头穴透刺法对急性期脑出血大鼠脑组织Beclin1和BNIP3L蛋白表达的影响
|
摘要
目的通过对急性期脑出血模型大鼠进行头穴透刺法干预,观察脑组织自噬蛋白Beclin1和BNIP3L的表达情况。方法选用雄性SD大鼠90只随机分为假手术组、模型组、3-MA组、针刺组、针刺+3-MA组,每组再分为6 h、1 d和7 d 3个亚组,每个亚组各6只。脑出血模型为自体血注入法,头穴透刺选择百会穴透曲鬓穴。造模成功后各组进行神经功能评分测定,并在各自时间点取材,采用免疫化分析法观察脑组织内Beclin1和BNIP3L的表达。结果头穴透刺法可以降低急性期脑出血模型动物的神经功能评分,并随着时间递减7 d达到最低。Beclin1和BNIP3L在大鼠脑出血后6 h表达开始增多,7 d达高峰。针刺组各时间点阳性细胞数均比模型组高(P <0.05)。针刺组与针刺+3-MA组比较差异有统计学意义(P <0.05)。结论头穴透刺法能够提高Beclin1和BNIP3L蛋白的表达,促进急性期脑出血后脑组织自噬水平,减轻脑出血后的继发性损伤,达到减轻神经功能缺损的目的。
Objective: To observe the expression of autophagic protein Beclin1 and BNIP3 L in brain tissue of rats with acute cerebral hemorrhage by scalp acupoint penetration therapy. Methods: 90 male SD rats were randomly divided into the sham operation group,the model group,the 3-MA group,acupuncture group and acupuncture+3-MA group. Each group was divided into three subgroups at 6 h,1 d and 7 d,6 rats in each. ICH model was injected with autologous blood. Baihui acupoint was selected to penetrate Qubi acupoint. After successful modeling,neurological function score was measured,and the expressions of Beclin1 and BNIP3 L in brain tissues were observed by immunohistochemical analysis in the sampling groups at their respective time points. Results:The scalp acupoint penetrating needling method could reduce the neurological function score of acute ICH model animals,and it decreased for 7 days to the lowest. The expression of Beclin1 and BNIP3 L began to increase in 6 hours after ICH in rats,peaking on 7 th day. The number of positive cells at each time point in the acupuncture group was higher than that in the model group,with significant difference(P < 0.05). There was significant difference between the acupuncture group and the acupuncture+3-MA group(P < 0.05). Conclusion: The scalp acupoint penetrating needling method can increase the expression of Beclin1 and BNIP3 L protein,promote the level of autophagy of brain tissue after acute ICH,alleviate the secondary injury after ICH,and alleviate the neurological deficit.
Objective: To observe the expression of autophagic protein Beclin1 and BNIP3 L in brain tissue of rats with acute cerebral hemorrhage by scalp acupoint penetration therapy. Methods: 90 male SD rats were randomly divided into the sham operation group,the model group,the 3-MA group,acupuncture group and acupuncture+3-MA group. Each group was divided into three subgroups at 6 h,1 d and 7 d,6 rats in each. ICH model was injected with autologous blood. Baihui acupoint was selected to penetrate Qubi acupoint. After successful modeling,neurological function score was measured,and the expressions of Beclin1 and BNIP3 L in brain tissues were observed by immunohistochemical analysis in the sampling groups at their respective time points. Results:The scalp acupoint penetrating needling method could reduce the neurological function score of acute ICH model animals,and it decreased for 7 days to the lowest. The expression of Beclin1 and BNIP3 L began to increase in 6 hours after ICH in rats,peaking on 7 th day. The number of positive cells at each time point in the acupuncture group was higher than that in the model group,with significant difference(P < 0.05). There was significant difference between the acupuncture group and the acupuncture+3-MA group(P < 0.05). Conclusion: The scalp acupoint penetrating needling method can increase the expression of Beclin1 and BNIP3 L protein,promote the level of autophagy of brain tissue after acute ICH,alleviate the secondary injury after ICH,and alleviate the neurological deficit.
引文
[1]Keep RF,Hua Y,Xi G.Intracerebral haemor rhage:mechanisms of injury and therapeutic targets[J].The Lancet Neurology,2012,11(8):720-731.
[2]Bobinger T,Burkardt P,Huttner HB,et al.Pro grammed cell death after intracerebral hemorrhage[J].Current Neuropharmacol,2018,16(9):1267-1281.
[3]牛明明,邹伟,于学平.百会透曲鬓针刺法对脑出血大鼠自噬相关蛋白LC3A/B的影响研究[J].中医临床研究,2016,8(12):15-18.
[4]Yorimitsu T,Klionsky DJ.Autophagy:molecular machinery for self-eating[J].Cell Death&Differentiation,2005,2(12):1542-1552.
[5]刘建浩,陈书俞,莫燕丽,等.头穴透刺治疗急性中风病针刺手法特异性临床研究[J].上海针灸杂志,2018,37(2):144-148.
[6]张树源,鲍春玲,东贵荣.头穴透刺治疗急性高血压性脑出血神经功能缺损的临床研究[J].针灸临床杂志,2005,3(3):12-13.
[7]Zheng GQ.Methodological standards for experimental research on stroke using scalp acupuncture[J].Acupunct Electrother Res,2009,34(1-2):1-13.
[8]Hua Y,Schallert T,Keep RF,et al.Behavioral tests after intracerebral hemorrhage in the rat[J].Stroke,2002,33(10):2478-2484.
[9]Paxinos G,Watson C.Bregma,lambda and the interaural midpoint in stereotaxic surgery with rats of different sex,strain and weight[J].J Neurosci Methods,1985,13(2):139-143.
[10]Longa EZ,Weinstein PR,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J]Stroke a journal of cerebral circulation,1989,20(1):84-91.
[11]Kim WY,Nam SA,Song HC,et al.The role of autophagy in unilateral ureteral obstruction rat model[J].Nephrology,2012,17(2):148-159.
[12]Berderson JB,Pitts LH,Tsuji M,et al.Rat middle cerebral artery occlusion:evaluation of the model and development of neurologic ex-amination[J].Stroke,1986,17(3):472-476.
[13]Feigin VL,Lawes CM,Bennett DA,et al.Worldwide stroke incidence and early case fatality reported in 56 populationbased studies:a systematic review[J].Lancet Neurol.2009,8(4):355-369.
[14]Jing CH.Autophagy activation is associated with neuroprotection against apoptosis via a mitochondrial pathway in a rat model of subarachnoid hemorrhage[J].Neuroscience,2012,213(2):144-153.
[15]Hirosako K,Imasato H,Hirota Y,et al.3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factorⅡreceptor from the early endosome to the TGN[J].Biochemical and biophysical research communications,2004,316(3):845-852.
[16]Yoshimori T.Autophagy:a regulated bulk degradation process inside cells[J].Biochemical and biophysical research communications,2004,313(2):453-458.
[17]Kim H,Rafiuddinshah M,Tu HC,et al.Hierar chical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies[J].Nature Cell Biology,2006,8(12):1348.
[18]Mclelland CL,Soubannier V,Chen CX,et al.Parkin and PIN-KI function in a vesicular trafficking pathway regulating mitochondria quality control[J].EMBO J,2014,33(4):282-295.
[19]Zhu Y,Massen S,Terenzio M,et al.Modulation of serines 17and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis[J].J Biol Chem,2013,288(2):1099-1113.
[20]Maiuri MC,Criollo A.Functional and physical interaction between Bcl-X(L)and a BH3-like domain in Beclin-1[J]EMBO J,2007,26(10):2527-2539.
[21]曲馨,邹伟,刘鹏,等.浅谈“百会”透“曲鬓”头针疗法对急性脑出血[J].中医临床研究,2014,29(6):44-45.
[22]邹伟,李丹,于学平,等.针刺治疗急性期高血压脑出血的临床研究[J].中医药信息,2009,5(7):104-105.
[23]Inoue I,Chen L,Zhou L,et al.Reproduction of scalp acupuncture therapy on strokes in the model rats,spontaneous hypertensive rats-stroke prone(SHR-SP)[J].Neuroscience letters,2002,333(3):191-194.
[2]Bobinger T,Burkardt P,Huttner HB,et al.Pro grammed cell death after intracerebral hemorrhage[J].Current Neuropharmacol,2018,16(9):1267-1281.
[3]牛明明,邹伟,于学平.百会透曲鬓针刺法对脑出血大鼠自噬相关蛋白LC3A/B的影响研究[J].中医临床研究,2016,8(12):15-18.
[4]Yorimitsu T,Klionsky DJ.Autophagy:molecular machinery for self-eating[J].Cell Death&Differentiation,2005,2(12):1542-1552.
[5]刘建浩,陈书俞,莫燕丽,等.头穴透刺治疗急性中风病针刺手法特异性临床研究[J].上海针灸杂志,2018,37(2):144-148.
[6]张树源,鲍春玲,东贵荣.头穴透刺治疗急性高血压性脑出血神经功能缺损的临床研究[J].针灸临床杂志,2005,3(3):12-13.
[7]Zheng GQ.Methodological standards for experimental research on stroke using scalp acupuncture[J].Acupunct Electrother Res,2009,34(1-2):1-13.
[8]Hua Y,Schallert T,Keep RF,et al.Behavioral tests after intracerebral hemorrhage in the rat[J].Stroke,2002,33(10):2478-2484.
[9]Paxinos G,Watson C.Bregma,lambda and the interaural midpoint in stereotaxic surgery with rats of different sex,strain and weight[J].J Neurosci Methods,1985,13(2):139-143.
[10]Longa EZ,Weinstein PR,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J]Stroke a journal of cerebral circulation,1989,20(1):84-91.
[11]Kim WY,Nam SA,Song HC,et al.The role of autophagy in unilateral ureteral obstruction rat model[J].Nephrology,2012,17(2):148-159.
[12]Berderson JB,Pitts LH,Tsuji M,et al.Rat middle cerebral artery occlusion:evaluation of the model and development of neurologic ex-amination[J].Stroke,1986,17(3):472-476.
[13]Feigin VL,Lawes CM,Bennett DA,et al.Worldwide stroke incidence and early case fatality reported in 56 populationbased studies:a systematic review[J].Lancet Neurol.2009,8(4):355-369.
[14]Jing CH.Autophagy activation is associated with neuroprotection against apoptosis via a mitochondrial pathway in a rat model of subarachnoid hemorrhage[J].Neuroscience,2012,213(2):144-153.
[15]Hirosako K,Imasato H,Hirota Y,et al.3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factorⅡreceptor from the early endosome to the TGN[J].Biochemical and biophysical research communications,2004,316(3):845-852.
[16]Yoshimori T.Autophagy:a regulated bulk degradation process inside cells[J].Biochemical and biophysical research communications,2004,313(2):453-458.
[17]Kim H,Rafiuddinshah M,Tu HC,et al.Hierar chical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies[J].Nature Cell Biology,2006,8(12):1348.
[18]Mclelland CL,Soubannier V,Chen CX,et al.Parkin and PIN-KI function in a vesicular trafficking pathway regulating mitochondria quality control[J].EMBO J,2014,33(4):282-295.
[19]Zhu Y,Massen S,Terenzio M,et al.Modulation of serines 17and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis[J].J Biol Chem,2013,288(2):1099-1113.
[20]Maiuri MC,Criollo A.Functional and physical interaction between Bcl-X(L)and a BH3-like domain in Beclin-1[J]EMBO J,2007,26(10):2527-2539.
[21]曲馨,邹伟,刘鹏,等.浅谈“百会”透“曲鬓”头针疗法对急性脑出血[J].中医临床研究,2014,29(6):44-45.
[22]邹伟,李丹,于学平,等.针刺治疗急性期高血压脑出血的临床研究[J].中医药信息,2009,5(7):104-105.
[23]Inoue I,Chen L,Zhou L,et al.Reproduction of scalp acupuncture therapy on strokes in the model rats,spontaneous hypertensive rats-stroke prone(SHR-SP)[J].Neuroscience letters,2002,333(3):191-194.