针药结合对阿尔茨海默病大鼠海马区Bcl-2和Bax蛋白表达的影响
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摘要
目的:探讨针药结合治疗阿尔茨海默病(Alzheimer’sdisease,AD)的机制。方法:将成年SD大鼠60只随机分为正常组、假手术组、模型组、电针组、天麻素组和针药结合组,每组10只。腹腔注射D-半乳糖并联合双侧海马注射Aβ1-40建立AD模型大鼠。造模2周后,电针组和针药结合组大鼠选取"百会""大椎"与双侧"足三里"进行电针治疗,每天1次,每次30 min,连续4周;天麻素组和针药结合组以天麻素注射液进行腹腔注射治疗,每天1次,连续4周;正常组、模型组和假手术组大鼠不予干预。采用HE染色观察各组大鼠海马神经元形态;免疫组化法检测各组大鼠海马CA1区B淋巴细胞瘤-2基因(Bcl-2)、B淋巴细胞瘤因子相关X蛋白(Bax)的表达;Western blot法检测各组大鼠海马区Bcl-2、Bax蛋白的表达。结果:HE染色结果显示,正常组与假手术组海马CA1区神经元排列规则,胞质与胞核清晰可见;模型组神经元出现严重损伤,细胞排列不紧密,细胞形态不完整;各干预组细胞形态较模型组有显著改善。免疫组化法结果显示,与正常组和假手术组比较,模型组大鼠海马CA1区Bcl-2的表达减弱,Bax的表达增强(均P<0.05);与模型组比较,各干预组Bcl-2的表达增强,Bax的表达减弱(均P<0.05);与电针组或天麻素组比较,针药结合组Bcl-2的表达增强,Bax的表达减弱(均P<0.05)。Western blot法检测结果与免疫组化结果趋于一致。结论:电针与天麻素可明显抑制Bax的表达,上调Bcl-2的表达,以针药结合作用最为显著,表明电针结合天麻素对抑制AD大鼠海马区神经元的凋亡具有协同促进作用,这可能是针药结合治疗AD病变的机制之一。
Objective To explore the mechanism of acupuncture plus medication on treatment of Alzheimer's disease(AD). Methods Sixty adult SD rats were randomly divided into a normal group, a sham operation group, a model group, an electroacupuncture(EA) group, a gastrodin group and an EA+gastrodin group, 10 rats in each one. The rat model of AD was established by intraperitoneal injection of D-galactose and bilateral hippocampal injection of Aβ1-40. Two weeks after modeling, the rats in the EA group and EA+gastrodin group were treated with EA at "Baihui"(GV 20) "Dazhui"(GV 14) and bilateral "Zusanli"(ST 36), 30 min per treatment, once a day for consecutive 4 weeks. The rats in the gastrodin group and EA+gastrodin group were treated with intraperitoneal injection of gastrodin, once a day for consecutive 4 weeks. The rats in the normal group, model group and sham operation group were not treated. The morphology of hippocampal neurons was observed by using HE staining. The expression of Bcl-2 and Bax in the hippocampal CA1 area was detected by using immunohistochemical method. The expression of Bcl-2 and Bax protein in hippocampus was detected by using Western blot.Results The HE staining results showed the arrangement of neurons in the hippocampal CA1 area was regular in the normal group and the sham operation group, and the cytoplasm and nucleus were clearly visible. The neurons in the model group were severely damaged; the cell arrangement was not close, and the cell morphology was incomplete. Compared with the model group, the cell morphology of each intervention group was significantly improved. The immunohistochemistry results showed that, compared with the normal group and the sham operation group, the expression of Bcl-2 in the hippocampal CA1 region in the model group was decreased(P<0.05), but the expression of Bax was enhanced(P<0.05); compared with the model group, the expression of Bcl-2 was increased(all P<0.05) and the expression of Bax was decreased(all P<0.05) in all intervention group; compared with the EA group or the gastrodin group, the expression of Bcl-2 was enhanced(P<0.05) and the expression of Bax was decreased(P<0.05) in the EA+gastrodin group. The result of Western blot method was consistent with that of immunohistochemistry method. Conclusion EA and gastrodin could significantly inhibit the expression of Bax and up-regulate the expression of Bcl-2, and the combination of EA and gastrodin has the most significant effect. This indicates that EA combined with gastrodin has synergistic effect on inhibiting the apoptosis of neurons in hippocampus in AD rats, which may be one of the mechanisms of EA plus medication on AD lesions.
Objective To explore the mechanism of acupuncture plus medication on treatment of Alzheimer's disease(AD). Methods Sixty adult SD rats were randomly divided into a normal group, a sham operation group, a model group, an electroacupuncture(EA) group, a gastrodin group and an EA+gastrodin group, 10 rats in each one. The rat model of AD was established by intraperitoneal injection of D-galactose and bilateral hippocampal injection of Aβ1-40. Two weeks after modeling, the rats in the EA group and EA+gastrodin group were treated with EA at "Baihui"(GV 20) "Dazhui"(GV 14) and bilateral "Zusanli"(ST 36), 30 min per treatment, once a day for consecutive 4 weeks. The rats in the gastrodin group and EA+gastrodin group were treated with intraperitoneal injection of gastrodin, once a day for consecutive 4 weeks. The rats in the normal group, model group and sham operation group were not treated. The morphology of hippocampal neurons was observed by using HE staining. The expression of Bcl-2 and Bax in the hippocampal CA1 area was detected by using immunohistochemical method. The expression of Bcl-2 and Bax protein in hippocampus was detected by using Western blot.Results The HE staining results showed the arrangement of neurons in the hippocampal CA1 area was regular in the normal group and the sham operation group, and the cytoplasm and nucleus were clearly visible. The neurons in the model group were severely damaged; the cell arrangement was not close, and the cell morphology was incomplete. Compared with the model group, the cell morphology of each intervention group was significantly improved. The immunohistochemistry results showed that, compared with the normal group and the sham operation group, the expression of Bcl-2 in the hippocampal CA1 region in the model group was decreased(P<0.05), but the expression of Bax was enhanced(P<0.05); compared with the model group, the expression of Bcl-2 was increased(all P<0.05) and the expression of Bax was decreased(all P<0.05) in all intervention group; compared with the EA group or the gastrodin group, the expression of Bcl-2 was enhanced(P<0.05) and the expression of Bax was decreased(P<0.05) in the EA+gastrodin group. The result of Western blot method was consistent with that of immunohistochemistry method. Conclusion EA and gastrodin could significantly inhibit the expression of Bax and up-regulate the expression of Bcl-2, and the combination of EA and gastrodin has the most significant effect. This indicates that EA combined with gastrodin has synergistic effect on inhibiting the apoptosis of neurons in hippocampus in AD rats, which may be one of the mechanisms of EA plus medication on AD lesions.
引文
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[16]黄新格,零佩东,赵利华.D-半乳糖AD模型与中医脑衰老证候病机关系的探讨及在针灸防治脑衰老中的应用[J].世界中医药,2015,10(12):1983-1986.
[17]赖凤娇,李向宇,徐乐,等.电针对异氟醚诱导的阿尔茨海默病小鼠行为学改变的作用及机制[J].广州中医药大学学报,2017,34(3):376-380.
[18]郑丹丹.天麻素在神经及精神系统疾病中的应用进展[J].中国临床药学杂志,2018,27(1):68-72.
[19]崔晶晶,高俊虹,王玉敏,等.针药结合增效机制研究的新思路探讨[J].针刺研究,2010,35(2):146-150.
[20]陈以国,贾维宁,成泽东,等.电针对佐剂性关节炎大鼠体内独活有效成分趋向性的影响[J].中国针灸,2004,24(1):59-61.
[2]李向宇,徐乐,柳垂亮,等.电针对异氟醚引起的APPswe/PS1dE9小鼠学习记忆能力及海马激活型Caspase-3、Bcl-2/Bax变化的影响[J].针刺研究,2016,41(1):24-30.
[3]冯超,姜霁芳,唐美霞,等.天麻素对Aβ1-42致痴呆大鼠模型的保护作用及机制探究[J].天然产物研究与开发,2018,30(1):89-96.
[4]黄锐,吴锋,赵健,等.电针联合天麻素对阿尔茨海默病大鼠海马CA1区沉默信息调节因子2同源蛋白1和过氧化物酶体增殖物激活受体γ辅激活子1ɑ表达的影响[J].针刺研究,2018,43(3):140-145.
[5]Sahuncu MR,Desikan TS,Sepulcre J.The Dynamics of Cortical and hippocampal Atrophy in Alzheimer Disease[J].Arch Neurol,2011,68(8):1040-1048.
[6]刘智斌,牛文民,杨晓航,等.“嗅三针”对阿尔茨海默病大鼠海马Bcl-2和Bax表达的干预效应[J].针刺研究,2011,36(1):7-11.
[7]华兴邦,李辞蓉,周浩良,等.大鼠穴位图谱的研制[J].实验动物与动物实验,1991(1):1-5.
[8]Renault TT,Chipuk JE.Death upon a kiss:mitochondrial outer membrane composition and organelle communication govern sensitivity to BAK/BAX-dependent apoptosis[J].Chem Biol,2014,21(1):114-123.
[9]Taits WG,Green DR.Mitochondria and cell death:outer membrane permeabilization and beyond[J].Nat Rev Mol Cell Biol,2010,11(9):621-632.
[10]Chen JH,Ke KF,Lu JH,et al.Protection of TGF-β1 against neuroinflammation and neurodegeneration in Aβ1-42-induced Alzheimer's disease model rats[J].PLoS One,2015,10(2):e0116549.
[11]Durairajan SS,Liu LF,Lu JH,et al.Berberine amelioratesβ-amyloid pathology,gliosis,and cognitive impairment in an Alzheimer's disease transgenic mouse model[J].Neurobiol Aging,2012,33(12):2903-2919.
[12]Lee MH,Hong SH,Park C,et al.Hwang-Heuk-San induces apoptosis in HCT116 human colorectal cancer cells through the ROS mediated activation of caspases and the inactivation of the PI3K/Akt signaling pathway[J].Oncol Rep,2016,36(1):205-214.
[13]Al-Qathama A,Gibbons S,Prieto JM.Differential modulation of Bax/Bcl-2 ratio and onset of caspase-3/7 activation induced by derivatives of Justicidin B in human melanoma cells A375[J].Oncotarget,2017,8(56):95999-96012.
[14]王康锋,张立娟,陈新勇.电针大椎及百会穴治疗老年性痴呆36例临床观察[J].中华中医药杂志,2015,30(3):784-786.
[15]孙国杰,罗磊,杜艳军,等.针灸对AD模型大鼠海马神经元线粒体保护机制研究[J].中国针灸,2014,34(2):157-162.
[16]黄新格,零佩东,赵利华.D-半乳糖AD模型与中医脑衰老证候病机关系的探讨及在针灸防治脑衰老中的应用[J].世界中医药,2015,10(12):1983-1986.
[17]赖凤娇,李向宇,徐乐,等.电针对异氟醚诱导的阿尔茨海默病小鼠行为学改变的作用及机制[J].广州中医药大学学报,2017,34(3):376-380.
[18]郑丹丹.天麻素在神经及精神系统疾病中的应用进展[J].中国临床药学杂志,2018,27(1):68-72.
[19]崔晶晶,高俊虹,王玉敏,等.针药结合增效机制研究的新思路探讨[J].针刺研究,2010,35(2):146-150.
[20]陈以国,贾维宁,成泽东,等.电针对佐剂性关节炎大鼠体内独活有效成分趋向性的影响[J].中国针灸,2004,24(1):59-61.