中华地方病学杂志
中華地方病學雜誌
중화지방병학잡지
Chinese Journal of Endemiology
2015年
4期
265-269
,共5页
刘蕾%王超%席玉慧%李弘%时飒%田野%王丽娜
劉蕾%王超%席玉慧%李弘%時颯%田野%王麗娜
류뢰%왕초%석옥혜%리홍%시삽%전야%왕려나
钙敏感受体%自噬%血管紧张素Ⅱ%大鼠%心肌肥大
鈣敏感受體%自噬%血管緊張素Ⅱ%大鼠%心肌肥大
개민감수체%자서%혈관긴장소Ⅱ%대서%심기비대
Calcium sensing receptor%Autophagy%Angiotensin Ⅱ%Rats%Cardiac hypertrophy
目的 探讨抑制钙敏感受体(CaSR)表达对血管紧张素Ⅱ(AngⅡ)诱导大鼠心肌H9c2细胞肥大的影响及作用机制.方法 采用细胞培养的方法,用AngⅡ处理H9c2细胞复制心肌肥大细胞模型,并在此基础上,应用CaSR激动剂三氯化钆(GdCl3)、CaSR抑制剂Calhex231或自噬抑制剂3-甲基腺嘌呤(3-MA)处理细胞.共分为5组:对照组、AngⅡ组、GdCl3+ AngⅡ组、GdCl3+ Calhex231+ AngⅡ组、GdCl3+ 3-MA+ AngⅡ组,每组6例.采用考马斯亮蓝蛋白试剂盒测定总蛋白含量;蛋白印迹法检测心肌肥大信号蛋白Ca2+/钙调蛋白依赖性蛋白激酶(CaMKⅡ)与其磷酸化形式(pCaMKⅡ)表达来评价细胞肥大情况;蛋白印迹法检测CaSR、自噬标志物[Beclin-1、微管相关蛋白轻链(LC3)Ⅱ/LC3Ⅰ、P62]和Ca2+/钙调蛋白依赖性蛋白激酶激酶β(CaMKKβ)-腺苷酸活化蛋白激酶(AMPK)-哺乳动物雷帕霉素靶蛋白(mTOR)通路的表达.结果 ①GdCl3能够促进AngⅡ所诱发的CaSR蛋白表达增多(对照组:0.22±0.04; AngⅡ组:0.43±0.02; GdCl3+ AngⅡ组:0.63±0.08,P均＜0.05)、细胞总蛋白含量增多[对照组:(2.52±0.84)g/L;AngⅡ组:(8.72±3.60)g/L;GdCl3+ AngⅡ组:(14.17±4.49)g/L,P均＜0.05]、pCaMKⅡ/CaMKⅡ比值升高(对照组:0.25±0.05; AngⅡ组:0.51±0.03;GdCl3+ AngⅡ组:0.77±0.06,P均＜0.05),而Calhex231则能够抑制GdCl3引起的上述指标的增加[GdCl3+Calhex231+AngⅡ组,CaSR:0.41±0.16;总蛋白含量:(9.92±2.54) g/L;pCaMK Ⅱ/CaMK Ⅱ:0.58±0.08,P均＜0.05].②GdCl3能够促进AngⅡ所诱发的自噬标志物Beclin-1蛋白表达增多(对照组:0.31±0.06; AngⅡ组:0.55±0.09;GdCl3+AngⅡ组:0.74±0.08,P均＜0.05)、LC3 Ⅱ/LC3 Ⅰ比值增加(对照组:0.28±0.06;AngⅡ组:0.56±0.10;GdCl3+AngⅡ组:1.00±0.15,P均＜0.05),P62蛋白表达减少(对照组:0.54±0.03;AngⅡ组:0.34±0.02; GdCl3+ AngⅡ组:0.15±0.03,P均＜0.05);Calhex231则能抑制GdCl3引起的自噬标志物指标的改变(GdCl3+Calhex231+AngⅡ组,Beclin-1:0.53±0.14;LC3Ⅱ/LC3Ⅰ:0.57±0.12;P62:0.28±0.05,P均＜ 0.05).③GdCl3可引起pCaMKKβ/CaMKKβ(对照组:0.43±0.09;AngⅡ组:0.76±0.12;GdCl3+ AngⅡ组:1.19±0.21)和pAMPK/AMPK(对照组:0.38±0.11;AngⅡ组:0.68±0.08;GdCl3+ AngⅡ组:1.18±0.08,P均＜0.05)比值升高,pmTOR/mTOR(对照组:0.90±0.10;AngⅡ组:0.54±0.04;GdCl3+AngⅡ组:0.29±0.09,P均＜ 0.05)比值降低;Calhex231则能抑制GdCl3引起的上述蛋白表达变化(GdCl3+ Calhex231+AngⅡ组,pCaMKKβ/CaMKKβ:0.75±0.06;pAMPK/AMPK:0.57±0.05;pmTOR/mTOR:0.51±0.08,P均＜0.05).结论 抑制CaSR表达可逆转AngⅡ诱导的H9c2细胞肥大,其机制可能与抑制自噬、抑制CaMKKβ-AMPK-mTOR通路有关.
目的 探討抑製鈣敏感受體(CaSR)錶達對血管緊張素Ⅱ(AngⅡ)誘導大鼠心肌H9c2細胞肥大的影響及作用機製.方法 採用細胞培養的方法,用AngⅡ處理H9c2細胞複製心肌肥大細胞模型,併在此基礎上,應用CaSR激動劑三氯化釓(GdCl3)、CaSR抑製劑Calhex231或自噬抑製劑3-甲基腺嘌呤(3-MA)處理細胞.共分為5組:對照組、AngⅡ組、GdCl3+ AngⅡ組、GdCl3+ Calhex231+ AngⅡ組、GdCl3+ 3-MA+ AngⅡ組,每組6例.採用攷馬斯亮藍蛋白試劑盒測定總蛋白含量;蛋白印跡法檢測心肌肥大信號蛋白Ca2+/鈣調蛋白依賴性蛋白激酶(CaMKⅡ)與其燐痠化形式(pCaMKⅡ)錶達來評價細胞肥大情況;蛋白印跡法檢測CaSR、自噬標誌物[Beclin-1、微管相關蛋白輕鏈(LC3)Ⅱ/LC3Ⅰ、P62]和Ca2+/鈣調蛋白依賴性蛋白激酶激酶β(CaMKKβ)-腺苷痠活化蛋白激酶(AMPK)-哺乳動物雷帕黴素靶蛋白(mTOR)通路的錶達.結果 ①GdCl3能夠促進AngⅡ所誘髮的CaSR蛋白錶達增多(對照組:0.22±0.04; AngⅡ組:0.43±0.02; GdCl3+ AngⅡ組:0.63±0.08,P均＜0.05)、細胞總蛋白含量增多[對照組:(2.52±0.84)g/L;AngⅡ組:(8.72±3.60)g/L;GdCl3+ AngⅡ組:(14.17±4.49)g/L,P均＜0.05]、pCaMKⅡ/CaMKⅡ比值升高(對照組:0.25±0.05; AngⅡ組:0.51±0.03;GdCl3+ AngⅡ組:0.77±0.06,P均＜0.05),而Calhex231則能夠抑製GdCl3引起的上述指標的增加[GdCl3+Calhex231+AngⅡ組,CaSR:0.41±0.16;總蛋白含量:(9.92±2.54) g/L;pCaMK Ⅱ/CaMK Ⅱ:0.58±0.08,P均＜0.05].②GdCl3能夠促進AngⅡ所誘髮的自噬標誌物Beclin-1蛋白錶達增多(對照組:0.31±0.06; AngⅡ組:0.55±0.09;GdCl3+AngⅡ組:0.74±0.08,P均＜0.05)、LC3 Ⅱ/LC3 Ⅰ比值增加(對照組:0.28±0.06;AngⅡ組:0.56±0.10;GdCl3+AngⅡ組:1.00±0.15,P均＜0.05),P62蛋白錶達減少(對照組:0.54±0.03;AngⅡ組:0.34±0.02; GdCl3+ AngⅡ組:0.15±0.03,P均＜0.05);Calhex231則能抑製GdCl3引起的自噬標誌物指標的改變(GdCl3+Calhex231+AngⅡ組,Beclin-1:0.53±0.14;LC3Ⅱ/LC3Ⅰ:0.57±0.12;P62:0.28±0.05,P均＜ 0.05).③GdCl3可引起pCaMKKβ/CaMKKβ(對照組:0.43±0.09;AngⅡ組:0.76±0.12;GdCl3+ AngⅡ組:1.19±0.21)和pAMPK/AMPK(對照組:0.38±0.11;AngⅡ組:0.68±0.08;GdCl3+ AngⅡ組:1.18±0.08,P均＜0.05)比值升高,pmTOR/mTOR(對照組:0.90±0.10;AngⅡ組:0.54±0.04;GdCl3+AngⅡ組:0.29±0.09,P均＜ 0.05)比值降低;Calhex231則能抑製GdCl3引起的上述蛋白錶達變化(GdCl3+ Calhex231+AngⅡ組,pCaMKKβ/CaMKKβ:0.75±0.06;pAMPK/AMPK:0.57±0.05;pmTOR/mTOR:0.51±0.08,P均＜0.05).結論 抑製CaSR錶達可逆轉AngⅡ誘導的H9c2細胞肥大,其機製可能與抑製自噬、抑製CaMKKβ-AMPK-mTOR通路有關.
목적 탐토억제개민감수체(CaSR)표체대혈관긴장소Ⅱ(AngⅡ)유도대서심기H9c2세포비대적영향급작용궤제.방법 채용세포배양적방법,용AngⅡ처리H9c2세포복제심기비대세포모형,병재차기출상,응용CaSR격동제삼록화구(GdCl3)、CaSR억제제Calhex231혹자서억제제3-갑기선표령(3-MA)처리세포.공분위5조:대조조、AngⅡ조、GdCl3+ AngⅡ조、GdCl3+ Calhex231+ AngⅡ조、GdCl3+ 3-MA+ AngⅡ조,매조6례.채용고마사량람단백시제합측정총단백함량;단백인적법검측심기비대신호단백Ca2+/개조단백의뢰성단백격매(CaMKⅡ)여기린산화형식(pCaMKⅡ)표체래평개세포비대정황;단백인적법검측CaSR、자서표지물[Beclin-1、미관상관단백경련(LC3)Ⅱ/LC3Ⅰ、P62]화Ca2+/개조단백의뢰성단백격매격매β(CaMKKβ)-선감산활화단백격매(AMPK)-포유동물뢰파매소파단백(mTOR)통로적표체.결과 ①GdCl3능구촉진AngⅡ소유발적CaSR단백표체증다(대조조:0.22±0.04; AngⅡ조:0.43±0.02; GdCl3+ AngⅡ조:0.63±0.08,P균＜0.05)、세포총단백함량증다[대조조:(2.52±0.84)g/L;AngⅡ조:(8.72±3.60)g/L;GdCl3+ AngⅡ조:(14.17±4.49)g/L,P균＜0.05]、pCaMKⅡ/CaMKⅡ비치승고(대조조:0.25±0.05; AngⅡ조:0.51±0.03;GdCl3+ AngⅡ조:0.77±0.06,P균＜0.05),이Calhex231칙능구억제GdCl3인기적상술지표적증가[GdCl3+Calhex231+AngⅡ조,CaSR:0.41±0.16;총단백함량:(9.92±2.54) g/L;pCaMK Ⅱ/CaMK Ⅱ:0.58±0.08,P균＜0.05].②GdCl3능구촉진AngⅡ소유발적자서표지물Beclin-1단백표체증다(대조조:0.31±0.06; AngⅡ조:0.55±0.09;GdCl3+AngⅡ조:0.74±0.08,P균＜0.05)、LC3 Ⅱ/LC3 Ⅰ비치증가(대조조:0.28±0.06;AngⅡ조:0.56±0.10;GdCl3+AngⅡ조:1.00±0.15,P균＜0.05),P62단백표체감소(대조조:0.54±0.03;AngⅡ조:0.34±0.02; GdCl3+ AngⅡ조:0.15±0.03,P균＜0.05);Calhex231칙능억제GdCl3인기적자서표지물지표적개변(GdCl3+Calhex231+AngⅡ조,Beclin-1:0.53±0.14;LC3Ⅱ/LC3Ⅰ:0.57±0.12;P62:0.28±0.05,P균＜ 0.05).③GdCl3가인기pCaMKKβ/CaMKKβ(대조조:0.43±0.09;AngⅡ조:0.76±0.12;GdCl3+ AngⅡ조:1.19±0.21)화pAMPK/AMPK(대조조:0.38±0.11;AngⅡ조:0.68±0.08;GdCl3+ AngⅡ조:1.18±0.08,P균＜0.05)비치승고,pmTOR/mTOR(대조조:0.90±0.10;AngⅡ조:0.54±0.04;GdCl3+AngⅡ조:0.29±0.09,P균＜ 0.05)비치강저;Calhex231칙능억제GdCl3인기적상술단백표체변화(GdCl3+ Calhex231+AngⅡ조,pCaMKKβ/CaMKKβ:0.75±0.06;pAMPK/AMPK:0.57±0.05;pmTOR/mTOR:0.51±0.08,P균＜0.05).결론 억제CaSR표체가역전AngⅡ유도적H9c2세포비대,기궤제가능여억제자서、억제CaMKKβ-AMPK-mTOR통로유관.
Objective To explore the effects and possible mechanism of calcium-sensing receptor(CaSR) in rat myocardial H9c2 cells hypertrophy model using angiotensin Ⅱ (Ang Ⅱ).Methods Cardiac hypertrophy model was established by treating cultured H9c2 cells with Ang Ⅱ in vitro.Hypertrophic H9c2 cells were treated with gadolinium chloride (GdCl3,a specific agonist of CaSR) and/or with Calhex231 (a specific inhibitor of CaSR) and 3-methyladenine (3-MA,a specific inhibitor of autophagy) to divided into 5 groups (six in each group):control,Ang Ⅱ,GdCl3 + Ang lⅡ,GdCl3 + Calhex231 + Ang Ⅱ,GdCl3 + 3-MA + Ang Ⅱ groups.To evaluate the status of H9c2 cells hypertrophy,protein content was determined through a coomassie brilliant blue protein kit and the expression of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and the phosphorylation form (pCaMK Ⅱ/CaMK Ⅱ) was analyzed by Western blotting.The protein expression of CaSR,autophagy maker [Beclin-1,micmtubule-associated protein 1 light chain 3(LC3)Ⅱ/LC3 Ⅰ,P62] and Ca2+/calmodulin-dependent-protein kinase-kinase-β (CaMKKβ)-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway was analyzed by Western blotting.Results ①GdCl3 further increased H9c2 cells protein content [control group:(2.52 ± 0.84) g/L,Ang Ⅱ group:(8.72 ± 3.60) g/L GdCl3 + Ang Ⅱ group:(14.17 ± 4.49) g/L,all P ＜ 0.05] and the expression of CaSR (control group:0.22 ± 0.04,Ang Ⅱ group:0.43 ± 0.02,GdCl3 + Ang Ⅱ group:0.63 ± 0.08,all P ＜ 0.05) and pCaMK Ⅱ/CaMKⅡ (control group:0.25 ± 0.05,AngⅡ group:0.51 ± 0.03,GdCl3 + AngⅡ group:0.77 ± 0.06,all P＜ 0.05) induced by Ang Ⅱ.Calhex231 suppressed the increasing of hypertrophy indicators induced by GdCl3 [GdCl3 + Calhex231 + AngⅡ group,CaSR:0.41 ± 0.16,protein content:(9.92 ± 2.54) g/L,pCaMK Ⅱ/CaMKⅡ:0.58 ± 0.08,all P ＜ 0.05].②GdCl3 promoted the effect of Ang Ⅱ in regulation of autophagy such as Beclin-1 protein increased (control group:0.31 ± 0.06,AngⅡ group:0.55 ± 0.09,GdCl3 + AngⅡ group:0.74 ± 0.08,all P ＜ 0.05),LC3 Ⅱ/LC3 Ⅰ increased (control group:0.28 ± 0.06,Ang Ⅱ group:0.56 ± 0.10,GdCl3 + Ang Ⅱ group:1.00 ± 0.15,all P ＜ 0.05) and P62 protein decreased (control group:0.54 ± 0.03,AngⅡ group:0.34 ± 0.02,GdCl3 + AngⅡ group:0.15 ± 0.03,all P ＜ 0.05).Moreover,Calhex231 suppressed autophagy induced by GdCl3 (GdCl3 + Calhex231 + Ang Ⅱ group,Beclin-1:0.53 ± 0.14,LC3 Ⅱ/LC3 Ⅰ:0.57 ± 0.12,P62:0.28 ± 0.05,all P ＜ 0.05).③GdCl3 increased pCaMKKβ/CaMKKβ (control group:0.43 ± 0.09,AngⅡ group:0.76 ± 0.12,GdCl3 + AngⅡ group:1.19 ± 0.21,all P ＜ 0.05),pAMPK/AMPK (control group:0.38 ± 0.11,AngⅡ group:0.68 ± 0.08,GdCl3 + AngⅡ group:1.18 ± 0.08,all P ＜ 0.05) and decreased pmTOR/mTOR (control group:0.90 ± 0.10,Ang Ⅱ group:0.54 ± 0.04,GdCl3 + AngⅡ group:0.29 ± 0.09,all P ＜ 0.05).Furthermore,Calhex231 blocked the effect of GdCl3 on the above-mentioned proteins changes (GdCl3 + Calhex231 + Ang Ⅱ group,pCaMKKβ/CaMKKβ:0.75 ± 0.06,pAMPK/AMPK:0.57 ± 0.05,pmTOR/mTOR:0.51 ± 0.08,all P ＜ 0.05).Conclusion Inhibiting calcium-sensing receptor expression has reversed H9c2 cell hypertrophy induced by Ang Ⅱ,which may be related to suppressing autophagy and suppressing CaMKKβ-AMPK-mTOR pathway.
