氯喹对大鼠脑缺血再灌注自噬相关蛋白的影响
Effects of chloroquine on the expressions of autophagy related proteins after focal cerebral ischemia-reperfusion in rats
目的 探讨氯喹对大鼠脑缺血再灌注自噬相关蛋白的影响及其机制。 方法 取75只大鼠随机分为假手术组（Sham），模型组（Model），氯喹（CQ)20、40、60 mg/kg组，每组15只。应用线栓法使右脑中动脉栓塞（MCAO)，栓塞2 h。3个药物组于栓塞后立即腹腔注射CQ 20、40、60 mg/kg，其余各组注射2 ml 0.9%氯化钠。再灌注24 h后，采用神经功能缺损评分、TTC染色判断模型是否制作成功，采用免疫荧光和免疫印迹法检测大鼠脑自噬相关蛋白的表达。 结果 ① Sham组, Model组, CQ 20、40、60 mg/kg组神经功能缺损评分，分别为0、2.67±0.49、2.93±0.26、2.40±0.51及1.93±0.70。通过TTC染色与Model组比较梗死面积，CQ 20 mg/kg组增大，CQ 40 mg/kg组无明显变化，CQ 60 mg/kg组减小。②免疫荧光和免疫印迹结果显示各组均有蛋白(Atg5、Atg7、Beclin1、Atg12）表达，与Model组比较,其中Sham组仅有极少量表达（P<0.05)，CQ 20 mg/kg组表达量无明显差异,CQ 60 mg/kg组的表达量明显减少（P<0.05)。 结论 氯喹对大鼠脑缺血再灌注损伤有双重作用，低剂量20 mg/kg加重损伤，高剂量60 mg/kg减轻损伤。其机制可能与某些脑自噬相关蛋白表达的量有关。
Objective To investigate the effects of chloroquine on the expressions of autophagy related proteins after focal cerebral ischemia-reperfusion in adult rats. Methods A total of 75 healthy SD rats were randomly allocated to sham-operation, model and chloroquine(CQ) 20,40 and 60 mg/kg groups. A model of middle cerebral artery occlusion (MCAO) for 2 h was induced by suture method. The CQ 20,40 and 60 mg/kg groups were injected intraperitoneally immediately following MCAO in the 3 medication groups. Neurological deficit scores and TTC staining were evaluated, and then the expressions of autophagy related proteins in area of cerebral ischemia-reperfusion were analyzed by immunofluorescence and Western blot technique at 24 h after reperfusion. Results ①Neurological deficit scores in the sham-operation, model, CQ 20, 40 and 60 mg/kg groups were 0, 2.67±0.49, 2.93±0.26, 2.40±0.51 and 1.93±0.7 respectively. Compared with the infarction area of the model group by TTC staining, the CQ 20 mg/kg group was significantly increased, the CQ 40 mg/kg group was almost like the model group，the CQ 60 mg/kg group was significantly decreased. ② Compared with the model group, the results of immunofluorescence and Western blot technique showed only a trace expressions of proteins(Atg5, Atg7, Beclin1 and Atg12 ) in the sham operation group (P<0.05), and the same pattern of expressions were observed in the CQ 20 mg/kg group. However there was significantly reduced expressions in the CQ 60 mg/kg group (P<0.05). Conclusion The CQ have has two fold effects, i.e., the CQ 20 mg/kg group can aggravate the damage, whereas the CQ 60 mg/kg can alleviatereduce the damage. The mechanism of CQ in the prevention and treatment of cerebral ischemia-reperfusion injury in rats may be associated with the dose of the expressions of some autophagy related proteins.
[1] Thornton C, Hagberg H. Role of mitochondria in apoptotic and necroptotic cell death in the developing brain[J].Clin Chim Acta, 2015,451(Pt A):35-38.
[2] Jiang P, Mizushima N. Autophagy and human diseases[J].Cell Res, 2014, 24(1): 69-79.
[3] Li L, Zhang Q, Tan J, et al. Autophagy and hippocampal neuronal injury [J].Sleep Breath, 2014, 18(2):243-249.
[4] Chen S, Rehman SK, Zhang W, et al. Autophagy is a therapeutictarget in anticancer drug resistance[J]. Biochim Biophys Acta, 2010, 1806(2):220-229.
[5] Denton D, Xu T, Kumar S. Autophagy as a pro-death pathway[J].Immunol Cell Biol, 2015, 93(1):35-42.
[6] Wang Y, Peng RQ, Li DD, et al. Chloroquine enhances the cytotoxicity of topotecan by inhibiting autophagy in lung cancer cells[J].Chin J Cancer, 2011, 30(10): 690-700.
[7] Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke, 1989, 20(1):84-91.
[8] Li L, Wang H Y, Chen BY, et al. Influence of different degrees of chronic intermittent hypoxia on c-fos protein and apoptosis in rats [J].Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi, 2012, 47(2):117-121.
[9] Xilouri M, Stefanis L. Autophagy in the central nervous system: implications for neurodegenerative disorders[J].CNS Neurol Disord Drug Targets, 2010, 9(6): 701-719.
[10]Mizushima N, Yoshimorim T, Levine B. Methods in mammalian autophagy research[J]. Cell, 2010, 140(3):313-326.
[11]Lim J, Lachenmayer ML, Wu S, et al. Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates[J].PLoS Genet, 2015, 11(2):e1004987.
[12]Zhou J, Wang J, Li X, et al. H2O2 mediates the crosstalk of brassinosteroid and abscisic acid in tomato responses to heat and oxidative stresses [J]. Exp Bot 2014, 65(15):4371-4383.
[13]Su H, Wang X. p62 Stages an interplay between the ubiquitin-proteasome system and autophagy in the heart of defense against proteotoxic stress [J].Trends Cardiovasc Med, 2011, 21(8):224-228.
[14]Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues[J]. Cell, 2011,147(4):728-741.
[15]Zheng W, Wei M, Li S, et al. Nanomaterial-modulated autophagy: underlying mechanisms and functional consequences [J]. Nanomedicine (Lond), 2016, 11 (11): 1417-1430.
[16]Fullgrabe J, Klionsky D J, Joseph B. Histone post-translational modifications regulate autophagy flux and outcome [J]. Autophagy, 2013, 9(10):1621-1623.
[17]Liu Q, Luo XY, Jiang H, et al. Hydroxychloroquine facilitates autophagosome formation but not degradation to suppress the proliferation of cervical cancer Si Ha cells[J]. Oncol Lett, 2014, 7(4):1057-1062.
[18]张树华,朱长庚,刘庆莹,等.氯喹对戊四氮致痫大鼠皮质和海马白细胞介素1β及肿瘤坏死因子α表达的影响[J].中国组织化学与细胞化学杂志, 2005, 14(5):491-495.
[19]吴淑华,李建民,李扬扬,等.不同剂量氯喹对戊四氮慢性致痫大鼠脑内nNOS 表达的影响[J].神经解剖学杂志, 2012, 28(4):392-396.
[20]Rajawat YS, Bossis I. Autophagy in aging and in neurodegenerative disorders[J]. Hormones (Athens), 2008, 7(1):46-61.
[21]Kimura T, Takabatake Y, Takahashi A, et al. Chloroquine in cancer therapy: a double-edged sword of autophagy[J].Cancer Res, 2013,73(1):3-7.
国家自然科学基金（81202783）；省科技厅计划项目（2015020696，20170540378）
京ICP备08002354号-3/
| 〈 |
|
〉 |
