基于核磁共振氢谱技术研究艾灸对胃热证、胃寒证大鼠胃组织代谢的影响

Endogenous metabolites involving effects of moxibustion on injured gastric mucosal tissue in rats with stomach heat or cold syndrome based on nuclear magnetic resonance spectroscopy

目的:采用核磁共振氢谱(1H-NMR)技术研究艾灸对胃热证、胃寒证大鼠胃组织代谢物的影响,阐述艾灸对胃热证、胃寒证代谢调节机制,并验证"热证可灸"的科学性。方法:将SD雄性大鼠随机分为空白组、胃热模型组、胃寒模型组、艾灸胃热组、艾灸胃寒组,每组6只。采用辣椒液加乙醇灌胃法复制胃热证大鼠模型,冰水加NaOH灌胃法复制胃寒证大鼠模型。艾灸组温和灸"足三里""梁门"穴,20 min/次,1次/d,连续干预7 d。观察大鼠一般状态并记录体质量,采用HE染色法观察大鼠胃组织病理形态,采用~1H-NMR技术对胃组织进行代谢组学检测,用模式识别方法筛选出差异代谢物,通过在线分析网站MetPA筛选相关代谢通路。结果:两模型组大鼠体质量逐渐下降,从第4天开始明显低于空白组(P<0.05,P<0.01);两艾灸组大鼠体质量逐渐增加,从第10天开始明显高于各自的模型组(P<0.05,P<0.01)。空白组胃黏膜皱襞完整,结构清晰;胃热模型组可见胃黏膜柱状上皮结构破坏严重,炎性细胞浸润,颜色晦暗;胃寒模型组可见炎性细胞浸润;艾灸组胃组织炎性细胞浸润程度较模型组有明显改善。经~1H-NMR技术共筛选出16种与胃热证胃黏膜损伤相关的代谢物,14种与胃寒证相关的代谢物。艾灸对胃热证大鼠胃黏膜中13个代谢物产生调节作用:上调异亮氨酸、肌酸酐、胆碱、乳酸盐(P<0.05),下调胆碱磷酸、甘氨酸、丙氨酸、尿嘧啶、酪氨酸、苯丙氨酸、次黄嘌呤、腺苷、烟酰胺(P<0.05);可对胃寒证大鼠胃黏膜中8个代谢物产生调节作用:上调肌酸酐、乙醇胺、胆碱、腺苷、烟酰胺(P<0.05),下降甘氨酸、磷酸肌酸、酪氨酸(P<0.05)。MetPA显示艾灸能调节胃热证大鼠胃黏膜中10条代谢通路、胃寒证大鼠胃黏膜中7条代谢通路,这些代谢物和代谢通路主要涉及氨基酸代谢、能量代谢和炎性反应。结论:"热证可灸"具有代谢物变化基础;艾灸对胃黏膜损伤修复涉及其对氨基酸代谢、能量平衡和炎性反应的调节作用;艾灸对不同证型大鼠胃黏膜的代谢调节具有共性和特异性。

Objective To investigate the profile of metabolites of gastric mucosa involving the effectiveness of moxibustion in the treatment of syndromes of stomach heat(SH)and stomach cold(SC)by 1H-nuclear magnetic resonance(1H-NMR)spectroscopy in rats,so as to reveal its mechanisms underlying improvement of gastric disorders.Methods Male SD rats were randomly divided into control,SH-model,SC-model,SH-moxibustion and SC-moxibustion groups(n=6 rats/group).The SH-model and SC-model were established by gavage of pepper liquid plus ethanol,and ice water plus NaOH,respectively.Moxibustion was applied to bilateral"Zusanli"(ST36)and"Liangmen"(ST21)for 20 min,once daily for 7 days.Histopathological changes of the gastric tissue were observed by H.E.staining.Differential metabolites in the gastric mucosal tissue were detected and the relevant metabolic pathways analyzed by using 1H-NMR,pattern recognition method,and online MetPA(http://www.metaboanalyst.ca).Results Compared with the control group,the body mass was decreased significantly from the 4th to 14th day after modeling(P<0.05,P<0.01).After the treatment,the body mass was obviously increased from the 10th day on in both SH-EA and SC-EA groups relevant to the SH and SC model group,respectively(P<0.05,P<0.01).H.E.staining showed severe damage of the columnar epithelial structure of the gastric mucosa and inflammatory cell infiltration in the SH group,and inflammatory cell infiltration in the SC model group,which were relatively milder in both moxibustion groups.1H-NMR analysis displayed a total of 16 potential biomarkers in the injured gastric mucosa of SH syndrome and 14 biomarkers for the SC syndrome after mode-ling,and 13 metabolites related to SH moxibustion and 8 metabolites related to SC moxibustion after moxibustion interventions,respectively.After moxibustion,among the 13 differential metabolites of the SH syndrome,the effectively up-regulated candidates were isoleucine,creatinine,choline and lactate(P<0.05),and the down-regulated ones were choline phosphate,glycine,alanine,urine pyrimidine,tyrosine,phenylalanine,hypoxanthine,adenosine and nicotinamide(P<0.05).Among the 8 metabolites related to the SC syndrome,creatinine,ethanolamine,choline,adenosine and nicotinamide were markedly increased(P<0.05),and glycine,creatine phosphate and tyrosine remarkably decreased in their levels after moxibustion(P<0.05).MetPA showed that moxibustion could regulate 10 metabolic pathways for SH syndrome and 7 metabolic pathways for SC syndrome.Metabolites and metabolic pathways are mainly involved in functions of amino acid metabolism,energy metabolism and inflammatory response.Conclusion The idea of"moxibustion could be used for heat syndrome"has metabolic substance basis,and its efficacy in repairing the injured gastric mucosa involves regulation of amino acid metabolism,energy balance and inflammation response,and moxibustion for SH and SC syndromes has both generality and specificity in regulating metabolic activities.