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钙网蛋白(calreticulin,CRT)是内质网(endoplasmic reticulum,ER)/肌浆网(sarcoplasmic reticulum,SR)中主要的Ca2+结合分子伴侣,参与调节细胞Ca2+稳态和协助蛋白质折叠,在内源性保护现象——缺血后处理(ischemic postconditioning,I-postC)过程中表达上调,但其发挥作用的分子机制尚未完全阐明。本工作在原代培养Sprague-Dawley(SD)乳鼠心肌细胞低氧/复氧(hypoxia/reoxygenation,H/R)模型上,采用反义寡核苷酸(antisense oligodeoxynucleotides,AS-ODNs)抑制CRT表达,观察低氧后处理(hypoxic postconditioning,H-postC)过程中CRT下游分子钙调神经磷酸酶(calcineurin,CaN)活性以及CaN、核转录因子κB(nuclear factor kappa B,NFκB)、凋亡相关分子Bcl-2、Bax、C/EBP同源蛋白(C/EBP homologous protein,CHOP)等蛋白表达变化,及其与细胞保护的关系。心肌细胞随机分为6组(n=4):对照组、低氧/复氧(H/R)组、低氧后处理(H-postC)组、AS-ODNs抑制CRT表达(AS)组、AS+H/R组和AS+H-postC组,采用台盼蓝排斥实验、培养基乳酸脱氢酶(lactate dehydrogenase,LDH)活性测定及流式细胞术检测细胞损伤;经Fluo-3/AM染色,采用激光共聚焦显微镜测定细胞浆游离Ca2+浓度;采用对硝基磷酸酚(p-nitrophenyl phosphate,PNPP)底物发色法测定CaN活性;Western blot检测相关蛋白表达。结果显示:(1)H-postC可减轻H/R诱导的心肌细胞损伤,与H/R组比较,细胞存活率升高17.1%,凋亡率和LDH漏出分别降低6.67%和27.9%(P均<0.05);(2)H-postC轻度上调CRT,AS-ODNs抑制CRT表达后,部分消除后处理的心肌保护作用,与H-postC组相比,AS+H-postC组细胞存活率降低8.98%、凋亡率和LDH漏出分别升高1.74%和13.6%(P均<0.05),而胞浆游离Ca2+浓度、CaN活性、CaN及NFκB表达均未发生明显变化(P>0.05),提示CRT参与后处理保护,但不是通过胞浆Ca2+-CaN途径发挥作用;(3)H-postC抑制促凋亡蛋白Bax、CHOP表达(与H/R组相比,分别下调54%和51%,P<0.05),诱导抗凋亡蛋白Bcl-2上调(与H/R组相比,上调99%,P<0.05),抑制CRT表达部分减弱H-postC诱导的上述凋亡相关蛋白变化,提示H-postC诱导CRT上调可能通过调节凋亡相关蛋白表达参与心肌保护。综上所述,H-postC可降低胞浆Ca2+超载,减轻心肌细胞H/R损伤;CRT通过调节凋亡相关蛋白表达参与H-postC心肌保护,而不是通过Ca2+-CaN途径发挥抗凋亡作用。
Calreticulin (CRT) is the major Ca2 + binding chaperone in the endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR), which is involved in the regulation of Ca2 + homeostasis and protein folding. Endogenous The phenomenon of sexual protection - ischemic postconditioning (ischemic postconditioning, I-postC) upregulation, but its molecular mechanism of action has not been fully elucidated. In this study, antisense oligodeoxynucleotides (AS-ODNs) were used to inhibit the expression of CRT in hypoxia / reoxygenation (H / R) model of primary cultured Sprague-Dawley , The activity of calcineurin (CaN) and the expression of CaN, nuclear factor kappa B (NFκB) and apoptosis related molecules in the process of hypoxic postconditioning (H-postC) Bcl-2, Bax, C / EBP homologous protein (CHOP) protein expression and its relationship with cell protection. Cardiac myocytes were randomly divided into 6 groups: control group, hypoxia / reoxygenation (H / R) group, H-postC group, AS-ODNs inhibition of CRT expression (AS) The cells were stained with Fluo-3 / AM (superscript +) in the AS + H / R group and the AS + H-postC group by trypan blue exclusion test, lactate dehydrogenase (LDH) activity assay and flow cytometry. The concentration of free Ca2 + in cytoplasm was determined by laser scanning confocal microscopy. The activity of CaN was determined by p-nitrophenylphosphate (PNPP) substrate chromogenic assay. The expression of related protein was detected by Western blot. The results showed that: (1) H-postC attenuated H / R-induced cardiomyocyte injury. Compared with H / R group, cell survival rate increased by 17.1% and apoptosis rate and LDH leakage decreased by 6.67% and 27.9% (P <0.05). (2) Compared with H-postC group, H-postC mildly up-regulated CRT and AS-ODNs inhibited the expression of CRT, (P <0.05). However, the apoptotic rate and LDH leakage increased by 1.74% and 13.6%, respectively. However, the changes of cytoplasmic free Ca2 + concentration, CaN activity, CaN and NFκB were not significantly different (P> (3) H-postC inhibited the expression of pro-apoptotic proteins Bax and CHOP (down-regulated 54% and 51% respectively compared with H / R group) (P <0.05, P <0.05). The up-regulation of Bcl-2 was induced by H-postC (up-regulated by 99% It is suggested that H-postC induced CRT up-regulation may play a role in myocardial protection by regulating apoptosis-related protein expression. In summary, H-postC can reduce the overload of cytoplasmic Ca2 + and reduce the H / R injury of cardiomyocytes. CRT regulates the myocardial protection of H-postC by regulating the expression of apoptosis-related proteins, but not by the Ca2 + -CaN pathway .