电针内关穴循经靶向调整心肌缺血Gi、Gs蛋白表达及情志相关性研究

发布时间：2018-06-30 09:52

本文选题：心肌缺血 + 内关　；参考：《辽宁中医药大学》2016年硕士论文

【摘要】：目的:实验一从分子水平研究电针内关穴对心肌缺血小鼠心肌细胞Gi、Gs蛋白表达的影响,实验二用慢性应激刺激,结合情志因素来加重心肌缺血的损伤,观察电针内关穴对炎症因子及心肌细胞Gi、Gs蛋白表达的影响。探讨内关穴治疗心肌缺血的机理和内关穴治疗慢性应激抑郁合并心肌缺血的机理,为内关穴与心以及经络与脏腑之间关联,及情志因素在心肌缺血的发病与治疗的相关性之中提供理论依据。材料与方法:在研究电针内关穴对心肌缺血小鼠心肌细胞Gi、Gs蛋白表达的影响的实验中,选取24只C57BL/6(SPF级)小鼠和21只同品系酸敏感离子通道3(SPF级)基因敲除小鼠,随机分为5组,为正常组、模型组、内关组、列缺组和非经非穴组,给予多点皮下注射异丙肾的方法建立心肌缺血模型,观察电针内关组与电针列缺组、电针非经非穴组比较心电图ST段值以及T波值的改变;用Western-blot法分析Gi、Gs蛋白表达,光镜观察心肌组织形态学的变化。在研究电针内关穴对慢性应激抑郁合并心肌缺血大鼠炎症因子IL-1、IL-2、IL-8及心肌细胞Gi、Gs蛋白表达的影响实验中,选取50只SD大鼠随机分为5组,为正常组、心肌缺血模型组、心肌缺血合并慢性应激抑郁模型组、内关治疗1组、内关治疗2组,给予多点皮下注射异丙肾上腺素和强制束缚加游泳的方法建立心肌缺血合并慢性应激抑郁模型,经电针内关治疗1组与内关治疗2组后,采用Western-blot法检测心肌细胞Gi、Gs蛋白表达的影响,采用ELISA法检测血浆中IL-1、IL-2、IL-8的含量。结果:1.C57BL/6和ASIC3~(-/-)两种小鼠造模后,除正常组外,各组小鼠ST段值与T波值都有不同程度抬高,差异有统计学意义(P0.05),表明心肌缺血模型成功建立。经过7天电针干预后,内关组、列缺组和非经非穴组ST段均有降低,各组与本组造模后比较差异有统计学意义(P0.05),与模型组7天电针干预后比较有差异(P0.05)。内关组ST段降低更加明显,与列缺组和非经非穴组比较差异有统计学意义(P0.05)。但未降低到正常水平。提示:电针内关穴,可明显改善小鼠急性心肌缺血损伤后的心电图ST段值与T波值的异常抬高,促进缺血心肌心电图ST段值与T波值的恢复,列缺穴和非经非穴组干预效果差于内关穴组。C57BL/6小鼠内关组与ASIC3~(-/-)小鼠内关组比较,C57BL/6小鼠组心电图ST段值与T波值降低更显著。2.通过光镜检测心肌缺血心肌组织形态改变,电针内关穴、列缺穴和非经非穴对心肌缺血的两种小鼠心肌细胞均有一定的作用,但是电针内关穴对C57BL/6与ASIC3~(-/-)小鼠的心肌保护作用更明显。3.C57BL/6和ASIC3~(-/-)两种小鼠造模后与正常组比较,模型组、内关组、列缺组和非经非穴组C57BL/6小鼠Gs蛋白表达均有所降低,Gi蛋白表达均有所升高,且具有统计学意义(P0.05);经电针干预后与模型组比较,内关组、列缺组Gs蛋白表达均有所上升,Gi蛋白表达均有所下降,且具有统计学意义(P0.05);内关组与列缺组比较,内关组Gs蛋白表达上升更明显,Gi蛋白表达下降更明显,且具有统计学意义(P0.05)。提示:电针内关穴可明显增加C57BL/6和ASIC3~(-/-)两种小鼠Gs蛋白表达,降低两种小鼠Gi蛋白表达,但没能恢复到正常水平,列缺组没内关组效果明显,且具有统计学意义(P0.05),非经非穴组效过不明显。C57BL/6小鼠内关组与ASIC3~(-/-)小鼠内关组比较,C57BL/6小鼠组Gi、Gs蛋白表达更明显。4.与正常组比较,心肌缺血模型组、心肌缺血合并慢性应激抑郁模型组,血清CK-MB、LDH都有所升高,且具有统计学意义(P0.05)。提示心肌缺血造模成功。与正常组、心肌缺血模型组以及内关治疗1组比较,心肌缺血合并慢性应激抑郁组和内关治疗2组大鼠体重增加减慢,蔗糖摄入量减少,差异具有统计学意义(P0.05),提示造模方法可诱发大鼠慢性应激抑郁的发生。与正常组比较,心肌缺血模型组与心肌缺血合并慢性应激抑郁模型组血清的IL-1、IL-2、IL-8含量升高,Gi蛋白表达升高、Gs蛋白表达降低,且具有统计学意义(P0.05);与心肌缺血模型组比较,心肌缺血合并慢性应激抑郁模型组的血清IL-1、IL-2、IL-8含量升高更明显、Gi蛋白表达升高更明显、Gs蛋白表达降低更明显,且具有统计学意义(P0.05),说明慢性应激抑郁能加重炎症因子含量、加重Gi蛋白表达升高、加重Gs蛋白表达下降。电针内关穴后,与心肌缺血模型组及心肌缺血合并慢性应激抑郁模型组比较,内关治疗1组与内关治疗2组的血清IL-1、IL-2、IL-8含量降低,Gi蛋白表达降低、Gs蛋白表达上升,且具有统计学意义(P0.05);与内关治疗2组比较,内关治疗1组IL-1、IL-2、IL-8含量降低更明显,Gi蛋白表达降低更明显、Gs蛋白表达上升更明显,且具有统计学意义(P0.05)。提示:电针内关穴可明显改善心肌缺血合并慢性应激抑郁大鼠血清中IL-1、IL-2、IL-8含量,改善Gi、Gs蛋白异常表达,但是电针内关穴对单纯心肌缺血大鼠各项指标的改善较心肌缺血合并慢性应激抑郁大鼠的效果更明显。结论:1.电针内关穴通过降低Gi蛋白表达及升高Gs蛋白表达,达到保护心肌细胞,从心电图的改善、组织形态学的改善中体现。2.针刺对C57BL/6小鼠治疗作用明显优于对ASIC3~(-/-)小鼠,可能的机制是ASIC3参与针刺作用的外周感受器传入神经机制,因此针刺内关穴可以激活ASIC3受体离子通道,从而抑制交感神经,缓解因异丙肾上腺素兴奋交感神经所引起的心肌缺血的症状。3.慢性应激抑郁加重了心肌缺血损伤,说明情志因素可以通过炎症因子改变影响心肌缺血的发生发展。电针内关穴可通过调节炎症因子表达和心肌细胞Gi、Gs蛋白来减轻心肌缺血合并慢性应激抑郁大鼠的心肌损伤,从而同时治疗情志病及心系病,说明内关穴是情志与心系病相互关联的经络基础。
[Abstract]:Objective: To study the effect of electroacupuncture Neiguan point on the expression of Gi and Gs protein in myocardial ischemia mice from electroacupuncture at molecular level. Experiment two with chronic stress stimulation, combined with emotional factors to aggravate myocardial ischemia injury, and observe the effect of electroacupuncture Neiguan point on the expression of inflammatory factors and Gi and Gs protein in cardiac myocytes. The mechanism of ischemia and the mechanism of Neiguan Acupoint in the treatment of chronic stress depression combined with myocardial ischemia provide a theoretical basis for the association between Neiguan acupoint and heart, the relationship between the meridian and the viscera, and the relationship between the emotional factors and the correlation between the pathogenesis and treatment of myocardial ischemia. Materials and methods: the study on the Gi, Gs egg of the myocardial ischemia mice by the electroacupuncture Neiguan point In the experiment of the effect of white expression, 24 C57BL/6 (SPF grade) mice and 21 same line acid sensitive ion channel 3 (SPF grade) gene knockout mice were randomly divided into 5 groups, which were divided into normal group, model group, Neiguan group, column deficiency group and non non acupoint group, and the myocardial ischemia model was established by multiple subcutaneous injection of isopropyl kidney. The ECG ST segment value and the change of T wave value were compared by Electroacupuncture without non acupoint group. The changes of Gi, Gs protein expression and myocardial histomorphology were observed by Western-blot method. The expression of IL-1, IL-2, IL-8 and myocardium cells Gi and Gs protein were studied in the electroacupuncture Neiguan point for the chronic stress depression with myocardial ischemia rats, IL-1, IL-2, IL-8 and cardiac myocytes. In the experiment, 50 SD rats were randomly divided into 5 groups, which were normal group, myocardial ischemia model group, myocardial ischemia combined with chronic stress depression model group, 1 groups of Neiguan treatment, 2 groups of Neiguan treatment, multi point subcutaneous injection of isoproterenol and compulsory shackle and swimming. After 1 groups and 2 groups of Neiguan treatment, the effect of Western-blot method was used to detect the expression of Gi and Gs protein in cardiac myocytes. The content of IL-1, IL-2 and IL-8 in plasma was detected by ELISA. Results: after two kinds of mice model of 1.C57BL/6 and ASIC3~ (- / -), the ST segment value and T wave value of all the mice were elevated in different degrees, and the difference was statistically significant. Learning significance (P0.05) showed that the model of myocardial ischemia was successfully established. After 7 days of electroacupuncture, the ST segment of Neiguan group, lacked group and non acupoint group decreased, and there was a significant difference between the groups and the group (P0.05), and the prognosis was different from that of the model group at 7 days (P0.05). The decrease of the ST segment in the Neiguan group was more obvious, and the group and the absence of the group. The difference between non acupoint group and non acupoint group was statistically significant (P0.05), but it did not decrease to normal level. It suggested that electroacupuncture Neiguan acupoint can obviously improve the abnormal elevation of ST segment value and T wave value of ECG after acute myocardial ischemia injury in mice, promote the recovery of ST segment value and T wave value of ischemic myocardium, and the intervention effect of column deficiency and non acupoint group is worse than that of the non acupoint group. Compared with the ASIC3~ (- / -) Neiguan group of Neiguan group.C57BL/6 mice and Neiguan group of ASIC3~ (- / -), the electrocardiogram of C57BL/6 mice and T wave value decreased more significantly by.2. to detect the changes of myocardial tissue morphology by light microscope, and the electroacupuncture Neiguan point, the lacing points and non non acupoints had a certain effect on the two kinds of myocardial cells of myocardial ischemia. The protective effect of electroacupuncture at Neiguan point on the myocardium of C57BL/6 and ASIC3~ (- / -) mice was more obvious than that of the two mice of.3.C57BL/6 and ASIC3~ (- / -). The expression of Gs protein in the model group, the Neiguan group, the absence group and the non non acupoint group of C57BL/6 mice decreased, and the expression of Gi protein increased, and was statistically significant (P0.05). Compared with the model group, the expression of Gs protein in the Neiguan group and the absence group were all increased, and the expression of Gi protein decreased and was statistically significant (P0.05). In Neiguan group, the expression of Gs protein in Neiguan group increased more obviously, and the expression of Gi protein decreased more clearly, and was statistically significant (P0.05). It suggested that the electroacupuncture Neiguan point was clear. Increase the expression of C57BL/6 and ASIC3~ (- / -) two mice Gs protein and reduce the expression of Gi protein in two mice, but did not recover to the normal level. The effect of the group without the Neiguan group was significant and had statistical significance (P0.05). The non acupoint group and the.C57BL/6 mice Neiguan group were compared with the ASIC3~ (- / -) mouse Neiguan group, and C57BL/6 mice group Gi, Gs. The expression of.4. was more obvious than that in the normal group. The myocardial ischemia model group, the myocardial ischemia combined with the chronic stress depression model group, the serum CK-MB, LDH all increased, and had statistical significance (P0.05). It suggested that the model of myocardial ischemia was successful. Compared with the normal group, the myocardial ischemia model group and the Neiguan treatment group, the myocardial ischemia combined with the chronic stress in 1 groups. In the 2 groups of depression group and Neiguan treatment, the weight gain decreased and the sucrose intake decreased, the difference was statistically significant (P0.05), suggesting that the modeling method could induce chronic stress depression in rats. Compared with the normal group, the serum IL-1, IL-2, and IL-8 levels in the myocardial ischemia model group and the myocardial ischemia combined with chronic irritable depression model group were increased, Gi The expression of protein expression increased, Gs protein expression decreased and had statistical significance (P0.05). Compared with myocardial ischemia model group, the levels of serum IL-1, IL-2, IL-8 in the group of myocardial ischemia combined with chronic stress depression were more obvious, the increase of Gi protein expression was more obvious, the decrease of Gs protein expression was more obvious, and there was statistical significance (P0.05), indicating chronic (P0.05). Stress and depression could aggravate the content of inflammatory factors, increase the expression of Gi protein and increase the expression of Gs protein. After electroacupuncture Neiguan point, compared with myocardial ischemia model group and myocardial ischemia combined with chronic stress depression model group, the content of serum IL-1, IL-2, IL-8 in Neiguan treatment group and Neiguan treatment group 2 were decreased, the expression of Gi protein was reduced, and the expression of Gs protein was expressed. The increase was statistically significant (P0.05). Compared with the 2 groups in the Neiguan treatment group, the content of IL-1, IL-2, IL-8 decreased more obviously in Neiguan treatment, the decrease of Gi protein expression was more obvious, the expression of Gs protein increased more obviously, and it had statistical significance (P0.05). It suggested that the electroacupuncture Neiguan point could obviously improve the serum IL in the myocardial ischemia combined with chronic stress depression rats. -1, IL-2, IL-8 content, improve the abnormal expression of Gi, Gs protein, but the effect of electroacupuncture Neiguan point on myocardial ischemia rats is more obvious than that of myocardial ischemia combined with chronic stress depression rats. Conclusion: 1. electroacupuncture Neiguan point can protect cardiomyocytes by reducing the expression of Gi protein and increasing the expression of Gs protein, and change the electrocardiogram from electrocardiogram. The improvement of the histomorphology shows that the therapeutic effect of.2. acupuncture on C57BL/6 mice is obviously better than that of ASIC3~ (- / -) mice. The possible mechanism is that ASIC3 participates in the peripheral receptor of the peripheral receptor of the acupuncture effect. Therefore, the acupuncture Neiguan point can activate the ASIC3 receptor ion channel, thus inhibiting the sympathetic nerve and relieving the isoprenaline. .3. chronic stress depression aggravates myocardial ischemia injury, indicating that emotional factors can affect the development of myocardial ischemia through the change of inflammatory factors. The electroacupuncture Neiguan point can reduce myocardial ischemia and chronic stress depression by regulating the expression of inflammatory factors and myocardial cells Gi, Gs egg white. Myocardial injury in rats, so as to treat emotional diseases and heart diseases at the same time, shows that Neiguan is the basis of meridians and collaterals, which is related to emotion and heart disease.
【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R245

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