加味涤痰汤对脑缺血再灌注损伤大鼠脑细胞自噬相关蛋白表达的影响
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摘要
目的:探讨加味涤痰汤对脑缺血再灌注损伤大鼠脑细胞自噬相关蛋白表达的影响。方法:采用可逆性脑中动脉闭塞线栓法栓塞右侧大脑中动脉,构建脑缺血再灌注(CIR)损伤模型,随机分为假手术组、模型组、加味涤痰汤高、低剂量组(0. 768,0. 384 g·kg~(-1))和吡拉西坦组(0. 1 g·kg~(-1))。假手术组和模型组灌胃生理盐水,加味涤痰汤高、低剂量组灌胃加味涤痰汤,吡拉西坦组灌胃吡拉西坦片,灌胃10 m L·kg~(-1);给药7 d。给药24 h内,处死后行组织病理学检查,比较脑梗死体积、神经细胞凋亡和血清炎症因子水平,蛋白免疫印迹法(Western blot)测定脑组织中自噬相关蛋白微管相关蛋白轻链3(LC3)Ⅱ,Beclin1,B淋巴细胞瘤-2(Bcl-2)和p62的表达。结果:模型组脑组织梗死灶内细胞和血管坏死,神经元肿胀,间质水肿;加味涤痰汤高、低剂量组及吡拉西坦组脑组织少数神经细胞死亡,水肿减轻,神经细胞肿胀减轻。加味涤痰汤高、低剂量组及吡拉西坦组脑梗死体积和神经细胞凋亡低于模型组(P <0. 05),加味涤痰汤高、低剂量组及吡拉西坦组肿瘤坏死因子-α(TNF-α),白细胞介素-2(IL-2)和白细胞介素-8(IL-8)水平降低(P <0. 05)。与假手术组比较,模型组LC3Ⅱ,Bcl-2和Beclin1蛋白及mRNA表达明显升高,p62蛋白及mRNA表达明显下降;与模型组比较,加味涤痰汤高、低剂量组及吡拉西坦组LC3Ⅱ和Beclin1蛋白及mRNA表达均明显降低,p62蛋白及mRNA表达明显升高,差异均具有明显性(P <0. 05)。结论:加味涤痰汤可改善MCAO/R脑组织损伤和自噬,减轻炎症反应,调节自噬活性,可能与下调LC3Ⅱ,Beclin1表达,上调p62表达有关。
Objective: To investigate the effect of modified Ditantang on autophagy and relevant proteins in brain cells of rats with cerebral ischemia reperfusion injury. Method: The cerebral ischemic reperfusion(CIR)injury model in rats was built by reversible middle cerebral artery occlusion artery suture of middle cerebral embolism method,and randomly divided into sham group,model group,high-dose modified Ditantang group(H-dose),low-dose modified Ditantang group(L-dose,0. 384 g·kg~(-1)) and PLXT group(0. 1 g·kg~(-1)). Sham and model groups were given normal saline by gastric perfusion,H-dose and L-dose groups were given modified Ditantang,and the PLXT group were given Piracetam tablets,intragastric volume 10 m L·kg~(-1). The treatment lasted for 7 d. Within 24 hours after administration,the histopathological examination was performed,the volume of cerebral infarction,neuronal apoptosis and serum levels of inflammatory factors were compared,and the expressions of autophagy related microtuble-associated protein 1 light chain 3(LC3) Ⅱ,Beclin1,B-cell lymphoma-2(Bcl-2) and p62 in brain tissues were determined. Result: Cells and blood vessel necrosis,neuron swelling and interstitial edema were observed in model group,a few neurons died,edema was reduced,swelling of nerve cells was alleviated in H-dose,L-dose and PLXT groups. The volume of cerebral infarction and neuronal apoptosis in Hdose,L-dose and PLXT groups were lower than those in model group(P < 0. 05). The levels of tumor necrosis factor(TNF)-α,intedeukin(IL)-2 and IL-8 in H-dose,L-dose and PLXT groups were lower than those in model group(P < 0. 05). Compared with sham group,protein and mRNA expressions of LC3 Ⅱ,Bcl-2 and Beclin1 in the ischemic brain tissue of model group were significantly increased,while protein and mRNA expressions of p62 in the ischemic brain tissue of model group were decreased significantly. Compared with the model group,protein and mRNA expressions of LC3 Ⅱ and Beclin1 in H-dose,L-dose and PLXT groups were significantly decreased,while protein and mRNA expressions of p62 were significantly increased,with significant differences(P < 0. 05). Conclusion: Modified Ditantang can improve brain injury and interfere with autophagy after MCAO/R,alleviate inflammation,and regulate autophagic activity,which may be related to the downregulation of expressions of LC3 Ⅱ,Beclin1 and the up-regulation of expression of p62.
Objective: To investigate the effect of modified Ditantang on autophagy and relevant proteins in brain cells of rats with cerebral ischemia reperfusion injury. Method: The cerebral ischemic reperfusion(CIR)injury model in rats was built by reversible middle cerebral artery occlusion artery suture of middle cerebral embolism method,and randomly divided into sham group,model group,high-dose modified Ditantang group(H-dose),low-dose modified Ditantang group(L-dose,0. 384 g·kg~(-1)) and PLXT group(0. 1 g·kg~(-1)). Sham and model groups were given normal saline by gastric perfusion,H-dose and L-dose groups were given modified Ditantang,and the PLXT group were given Piracetam tablets,intragastric volume 10 m L·kg~(-1). The treatment lasted for 7 d. Within 24 hours after administration,the histopathological examination was performed,the volume of cerebral infarction,neuronal apoptosis and serum levels of inflammatory factors were compared,and the expressions of autophagy related microtuble-associated protein 1 light chain 3(LC3) Ⅱ,Beclin1,B-cell lymphoma-2(Bcl-2) and p62 in brain tissues were determined. Result: Cells and blood vessel necrosis,neuron swelling and interstitial edema were observed in model group,a few neurons died,edema was reduced,swelling of nerve cells was alleviated in H-dose,L-dose and PLXT groups. The volume of cerebral infarction and neuronal apoptosis in Hdose,L-dose and PLXT groups were lower than those in model group(P < 0. 05). The levels of tumor necrosis factor(TNF)-α,intedeukin(IL)-2 and IL-8 in H-dose,L-dose and PLXT groups were lower than those in model group(P < 0. 05). Compared with sham group,protein and mRNA expressions of LC3 Ⅱ,Bcl-2 and Beclin1 in the ischemic brain tissue of model group were significantly increased,while protein and mRNA expressions of p62 in the ischemic brain tissue of model group were decreased significantly. Compared with the model group,protein and mRNA expressions of LC3 Ⅱ and Beclin1 in H-dose,L-dose and PLXT groups were significantly decreased,while protein and mRNA expressions of p62 were significantly increased,with significant differences(P < 0. 05). Conclusion: Modified Ditantang can improve brain injury and interfere with autophagy after MCAO/R,alleviate inflammation,and regulate autophagic activity,which may be related to the downregulation of expressions of LC3 Ⅱ,Beclin1 and the up-regulation of expression of p62.
引文
[1]吴志元,曹柱,黄金凤,等.青年缺血性脑卒中与老年缺血性脑卒中的危险因素研究[J].现代诊断与治疗,2017,28(6):983-985.
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[10]彭静,刘玲,陈钢,等.涤痰汤改善痰浊型老龄认知障碍大鼠空间认知能力及海马神经元突触可塑性[J].华中科技大学学报:医学版,2017,46(2):175-179.
[11]魏砚明,任晋宏,栾智华,等.多种细胞自噬调节剂对自噬标记物LC3Ⅱ及p62表达的影响[J].中国药科大学学报,2018,49(3):341-347.
[12]李建荣,蒋晓帆,张磊,等.脑缺血模型中皮层Sirt3与自噬蛋白LC3表达的相关性[J].中华神经外科疾病研究杂志,2018,17(5):387-390.
[13]李丹丹,肖学进,秦书俭,等.脑缺血再灌注损伤程度与自噬相关基因LC3、m TOR表达的相关性研究[J].中国临床解剖学杂志,2018,36(2):182-186.
[14]谭成富,王超,杜琳,等.电针、艾灸预处理对心肌缺血再灌注损伤大鼠心肌自噬相关蛋白LC3、Beclin1表达的影响[J].针刺研究,2018,43(1):1-7.
[15]孔凡奇,赵书杰,凡进,等.Beclin-1的泛素化在自噬中的研究进展[J].江苏医药,2018,44(10):1189-1192.
[16]张丽颖,胡昔权,郑海清,等.运动训练对脑梗死大鼠梗死边缘区神经细胞自噬及凋亡的影响[J].中国康复医学杂志,2017,32(8):863-868.
[17]李春明,舒适,钱小路,等.电针水沟穴对脑缺血再灌注大鼠自噬相关蛋白p62表达的影响[J].辽宁中医杂志,2016,43(11):2439-2441.
[2]樊文香.缺血性脑卒中的机制研究进展[J].中国药科大学学报,2018,49(6):751-759.
[3]王天磊,刘建浩,郑杨杨,等.加味补阳还五汤联合头针对气虚血瘀证脑卒中后单侧空间忽略的疗效观察[J].中国实验方剂学杂志,2018,24(13):196-201.
[4]王彦华,路永坤,张燕平,等.涤痰汤加减治疗中青年急性缺血性脑卒中合并OSAHS痰瘀互结证的疗效观察[J].中国实验方剂学杂志,2018,24(15):204-209.
[5]卢巧喜,杨云华,李欣,等.涤痰汤对中风急性期患者脑损伤及炎症因子的影响[J].中国中医急症,2017,26(3):490-492.
[6]仇志富,吴晓光.SDF-1/CXCR4生物学自噬轴与脑缺血损伤研究进展[J].中国老年学杂志,2018,38(9):2295-2298.
[7]Sehara Y,Inaba T,Urabe T,et al.Survivin overexpression via adeno-associated virus vector Rh10ameliorates ischemic damage after middle cerebral artery occlusion in rats[J].Eur J Neurosci,2018,48(12):3466-3476.
[8]徐振宇,陆斌,李广平,等.高压氧治疗模型大鼠颅脑损伤性神经功能障碍的研究[J].神经解剖学杂志,2018,34(4):504-508.
[9]刘晓婷,陈维达,陈泽涛,等.加味涤痰汤灌胃对脑缺血再灌注损伤大鼠神经功能的影响[J].山东医药,2016,56(41):36-38.
[10]彭静,刘玲,陈钢,等.涤痰汤改善痰浊型老龄认知障碍大鼠空间认知能力及海马神经元突触可塑性[J].华中科技大学学报:医学版,2017,46(2):175-179.
[11]魏砚明,任晋宏,栾智华,等.多种细胞自噬调节剂对自噬标记物LC3Ⅱ及p62表达的影响[J].中国药科大学学报,2018,49(3):341-347.
[12]李建荣,蒋晓帆,张磊,等.脑缺血模型中皮层Sirt3与自噬蛋白LC3表达的相关性[J].中华神经外科疾病研究杂志,2018,17(5):387-390.
[13]李丹丹,肖学进,秦书俭,等.脑缺血再灌注损伤程度与自噬相关基因LC3、m TOR表达的相关性研究[J].中国临床解剖学杂志,2018,36(2):182-186.
[14]谭成富,王超,杜琳,等.电针、艾灸预处理对心肌缺血再灌注损伤大鼠心肌自噬相关蛋白LC3、Beclin1表达的影响[J].针刺研究,2018,43(1):1-7.
[15]孔凡奇,赵书杰,凡进,等.Beclin-1的泛素化在自噬中的研究进展[J].江苏医药,2018,44(10):1189-1192.
[16]张丽颖,胡昔权,郑海清,等.运动训练对脑梗死大鼠梗死边缘区神经细胞自噬及凋亡的影响[J].中国康复医学杂志,2017,32(8):863-868.
[17]李春明,舒适,钱小路,等.电针水沟穴对脑缺血再灌注大鼠自噬相关蛋白p62表达的影响[J].辽宁中医杂志,2016,43(11):2439-2441.