中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2015年
6期
980-987
,共8页
张军%代文静%周敬群%张家俊%曹志刚
張軍%代文靜%週敬群%張傢俊%曹誌剛
장군%대문정%주경군%장가준%조지강
Klotho蛋白%缺氧/复氧%氧化应激%内质网应激%细胞凋亡
Klotho蛋白%缺氧/複氧%氧化應激%內質網應激%細胞凋亡
Klotho단백%결양/복양%양화응격%내질망응격%세포조망
KEY WORDS] Klotho protein%Hypoxia/reoxygenation%Oxidative stress%Endoplasmic reticulum stress%Apoptosis
目的：观察抗衰老Klotho蛋白对大鼠乳鼠原代心肌细胞缺氧／复氧（ H／R）损伤的作用并探讨其作用机制。方法：建立大鼠乳鼠心肌细胞H／R模型，并将心肌细胞分为正常对照组、H／R模型组和不同浓度（0．1μmol／L、1μmol／L和10μmol／L） Klotho作用H／R组。观察各组心肌细胞H／R前后搏动频率变化，利用MTT方法检测细胞存活率；测定各组心肌细胞H／R后LDH、CK、AST的漏出量及MDA含量、SOD活性；流式细胞术检测各组心肌细胞的凋亡率；real－time PCR检测各组心肌细胞中内质网应激标记及凋亡相关分子GRP78、CRT和CHOP和caspase－12 mRNA的表达情况；Western blot法检测心肌细胞内内质网应激凋亡蛋白CHOP和caspase－12蛋白表达及Akt磷酸化水平。结果：与正常对照组相比，H／R模型组中心肌细胞搏动频率和细胞存活率显著下降，细胞凋亡率显著升高（P＜0．05）；LDH、CK、AST和MDA含量升高而SOD活性显著降低（P＜0．05）；GRP78、CRT、CHOP和caspase－12 mRNA表达显著增高（ P＜0．05）；CHOP和caspase－12蛋白表达随之增高而Akt的磷酸化水平显著降低（P＜0．05）。与H／R模型组相比，抗衰老Klotho蛋白作用H／R心肌细胞后，心肌细胞搏动频率和细胞存活率显著升高，细胞凋亡率逐渐降低（P＜0．05），LDH、CK、AST和MDA含量下降而SOD活性显著增高（P＜0．05），GRP78、CRT、CHOP和caspase－12 mRNA的表达逐渐降低（ P＜0．05），CHOP和caspase－12蛋白表达也随之降低，而Akt磷酸化水平显著增加（ P＜0．05）。结论：抗衰老Klotho蛋白能够提升H／R损伤后心肌细胞的存活率，抑制细胞凋亡，通过抵抗氧化应激和过度内质网应激反应发挥作用，并与激活Akt磷酸化有关。
目的：觀察抗衰老Klotho蛋白對大鼠乳鼠原代心肌細胞缺氧／複氧（ H／R）損傷的作用併探討其作用機製。方法：建立大鼠乳鼠心肌細胞H／R模型，併將心肌細胞分為正常對照組、H／R模型組和不同濃度（0．1μmol／L、1μmol／L和10μmol／L） Klotho作用H／R組。觀察各組心肌細胞H／R前後搏動頻率變化，利用MTT方法檢測細胞存活率；測定各組心肌細胞H／R後LDH、CK、AST的漏齣量及MDA含量、SOD活性；流式細胞術檢測各組心肌細胞的凋亡率；real－time PCR檢測各組心肌細胞中內質網應激標記及凋亡相關分子GRP78、CRT和CHOP和caspase－12 mRNA的錶達情況；Western blot法檢測心肌細胞內內質網應激凋亡蛋白CHOP和caspase－12蛋白錶達及Akt燐痠化水平。結果：與正常對照組相比，H／R模型組中心肌細胞搏動頻率和細胞存活率顯著下降，細胞凋亡率顯著升高（P＜0．05）；LDH、CK、AST和MDA含量升高而SOD活性顯著降低（P＜0．05）；GRP78、CRT、CHOP和caspase－12 mRNA錶達顯著增高（ P＜0．05）；CHOP和caspase－12蛋白錶達隨之增高而Akt的燐痠化水平顯著降低（P＜0．05）。與H／R模型組相比，抗衰老Klotho蛋白作用H／R心肌細胞後，心肌細胞搏動頻率和細胞存活率顯著升高，細胞凋亡率逐漸降低（P＜0．05），LDH、CK、AST和MDA含量下降而SOD活性顯著增高（P＜0．05），GRP78、CRT、CHOP和caspase－12 mRNA的錶達逐漸降低（ P＜0．05），CHOP和caspase－12蛋白錶達也隨之降低，而Akt燐痠化水平顯著增加（ P＜0．05）。結論：抗衰老Klotho蛋白能夠提升H／R損傷後心肌細胞的存活率，抑製細胞凋亡，通過牴抗氧化應激和過度內質網應激反應髮揮作用，併與激活Akt燐痠化有關。
목적：관찰항쇠로Klotho단백대대서유서원대심기세포결양／복양（ H／R）손상적작용병탐토기작용궤제。방법：건립대서유서심기세포H／R모형，병장심기세포분위정상대조조、H／R모형조화불동농도（0．1μmol／L、1μmol／L화10μmol／L） Klotho작용H／R조。관찰각조심기세포H／R전후박동빈솔변화，이용MTT방법검측세포존활솔；측정각조심기세포H／R후LDH、CK、AST적루출량급MDA함량、SOD활성；류식세포술검측각조심기세포적조망솔；real－time PCR검측각조심기세포중내질망응격표기급조망상관분자GRP78、CRT화CHOP화caspase－12 mRNA적표체정황；Western blot법검측심기세포내내질망응격조망단백CHOP화caspase－12단백표체급Akt린산화수평。결과：여정상대조조상비，H／R모형조중심기세포박동빈솔화세포존활솔현저하강，세포조망솔현저승고（P＜0．05）；LDH、CK、AST화MDA함량승고이SOD활성현저강저（P＜0．05）；GRP78、CRT、CHOP화caspase－12 mRNA표체현저증고（ P＜0．05）；CHOP화caspase－12단백표체수지증고이Akt적린산화수평현저강저（P＜0．05）。여H／R모형조상비，항쇠로Klotho단백작용H／R심기세포후，심기세포박동빈솔화세포존활솔현저승고，세포조망솔축점강저（P＜0．05），LDH、CK、AST화MDA함량하강이SOD활성현저증고（P＜0．05），GRP78、CRT、CHOP화caspase－12 mRNA적표체축점강저（ P＜0．05），CHOP화caspase－12단백표체야수지강저，이Akt린산화수평현저증가（ P＜0．05）。결론：항쇠로Klotho단백능구제승H／R손상후심기세포적존활솔，억제세포조망，통과저항양화응격화과도내질망응격반응발휘작용，병여격활Akt린산화유관。
[ ABSTRACT] AIM:To study the effects of anti-aging Klotho protein on neonatal rat myocardial cells with hypo-xia/reoxygenation ( H/R) injury.METHODS:The cardiomyocytes of neonatal SD rats were cultured to establish hypoxia/reoxygenation model.The myocardial cells were divided into normal control group, H/R model group, different concentra-tions of Klotho protein (0.1μmol/L, 1μmol/L and 10μmol/L) pretreatment groups.The myocardial cells pulse frequen-cy was observed before and after H/R.The cell viability was measured by MTT assay.The leakages of LDH, CK and AST, the content of MDA and the activity of SOD were detected.The apoptotic rate of the myocardial cells was analyzed by flow cytometry.The mRNA expression of endoplasmic reticulum stress markers and apoptosis-related molecules GRP78, CRT, CHOP and caspase-12 was measured by real-time PCR.The protein levels of CHOP, caspase-12 and phosphorylated Akt in the myocardial cells were determined by Western blot.RESULTS: Compared with normal control group, the pulse fre-quency, cell viability rate and SOD activity of myocardial cells were significantly decreased, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were increased in H/R model group.The mRNA expressions of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were increased, whereas p-Akt level was decreased obviously.Compared with H/R model group, the pulse frequency, cell viability rate and SOD activity were in-creased significantly, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were decreased in Klotho pretreated group.The mRNA expression of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and <br> caspase-12 were decreased, while p-Akt level increased significantly.CONCLUSION:Anti-aging Klotho protein improves the myocardial cell survival and inhibits the apoptosis by increasing the resistance of the cells to oxidative stress and exces-sive endoplasmic reticulum stress response, which is related with the activation of Akt phosphorylation in H/R-injured my-cardial cells.
