电针胃经(穴)大鼠胃黏膜修复相关蛋白磷酸化信号转导通路的多途径变化

Changes of multiple protein phosphorylation signal transduction pathways in the process of repairing rat gastric mucosa through stimulation of stomach meridian with electro-acupuncture

背景:近年来研究认为,胃黏膜损伤修复过程有多条信号转导通路参与,并与蛋白质广泛的发生磷酸化与去磷酸化有关,多个实验均从单个通路的角度验证这些观点。目的:课题组设计了全面、动态、定量观察电针足阳明胃经(穴)大鼠胃黏膜修复过程中是否有多个通路的参与的方案,并应用蛋白芯片技术观察相关蛋白磷酸化信号转导通路的多途径变化情况。设计、时间及地点:随机对照动物实验,于2006-11/2008-01在湖南中医药大学实验动物室和国家中医药管理局三级实验室、经穴与脏腑相关重点研究室完成。材料:健康雄性SD大鼠20只,随机选15只用无水乙醇灌胃法制备胃黏膜损伤大鼠模型,剩余5只为空白对照。方法:①空白组与模型组每天只捆绑不针刺。②针刺治疗组针刺大鼠双侧足三里、梁门、四白穴,接上G6805治疗仪,疏密波,频率4/50Hz,输出电压2～4V,强度以肢体轻微颤抖为度,30min/次,1次/d,连续7d。③针刺对照组则在同一时间针刺双侧的足三里、梁门、四白穴内侧约0.3cm的非经非穴点,其余的处理均同治疗对照组。主要观察指标:参照GUTH法行胃黏膜损伤指数;各组间上下调(磷酸化水平变化≥1.5倍)蛋白的归属通路的比较。结果:①模型组胃黏膜损伤指数高于空白组(P<0.01);针刺治疗组胃黏膜损伤指数值显著低于模型组和针刺对照组(P<0.01),而与空白组比较差异无显著性意义。②蛋白芯片分析示:针刺治疗组与针刺对照组均可上调或下调模型组的蛋白磷酸化的水平(≥1.5倍)。针刺治疗组与针刺对照组差异通路比较,磷酸化水平上调1.5倍蛋白中,针刺治疗组激活了6条通路,而针刺对照组只激活了5条通路,其中针刺对照组MAPKS通路缺如,磷酸化水平下调1.5倍蛋白中,针刺治疗组激活了3条通路,而针刺对照组只激活了一条通路,且无同名通路。结论:电针足阳明胃经(穴)修复胃黏膜引起大鼠胃黏膜损伤后修复信号蛋白的多个信号通路的激活,提示电针促胃黏膜修复机制是一个多通路、多水平的调节过程,其修复过程也是多通路、多靶点、多途径共同作用的结果。

BACKGROUND： Researches in the past few years have revealed that multiple signal transduction pathways are involved in the repairing of gastric mucosa in which process the phosphorylation and dephosphorylation of proteins also occur generally, and this has been verified by many experiments aiming at single pathway. OBJECTIVE： A complete, dynamic and quantitative observation plan was designed to verify whether multiple signal transduction pathways take part in the repairing of rat gastric mucosa by stimulation of the Stomach Channel of Foot-Yangming （St） with electro-acupuncture （EA）, and to observe the changes of multiple signal transduction pathways of related protein phosphorylation with protein biochip technology. DESIGN, TIME AND SETTING： The randomized control animal experiment was accomplished in the Experimental Animal Room of Hunan University of Chinese Medicine and the Tertiary Laboratory of State Administration of Traditional Chinese Medicine of China, the Key Laboratory of Correlation between Points on Meridian and Viscera between November 2006 and January 2008. MATERIALS： Twenty healthy male SD rats, out of which 15 ones were used to prepare the models of gastric mucosa injury through administration of absolute alcohol by gavage, and the other 5 were used as controls. METHODS： ①Rats in the blank group and the model group were bound only without any acupuncture stimulation. ②ln the acupuncture treatment （AT） group, rats received 30 minutes of stimulation at their bilateral Zusanli, Liangmen and Sibai via G6805 therapeutic instrument （dilatational wave, 4/50 Hz frequency, 2 4 V output voltage, strong enough to lead to light shiver of limbs） once a day, for 7 days in total. ③In the acupuncture control （AC） group, rats received the same treatment with the treatment control group, except stimulation at the non-points about 0.3 cm inside Zusanfi, Liangrnen and Sibai. MAIN OUTCOME MEASURES： The injury index of gastric mucosa measured by GUTH; Comparisons on pathways of the up-regulated and down-regulated proteins （phosphorylation level changes ≥1.5 times） between the groups. RESULTS：①The injury index of gastric mucosa in the model group was significantly higher than that in the blank group （P 〈 0.01）;The injury index of gastric mucosa in the AT group was obviously lower than that in the model group and the AC group （P 〈 0.01 ）. However, there was no significant difference in the injury index of gastric mucosa between the AT group and the blank group. ②The protein chip analysis indicated that the level of protein phosphorylation in both the AT group and the AC group was either up-regulated or down-regulated （≥1.5 times） compared with the model group. But according to the comparison on the activated pathways in the proteins whose phosphorylation levels were 1.5 times up-regulated, there were 6 pathways activated in the AT group but 5 in the AC group with MAPKS pathway absent. Among many pathways there was only one protein activated which was unable to form the signal pathway. As to the proteins whose phosphorylation levels were 1.5 times down-regulated, 3 pathways were activated in the AT group and only one in the AC group which at the same time was different from that 3 in the AT group. CONCLUSION： Multiple signal pathways of signal proteins get activated when repairing gastric mucosa injury through the stimulation of St with EA, which indicates that the mechanism under repairing gastric mucosa injury with EA involves the regulation of multi-levels and multi-pathways, and the repairing effect is also achieved through multi-pathways, multi-targets and multi-ways.