肾气丸对2型糖尿病模型小鼠认知功能及海马葡萄糖能量代谢的影响

Effect of Shenqi Pills(肾气丸)on Cognitive Function and Hippocampal Glucose Energy Metabolism in Type 2 Diabetes Mellitus Model Mice

目的探讨肾气丸对2型糖尿病(T2DM)认知障碍的影响及海马葡萄糖能量代谢方面的可能作用机制。方法60只C57BL/6小鼠随机分为对照组、模型组、罗格列酮组和肾气丸低、中、高剂量组,每组10只。除对照组外,其余各组小鼠采用高脂饲料喂养联合链脲佐菌素腹腔注射构建T2DM小鼠模型。造模成功后,肾气丸高、中、低剂量组分别予肾气丸颗粒2.08、1.04、0.52 g/(kg·d)灌胃,罗格列酮组予罗格列酮片3 mg/(kg·d)灌胃,对照组和模型组予蒸馏水10 ml/(kg·d)灌胃,连续灌胃8周。给药开始后每2周记录各组小鼠体质量、空腹血糖(FBG)水平。给药第8周开始进行Morris水迷宫实验;给药8周结束后进行口服葡萄糖耐量试验(OGTT)及空腹胰岛素水平检测,液相色谱串联质谱靶向定量检测海马葡萄糖能量代谢相关产物,并进行KEGG注释分析。结果与对照组同时间比较,模型组在第6、8周时体质量明显升高,第2、4、6、8周时FBG水平升高;OGTT检测中0、30、60、120 min时血糖水平均升高,8周后空腹胰岛素水平降低;Morris水迷宫实验中逃避潜伏期在第3、4天时延长,逃避潜伏时间增加,游泳总距离、平台象限停留时间、穿越平台次数减少(P<0.05或P<0.01)。与模型组同时间比较,肾气丸低剂量组第6周时体质量降低,肾气丸中、高剂量组第6、8周时体质量降低,罗格列酮组第8周时体质量降低;肾气丸各剂量组及罗格列酮组第6、8周时FBG水平均降低,空腹胰岛素水平均升高;OGTT检测中肾气丸中剂量组各时间点的血糖均降低;Morris水迷宫实验中肾气丸中、高剂量组第3、4天时逃避潜伏期缩短,肾气丸中剂量组逃避潜伏时间减少,游泳总距离、平台象限停留时间、穿越平台次数增加(P<0.05或P<0.01)。肾气丸中剂量组改善各指标效果最优,故代谢物靶向定量检测选用肾气丸中剂量组。肾气丸中剂量组可调节T2DM模型小鼠海马脑区葡萄糖能量代谢途径紊乱,共发现13个差异代谢物,可上调α-酮戊二酸、3-磷酸甘油酸含量,下调富马酸、谷氨酸、乳酸、肌苷、苹果酸、腺嘌呤、果糖1,6-二磷酸等含量。通过对差异代谢物进行KEGG注释分析,提示肾气丸调控T2DM模型小鼠海马脑区葡萄糖能量代谢紊乱与改善胰岛素抵抗、神经退行性病变及调控ABC转运、缺氧诱导因子1(HIF-1)、叉头转录因子(FoxO)、环磷酸腺苷(cAMP)等信号通路密切相关。结论肾气丸可改善T2DM模型小鼠学习记忆能力,改善认知障碍,可能通过参与调控海马葡萄糖能量代谢而发挥作用。

Objective To explore the effect and possible mechanism of Shenqi Pills(肾气丸)on cognitive impairment and hippocampal glucose energy metabolism in type 2 diabetes mellitus(T2DM).Methods Sixty C57BL/6 mice were randomly divided into control group,model group,rosiglitazone group and Shenqi Pills low-,medium-and high-dose groups,with 10 mice in each group.T2DM model was induced by a high-fat diet combined with intraperitoneal injection of streptozotocin in all the groups except for the control group.After successful modeling,the high-,medium-,and low-dose Shenqi Pills groups were given 2.08,1.04,and 0.52 g/(kg·d)of Shenqi Pills granules by gavage respectively,while the rosiglitazone group was given 3 mg/(kg·d)of rosiglitazone tablets by gavage,and the control group and model group were gavaged with 10 ml/(kg·d)of distilled water,all for 8 consecutive weeks.The body weight and fasting blood glucose(FBG)level were recorded every two weeks.The Morris water maze test was performed on the 8th week of medication.After 8-week medication,oral glucose tolerance test(OGTT)and fasting insulin level were measured,hippocampal glucose energy metabolism-related products were quantitatively detected by liquid chromatography tandem mass spectrometry,and KEGG annotation analysis was performed.Results Compared to those measured at the same timepoints in the control group,the body mass on week 6 and 8,as well as the FBG level on week 2,4,6 and 8 in the model group increased;the blood glucose level at 0,30,60 and 120 minutes of the OGTT test increased,while fasting insulin level after 8-week medication decreased.The escape latency of the model group was significantly prolonged on the 3rd and 4th days,and the escape latency time increased,while the total swimming distance,platform quadrant residence time and the number of platform crossings decreased(P<0.05 or P<0.01).Compared to those measured at the same timepoints in the model group,the body mass on week 6 in the low-dose Shenqi Pills group,on week 6 and 8 in the medium-and high-dose groups,and on week 8 in the rosiglitazone group were significantly reduced;the FBG levels in all the Shenqi Pills groups and rosiglitazone group on week 6 and 8 decreased,while fasting insulin levels increased.In the OGTT test,blood glucose in the medium-dose group of Shenqi Pills at all timepoints decreased;in the Morris water maze test,the escape latency period of the medium-and high-dose Shenqi Pills groups was shortened on the 3rd and 4th days,while the escape latency time was reduced,and the total swimming distance,platform quadrant residence time,and number of platform crossings increased in the medium-dose Shenqi Pills group(P<0.05 or P<0.01).The medium-dose Shenqi Pills showed best effect,therefore it was selected for the targeted quantitative detection of metabolites.The medium-dose Shenqi Pills group could regulate the disorder of glucose metabolism in the hippocampus of T2DM mice,and 13 differential metabolites were found,up-regulatingα-ketoglutarate and 3-phosphoglyceric acid,and down-regulating fumaric acid,glutamatic acid,lactatic acid,inosine,malic acid,adenine,fructose 1,6-diphosphate and others.KEGG annotation of differential metabolites suggested that Shenqi Pills was closely related to the regulation of glucose metabolism disorder and insulin resistance in the hippocampus region of T2DM model mice,as well as neurodegenerative diseases and ABC transport,hypoxia-inducible factor 1(HIF),forkhead transcription factor(FoXO)and cyclic adenosine monophosphate(cAMP)signaling pathways.Conclusion Shenqi Pills can improve learning and memory abilities and cognitive impairment in T2DM mice,and may act its role by regulating glucose energy metabolism in the hippocampus of T2DM.