艾灸通过诱导热休克蛋白70表达抑制大鼠胃黏膜细胞凋亡线粒体信号转导途径的研究
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摘要
目的:①通过观察艾灸预处理对健康大鼠不同组织器官HSP70表达的影响,探讨艾灸防病保健理论的分子生物学基础;②通过观察槲皮素灌胃阻断HSP70合成后,艾灸预保护作用是否依然存在,进一步明确艾灸保护作用与HSP70表达的关系;③通过观察艾灸诱导的HSP70对急性胃黏膜损伤大鼠胃黏膜细胞凋亡及其线粒体信号转导途径中关键物质细胞色素C(Cyt-c)、凋亡激活因子-1(Apaf-1)、Caspase-9和Caspase-3的影响,探讨艾灸预处理抑制胃黏膜细胞凋亡、保护胃黏膜损伤作用的内在机制。
方法:本研究分为三个部分。第一部分实验:32只wister健康大鼠随机分为4组,即空白组、捆绑组、艾灸穴位组、艾灸非穴组,8只/组。采用免疫组化方法检测大鼠心肌、脑组织、胃黏膜HSP70蛋白表达的变化,实时定量RT-PCR方法检测大鼠胃黏膜HSP70mRNA的表达。第二部分实验:40只wister健康大鼠随机分为5组,即捆绑组、模型组、艾灸穴位+模型组、槲皮素+艾灸穴位+模型组、槲皮素+模型组,8只/组。采用无水乙醇灌胃方法制备急性胃黏膜损伤大鼠模型,Guth评分法计算大鼠胃黏膜损伤指数(UI),免疫印迹(Western-blot)方法检测大鼠胃黏膜HSP70表达的变化,原位末端标记(TUNEL)方法检测大鼠胃黏膜细胞凋亡。第三部分实验:32只wister健康大鼠随机分为4组,即捆绑组、模型组、艾灸穴位+模型组、艾灸非穴+模型组,8只/组。采用Western-blot方法检测大鼠胃黏膜Cyt-c的表达,免疫组化方法检测大鼠胃黏膜HSP70、细胞凋亡、Apaf-1、Caspase-9和Caspase-3的表达。
结果:
(1)艾灸足三里、梁门穴可诱导健康大鼠心肌、脑组织、胃黏膜HSP70蛋白表达及胃黏膜HSP70mRNA表达上调,与空白组、捆绑组和艾灸非穴组相比,差异具有显著性或非常显著性意义(P＜0.05或P＜0.01)。
(2)与模型组相比,艾灸穴位+模型组大鼠胃黏膜损伤指数(UI)和细胞凋亡均明显减小,HSP70表达则显著升高,差异非常明显(P＜0.05或P＜0.01)。
(3)槲皮素阻断HSP70合成后,槲皮素+艾灸+模型组大鼠胃黏膜UI和AI明显高于艾灸+模型,且HSP70表达明显低于艾灸+模型组,差异具有非常显著性意义(P＜0.01)。
(4)艾灸足三里、梁门穴可以下调急性胃黏膜损伤大鼠胃黏膜细胞胞浆Cyt-c和Apaf-1含量及Caspase-9和Caspase-3的表达,同时上调HSP70表达,降低大鼠胃黏膜细胞凋亡指数(AI),与模型组和艾灸非穴+模型组比较具有显著性或非常显著性差异(P＜0.05或P＜0.01)。
结论:
(1)艾灸预处理可诱导保护性蛋白HSP70在不同组织器官表达上调可能是艾灸“防病保健”作用的物质基础。
(2)艾灸预处理可以抑制大鼠损伤胃黏膜细胞凋亡、减轻急性胃黏膜损伤,对胃黏膜产生预保护作用与艾灸诱导的HSP70表达直接相关。
(3)艾灸通过诱导HSP70表达抑制细胞凋亡线粒体信号转导途径中的不同靶点是艾灸预处理抑制急性胃黏膜损伤细胞凋亡、保护胃黏膜损伤作用的内在机制。
(4)艾灸对胃黏膜保护作用具有一定的穴位特异性,反映了艾灸-穴位-经络的“综合效用”结果。
Objective:To observe the effects of pre-moxibustion on the expression of heat shock protein 70(HSP70) in gastric mocosa of acute damnification of gastric rats.To observe the effects about HSP70 of moxibustion on apoptosis mitochondrial pathway and the content of Cytochrome C(Cyt-c)、apoptotic protease-activating factor-1(Apaf-1)、cysteine aspartic acic specific protease-9(Caspase -9)、Caspase-3.To explore the relationship between the apoptosis mitochondrial pathway and HSP70,and the mechanism of pre-moxibustion at the acupoints of Foot-Yangming meridian in preventing gastric mucosa systemly.
Methods:There are three sections of this problem.
The first section:Thirty-two healthy Wister rats were randomly assigned to blank,binding,moxibustion at "Zusanli" and "Liangmen" and moxibustion at control points groups.Assaying the content of HSP70 in heart、brain、gastric mucosa by immunohistochemical method and observing the express HSP70mRNA in gastric mucosa by Real-Time PCR method.
The second section:Forty healthy Wister rats were randomly assigned to binding,model,moxibustion+model,quercetin+moxibustion+model,quercetin +model groups.Ethanol lavage in stomach method was adopted for acute damnification of gastric mucosa rat model.After quercetin blocking HSP70 synthesis,we observed the expression of ulser index(UI) by Guth method, apoptosim index(AI) by TUNEL method,and HSP70 by western-blot method.
The third section:Thirty-two healthy Wister rats were randomly assigned to binding,model,moxibustion point+model,moxibustion control point+model groups.Assaying the content of Cyt-c by western-blot method and observing HSP70、AI、Apaf-1、Caspase-9、Caspase-3 by immunohistochemical method in gastric mucosa.
Results:After treated with ethanol,the UI and AI increased,the expression of HSP70 in gastric mucosa decreased(P＜0.05 or P＜0.01).At the same time, the content of Cyt-c、Apaf-1、Caspase-9、Caspase-3 increased highly(P＜0.05 or P＜0.01) compared with binding group.
While treated with pre-moxibustion at"Zusanli" "Liangmen",the UI and AI in gastric mucosa significantly decreased,the expression of HSP70 in gastric mucosa increased clearly(P＜0.05 or P＜0.01).At the same time,the content of Cyt-c、Apaf-1、Caspase-9、Caspase-3 decreased highly(P＜0.05 or P＜0.01) compared with model and moxibustion control point+model group.
Conclusion:Moxibustion at "Zusanli" "Liangmen" had the protective effects on gastric mucosa of acute damnification in rat.And the mechanism of inhibition apoptosis may be promoting the synthesis HSP70,decreasing the content of Cyt-c、Apaf-1、Caspase-9、Caspase-3 so as to suppressing the cell apoptosis mitochondrial pathway.
方法:本研究分为三个部分。第一部分实验:32只wister健康大鼠随机分为4组,即空白组、捆绑组、艾灸穴位组、艾灸非穴组,8只/组。采用免疫组化方法检测大鼠心肌、脑组织、胃黏膜HSP70蛋白表达的变化,实时定量RT-PCR方法检测大鼠胃黏膜HSP70mRNA的表达。第二部分实验:40只wister健康大鼠随机分为5组,即捆绑组、模型组、艾灸穴位+模型组、槲皮素+艾灸穴位+模型组、槲皮素+模型组,8只/组。采用无水乙醇灌胃方法制备急性胃黏膜损伤大鼠模型,Guth评分法计算大鼠胃黏膜损伤指数(UI),免疫印迹(Western-blot)方法检测大鼠胃黏膜HSP70表达的变化,原位末端标记(TUNEL)方法检测大鼠胃黏膜细胞凋亡。第三部分实验:32只wister健康大鼠随机分为4组,即捆绑组、模型组、艾灸穴位+模型组、艾灸非穴+模型组,8只/组。采用Western-blot方法检测大鼠胃黏膜Cyt-c的表达,免疫组化方法检测大鼠胃黏膜HSP70、细胞凋亡、Apaf-1、Caspase-9和Caspase-3的表达。
结果:
(1)艾灸足三里、梁门穴可诱导健康大鼠心肌、脑组织、胃黏膜HSP70蛋白表达及胃黏膜HSP70mRNA表达上调,与空白组、捆绑组和艾灸非穴组相比,差异具有显著性或非常显著性意义(P＜0.05或P＜0.01)。
(2)与模型组相比,艾灸穴位+模型组大鼠胃黏膜损伤指数(UI)和细胞凋亡均明显减小,HSP70表达则显著升高,差异非常明显(P＜0.05或P＜0.01)。
(3)槲皮素阻断HSP70合成后,槲皮素+艾灸+模型组大鼠胃黏膜UI和AI明显高于艾灸+模型,且HSP70表达明显低于艾灸+模型组,差异具有非常显著性意义(P＜0.01)。
(4)艾灸足三里、梁门穴可以下调急性胃黏膜损伤大鼠胃黏膜细胞胞浆Cyt-c和Apaf-1含量及Caspase-9和Caspase-3的表达,同时上调HSP70表达,降低大鼠胃黏膜细胞凋亡指数(AI),与模型组和艾灸非穴+模型组比较具有显著性或非常显著性差异(P＜0.05或P＜0.01)。
结论:
(1)艾灸预处理可诱导保护性蛋白HSP70在不同组织器官表达上调可能是艾灸“防病保健”作用的物质基础。
(2)艾灸预处理可以抑制大鼠损伤胃黏膜细胞凋亡、减轻急性胃黏膜损伤,对胃黏膜产生预保护作用与艾灸诱导的HSP70表达直接相关。
(3)艾灸通过诱导HSP70表达抑制细胞凋亡线粒体信号转导途径中的不同靶点是艾灸预处理抑制急性胃黏膜损伤细胞凋亡、保护胃黏膜损伤作用的内在机制。
(4)艾灸对胃黏膜保护作用具有一定的穴位特异性,反映了艾灸-穴位-经络的“综合效用”结果。
Objective:To observe the effects of pre-moxibustion on the expression of heat shock protein 70(HSP70) in gastric mocosa of acute damnification of gastric rats.To observe the effects about HSP70 of moxibustion on apoptosis mitochondrial pathway and the content of Cytochrome C(Cyt-c)、apoptotic protease-activating factor-1(Apaf-1)、cysteine aspartic acic specific protease-9(Caspase -9)、Caspase-3.To explore the relationship between the apoptosis mitochondrial pathway and HSP70,and the mechanism of pre-moxibustion at the acupoints of Foot-Yangming meridian in preventing gastric mucosa systemly.
Methods:There are three sections of this problem.
The first section:Thirty-two healthy Wister rats were randomly assigned to blank,binding,moxibustion at "Zusanli" and "Liangmen" and moxibustion at control points groups.Assaying the content of HSP70 in heart、brain、gastric mucosa by immunohistochemical method and observing the express HSP70mRNA in gastric mucosa by Real-Time PCR method.
The second section:Forty healthy Wister rats were randomly assigned to binding,model,moxibustion+model,quercetin+moxibustion+model,quercetin +model groups.Ethanol lavage in stomach method was adopted for acute damnification of gastric mucosa rat model.After quercetin blocking HSP70 synthesis,we observed the expression of ulser index(UI) by Guth method, apoptosim index(AI) by TUNEL method,and HSP70 by western-blot method.
The third section:Thirty-two healthy Wister rats were randomly assigned to binding,model,moxibustion point+model,moxibustion control point+model groups.Assaying the content of Cyt-c by western-blot method and observing HSP70、AI、Apaf-1、Caspase-9、Caspase-3 by immunohistochemical method in gastric mucosa.
Results:After treated with ethanol,the UI and AI increased,the expression of HSP70 in gastric mucosa decreased(P＜0.05 or P＜0.01).At the same time, the content of Cyt-c、Apaf-1、Caspase-9、Caspase-3 increased highly(P＜0.05 or P＜0.01) compared with binding group.
While treated with pre-moxibustion at"Zusanli" "Liangmen",the UI and AI in gastric mucosa significantly decreased,the expression of HSP70 in gastric mucosa increased clearly(P＜0.05 or P＜0.01).At the same time,the content of Cyt-c、Apaf-1、Caspase-9、Caspase-3 decreased highly(P＜0.05 or P＜0.01) compared with model and moxibustion control point+model group.
Conclusion:Moxibustion at "Zusanli" "Liangmen" had the protective effects on gastric mucosa of acute damnification in rat.And the mechanism of inhibition apoptosis may be promoting the synthesis HSP70,decreasing the content of Cyt-c、Apaf-1、Caspase-9、Caspase-3 so as to suppressing the cell apoptosis mitochondrial pathway.
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