴장소Ⅱ(angiotensinⅡ,AngⅡ)유도적심기세포비대적영향,병탐토기작용궤제.방법 SD대서,자웅불구,2～3천령.체외배양대서유서적심기세포.실험분위대조조、CSA(1μmol/L)조、AngⅡ(1μmol/L)조、AngⅡ(1μmol/L)+U50488H(1 μmol/L)조、AngⅡ(1μmol/L)+ CSA(1μmol/L)조、AngⅡ(1μmol/L)+Rp-cAMPS(1μmol/L)、AngⅡ(1μmol/L)+ CSA(1μmol/L)+U50488H(1μmol/L)조화AngⅡ(1μmol/L) +PTX(5 mg/L)+U50488H(1 μmol/L)조,공8조.이AngⅡ1 μmol/L유도심기세포비대,관찰κ아편수체격동제U50488H 1 μmol/L대기적작용,병진일보채용개조신경린산매(calcineurin,CaN)특이성억제제배포균소A(cyclosporin A,CSA)l μmol/L、PKA억제제cAMP삼을알염(RP-cAMPS)1μmol/L、백일해독소(pertussis toxin,PTX)5 mg/L분별진행간예,관찰상술간예인소대κ아편수체격활대심기세포비대적영향.통과Lowry법측심기세포단백함량.Fluo-3/AM위형광탐침,공취초현미경하측량심기세포내유리개리자농도([Ca2+]i)순변.단백면역인적법측심기세포CaN적상대표체수평.결과 (1)각조대서유서심기세포총단백함량적검측결과:AngⅡ조유서심기세포적총단백함량명현고우대조조(P＜0.01).U50488H조、CSA조화Rp-cAMPS조유서심기세포적총단백함량균명현저우AngⅡ조(P균＜0.01),차삼조간차이무통계학의의.AngⅡ+PTX+ U50488H조유서심기세포적총단백함량여AngⅡ조비교차이칙무통계학의의.(2)각조대서유서심기세포[Ca2+]i적검측결과:AngⅡ조유서심기세포[Ca2+]i명현고우대조조(P＜0.01).U50488H조、CSA조화Rp-cAMPS조유서심기세포[Ca2+]i균명현저우AngⅡ조(P＜0.01),차삼조간차이무통계학의의.AngⅡ+PTX+ U50488H조유서심기세포내[Ca2+]i여AngⅡ조비교차이무통계학의의.(3)각조대서유서심기세포CaN표체적검측결과:AngⅡ조유서심기세포CaN적표체수평명현고우대조조(P＜0.01).U50488H조、CSA조화Rp-cAMPS조유서심기세포CaN적표체수평균명현저우AngⅡ조(P＜0.01),차삼조간비교차이무통계학의의.AngⅡ+PTX+ U50488H조여AngⅡ조비교차이무통계학의의.결론 κ아편수체가통과CaN통로억제AngⅡ유도적심기비대,기궤제여강저세포내[Ca2+]i급CaN표체유관.
Objective To observe the effect of κ-opioid receptor(κ-OR) stimulation on AngiotensinⅡ (AngⅡ)-induced cardiomyocyte hypertrophy in vitro cultured myocardial cells from neonatal rats and on calcineurin (CaN) signal pathways.Methods Cultured myocardial cells of neonatal rats were divided into control group,CSA (1 μmol/L) group,Ang Ⅱ (1 μ mol/L) group,Ang Ⅱ (1 μ mol/L) + U50488H (1 μmol/L) group,Ang Ⅱ (1 μmol/L) + CSA (1 μmol/L) group,Ang Ⅱ (1 μmol/L) + Rp-cAMPS (1 μmol/L) group,Ang Ⅱ (1 μmol/L) + CSA(1 μmol/L) + U50488H(1 μmol/L) group and Ang Ⅱ (1 μmol/L) + PTX5 mg/L + U50488H(1 μmol/L)group.The hypertrophic myocytes were induced by Ang Ⅱ 1μmol/L before κ-OR agonist U50488H 1 μmol/L was administered.The antihypertrophic effect of κ-OR stimulation was observed in the presence of ciclosporine A (CsA) 1 μmol/L,cAMP triethyl-ammonium salt (Rp-cAMPS) 1 μmol/L,and pertussistoxin (PTX) 5 mg/L.The total protein content was assayed by the method of Lowry.The [Ca2+] i was measured by confocal microscope using Fluo-3/AM as flouresecent indicator.The relative expression of CaN was determined by Western blot.Results (1) The total protein content of Ang Ⅱ group was significantly higher than that in control group (P ＜ 0.01),which could be equally reduced by cotreatment with U50488H,CSA and Rp-cAMPS (P ＜ 0.01).Total protein content of the Ang Ⅱ + PTX + U50488H group and the Ang Ⅱ group was similar.(2) The [Ca2+] i was significantly higher in Ang Ⅱ group of neonatal rat cardiomyocytes than that in control group (P ＜ 0.01),which could be reduced by cotreatment with U50488H,CSA and Rp-cAMPS (P ＜ 0.01).[Ca2+] i was similar between the Ang Ⅱ + PTX + U50488H group and the Ang Ⅱ group.(3) The expression of CaN was significantly higher in Ang Ⅱ group than that in control group (P ＜ 0.01),which could be significantly reduced by cotreatment with U50488H,CSA and Rp-cAMPS (P ＜ 0.01).CaN was similar between the Ang Ⅱ + PTX + U50488H group and the Ang Ⅱ group.Conclusion κ-opioid receptor activation could attenuate Ang Ⅱ induced cardiomyocytes hypertrophy via reducing [Ca2+]i and downreglating CaN.