合募配穴对胃溃疡大鼠代谢物谱表达调节的研究

【作者】 周丹；

【导师】 王富春；

【作者基本信息】 长春中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 目的:通过研究合募配穴对胃溃疡大鼠的血清、尿液代谢物谱的影响,从代谢物谱表达角度分析合募配穴防治胃溃疡的生物学机制,为合募配穴的临床推广提供实验依据。方法:72只Wistar大鼠随机分为6组:空白对照组(A)、捆绑对照组(B)、应激性胃溃疡模型组(C)、募穴组(D)、下合穴组(E)、合募配穴组(F)。各组大鼠依照分组,A组正常喂养,不予处理,B组只捆绑,不针刺,不造模;C组每日只捆绑不针刺;D、E、F组每日捆绑针刺;C、D、E、F组在连续针刺7天后,针刺后采用水浸-束缚应激的方法进行模型复制。观察胃黏膜损伤情况,并对各组大鼠血清和尿液利用LC-MS技术进行不同组间的差异代谢组分分析。结果:1肉眼观察各组大鼠胃黏膜的损伤情况,应激性胃溃疡模型组有数量不等、大小不一的溃疡灶出现。捆绑组与正常组比较有显著性差异(P<0.01),提示捆绑也是一种应激性刺激;模型组与捆绑组比较有差异(P<0.05),提示造模成功;下合穴组、募穴组与模型组比较有差异(P<0.05),合募组穴组与模型组比较有显著性差异(P<0.01),提示单纯针刺足三里穴或中脘穴对胃溃疡模型的治疗有一定防治作用,募穴组、下合穴组与合募配穴组比较有显著性差异(P<0.01),提示合募配穴组防治胃溃疡效果优于募穴组、下合穴组。2通过比较血清代谢物谱发现:捆绑组与空白组比较共有谱峰14个,有10个代谢产物的峰面积(1、5、6、7、8、9、11、12、15、17号峰)有显著性差异(P<0.01)。模型组与捆绑组比较共有谱峰16个,有5个代谢产物的峰面积(4、5、12、13、14号峰)有显著性差异(P<0.01,P<0.05);提示捆绑也是一种应激刺激。三个针刺组与模型组比较存在不同程度的差异:募穴组与与模型组比较共有峰16个,有4个代谢产物的峰面积(2、3、6、8号峰)有显著性差异(P<0.01)。下合穴组与模型组比较共有峰16个,有4个代谢产物的峰面积(2、3、7、8号峰)有显著性差异(P<0.01)16号峰为特异性峰,有可能是新物质。合募配穴组与模型组比较共有峰16个,差异峰1个,有8个代谢产物的峰面积(2、3、5、6、7、8、12、13号峰)有显著性差异(P<0.01),16号峰为特异性峰,有可能是新物质。三个针刺组之间代谢图谱比较:募穴组与下合穴组比较共有峰16个,有5个代谢产物的峰面积(2、3、6、7、15号峰)有显著性差异(P<0.01,P<0.05)。募穴组与合募配穴组比较有9个代谢产物的峰面积(2、3、5、7、8、12、13、14、15号峰)有显著性差异(P<0.01,P<0.05)。下合穴组与合募配穴组比较有9个代谢产物的峰面积(2、3、5、7、8、12、13、14、15号峰)有显著性差异(P<0.01,P<0.05)。提示模型复制后产生应激性损伤后,胃溃疡模型大鼠血清代谢产物与空白组相比有大量新的代谢产物产生,也有消失的代谢产物。针刺对胃溃疡大鼠血清代谢产物有影响,存在新产生的峰、消失的峰及峰面积有差异的代谢产物与针刺防治胃溃疡相关的血清标志性代谢产物。三个针刺组中,以合募配穴组的针刺相关代谢产物最多,提示合募配穴与针刺单穴相比特异性表达明显,有一定的协同效应。3通过比较尿液代谢物谱发现:捆绑组与空白组比较共有谱峰31个,有17个代谢产物的峰面积(1、3、4、7、8、10、13、15、17、18、19、28、34、37、40、41、42号峰)有显著性差异(P<0.01)。9、20号峰为新出现的峰,16、25、33、35、38号峰为消失的峰,这些特异性峰,有可能是捆绑刺激产生的新物质。捆绑组与模型组比较共有谱峰30个,有16个代谢产物的峰面积(1、3、7、10、13、15、17、18、19、20、21、24、28、32、40、41号峰)有差异或显著性差异(P<0.05,P<0.01),其中12个代谢产物与空白组和模型组均有差异,提示捆绑也是一种应激刺激,这12个代谢产物可能是由于应激引起的改变。三个针刺组与模型组比较存在不同程度的差异。募穴组与与模型组比较共有峰共有峰34个,有12个代谢产物的峰面积(1、2、6、7、10、13、15、17、20、22、23、24、29、30、32、36、37、39、40、41号峰)有显著性差异(P<0.01)。5、31、33号峰为新出现的峰,28号峰为消失的峰,这些特异性峰,有可能是新的代谢产物。下合穴组与模型组比较共有峰35个,有13个代谢产物的峰面积(1、7、8、15、18、20、22、25、32、36、37、41、42号峰)有显著性差异(P<0.01),5号峰为新出现的峰,有可能是新的代谢产物。合募配穴组与模型组比较共有峰31个,有10个代谢产物的峰面积(2、10、12、13、17、18、24、37、40、41号峰)有显著性差异(P<0.01),5、9、16、31、33、35、38号峰为新出现的峰,8、15、19、22、25号峰为消失的峰,这些特异性峰,有可能是新的代谢产物。三个针刺组与模型组相比,都新出现了5号峰,考虑是针刺后产生的特异性代谢产物,与针刺防治应激性胃溃疡的效应机制相关。三个针刺组比较:募穴组与下合穴组比较共有峰34个,有16个代谢产物的峰面积(1、2、10、12、13、17、18、21、22、23、24、25、29、30、37、40号峰)有显著性差异(P<0.01,P<0.05)。28号峰为新出现的峰,31、33号峰为消失的峰,这些特异性峰,有可能是新的代谢产物。募穴组与合募配穴组比较共有峰32个,有13个代谢产物的峰面积(1、2、7、10、13、17、18、21、24、32、33、37、40号峰)有显著性差异(P<0.01,P<0.05)。9、16、28号峰为新出现的峰,8、15、19、22、25、35、38号峰为消失的峰,这些特异性峰,有可能是新的代谢产物。下合穴组与合募配穴组比较共有峰31个,同时有11个代谢产物的峰面积(1、2、7、10、13、17、18、21、32、37、40号峰)有显著性差异(P<0.01,P<0.05)。9、16、31、33、35、38号峰为新出现的峰,8、15、19、22、25号峰为消失的峰,这些特异性峰,有可能是新的代谢产物。提示针刺后对应激性胃溃疡大鼠的尿液代谢产物谱表达有调节作用。模型复制对大鼠尿液代谢物谱有影响,有新产生的峰,消失的峰。可能是应激导致的尿液代谢产物改变。考虑新出现的峰、消失的峰及峰面积有差异的代谢产物为与针刺防治胃溃疡相关的尿液标志性代谢产物。三个针刺组的比较,以合募配穴组的针刺相关代谢产物最多,合募配穴对尿液代谢物谱表达的调节最为显著,表明合募配穴与单穴相比具有一定的相对特异性,可能是合募配穴与单穴相比协同效应机制所在。4通过比较发现在血清及尿液针刺相关代谢产物中有分别3个代谢物是三个针刺组共同拥有的,相应谱峰的主要成分也是一致的,但峰面积却有不同。而其他各代谢产物分布在不同的针刺组中,提示针刺不同腧穴可产生不同的标志性代谢物。结论:1针刺对胃黏膜损伤有修复作用。2应激对大鼠血清和尿液代谢物谱的表达有影响。3针刺对应激性胃溃疡大鼠血清代谢物谱有特异性表达,有新的谱峰出现,且针刺合募配穴的特异性表达优于针刺单穴。4针刺对应激性胃溃疡大鼠尿液代谢物谱有特异性表达,有新的及新消失的谱峰,且针刺合募配穴的特异性表达优于针刺单穴。5合募配穴对应激性胃溃疡大鼠的血清、尿液代谢物谱有一定的特异性协同作用。更多还原

【Abstract】 Purpose: Analyze the theory from metabonomic side,by researching how He-Mu points effects on the serum and urine metabolic spectrums of the gastric ulcer mice.Method: Divide 72 Wistar mice into 6 groups: the normal group,the bundled group,the model group,“He-sea points”group,“Mu points”group,He-Mu points group.Gastric ulcer mouse model uses the water- immersion and restraint stress method to copy the model.The normal group shoud be only fed without any treatment;the bundled group shoud be bind everyday without acupuncture and copy model; the model group shoud be bind everyday and copied the model without acupuncture; the Mu points group shoud be acupunctured on“zhong wan”everyday and copied the model; the He points group shoud be acupunctured on“zu san-li”everyday and copied the model; the He-Mu points group shoud be acupunctured on both“zhong wan”and“zu san-li”everyday and copied the model. After 7 continuous treatments, observing the injuries of the gastric mucosa, analyze the differences between each groups, which the serum and urine metabolic spectrums of the gastric ulcer mice by LC-MS.Result:1 Visual observation on gastric mucosa,there are varying amounts and sizes of ulcer lesions appeared in the model group.Bundled group compared with the normal group has significant difference (P<0.01), showed bundle is also a stress stimulus; the model group compared with the bundled group has difference (P<0.05), showed the model had been successfully copied; the He-sea points group or the Mu points group compared with the model group has difference (P<0.05), He-Mu points group compared with the model group has significant difference (P<0.01), showed that simple acupuncture Zusanli or Zhongwan Point on the treatment of gastric ulcer model has some role in prevention and treatment, Mu points group,He-Mu points group compared with He-Mu points group has significant difference (P<0.01), showed He-Mu points group effect to surpass the He-sea points group, Mu points group.2 After the comparision of serum metabolic spectrum:There are 14 common peaks, 10 peak areas (No. 1, 5, 6, 7, 8, 9, 11, 12, 15, 17) have prominent difference between the normal group and bundled group(P<0.01). There are 16 common peaks, 5 peck areas (No. 4, 5, 12, 13, 14) have significant difference between the model group and bundled group(P<0.01, P<0.05), point out binding is also a kind of stress stimulation. There are 16 common peaks, 4 peak areas (No. 2, 3, 6, 8) have significant difference between the Mu points group and the model group (P<0.01). There are 16 common peaks, 4 peak areas (No. 2, 3, 7, 8) have significant difference between the He-sea points group and the model group (P<0.01), and No. 16 peak is a specific peak, may be a new product. There are 16 common peaks, 8 peak areas (No. 2, 3, 5, 6, 7, 8, 12, 13) have significant difference between the He-Mu points group and the model group (P<0.01), and No. 16 peak is a specific peak, may be a new product. Compared between the three treatment groups, there are 16 common peaks, 1 different peak, 5 peak areas (No. 2, 3, 6, 7, 15) have significant difference between the Mu points group and He-sea group (P<0.01, P<0.05); there are 9 peak areas (No. 2, 3, 5, 7, 8, 12, 13, 14, 15) have significant difference between the Mu points group and He-Mu points group (P<0.01, P<0.05). There are 9 peak areas (No. 2, 3, 5, 7, 8, 12, 13, 14, 15) have significant difference between the He-sea points group and He-Mu points group (P<0.01, P<0.05). Point out that acupuncture has effect on the serum metabolic products of gastric ulcer mice, the new peak and the peaks which have different peak areas may be the metabolic products are specific metabolic products of the prevention and treatment related to acupuncture the gastric ulcer mice serum. During three acupuncture groups , He-Mu points group has the most interrelated metabolic products. As a result, He-Mu points compared with single points has specific expression, there is a certain synergy.3 After the comparision of urine metabolic spectrum:There are 31 common peaks, 17 peak areas (No. 1, 3, 4, 7, 8, 10, 13, 15, 17, 18, 19, 28, 34, 37, 40, 41, 42) have significant difference between the normal group and bundled group (P<0.01). No. 9 and 20 are the new peaks, No. 16, 25, 33, 35, 38 peaks are the disappeared peaks, these special peaks may be the new products after the bundled stimulate. There are 30 common peaks, 16 peck areas (No. 1, 3, 7, 10, 13, 15, 17, 18, 19, 20, 21, 24, 28, 32, 40, 41) have significant difference between the model group and bundled group(P<0.01,P<0.05),and there are 12 metabolic products have difference with normal group and model group, point out binding is also a kind of stress stimulation. There are 34 common peaks, 12 peak areas (No. 1, 2, 6, 7, 10, 13, 15, 17, 20, 22, 23, 24, 29, 30, 32, 36, 37, 39, 40, 41) have significant difference between the Mu points group and the model group (P<0.01), No. 5, 31, 33 peaks are the new peaks, No. 28 peak is the disappeared peak, these metabolic products may be the new products. There are 35 common peaks, 13 peak areas (No. 2, 10, 12, 13, 17, 18, 24, 37, 40, 41) have significant difference between the He-sea points group and the model group (P<0.01), and No. 5 peak is a new peak, may be a new product. There are 31 common peaks, 10 peak areas (No. 2, 10, 12, 13, 17, 18, 24, 37, 40, 41 ) have significant difference between the He-Mu points group and the model group (P<0.01), and No. 5, 9, 16, 31, 33, 5, 38 peaks are the new peaks, No. 8, 15, 19, 22, 25 peaks are the disappeared peaks, these peaks are the specific peaks, may be are new products. Compared between the three treatment groups, there are 34 common peaks, 16 peak areas (No. 1, 2, 10, 12, 13, 17, 18, 21, 22, 23,24, 25, 29, 30, 37,40) have significant difference between the Mu points group and He-sea group (P<0.01, P<0.05), No. 28 peak is the new peak, No. 31, 33 are the disappeared peaks. There are 32 common peaks, 13 peak areas (No. 1, 2, 7, 10, 13, 17, 18, 21, 24, 32, 33, 37, 40) have significant difference between the Mu points group and He-Mu points group (P<0.01, P<0.05), No. 9, 16, 28 peaks are the new peaks, No. 8, 15, 19, 22, 25, 35, 38 peaks are the disappeared peaks. There are 31 common peaks, 11 peak areas (No. 1, 2, 7, 10, 13, 17, 18, 21, 32, 37, 40) have significant difference between the He-sea points group and He-Mu points group (P<0.01, P<0.05), No. 9, 16, 31, 33, 35, 38 peaks are the new peaks, No. 8, 15, 18 , 22, 25 peaks are the disappeared peaks. Point out that acupuncture has effect on the serum metabolic products of gastric ulcer mice. During three acupuncture groups, He-Mu points group has the most interrelated metabolic products. So as a result, He-Mu points compared with single points has specific expression, there is a certain synergy.4 There are 3 same metabolic products during the three acupuncture groups separately in serum and urine of gastric ulcer mice, and the correspounding peaks have the same bases with different peak areas. Point out that acupunctured on different points could generate different symbolical metabolic products.Conclusion:1 Acupuncture can effect the gastric injury restoration.2 Stress can influence the metabolite expression of mice’s serum and urine. 3 Effects of acupuncture on stress ulcer serum metabolite profiling have specific expression, with new peaks appeared, and the specific expression of acupuncture on He-Mu point better than acuncture on a single point.4 Effects of acupuncture on stress ulcer urine metabolite profiling have specific expression, with new peaks appeared and new peaks disappeared, and the specific expression of acupuncture on He-Mu point better than acuncture on a single point.5 He-Mu points have some specific synergy on serum and urine metabolite profiling of the stress ulcer mice.更多还原