眼针与体针对脑缺血再灌注损伤大鼠24 h脑组织水通道蛋白4表达的差异研究
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摘要
目的观察眼针与体针对24 h脑缺血再灌注损伤模型(CIRI)大鼠脑组织水通道蛋白4(AQP4)表达的影响,探讨眼针与体针治疗急性脑缺血再灌注损伤机制的差异。方法 SD大鼠48只,采用随机数字法分为6组,分别为:正常对照组,假手术组,模型组,穴区组,体针组,穴区外组。模型组、穴区组、体针组、穴区外组大鼠采用线栓法制备脑缺血再灌注损伤模型。缺血1 h,拔出栓塞线,令其再灌注。再灌注后1 h,大鼠完全苏醒后,采用ZeaLonga评分法进行大鼠神经功能评分,并开始给予针刺干预,再灌注24 h,处死大鼠,采用电镜观察脑组织神经细胞形态学变化,采用Western blot方法和实时荧光定量聚合酶链反应(RQ-PCR)方法检测脑组织中水通道蛋白4(AQP4)以及mRNA表达水平。结果与模型组比较,穴区组大鼠神经功能缺损评分明显下降;穴区组大脑皮质神经元细胞电镜下体积增大,核大,胞质丰富,线粒体、粗面内质网等肿胀明显减轻。脑组织AQP4蛋白及mRNA表达明显下调(P<0.05)。眼针与体针比较差异无统计学意义。结论眼针与体针均具有改善大鼠24 h脑缺血再灌注损伤的作用;其机制与脑组织AQP4表达下调有关,眼针与体针差异不明显。
Objective To observe the influence of eye acupuncture and body acupuncture on 24 h expression of cerebral aquaporin 4(AQP4) in rats with cerebral ischemia-reperfusion injury(CIRI), and discuss the difference in mechanism of treating CIRI between eye acupuncture and body acupuncture. Methods SD rats(n=48) were divided into 6 groups by using random digital method: normal control group, 24 h sham-operation group, 24 h model group, 24 h eye acupuncture group, 24 h body acupuncture group and 24 h outside area group. The model of CIRI was established by using thread approach in 24 h model group, 24 h eye acupuncture group, 24 h body acupuncture group and 24 h outside area group. After making ischemia for 1 s, the thread was pulled out to make reperfusion. After 1 h and rats revived fully, the neurologic function was scored by applying ZeaLonga scoring method, and acupuncture intervention was given to rats. Then reperfusion was continued for 24 h and rats were executed. The changes of cerebral neurocyte morphology were observed by using microscope, and protein and mRNA expressions of AQP4 were detected by using Western blotting assay and RQ-PCR. Results The neurological functional deficit scores decreases significantly in eye acupuncture group compared with model group. In eye acupuncture group cerebral cortex neurons increased in volume with large cores and abundant cytoplasm, and swelling of mitochondria and rough endoplasmic reticulum were significantly relieved. The protein and mRNA expressions of AQP4 decreased significantly(P<0.05). The comparison showed that there was no difference between eye acupuncture group and the body acupuncture group. Conclusion Eye acupuncture and body acupuncture have relieving effects on 24 h CIRI, and the mechanism is related to decrease of brain AQP4 expression. The difference between eye acupuncture and body acupuncture is not significant.
Objective To observe the influence of eye acupuncture and body acupuncture on 24 h expression of cerebral aquaporin 4(AQP4) in rats with cerebral ischemia-reperfusion injury(CIRI), and discuss the difference in mechanism of treating CIRI between eye acupuncture and body acupuncture. Methods SD rats(n=48) were divided into 6 groups by using random digital method: normal control group, 24 h sham-operation group, 24 h model group, 24 h eye acupuncture group, 24 h body acupuncture group and 24 h outside area group. The model of CIRI was established by using thread approach in 24 h model group, 24 h eye acupuncture group, 24 h body acupuncture group and 24 h outside area group. After making ischemia for 1 s, the thread was pulled out to make reperfusion. After 1 h and rats revived fully, the neurologic function was scored by applying ZeaLonga scoring method, and acupuncture intervention was given to rats. Then reperfusion was continued for 24 h and rats were executed. The changes of cerebral neurocyte morphology were observed by using microscope, and protein and mRNA expressions of AQP4 were detected by using Western blotting assay and RQ-PCR. Results The neurological functional deficit scores decreases significantly in eye acupuncture group compared with model group. In eye acupuncture group cerebral cortex neurons increased in volume with large cores and abundant cytoplasm, and swelling of mitochondria and rough endoplasmic reticulum were significantly relieved. The protein and mRNA expressions of AQP4 decreased significantly(P<0.05). The comparison showed that there was no difference between eye acupuncture group and the body acupuncture group. Conclusion Eye acupuncture and body acupuncture have relieving effects on 24 h CIRI, and the mechanism is related to decrease of brain AQP4 expression. The difference between eye acupuncture and body acupuncture is not significant.
引文
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[2] Crane JM, Rossi A, Gupta T, et al. Orthogonal array formation by human aquaporin-4: Examination of neuromyelitis optica-associated aquaporin-4 poly morphisms[J]. Journal of Neuroimmunology, 2011, 236(1-2): 93-98.
[3] Chu HL, Tang YP, Dong Q. Protection of vascular endothelial growth factor to brain edema followong intracerebral hemorrhage and its involved mechanisms: effect of aquaporin-4[J]. PLos One, 2013, 8(6): 65-65.
[4] 马贤德,孙宏伟,柴纪严,等. 线栓法制备大鼠脑缺血再灌注模型的方法研究[J]. 中华中医药学刊,2009,27(6):1200-1201.Ma XD, Sun HW, Chai JY, et al. Establishment a model of rat ischemia-reperfusion injury with intraluminal suture[J]. Chinese Archives of Traditional Chinese Medicine, 2009, 27(6): 1200-1201.
[5] Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84-91.
[6] 彭静山. 眼针疗法[M]. 沈阳:辽宁科学技术出版社.1990:15-27.Peng JS. Eye Acupuncture Therapy[M]. Shenyang: Liaoning Science and Technology Publishing House, 1990: 15-27.
[7] Toscano EC, Silva BC, Victori EC. Platelet-activating factor receptor(PAFR) plays a crucial role in experimental global cerebral ischemia and reperfusion[J]. Brain Res Bull, 2016, 124(6): 55-61.
[8] 吴川丽,陈国宁,张冠壮,等. RNA干扰水通道蛋白4抑制创伤性脑水肿的效果及相关机制[J]. 中国老年学杂志,2017,37(18):4493-4495.Wu CL, Chen GN, Zhang GZ, et al. Effect and related mechanism of AQP4 RNAi inhibiting traumatic brain edema[J]. Chinese Journal of Gerontology, 2017, 37(18): 4493-4495.
[9] 赵薇,王树,李方江. 大黄酚对小鼠脑缺血/再灌注脑组织抗氧化应激和AQP4的影响[J]. 中国药理学通报,2015,31(10):1477-1478. Zhao W, Wang S, Li FJ. Effects of chrysophanol on antioxidative stress and AQP4 in brain tissue of mice induced by ischemia-reperfusion injury[J]. China Pharmacological Bulletin, 2015, 31 (10): 1477-1478.
[10] Liang FY, Luo CM, Xu GQ, et al. Deletion of aquaporin-4 is neuroprotective during the acute stage of micro traumatic brain injury in mice[J]. Neurosci Lett, 2015, 598: 29-35.
[11] Yuan F, Xu ZM, Lu LY, et al. SIRT2 inhibition exacerbates neuro inflammation and blood -brain barrier disruption in experimental traumatic brain injury by enhancing NF-κB p65 acetylation and activation[J]. J Neurochem. 2016, 136(3), 581-593 .
[12] 王秀辉,郑咏秋,姚明江,等. 大鼠脑缺血再灌注后AQP4的表达与脑水肿的关系[J]. 中国实验方剂学杂志,2013,19(15):182-185. Wang XH, Zheng YQ, Yao MJ, et al. Relationship between AQP4expression and brain water content after cerebral ischemia/reperfusion in rats[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2013, 19(15): 182-185.
[13] Chen HM, Huang HS, Ruan L, et al. Ulinastatin attenuates cerebral ischemia-reperfusion injury in rats[J]. Int J Clin Exp Med, 2014, 7(5): 1483-1489.
[14] 陆立和,黄李平. 中药怀牛膝及黄芪对重型颅脑损伤大鼠脑水肿及脑组织AQP4表达的影响[J]. 时珍国医国药,2014,25(10):2359-2361.Lu LH, Huang LP. Effects of Chinese medicinal-Huainiuxi and Huangqi on brain edema and AQP4 expression of in brain tissue in rats with severe brain injury[J]. Lishizhen Med Mater Med Res, 2014, 25(10): 2359-2361.
[2] Crane JM, Rossi A, Gupta T, et al. Orthogonal array formation by human aquaporin-4: Examination of neuromyelitis optica-associated aquaporin-4 poly morphisms[J]. Journal of Neuroimmunology, 2011, 236(1-2): 93-98.
[3] Chu HL, Tang YP, Dong Q. Protection of vascular endothelial growth factor to brain edema followong intracerebral hemorrhage and its involved mechanisms: effect of aquaporin-4[J]. PLos One, 2013, 8(6): 65-65.
[4] 马贤德,孙宏伟,柴纪严,等. 线栓法制备大鼠脑缺血再灌注模型的方法研究[J]. 中华中医药学刊,2009,27(6):1200-1201.Ma XD, Sun HW, Chai JY, et al. Establishment a model of rat ischemia-reperfusion injury with intraluminal suture[J]. Chinese Archives of Traditional Chinese Medicine, 2009, 27(6): 1200-1201.
[5] Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84-91.
[6] 彭静山. 眼针疗法[M]. 沈阳:辽宁科学技术出版社.1990:15-27.Peng JS. Eye Acupuncture Therapy[M]. Shenyang: Liaoning Science and Technology Publishing House, 1990: 15-27.
[7] Toscano EC, Silva BC, Victori EC. Platelet-activating factor receptor(PAFR) plays a crucial role in experimental global cerebral ischemia and reperfusion[J]. Brain Res Bull, 2016, 124(6): 55-61.
[8] 吴川丽,陈国宁,张冠壮,等. RNA干扰水通道蛋白4抑制创伤性脑水肿的效果及相关机制[J]. 中国老年学杂志,2017,37(18):4493-4495.Wu CL, Chen GN, Zhang GZ, et al. Effect and related mechanism of AQP4 RNAi inhibiting traumatic brain edema[J]. Chinese Journal of Gerontology, 2017, 37(18): 4493-4495.
[9] 赵薇,王树,李方江. 大黄酚对小鼠脑缺血/再灌注脑组织抗氧化应激和AQP4的影响[J]. 中国药理学通报,2015,31(10):1477-1478. Zhao W, Wang S, Li FJ. Effects of chrysophanol on antioxidative stress and AQP4 in brain tissue of mice induced by ischemia-reperfusion injury[J]. China Pharmacological Bulletin, 2015, 31 (10): 1477-1478.
[10] Liang FY, Luo CM, Xu GQ, et al. Deletion of aquaporin-4 is neuroprotective during the acute stage of micro traumatic brain injury in mice[J]. Neurosci Lett, 2015, 598: 29-35.
[11] Yuan F, Xu ZM, Lu LY, et al. SIRT2 inhibition exacerbates neuro inflammation and blood -brain barrier disruption in experimental traumatic brain injury by enhancing NF-κB p65 acetylation and activation[J]. J Neurochem. 2016, 136(3), 581-593 .
[12] 王秀辉,郑咏秋,姚明江,等. 大鼠脑缺血再灌注后AQP4的表达与脑水肿的关系[J]. 中国实验方剂学杂志,2013,19(15):182-185. Wang XH, Zheng YQ, Yao MJ, et al. Relationship between AQP4expression and brain water content after cerebral ischemia/reperfusion in rats[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2013, 19(15): 182-185.
[13] Chen HM, Huang HS, Ruan L, et al. Ulinastatin attenuates cerebral ischemia-reperfusion injury in rats[J]. Int J Clin Exp Med, 2014, 7(5): 1483-1489.
[14] 陆立和,黄李平. 中药怀牛膝及黄芪对重型颅脑损伤大鼠脑水肿及脑组织AQP4表达的影响[J]. 时珍国医国药,2014,25(10):2359-2361.Lu LH, Huang LP. Effects of Chinese medicinal-Huainiuxi and Huangqi on brain edema and AQP4 expression of in brain tissue in rats with severe brain injury[J]. Lishizhen Med Mater Med Res, 2014, 25(10): 2359-2361.