腺苷受体激动剂对心肌缺血再灌注损伤大鼠内质网应激的影响及其作用机制研究
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摘要
目的:探讨腺苷受体激动剂对心肌缺血再灌注损伤(MIRI)大鼠内质网应激(ERS)的影响及其作用机制。方法:选取雄性成年Wistar大鼠56只,利用Langendorff装置制成大鼠离体心脏MIRI模型。随机分为四组(n=14):假手术组(Sham组)、心肌缺血再灌注组(MIRI组)、腺苷受体激动剂组(NECA组)和内质网应激抑制剂组(TUDCA组)。利用透射电镜观察四组心肌超微结构的变化;免疫组化观察心肌肌醇依赖酶1α(IRE1α)的表达情况;Western blot方法检测心肌ERS中IRE1-XBP1信号通路标志蛋白IRE1α、X盒结合蛋白1s(XBP1s)的表达水平;TUNEL检测心肌细胞凋亡情况。结果:透射电镜结果显示,Sham组肌丝排列规则致密,嵴排列整齐,外膜和肌节形态完整;MIRI组大部分肌丝断裂,肌节挛缩变形,嵴排列稀疏结构破坏,间隙增宽,可见线粒体空泡变性;NECA组及TUDCA组较MIRI组损伤减轻,内质网轻度扩张或者正常,肌丝排列较整齐。免疫组化结果发现,Sham组心肌纤维呈细长圆柱形,形态正常,少量结缔组织存在,基本无IRE1α阳性染色;MIRI组细胞排列紊乱,有许多断裂的细胞出现,IRE1α阳性染色区域显著增加,而NECA和TUDCA组细胞病理的变化较轻,相对于MIRI组,IRE1α阳性染色部位明显减少。Western blot结果显示,与Sham组相比,MIRI组的IRE1α和XBP1s蛋白的表达水平明显上升(P<0.05);而与MIRI组相比,TUDCA组及NECA组IRE1α和XBP1s的蛋白表达水平显著降低(P<0.05)。TUNEL结果显示,MIRI组细胞凋亡明显,Sham组基本没有发生心肌细胞凋亡,MIRI组较NECA组及TUDCA组的凋亡细胞数更多。结论:NECA可通过抑制IRE1-XBP1信号通路来减轻ERS反应,达到保护心肌组织细胞的目的。
Objective: To investigate the effect and mechanism of adenosine receptor agonist on endoplasmic reticulum stress(ERS) in rats with myocardial ischemia-reperfusion injury(MIRI). Methods: 56 male adult Wistar rats were selected, and the rat models of Isolated heart MIRI were established by Langendorff. The rats were randomly divided into four groups(n=14): sham operation group(Sham group), myocardial ischemia reperfusion group(MIRI group), adenosine receptor agonist group(NECA group) and endoplasmic reticulum stress inhibitor Group(TUDCA group). The ultrastructural changes of myocardium in four groups were observed by transmission electron microscopy. The expression of myo inositol dependent enzyme 1α(IRE1α) was observed by immunohistochemistry. The expression of IRE1-XBP1 signal pathway marker protein IRE1α and X box binding protein 1 s(XBP1 s) in ERS of myocardium were detected by Western blot. The cardiomyocyte apoptosis was detected by TUNEL. Results: The results of transmission electron microscopy showed that the arrangement of muscle filament was regular and compact, the cristae were arranged neatly and the outer membrane and sarcomere morphological integrity in the Sham group. MIRI group showed most of the myofilament rupture, sarcomere contracture deformation, crista sparse structure damage, the gap widening, visible mitochondrial vacuolar degeneration. Compared with MIRI group, the injury was mild, the endoplasmic reticulum dilated slightly or normal, and the arrangement of muscle filament was neat in NECA group and TUDCA group. Immunohistochemistry showed that the Sham group showed a slender, cylindrical shape, normal morphology, and a small amount of connective tissue, and there was no IRE1α positive staining. The cell arrangement of MIRI group was disorder, and many broken cells appeared, IRE1α positive staining area increased significantly,the pathological changes of NECA and TUDCA group were less than that of MIRI group, and the positive staining sites of IRE1α were significantly decreased. The expression levels of IRE1αand XBP1 s protein in MIRI group were significantly increased compared with Sham group by Western blot(P<0.05), the protein expression levels of IRE1α and XBP1 s in TUDCA group and NECA group were significantly decreased when compared with the MIRI group(P<0.05). The results of TUNEL showed that there was obvious apoptosis in the MIRI group, but there was no apoptosis in the Sham group, the apoptotic cells in the MIRI group were higher than those in the NECA group and the TUDCA group. Conclusion: NECA can alleviate the ERS response by inhibiting the IRE1-XBP1 signaling pathway and protect the cardiac muscle tissue cells.
Objective: To investigate the effect and mechanism of adenosine receptor agonist on endoplasmic reticulum stress(ERS) in rats with myocardial ischemia-reperfusion injury(MIRI). Methods: 56 male adult Wistar rats were selected, and the rat models of Isolated heart MIRI were established by Langendorff. The rats were randomly divided into four groups(n=14): sham operation group(Sham group), myocardial ischemia reperfusion group(MIRI group), adenosine receptor agonist group(NECA group) and endoplasmic reticulum stress inhibitor Group(TUDCA group). The ultrastructural changes of myocardium in four groups were observed by transmission electron microscopy. The expression of myo inositol dependent enzyme 1α(IRE1α) was observed by immunohistochemistry. The expression of IRE1-XBP1 signal pathway marker protein IRE1α and X box binding protein 1 s(XBP1 s) in ERS of myocardium were detected by Western blot. The cardiomyocyte apoptosis was detected by TUNEL. Results: The results of transmission electron microscopy showed that the arrangement of muscle filament was regular and compact, the cristae were arranged neatly and the outer membrane and sarcomere morphological integrity in the Sham group. MIRI group showed most of the myofilament rupture, sarcomere contracture deformation, crista sparse structure damage, the gap widening, visible mitochondrial vacuolar degeneration. Compared with MIRI group, the injury was mild, the endoplasmic reticulum dilated slightly or normal, and the arrangement of muscle filament was neat in NECA group and TUDCA group. Immunohistochemistry showed that the Sham group showed a slender, cylindrical shape, normal morphology, and a small amount of connective tissue, and there was no IRE1α positive staining. The cell arrangement of MIRI group was disorder, and many broken cells appeared, IRE1α positive staining area increased significantly,the pathological changes of NECA and TUDCA group were less than that of MIRI group, and the positive staining sites of IRE1α were significantly decreased. The expression levels of IRE1αand XBP1 s protein in MIRI group were significantly increased compared with Sham group by Western blot(P<0.05), the protein expression levels of IRE1α and XBP1 s in TUDCA group and NECA group were significantly decreased when compared with the MIRI group(P<0.05). The results of TUNEL showed that there was obvious apoptosis in the MIRI group, but there was no apoptosis in the Sham group, the apoptotic cells in the MIRI group were higher than those in the NECA group and the TUDCA group. Conclusion: NECA can alleviate the ERS response by inhibiting the IRE1-XBP1 signaling pathway and protect the cardiac muscle tissue cells.
引文
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[6] Ding S, Jiang J, Zhang G, et al. Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats[J].PLo S One, 2017, 12(8):e0183541
[7] Tada Y, Yang PC. Myocardial Edema on T2-Weighted MRI:New Marker of Ischemia Reperfusion Injury and Adverse Myocardial Remodeling[J]. Circ Res, 2017, 121(4):326-328
[8] Xue Q, Pei H, Liu Q, et al. MICU1 protects against myocardial ischemia/reperfusion injury and its control by the importer receptor Tom70[J]. Cell Death Dis, 2017, 8(7):e2923
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[11]富旗,周正芳,李小辉,等.腺苷预处理对大鼠脊髓缺血再灌注损伤的保护作用[J].南方医科大学学报, 2014, 34(1):92-95Fu Qi, Zhou Zheng-fang, Li Xiao-hui, et al. Protective effect of adenosine preconditioning against spinal cord ischemia-reperfusion injury in rats[J]. Journal of Southern Medical University, 2014, 34(1):92-95
[12] Xia D, Ji W, Xu C, et al. Knockout of MARCH2 inhibits the growth of HCT116 colon cancer cells by inducing endoplasmic reticulum stress[J]. Cell Death Dis, 2017, 8(7):e2957
[13]王晴,周岩,韩会,等.NECA抑制内质网应激抗大鼠心肌缺血再灌注损伤的作用机制[J].医学研究生学报, 2017, 30(6):574-578Wang Qing, Zhou Yan, Han Hui, et al. Effects and mechanisms of NECA inhibit endoplasmic reticulum stress to against myocardial ischemia reperfusion injury in rats[J]. Journal of Medical Postgraduates,2017, 30(6):574-578
[14]刘丽娟,范蕾,常福厚,等.冠心十味丸对离体大鼠心肌缺血再灌注损伤后的保护作用[J].中国生化药物杂志, 2015, 35(3):21-24Liu Li-juan, Fan Lei, Chang Fu-hou, et al. Protective effect of GuanXinShiWeiWan on isolated rat hearts with ischemia reperfusion injury[J]. Chinese Journal of Biochemical Pharmaceutics, 2015, 35(3):21-24
[15] Yang Y, Sun Y, Yi W, et al. A review of melatonin as a suitable antioxidant against myocardial ischemia-reperfusion injury and clinical heart diseases[J]. J Pineal Res, 2014, 57(4):357-366
[16]张志坚,吴灿,田黎,等.沙苑子总黄酮对内质网应激诱导的细胞凋亡在百草枯致大鼠肺损伤中的保护作用[J].医学研究生学报,2014, 27(8):806-809Zhang Zhi-jian, Wu Can, Tian Li, et al. Effects of total flavonoids from astragalus complanatus and endoplasmic reticulum stress-induced cell apop-tosis in acute lung injury following paraquat poisoning in rats[J]. Journal of Medical Postgraduates, 2014, 27(8):806-809
[17] Rajamani U, Gross AR, Ocampo C, et al. Endocrine disruptors induce perturbations in endoplasmic reticulum and mitochondria of human pluripotent stem cell derivatives[J]. Nat Commun, 2017, 8(1):219
[18] Hu YB, Wu X, Qin XF, et al. Role of Endoplasmic Reticulum Stress in Silica-induced Apoptosis in RAW264.7 Cells[J]. Biomed Environ Sci, 2017, 30(8):591-600
[19] Chen Z, Wu Q, Ding Y, et al. YD277 Suppresses Triple-Negative Breast Cancer Partially Through Activating the Endoplasmic Reticulum Stress Pathway[J]. Theranostics, 2017, 7(8):2339-2349
[20] Huang HC, Tang D, Lu SY, et al. Endoplasmic reticulum stress as a novel neuronal mediator in Alzheimer's disease[J]. Neurol Res, 2015,37(4):366-374
[21] Bailey KA, Haj FG, Simon SI, et al. Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation[J]. Sci Rep, 2017, 7(1):8196
[22]郭智东,王弋,许国根,等.心肌细胞内质网应激的研究进展[J].心脑血管病防治, 2014, 14(1):48-50Guo Zhi-dong, Wang Yi, Xu Guo-gen, et al. Progress in endoplasmic reticulum stress in cardiomyocytes[J]. Prevention and Treatment of Cardio-Cerebral-Vascular Disease, 2014, 14(1):48-50
[23] Mottis A, Jovaisaite V, Auwerx J. The mitochondrial unfolded protein response in mammalian physiology[J]. Mamm Genome, 2014, 25(9-10):424-433
[24] Liao HY, Kao CM, Yao CL, et al. 2,4,6-Trinitrotoluene Induces Apoptosis via ROS-Regulated Mitochondrial Dysfunction andEndoplasmic Reticulum Stress in HepG2 and Hep3B Cells[J]. Sci Rep,2017, 7(1):8148
[25]谷晓峰,邓学峰,马群风,等.RNF13通过IRE1/XBP-1通路调控内质网应激介导的细胞凋亡[J].现代生物医学进展, 2016, 16(5):801-805Gu Xiao-feng, Deng Xue-feng, Ma Qun-feng, et al. RNF13 Mediates ER Stress induced Apoptosis through IRE1/XBP-1 Pathway[J].Progress in Modern Biomedicine, 2016, 16(5):801-805
[26] Homma T, Fujii J. Heat stress promotes the down-regulation of IRE1αin cells:An atypical modulation of the UPRpathway[J]. Exp Cell Res, 2016, 349(1):128-138
[27] Cameron TL, Bell KM, Gresshoff IL, et al. XBP1-Independent UPR Pathways Suppress C/EBP-βMediated Chondrocyte Differentiation inER-Stress Related Skeletal Disease[J]. PLoS Genet, 2015, 11(9):e1005505
[28] Jiao FJ, Wang QZ, Zhang P, et al. CDK5-mediated phosphorylation of XBP1s contributes to its nuclear translocation and activa tion in MPP+-induced Parkinson's disease model[J]. Sci Rep, 2017, 7(1):5622
[29] Wang S, Wang Z, Fan Q, et al. Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol-requiring enzyme 1α/X box-binding protein-1 pathway[J]. Br J Pharmacol,2016, 173(15):2402-2418
[30] Kalogeris T, Bao Y, Korthuis RJ. Mitochondrial reactive oxy gen species:a double edged sword in ischemia/reperfusion vs preconditioning[J]. Redox Biol, 2014, 2(1):702-714
[2] He XM, Chen L, Luo JB, et al. Effects of rhBNP after PCI on non-invasive hemodynamic in acute myocardial infarction patients with left heart failure[J]. Asian Pac J Trop Med, 2016, 9(8):791-795
[3]高妮妮,王芳,廉哲勋,等.PI3K-Akt-eNOS信号通路在三磷酸腺苷后处理减轻兔心肌缺血再灌注损伤中的作用[J].中国循环杂志,2014, 29(1):59-63Gao Ni-ni, Wang Fang, Lian Zhe-xun, et al. ATP Post Conditioning of PI3K-Akt-eNOS Signaling Pathway Reducing the Myocardial Ischemia Reperfusion Injury in Experimental Rabbits[J]. Chinese Circulation Journal, 2014, 29(1):59-63
[4] Zhou YH, Han QF, Wang LH, et al. High mobility group box 1 protein attenuates myocardial ischemia reperfusion injury via inhibition of the p38 mitogen-activated protein kinase signaling pathway[J]. Exp Ther Med, 2017, 14(2):1582-1588
[5] Cai X, Wang X, Li J, et al. Protective effect of glycyrrhizin on myocardial ischemia/reperfusion injury-induced oxidative stress, inducible nitric oxide synthase and inflammatory reactions through high-mobility group box 1 and mitogen-activated protein kinase expression[J].Exp Ther Med, 2017, 14(2):1219-1226
[6] Ding S, Jiang J, Zhang G, et al. Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats[J].PLo S One, 2017, 12(8):e0183541
[7] Tada Y, Yang PC. Myocardial Edema on T2-Weighted MRI:New Marker of Ischemia Reperfusion Injury and Adverse Myocardial Remodeling[J]. Circ Res, 2017, 121(4):326-328
[8] Xue Q, Pei H, Liu Q, et al. MICU1 protects against myocardial ischemia/reperfusion injury and its control by the importer receptor Tom70[J]. Cell Death Dis, 2017, 8(7):e2923
[9] Tohmonda T, Yoda M, Iwawaki T, et al. IRE1α/XBP1-mediated branch of the unfolded protein response regulates osteoclastogenesis[J]. J Clin Invest, 2015, 125(8):3269-3279
[10]肖晗,马晓伟,付勇南,等.异丙基肾上腺素不同给药模式对小鼠心脏腺苷酸活化蛋白激酶活性的影响[J].中国病理生理杂志, 2016,32(12):2177-2183Xiao Han, Ma Xiao-wei, Fu Yong-nan, et al. Distinct effects of differentβ-adrenoceptor stimulation patterns on car-diac AMP-acti vated protein kinase activity[J]. Chinese Journal of Pathophysiology, 2016, 32(12):2177-2183
[11]富旗,周正芳,李小辉,等.腺苷预处理对大鼠脊髓缺血再灌注损伤的保护作用[J].南方医科大学学报, 2014, 34(1):92-95Fu Qi, Zhou Zheng-fang, Li Xiao-hui, et al. Protective effect of adenosine preconditioning against spinal cord ischemia-reperfusion injury in rats[J]. Journal of Southern Medical University, 2014, 34(1):92-95
[12] Xia D, Ji W, Xu C, et al. Knockout of MARCH2 inhibits the growth of HCT116 colon cancer cells by inducing endoplasmic reticulum stress[J]. Cell Death Dis, 2017, 8(7):e2957
[13]王晴,周岩,韩会,等.NECA抑制内质网应激抗大鼠心肌缺血再灌注损伤的作用机制[J].医学研究生学报, 2017, 30(6):574-578Wang Qing, Zhou Yan, Han Hui, et al. Effects and mechanisms of NECA inhibit endoplasmic reticulum stress to against myocardial ischemia reperfusion injury in rats[J]. Journal of Medical Postgraduates,2017, 30(6):574-578
[14]刘丽娟,范蕾,常福厚,等.冠心十味丸对离体大鼠心肌缺血再灌注损伤后的保护作用[J].中国生化药物杂志, 2015, 35(3):21-24Liu Li-juan, Fan Lei, Chang Fu-hou, et al. Protective effect of GuanXinShiWeiWan on isolated rat hearts with ischemia reperfusion injury[J]. Chinese Journal of Biochemical Pharmaceutics, 2015, 35(3):21-24
[15] Yang Y, Sun Y, Yi W, et al. A review of melatonin as a suitable antioxidant against myocardial ischemia-reperfusion injury and clinical heart diseases[J]. J Pineal Res, 2014, 57(4):357-366
[16]张志坚,吴灿,田黎,等.沙苑子总黄酮对内质网应激诱导的细胞凋亡在百草枯致大鼠肺损伤中的保护作用[J].医学研究生学报,2014, 27(8):806-809Zhang Zhi-jian, Wu Can, Tian Li, et al. Effects of total flavonoids from astragalus complanatus and endoplasmic reticulum stress-induced cell apop-tosis in acute lung injury following paraquat poisoning in rats[J]. Journal of Medical Postgraduates, 2014, 27(8):806-809
[17] Rajamani U, Gross AR, Ocampo C, et al. Endocrine disruptors induce perturbations in endoplasmic reticulum and mitochondria of human pluripotent stem cell derivatives[J]. Nat Commun, 2017, 8(1):219
[18] Hu YB, Wu X, Qin XF, et al. Role of Endoplasmic Reticulum Stress in Silica-induced Apoptosis in RAW264.7 Cells[J]. Biomed Environ Sci, 2017, 30(8):591-600
[19] Chen Z, Wu Q, Ding Y, et al. YD277 Suppresses Triple-Negative Breast Cancer Partially Through Activating the Endoplasmic Reticulum Stress Pathway[J]. Theranostics, 2017, 7(8):2339-2349
[20] Huang HC, Tang D, Lu SY, et al. Endoplasmic reticulum stress as a novel neuronal mediator in Alzheimer's disease[J]. Neurol Res, 2015,37(4):366-374
[21] Bailey KA, Haj FG, Simon SI, et al. Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation[J]. Sci Rep, 2017, 7(1):8196
[22]郭智东,王弋,许国根,等.心肌细胞内质网应激的研究进展[J].心脑血管病防治, 2014, 14(1):48-50Guo Zhi-dong, Wang Yi, Xu Guo-gen, et al. Progress in endoplasmic reticulum stress in cardiomyocytes[J]. Prevention and Treatment of Cardio-Cerebral-Vascular Disease, 2014, 14(1):48-50
[23] Mottis A, Jovaisaite V, Auwerx J. The mitochondrial unfolded protein response in mammalian physiology[J]. Mamm Genome, 2014, 25(9-10):424-433
[24] Liao HY, Kao CM, Yao CL, et al. 2,4,6-Trinitrotoluene Induces Apoptosis via ROS-Regulated Mitochondrial Dysfunction andEndoplasmic Reticulum Stress in HepG2 and Hep3B Cells[J]. Sci Rep,2017, 7(1):8148
[25]谷晓峰,邓学峰,马群风,等.RNF13通过IRE1/XBP-1通路调控内质网应激介导的细胞凋亡[J].现代生物医学进展, 2016, 16(5):801-805Gu Xiao-feng, Deng Xue-feng, Ma Qun-feng, et al. RNF13 Mediates ER Stress induced Apoptosis through IRE1/XBP-1 Pathway[J].Progress in Modern Biomedicine, 2016, 16(5):801-805
[26] Homma T, Fujii J. Heat stress promotes the down-regulation of IRE1αin cells:An atypical modulation of the UPRpathway[J]. Exp Cell Res, 2016, 349(1):128-138
[27] Cameron TL, Bell KM, Gresshoff IL, et al. XBP1-Independent UPR Pathways Suppress C/EBP-βMediated Chondrocyte Differentiation inER-Stress Related Skeletal Disease[J]. PLoS Genet, 2015, 11(9):e1005505
[28] Jiao FJ, Wang QZ, Zhang P, et al. CDK5-mediated phosphorylation of XBP1s contributes to its nuclear translocation and activa tion in MPP+-induced Parkinson's disease model[J]. Sci Rep, 2017, 7(1):5622
[29] Wang S, Wang Z, Fan Q, et al. Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol-requiring enzyme 1α/X box-binding protein-1 pathway[J]. Br J Pharmacol,2016, 173(15):2402-2418
[30] Kalogeris T, Bao Y, Korthuis RJ. Mitochondrial reactive oxy gen species:a double edged sword in ischemia/reperfusion vs preconditioning[J]. Redox Biol, 2014, 2(1):702-714