基于血清代谢组学探究山茱萸多糖对2型糖尿病大鼠的干预机制

Mechanism of intervention of Corni Fructus polysaccharides in type 2 diabetes mellitus rats based on serum metabolomics

目的基于代谢组学探究山茱萸多糖干预2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠的作用机制。方法取10只SD雄性大鼠作为对照组,其余大鼠采用高糖高脂饲料结合ip链脲佐菌素(streptozocin,STZ)诱导构建T2DM模型。造模成功的大鼠随机分为模型组、二甲双胍(200 mg/kg)组和山茱萸多糖低、中、高剂量(75、150、300 mg/kg)组,各组大鼠每天上午ig药物,连续给药4周。末次给药前大鼠禁食10 h,麻醉后腹主动脉取血并分离血清,同时取肝脏组织。采用试剂盒检测大鼠血清中总胆固醇(total cholesterol,TC)、三酰甘油(triglyceride,TG)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、白细胞介素-1β(interleukin1β,IL-1β)、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、丙二醛(malondialdehyde,MDA)水平和丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、超氧化物歧化酶(superoxide dismutase,SOD)活性;采用苏木素-伊红(HE)染色观察肝脏组织病理变化;采用超高效液相色谱-线性离子阱串联静电场轨道高分辨质谱(UHPLC-LTQ-Orbitrap-MS/MS)技术检测大鼠血清内源性代谢物水平,并结合主成分分析和正交偏最小二乘法-判别分析对差异代谢物进行筛选和鉴定,利用MetaboAnalyst平台进行代谢通路分析。结果与模型组比较,山茱萸多糖给药组大鼠体质量和血糖水平均降低,肝损伤明显改善;血清中TC、TG、LDL-C、IL-6、IL-1β、TNF-α、MDA水平和AST、ALT活性均显著降低(P<0.05、0.01),HDL-C水平和SOD活性显著升高(P<0.05)。血清代谢组学共筛选出35个与T2DM相关的潜在生物标志物,山茱萸多糖给药组可回调29个生物标志物,主要涉及苯丙氨酸的代谢和生物合成、牛磺酸和次牛磺酸代谢、醚脂质代谢和丙酮酸代谢等22条代谢通路。结论山茱萸多糖能有效改善T2DM引起的糖脂代谢紊乱,减轻肝损伤,达到治疗T2DM作用,可能与调节氨基酸代谢、糖代谢、脂质代谢等多条代谢通路有关。

Objective To explore the mechanism of intervention of Shanzhuyu(Corni Fructus)polysaccharides in type 2 diabetes mellitus(T2DM)rats based on metabolomics.Methods A total of 10 male SD rats were selected as the control group,while the remaining rats were induced to construct a T2DM model using a high sugar and high-fat diet combined with streptozocin(STZ).The successfully modeled rats were randomly divided into model group,metformin(200 mg/kg)group,and Corni Fructus polysaccharides low-,medium-,and high-dose(75,150,300 mg/kg)groups,rats in each group were given drugs in the morning for four consecutive weeks.Before the last administration,the rats were fasted for 10 h.After anesthesia,blood was collected from the abdominal aorta and serum was separated,while liver tissue was taken.Kit was used to detect levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),malondialdehyde(MDA)and activities of alanine aminotransferase(ALT),aspartate aminotransferase(AST),superoxide dismutase(SOD).Hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue.Ultra-high performance liquid chromatography linear ion trap orbital trap tandem mass spectrometry(UHPLC-LTQ-Orbitrap-MS/MS)technology was used to detect the level of endogenous metabolites in serum of rats,and principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis were used to screen and identify differential metabolites.MetaboAnalyst platform was used for metabolic pathway analysis.Results Compared with model group,the body weight and blood glucose levels of rats in Corni Fructus polysaccharides administration group were reduced,and the liver injury was significantly improved.The levels of TC,TG,LDL-C,IL-6,IL-1β,TNF-α,MDA and activities of AST,ALT in serum were decreased(P<0.05,0.01),HDL-C level and SOD activity were increased(P<0.05).A total of 35 potential biomarkers related to T2DM were screened by serum metabolomics,and 29 biomarkers were regulated in Corni Fructus polysaccharides administration group,which mainly involved 22 metabolic pathways including phenylalanine metabolism and biosynthesis,taurine and hypotaurine metabolism,ether lipid metabolism and pyruvate metabolism.Conclusion Corni Fructus polysaccharides can effectively improve the glucose and lipid metabolism disorders caused by T2DM,alleviate liver damage,and achieve the effect of treating T2DM,which may be related to the regulation of amino acid metabolism,glucose metabolism,lipid metabolism and other metabolic pathways.