發(fā)布時間：2017-12-28 06:41

  本文關(guān)鍵詞：電針“內(nèi)關(guān)”預(yù)處理對心肌缺血再灌注大鼠p38MAPK信號通路的影響及機(jī)制研究　出處：《湖北中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 電針預(yù)處理 心肌缺血再灌注 p38絲裂原激活蛋白激酶信號通路

【摘要】：目的利用電針的低創(chuàng)傷、易操作和可控性強(qiáng)的優(yōu)勢,結(jié)合針灸經(jīng)典理論“心胸內(nèi)關(guān)謀”,并借助已有的關(guān)于內(nèi)關(guān)-心臟相關(guān)性的研究基礎(chǔ),觀察電針“內(nèi)關(guān)”預(yù)處理對心肌缺血再灌注大鼠早期心功能參數(shù)、心室重構(gòu)、心肌梗死情況,血清及心肌相關(guān)因子、線粒體呼吸功能的影響,并借用p38MAPK通路阻滯劑SB203580從p38MAPK信號通路的角度分析其作用機(jī)制,為臨床各型I/R的防治方法提供新的理論探討和實(shí)驗基礎(chǔ)。方法選用SPF級雄性Wistar大鼠200只,取材前體重180g-220g,采用隨機(jī)數(shù)字表法,將大鼠隨機(jī)分為五組:假手術(shù)組(Shame operation group,S組)、模型組(Model group,M組)、電針預(yù)處理組(electroacupuncture pretreatment group,EA組)、抑制劑預(yù)處理組(SB203580 group,SB組)和電針+抑制劑預(yù)處理組(electroacupuncture pretreatment+SB203580 group,EA+SB組)。假手術(shù)組:常規(guī)飼養(yǎng),捆綁1次/天,共7天,第8天手術(shù)過程中,只開胸穿線,不結(jié)扎左冠狀動脈前降支;模型組:捆綁1次/天,共7天,第8天通過采用推管法行左冠狀動脈缺血再灌注術(shù),制備在體心肌缺血再灌注模型;抑制劑預(yù)處理組:捆綁1次/天,共7天,第8天將p38MAPK抑制劑SB203580溶于二甲基亞砜成20%溶液,在造模前給予0.3mg/kg皮下注射,后造模過程同模型組;電針預(yù)處理組:捆綁1次/天。電針雙側(cè)內(nèi)關(guān)穴操作,直刺深度約5mm,采用HANS-200型電針治療儀,選用連續(xù)波,頻率2Hz,強(qiáng)度1mA,電針預(yù)處理1次/天,通電治療20min。共7天,后造模過程同模型組;電針+SB203580預(yù)處理組:捆綁1次/天,共7天,電針預(yù)處理過程同電針預(yù)處理組,第8天造模前給予抑制劑SB203580皮下注射,過程同抑制劑預(yù)處理組,后造模過程同模型組。復(fù)灌即刻,利用生物信號及壓力測試系統(tǒng)(BL-Newcentury 410)測定大鼠左心室心功能指標(biāo)(舒張末期壓力、左室收縮期平均壓、短軸縮短率和射血分?jǐn)?shù)),后處死大鼠,取血清待檢,剪取心臟,稱取梗死區(qū)重量和全心重量,分別計算心肌梗死區(qū)重量和全心重量的比值。ttc染色法分析心肌梗死面積,采用全自動生化分析儀檢測血清肌酸激酶(ck)、乳酸脫氫酶(ldh)、肌鈣蛋白i(ctni),采用比色法檢測ros含量,采用黃嘌呤氧化法檢測心肌超氧化物歧化酶(sod)活性,硫代巴比妥酸法檢測心肌丙二醛(mda)含量,高效液相色譜法計算心肌組織中atp的含量。利用激光共聚焦顯微鏡、he染色和透射電鏡觀察大鼠心肌細(xì)胞尤其是線粒體的形態(tài)變化。結(jié)果(1)m組的梗死面積和梗死程度的比值,與s組相比較,均顯著升高(p0.05),sb組、ea組和ea+sb組的梗死面積和梗死程度的比值均較m組有降低,差異有統(tǒng)計學(xué)意義(p0.05),ea組、ea+sb組和sb組之間無顯著性差異(p0.05)。(2)m組的lvedp與s組相比較,顯著升高(p0.05),sb組、ea組和ea+sb組的lvedp均較m組顯著降低,差異有統(tǒng)計學(xué)意義(p0.05),ea組、ea+sb組和sb組之間無顯著性差異(p0.05)。m組的lvsp、fs、ef值與s組相比較,顯著升高(p0.05),sb組、ea組和ea+sb組的lvsp、fs、ef值均較m組顯著升高,差異有統(tǒng)計學(xué)意義(p0.05),ea組、ea+sb組和sb組之間無顯著性差異(p0.05)。(3)m組的ldh、ck及ctni與s組相比較,顯著升高(p0.05),sb組、ea組和ea+sb組的ldh、ck及ctni均較m組顯著降低,差異有統(tǒng)計學(xué)意義(p0.05),ea組的ldh、ck與ea+sb組和sb組之間無顯著性差異(p0.05)。ea組和ea+sb組的ctni與sb組比較,有顯著降低,差異有統(tǒng)計學(xué)意義(p0.05)。(4)m組的tnf-α、il-1β和il-6與s組相比較,顯著升高(p0.05),sb組、ea組和ea+sb組的tnf-α、il-1β和il-6均較m組顯著降低,差異有統(tǒng)計學(xué)意義(p0.05),ea組的tnf-α、il-1β和il-6與ea+sb組和sb組比較,顯著降低,差異有統(tǒng)計學(xué)意義(p0.05)。m組的il-4和il-10與s組比較,差異無統(tǒng)計學(xué)意義(p0.05)。sb組、ea組和ea+sb組的il-4和il-10與m組比較,有顯著性差異(p0.05)。各治療組之間差異性無統(tǒng)計學(xué)意義(p0.05)。(5)m組的icam-1和vcam-1與s組相比較,顯著升高(p0.05),sb組、ea組和ea+sb組的icam-1和vcam-1均較m組顯著降低,差異有統(tǒng)計學(xué)意義(p0.05),ea組的icam-1和vcam-1明顯低于ea+sb組和sb組,差異有統(tǒng)計學(xué)意義(p0.05)。(6)m組的ros和mda與s組相比較,顯著升高(p0.05),sb組、ea組和ea+sb組的ros和mda均較m組顯著降低,差異有統(tǒng)計學(xué)意義(p0.05),ea組的ros和mda明顯低于ea+sb組和sb組,差異有統(tǒng)計學(xué)意義(p0.05)。m組的sod和atp與s組相比較,顯著降低(p0.05),sb組、ea組和ea+sb組的sod和atp均較m組顯著升高,差異有統(tǒng)計學(xué)意義(p0.05),ea組的ros和mda明顯高于ea+sb組和sb組,差異有統(tǒng)計學(xué)意義(p0.05)。(7)與s組比較,m組、ea組、sb組及ea+sb組大鼠心肌細(xì)胞na+-k+-atpase和ca2+-atpase的活性明顯下降(p0.05),ea組、sb組及ea+sb組大鼠心肌細(xì)胞na+-k+-atpase和ca2+-atpase的活性較m組明顯提高(p0.05),ea組中na+-k+-atpase和ca2+-atpase的改善明顯優(yōu)于sb組及ea+sb組,差異具有統(tǒng)計學(xué)意義(p0.05)。(8)m組的mkk3/6、p38及p-p38蛋白表達(dá)與s組相比較,顯著升高(p0.05),sb組、ea組和ea+sb組的mkk3/6、p38及p-p38蛋白表達(dá)均較m組顯著降低,差異有統(tǒng)計學(xué)意義(p0.05),ea組的mkk3/6、p38及p-p38蛋白表達(dá)數(shù)值低于ea+sb組和sb組,但差異無統(tǒng)計學(xué)意義(p0.05)。(9)s組正常心肌纖維排列規(guī)則,細(xì)胞結(jié)構(gòu)完整,細(xì)胞核及線粒體形態(tài)和數(shù)量正常,線粒體嵴清晰,心肌間未見異常。m組心肌肌纖維出現(xiàn)溶解斷裂甚至壞死,間質(zhì)增大,腫脹明顯。部分線粒體固縮變小,部分線粒體腫脹、或外膜破損。線粒體嵴不規(guī)則、斷裂成絮狀或缺失。EA組、SB組及EA+SB組肌纖維間隙輕度增寬,壞死灶減輕,心肌細(xì)胞輕微腫脹,細(xì)胞核及線粒體結(jié)構(gòu)基本完整,小部分線粒體仍存在空泡樣病變。結(jié)論1.電針“內(nèi)關(guān)”預(yù)處理可使I/R模型大鼠心肌梗死面積和梗死程度明顯下降,可減輕線粒體腫脹和壞死程度,減輕心肌細(xì)胞間炎癥因子浸潤,病理形態(tài)學(xué)檢測提示電針可顯著改善受損心肌組織的病理損害程度。2.電針“內(nèi)關(guān)”預(yù)處理可使I/R模型大鼠LVEDP降低,LVSP、FS和EF升高,可降低血清中LDH、CK及cTnI的含量,保護(hù)大鼠心肌組織,改善大鼠心功能。3.電針“內(nèi)關(guān)”預(yù)處理可以抑制TNF-a、IL-1β、IL-6等炎性因子的表達(dá),增加IL-4和IL-10等抗炎因子的表達(dá),可以下調(diào)心肌粘附分子ICAM-1和VCAM-1的蛋白表達(dá),通過調(diào)控炎癥反應(yīng)相關(guān)因子保護(hù)受損心肌組織。4.電針“內(nèi)關(guān)”預(yù)處理可以減少I/R模型大鼠心肌細(xì)胞ROS及MDA的含量,提高的ATP含量,激活SOD和ATP酶的活性,改善RCR,通過保護(hù)線粒體結(jié)構(gòu)和功能的完整性來減輕I/R中心肌損傷。5.MKK3/6/p38MAPK信號傳導(dǎo)通路參與在體I/R模型大鼠的病理過程,I/R中大鼠MKK3/6和p38MAPK表達(dá)上調(diào),電針“內(nèi)關(guān)”預(yù)處理可以明顯抑制I/R中MKK3/6、p38MAPK的蛋白表達(dá),抑制p38MAPK的磷酸化,對心肌細(xì)胞p38信號轉(zhuǎn)導(dǎo)通路的調(diào)節(jié)可能只是電針“內(nèi)關(guān)”預(yù)處理保護(hù)I/R,發(fā)揮其抗心肌缺血的作用機(jī)制之一。6.電針組與p38抑制劑組和電針+p38抑制劑組相比較,對I/R大鼠模型相關(guān)指標(biāo)結(jié)果的調(diào)控上可見明顯差異,但p38抑制劑組和電針+p38抑制劑組之間并未出現(xiàn)明顯疊加效應(yīng),說明電針內(nèi)關(guān)預(yù)處理對I/R的保護(hù)機(jī)制可能是多層次、多途徑的綜合效應(yīng)。
[Abstract]:Objective to use electroacupuncture low trauma, easy operation and high controllability advantages, combined with the classical theory of "mind in Guan acupuncture plan, and with existing on the acupoint Neiguan-heart correlation research foundation, to observe the effect of electroacupuncture at Neiguan on myocardial ischemia reperfusion in rats with early heart function, ventricular remodeling, myocardial infarction the situation, related factors, effects of serum and myocardial mitochondrial respiratory function, and p38MAPK pathway blocker SB203580 to analyze the mechanism of p38MAPK signaling pathway from the angle, to provide theoretical and experimental basis for the new control methods of various types of clinical I/R. Methods male Wistar SPF rats were taken before 200, weight 180g-220g, were randomly, the rats were randomly divided into five groups: sham operation group (Shame operation, group, S group), model group (Model group, M group), electroacupuncture pretreatment group (electroacupuncture pretreatment, group, EA group), preconditioning group (SB203580 inhibitor group, SB group) and electroacupuncture + inhibitor pretreatment group (electroacupuncture pretreatment+SB203580, group, EA+SB group). Sham operation group: conventional breeding, tied 1 times / day, 7 days, eighth days during the surgery, only thoracotomy threading, without ligating the left anterior descending coronary artery; model group: bind 1 times / day, 7 days, eighth days by pushing pipe for the left coronary artery ischemia perfusion, preparation of myocardial ischemia reperfusion model; inhibitor pretreatment group: bind 1 times / day, a total of 7 days, the p38MAPK inhibitor SB203580 dissolved in two dimethyl sulfoxide into 20% solution for eighth days, given subcutaneous injection of 0.3mg/kg before modeling, after modeling process is the same as the model group; Electroacupuncture pretreatment group: bind 1 times / day. Electroacupuncture at Neiguan point operation, puncture depth of about 5mm, using HANS-200 type electric acupuncture apparatus, using continuous wave, frequency of 2Hz and intensity 1mA, electroacupuncture pretreatment 1 times / day, electricity treatment 20min. A total of 7 days later, the modeling process was the same as the model group. The electroacupuncture +SB203580 pretreatment group was bound for 1 days / day for 7 days, the EA pretreatment process was combined with the electro acupuncture pretreatment group, and eighth days before the injection, the inhibitor SB203580 was injected subcutaneously, the process was the same as the inhibitor pretreatment group, and the modeling process was the same as the model group. Reperfusion immediately, using biological signal and pressure test system (BL-Newcentury 410) to measure the cardiac function indexes of rats (left ventricular end diastolic pressure, left ventricular systolic pressure, average shortening and ejection fraction), after the rats were killed, serum to be detected, their heart, weigh the infarction area and heart weight the weight ratio were calculated infarct weight and heart weight. Analysis of myocardial infarction area by TTC staining, serum creatine kinase detection automatic biochemical analyzer (CK), lactate dehydrogenase (LDH), troponin I (cTnI), the ratio of ROS content detection method to detect myocardial superoxide dismutase by xanthine oxidation (SOD) activity, detection of myocardial malondialdehyde thiobarbituric acid method (MDA) content, calculate the contents of myocardial ATP by hplc. The morphological changes of rat cardiac myocytes, especially mitochondria, were observed by laser confocal microscopy, he staining and transmission electron microscopy. Results (1) ratio of M group, infarct size and degree of infarction, compared with the s group, were significantly increased (P0.05), the ratio of sb group, EA group and ea+sb group the infarct size and degree of infarction were compared with the M group decreased, the difference was statistically significant (P0.05), no significant difference between EA group, ea+sb group and Sb group (P0.05). (2) LVEDP in group M increased significantly compared with group s (P0.05), LVEDP in sb group, EA group and ea+sb group was significantly lower than that in M group, the difference was statistically significant (P0.05), but there was no significant difference between the EA group, the M group and the control group. The values of LVSP, FS and EF in group M were significantly higher than those in s group (P0.05), and the values of LVSP, FS and FS in sb group, EA group and ea+sb group were significantly higher than those in group A. There was no significant difference between them. (3) LDH, CK and cTnI in group M were significantly higher than those in s group (P0.05), and the LDH, CK and LDH in sb group, EA group and ea+sb group were significantly lower than those in group A. There was no significant difference between them. The cTnI and Sb in group EA and group ea+sb were significantly lower than those in group sb, and the difference was statistically significant (P0.05). (4) M group tnf- alpha, IL-1 beta and IL-6 compared with the s group, was significantly increased (P0.05), Sb group, EA group and ea+sb group of tnf- alpha, IL-1 beta and IL-6 were significantly lower than those in M group, the difference was statistically significant (P0.05), EA group, tnf- alpha, IL-1 beta and IL-6 compared with ea+sb group and Sb group significantly decreased, the difference was statistically significant (P0.05). There was no significant difference in IL-4 and IL-10 between group M and group s (P0.05). There were significant differences in IL-4 and IL-10 between group sb, group EA and group ea+sb compared with that of group M (P0.05). There was no significant difference between the treatment groups (P0.05). (5) ICAM-1 and VCAM-1 in group M increased significantly compared with group s (P0.05), and ICAM-1 and VCAM-1 in sb group, EA group and ea+sb group were significantly lower than those in group C. The difference was statistically significant (P0.05). (6) ROS and MDA in group M increased significantly compared with group s (P0.05), and ROS and MDA in sb group, EA group and ea+sb group were significantly lower than those in group C. The difference was statistically significant (P0.05). The SOD and ATP in group M were significantly lower than those in group s (P0.05). The SOD and ATP in sb group, EA group and ea+sb group were significantly higher than those in group C. The difference was statistically significant (P0.05). (7) compared with group s, the activities of na+-k+-atpase and ca2+-atpase in M group, EA group, Sb group and ea+sb group were significantly decreased (P0.05), and the activities of myocardial cells in EA group, Sb group and ea+sb group were significantly higher than those in group A.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R245

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