电针“足三里”“曲池”穴对高血压前期大鼠肾脏p38MAPK通路影响的研究

发布时间：2018-06-29 03:03

本文选题：高血压前期 + 肾脏细胞外基质沉积　；参考：《辽宁中医药大学》2017年硕士论文

【摘要】：目的:本实验采用实时定量PCR及蛋白免疫印迹法(Western boltting)的方法,检测盐敏感大鼠建立的高血压前期模型中,肾脏组织中转化生长因子-β1(transforming growth factor-beta1,TGF-β1)介导的p38MAPK信号传导通路相关基因和蛋白的表达,主要有p38丝裂素活化蛋白激酶(p38mitogen-activated protein kinase,p38MAPK)、c AMP反应元件结合蛋白(c AMP-responsive element binding protein,CREB),及α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维黏连蛋白(fibronectin,FN)基因的表达,探讨针刺对高血压前期大鼠延缓肾脏细胞外基质沉积的作用原理,从更深层次上理解高血压病的发生发展过程。材料与方法:本次实验按照实验目的将实验动物分为四组,分别为正常对照组,模型组,针刺组以及非经非穴组。全部实验动物为40只健康雄性大鼠,其中有30只为Dahl盐敏感大鼠(Dahl salt sensitive rats,DS),给予高盐饲料喂养后血压升高。将它们按照随机数字表法平均分为三组,每组10只,分别记为模型、针刺及非经非穴组。另外10只为Dahl盐抵抗大鼠(Dahl salt resistant rats,DR),给予高盐饲料喂养后血压基本稳定不变。将每组大鼠分笼饲养,适应性喂养7天,无异常后给予高盐饲料喂养并每天检测血压变化,待盐敏感大鼠血压升高10-20mm Hg后停止高盐喂养改为普通饲料喂养,即造模成功并开始治疗。其中模型组大鼠仅每日捆绑20min静置;针刺组针刺双侧“足三里”“曲池”穴3-5mm,接通电针20min,采用疏密波,频率以针柄轻微震颤为度,每日1次,每周6次,治疗五周;非经非穴组针刺大鼠双侧髂嵴上15mm、后正中线旁开20mm这一固定对照点,同样采用疏密波,接通电针20min,频率以针柄轻微震颤为度,治疗日期与针刺组相同。治疗时每周检测大鼠血压一次并记录。停止针刺治疗结束。取材时,捆绑固定大鼠后注射10%水合氯醛(每100g大鼠给药0.35ml),待大鼠完全麻醉后,腹部正中切口,充分暴露肾脏后切除双肾,去包膜,沿最大冠状切面纵行切开,取其中一部分肾组织做匀浆液,提取肾脏组织总RNA,采用实时定量PCR方法检测肾脏中TGF-β1、p38MAPK、CREB、FN及α-SMAm RNA的表达;用蛋白免疫印迹法检测肾脏组织中p38MAPK、CREB的磷酸化蛋白表达量。结果:1.电针对Dahl盐敏感大鼠肾组织TGF-β1基因表达的影响模型组大鼠与正常组大鼠相比,肾组织中TGF-β1基因表达升高(P0.05)。经过五周治疗后,针刺组及非经非穴组大鼠与模型组相比,TGF-β1基因表达量均有所降低(P0.05),与非经非穴组相比,针刺组降低明显,具有统计学意义(P0.05)。2.电针对Dahl盐敏感大鼠肾组织p38MAPK信号通路的影响模型组大鼠与正常组大鼠相比,肾组织中p38MAPK、CREB基因表达及其磷酸化蛋白表达升高(P0.05)。经过五周治疗后,针刺组及非经非穴组大鼠与模型组相比,p38MAPK、CREB基因表达及其磷酸化蛋白表达量均有所降低(P0.05),与非经非穴组相比,针刺组降低明显,具有统计学意义(P0.05)。3.电针对Dahl盐敏感大鼠肾组织α-SMA、FN基因表达的影响模型组大鼠与正常组大鼠相比,肾组织中α-SMA、FN基因表达均升高(P0.05)。经过五周治疗后,针刺组及非经非穴组大鼠与模型组相比,α-SMA、FN基因表达均有所降低(P0.05),与非经非穴组相比,针刺组降低明显,具有统计学意义(P0.05)。结论:1.电针双侧“足三里”“曲池”穴有延缓高血压前期大鼠肾脏细胞外基质沉积的作用。2.延缓的作用机制可能与抑制TGF-β1介导的p38MAPK信号传导通路上的相关基因及磷酸化蛋白的表达有关。3.其中TGF-β1、p38MAPK、CREB、FN及α-SMA是其关键的作用点。
[Abstract]:Objective: to detect the expression of genes and proteins related to p38MAPK signal transduction pathway mediated by transforming growth factor - beta 1 (transforming growth factor-beta1, TGF- beta 1) in the prehypertensive rat model of salt sensitive rats by real-time quantitative PCR and Western boltting. 8 p38mitogen-activated protein kinase (p38MAPK), C AMP reactive element binding protein (C AMP-responsive element binding protein, CREB), and the expression of alpha smooth muscle actin, fibronectin gene, and the effect of acupuncture on prehypertensive rats The mechanism of the inhibition of extracellular matrix deposition in the kidney and understanding the process of the occurrence and development of hypertension from a deeper level. Materials and methods: the experimental animals were divided into four groups according to the purpose of the experiment, which were the normal control group, the model group, the acupuncture group and the non non acupoint group. All the experimental animals were 40 healthy male rats, including 3 of the healthy male rats. 0 Dahl salt sensitive rats (Dahl salt sensitive rats, DS) were given high salt diet after feeding their blood pressure. They were divided into three groups according to the random number table method. 10 rats in each group were recorded as model, acupuncture and non non acupoint group. The other 10 were Dahl salt resistant rats (Dahl salt resistant rats, DR), and given high salt feed after feeding blood. The pressure was stable and constant. The rats in each group were kept in cage and fed for 7 days, and the high salt feed was fed without abnormality and the blood pressure was measured every day. After the blood pressure of the salt sensitive rats was raised 10-20mm Hg, the high salt feeding was stopped and the diet was changed to the ordinary feed. The acupuncture group was treated with the acupuncture of "Zusanli" "Zusanli" "Qu Chi" (3-5mm) and electroacupuncture (20min). The needle was used as an electric needle for a slight tremor of the needle handle, 1 times a day, 6 times a week for five weeks, and five weeks in the treatment of non acupoint acupuncture rats on the bilateral iliac crest on the bilateral iliac crest, and the 20mm fixed control point beside the middle line, and the same density wave was used to connect the electroacupuncture 20min, frequency. The treatment date was the same as that in the acupuncture group with the slight tremor of the needle handle. The blood pressure of the rats was detected once a week and the end of the acupuncture treatment was stopped. When the material was taken, 10% chloral chloral hydrate (0.35ml) was injected into the rats. After the rats were completely anaesthetized, the abdominal median incision was fully exposed and the kidneys were fully exposed and the double kidneys were excised after the kidney was excised and went to the bag. The membrane was cut along the maximum coronal section, and a part of the kidney tissue was taken as homogenate to extract the total RNA of kidney tissue. The expression of TGF- beta 1, p38MAPK, CREB, FN and alpha -SMAm RNA in kidney was detected by real-time quantitative PCR. The expression of p38MAPK and CREB phosphorylated protein in renal tissue was detected by protein immunoblotting. Results: 1. electroacupuncture was used for Dahl. The expression of TGF- beta 1 gene in renal tissue of salt sensitive rats was affected by the increase of TGF- beta 1 gene expression in the renal tissue (P0.05). After five weeks of treatment, the expression of TGF- beta 1 in the acupuncture group and the non non acupoint group was lower than that in the model group (P0.05). Compared with the non non acupoint group, the acupuncture group decreased the brightness. The effect of (P0.05).2. electroacupuncture on the p38MAPK signaling pathway of renal tissue in Dahl salt sensitive rats model group, compared with normal rats, the p38MAPK, CREB gene expression and phosphorylated protein expression increased (P0.05) in the renal tissue. After five weeks of treatment, the acupuncture group and the non non acupoint group were compared with the model group, p38MAPK, The expression of CREB gene and the expression of phosphorylated protein decreased (P0.05). Compared with the non acupoint group, the acupuncture group decreased obviously and had statistically significant (P0.05).3. electroacupuncture on the renal tissue of the Dahl salt sensitive rat renal tissue alpha -SMA, and the expression of FN gene in the model group was higher than that in the normal group, and the expression of alpha -SMA and FN gene in the renal tissue increased (P0). .05) after five weeks of treatment, the expression of alpha -SMA and FN genes in the acupuncture group and the non acupoint group were lower than that in the model group (P0.05). Compared with the non acupoint group, the acupuncture group decreased obviously and had statistical significance (P0.05). Conclusion: 1. electroacupuncture bilateral "foot three li" "Qu Chi" point postpones the renal extracellular base of prehypertensive rats. The mechanism of.2. delayed action may be related to the inhibition of the related genes and the expression of phosphorylated protein on the p38MAPK signaling pathway mediated by TGF- beta 1, and.3. in which TGF- beta 1, p38MAPK, CREB, FN and alpha -SMA are the key points of action.
【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】：2017
【分类号】：R245

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