摘要
目的：以中医经络学说中经脉-脏腑相关理论为指导,观察电针足阳明经(穴)对大鼠胃黏膜损伤的修复效应、大鼠胃黏膜细胞表皮生长因子受体(EGFR)、细胞外调节蛋白激酶(ERK)和c-myc表达的影响,从器官、细胞、分子水平多层次系统地探讨“足阳明经与胃相关”的物质基础以及电针足阳明经(穴)参与胃黏膜损伤修复的信号转导途径。
方法：采用水浸束缚应激法(WRS)制作应激性大鼠胃黏膜损伤模型,以电针足阳明经“足三里”、“梁门”、“四白”穴为施治因素,按Guth等标准记录大鼠胃黏膜损伤指数,光镜下观察胃黏膜组织形态学变化；采用免疫组化和逆转录聚合酶链反应法(RT-PCR)检测胃黏膜细胞EGFR蛋白和基因的表达；免疫组化和免疫印迹法(Western blot)检测胃黏膜细胞ERK磷酸化水平；RT-PCR法检测胃黏膜细胞c-myc基因的表达。
结果：①经造模后,模型组大鼠胃黏膜损伤指数最高,正常组大鼠胃黏膜损伤指数最低,两组间差异有非常显著性意义(p<0.01),说明造模是成功的；电针胃经组和电针胆经组大鼠胃黏膜损伤指数均明显降低,与模型组比较有非常显著性差异(P＜0.01),其中电针胃经组大鼠胃黏膜损伤指数降低最为明显,与电针胆经组比较有非常显著性差异(P＜0.01)；
②在电针足阳明经(穴)后提取的不同稀释度血清对大鼠胃黏膜细胞EGFR表达差异影响中,1／10胃经血清组大鼠胃黏膜细胞EGFR表达与1／2、1／5和1／20胃经血清组比较均有非常显著性差异(P<0.01),大鼠胃黏膜细胞EGFR表达强弱依次为：1／10胃经血清组>1／20胃经血清组>1／5胃经血清组>1／2胃经血清组；
③在与电针足阳明经(穴)或足少阳经(穴)提取的血清孵育后,胃经组和胆经组大鼠胃黏膜细胞EGFR及其基因的表达均明显增强,与正常组、模型组比较均有非常显著性差异(P<0.01)；其中胃经组大鼠胃黏膜细胞EGFR及其基因的表达又最为明显,与胆经组比较有显著性差异(P<0.05)；
④在与电针足阳明经(穴)或电针足少阳经(穴)提取的血清孵育后,胃经组和胆经组大鼠胃黏膜细胞ERK磷酸化、c-myc基因表达均明显升高,与正常组、模型组比较均有非常显著性差异(P<0.01)；胃经组大鼠胃黏膜细胞ERK磷酸化、c-myc基因表达升高最为明显,与胆经组比较均有非常显著性差异(P<0.01)；当用PD153035阻断EGFR后,胃经+PD153035组大鼠胃黏膜细胞ERK磷酸化、c-myc基因表达均明显降低,与胃经组比较均有非常显著性差异(P<0.01)。
结论：①电针足阳明经(穴)可促进大鼠胃黏膜损伤的修复,足阳明经与胃具有相对的特异性联系；
②电针足阳明经(穴)后提取的不同稀释度血清皆能使大鼠胃黏膜细胞EGFR蛋白的表达增强,但存在一定的浓度效应相关性,1／10稀释度血清的效应最为明显；
③电针足阳明经(穴)后提取的血清可提高大鼠胃黏膜细胞EGFR及其基因的表达,EGFR是足阳明经与胃相关的重要物质基础；
④电针足阳明经(穴)后提取的血清可提高大鼠胃黏膜细胞ERK磷酸化和c-myc基因表达水平,并且上述效应可随EGFR被阻断而减弱,EGFR是电针足阳明经(穴)对胃黏膜损伤修复的调节位点,ERK和c-myc是足阳明经与胃相关的重要物质基础；
⑤电针足阳明经(穴)对胃黏膜损伤修复的细胞信号转导可能是通过激活EGFR及其ERK信号转导途径而实现的；
⑥利用针刺穴位后所提取的血清与离体器官、组织、细胞相互作用,以探讨针刺效应及其作用机制,值得做进一步更深入的研究。
Objective:Guided by the meridian and viscera relation theory, to observe the electroacupuncture's healing effect on gastric mucosa lesion, EGFR and related signaling molecular ERK, c-myc gene expression in gastric mucosa cell, and to reveal the related material foundation on the theory "A relative between Foot-Yangming meridian and stomach" and the signal transductional approach of acupuncture-related gastric mucosal protective effect.
Methods:Water-immersion and restrained stress methods was adopted for stress-induced gastric mucosa lesion rat model, following that it was respectively to detect:Gastric mucosa lesion index; changes of microstructure histopathologic gastric mucosal tissue; the EGFR expression on gastric mucosal cell was tested by immunohistochemistry and reverse transcription polymerase chain reaction assay(RT-PCR) methods; ERK phosphorylation in gastric mucosal cell was tested by immunohistochemistry and Western blotting methods; the c-myc gene expression was tested in gastric mucosal cell by RT-PCR.
Results:1, Compared with Normal group, the increase of the gastric mucosa lesion index in Model group rats was significant (P<0.01). In both the Stomach meridian group and Gallbladder meridian group, compared with Model group, the reduction of the gastric mucosa lesion index and the rehabilitation of microstructure of gastric mucosal tissue in stress-induced rats were more evident (P<0.01). Furthermore, the effect of electroacupuncture at Foot-Yangming Meridian acupoints were better than that of electroacupuncture at Foot-Shaoyang Meridian acupoints(P<0.01).
2, Compared with 1/10 Stomach meridian serum group, the EGFR proteinic expression on the gastric mucosal cell in the 1/2,1/5 and 1/20 Stomach meridian serum group were more attenuated; The sequence of the EGFR proteinic expression among the groups were as follows:1/10 Stomach meridan serum group> 1/20 Stomach meridan serum group> 1/5 Stomach meridan serum group > 1/2 Stomach meridan serum group.
3, In both Stomach meridian group and Gallbladder meridian group, compared with the Normal group or Model group, the EGFR proteinic and gene expression on the gastric mucosal cell were more reinforcement (P<0.01).Furthermore, the effects in Stomach meridian group were more obvious than that in Gallbladder meridian group(P<0.05).
4, In both Stomach meridian group and Gallbladder meridian group, compared with the Normal group or Model group, the ERK phosphorylation and the c-myc gene expression in the gastric mucosal cell were more reinforcement (P<0.01). Furthermore, the effects in Stomach meridian group were more obvious than those in Gallbladder meridian group(P<0.01). Compared with Stomach meridian group, the reduction of the ERK phosphorylation and the c-myc gene expression in Stomach meridian+PD153035 group were more significant (P<0.01).
Conclusions:1,Electroacupuncture at the acupoints of the Stomach Meridian could have repairing effect on gastric mucosal lesion in stress-induced rats. There is a relative particularity between Foot-Yangming meridian and stomach.
2, The different diluted serum derived from the stress-induced rats treated with electroacupuncture at the acupoints of Foot-Yangming meridian could accelerate the EGFR expression on gastric mucous cell, and there was the correlation of density and effect, compared with the EGFR expression,1/10 stomach meridan serum group brought about the optimal expression.
3, The serum derived from the stress-induced rats treated with electroacupuncture at the acupoints of Foot-Yangming meridian could accelerate the EGFR proteinic and gene expression, and EGFR is the important material basis of "relative particulariy between Foot-Yangming meridian and stomach.
4, The serum derived from the stress-induced rats treated with electroacupuncture at the acupoints of Foot-Yangming meridian could accelerate the ERK phosphorylation and the c-myc gene expression in gastric mucous cell, and the above effects could be attenuated by the PD153035 which is EGFR blocking agent. ERK and c-myc are the important material basis of "relative particulariy between Foot-Yangming meridian and stomach.
5, Thus it indicates that the signaling transduction pathway of the electroacupuncture's healing effect on gastric mucosa lesion is by the approach of EGFR and ERK signaling transduction pathway.
6, It should to have further study of the effect and mechanism of acupuncture by the means of acupuncture serum's reaction on isolated cell, tissure and organ.
引文
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