左归降糖解郁方调控SIRT1/c-Myc/LDHA信号通路影响糖尿病并发抑郁症海马乳酸代谢的机制研究

Mechanism of Zuogui Jiangtang Jieyu Formula on treatment of hippocampal lactate metabolism in diabetes-related depression based on SIRT1/c-Myc/LDHA signaling pathway

目的 研究左归降糖解郁方调控糖尿病并发抑郁症海马乳酸代谢的作用及机制。方法 SD大鼠随机分为对照组、模型组、阳性对照(二甲双胍0.18 mg/kg+氟西汀1.8 mg/kg)组和左归降糖解郁方高、中、低剂量(20.52、10.26、5.13 g/kg)组,每组8只。除对照组外,其余大鼠采用高脂饲料饲养、尾iv链脲佐菌素(streptozotocin,STZ)、慢性温和不可预知应激法进行糖尿病并发抑郁症疾病构建糖尿病并发抑郁症大鼠模型。给予药物干预28 d,采用强迫游泳实验和水迷宫实验评价大鼠的抑郁行为;采用血糖试纸测定空腹血糖,采用试剂盒检测大鼠空腹胰岛素和海马丙酮酸、乳酸、烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide+,NAD+)含量,并计算胰岛素敏感性和胰岛素抵抗指数;采用液相色谱-质谱联用仪(liquid chromatography tandem mass spectrometry,LC-MS/MS)以及多元统计分析方法对海马进行代谢组学分析并筛选差异代谢物;采用Western blotting检测沉默调节蛋白1(sirtuin 1,SIRT1)/Myc原癌基因(cancer-Myc,c-Myc)/乳酸脱氢酶A(lactate dehydrogenase A,LDHA)信号通路蛋白表达。给予SIRT1抑制剂干预后,考察左归降糖解郁方对糖尿病并发抑郁症大鼠的影响。结果 左归降糖解郁方高剂量组可显著降低糖尿病并发抑郁症大鼠的空腹血糖和胰岛素含量(P<0.05、0.01),升高胰岛素敏感指数(P<0.01),降低胰岛素抵抗指数(P<0.01),缩短大鼠在强迫游泳实验中的不动时间和水迷宫实验中的登台时间(P<0.05、0.01),增加在目标象限的路程比(P<0.01)。代谢组学研究发现,乳酸对于区分模型组和左归降糖解郁方高剂量组代谢差异的贡献值最大。Western blotting结果显示,左归降糖解郁方高剂量组可显著增加模型大鼠海马SIRT1、c-Myc、LDHA蛋白表达(P<0.01)。给予SIRT1抑制剂干预后,左归降糖解郁方对模型大鼠血糖、胰岛素抵抗以及抑郁样行为的改善作用被抑制(P<0.05、0.01)。对乳酸相关代谢物的研究发现,SIRT1抑制剂可抑制左归降糖解郁方对SIRT1/c-Myc/LDHA信号通路的激活作用(P<0.01),从而抑制乳酸的生成,减少海马乳酸含量(P<0.05)。结论 左归降糖解郁方通过调控海马SIRT1/c-Myc/LDHA信号通路,催化丙酮酸转化为乳酸,从而改善糖尿病并发抑郁症大鼠海马乳酸代谢。

Objective To study the effect and mechanism of Zuogui Jiangtang Jieyu Formula(左归降糖解郁方)on regulating lactate metabolism in hippocampus of diabetes-related depression.Methods SD rats were randomly divided into control group,model group,positive control group(metformin 0.18 mg/kg+fluoxetine 1.8 mg/kg),and Zuogui Jiangtang Jieyu Formula high-,medium-,and low-dose(20.52,10.26,5.13 g/kg)groups,with eight rats in each group.In addition to control group,other rats were fed with high-fat diet,tail iv streptozotocin(STZ)and receipt chronic mild unpredictable stress to establish diabetes-related depression model.Drug intervention was administered for 28 d,and the depression like behavior of rats was evaluated using forced swimming and water maze experiments;Fasting blood glucose was measured using blood glucose test strips,fasting insulin and levels of hippocampal pyruvate,lactate,and nicotinamide adenine dinucleotide+(NAD+)in rats were detected using a reagent kit,insulin sensitivity and insulin resistance index were calculated;Metabolomics analysis and screening of differential metabolites in hippocampus were performed using liquid chromatography tandem mass spectrometry(LC-MS/MS)and multivariate statistical analysis methods;Western blotting was used to detect the expressions of sirtuin 1(SIRT1)/cancer-Myc(c-Myc)/lactate dehydrogenase A(LDHA)signaling pathway proteins.After the intervention of SIRT1 inhibitor,the effect of Zuogui Jiangtang Jieyu Formula on diabetes-related depression were investigated.Results Zuogui Jiangtang Jieyu Formula high-dose group significantly reduced the fasting blood glucose and insulin contents(P<0.05,0.01),increased insulin sensitivity index(P<0.01),reduced insulin resistance index(P<0.01),shorten the immobility time of rats in forced swimming experiments and the stage time in water maze experiments(P<0.05,0.01),and increased the distance ratio in the target quadrant(P<0.01).Metabolomics studies found that the lactate had the greatest contribution in distinguishing metabolic differences between the model group and Zuogui Jiangtang Jieyu Formula high-dose group.Western blotting results showed that Zuogui Jiangtang Jieyu Formula high-dose group significantly increased the expressions of SIRT1,c-Myc and LDHA proteins in hippocampus of model rats(P<0.01).After intervention with SIRT1 inhibitor,the improvement effect of Zuogui Jiangtang Jieyu Formula on blood glucose,insulin resistance and depression like behavior in model rats was inhibited(P<0.05,0.01).Research on lactate related metabolites found that SIRT1 inhibitor could inhibit the activation of SIRT1/c-Myc/LDHA signaling pathway by Zuogui Jiangtang Jieyu Formula(P<0.01),thereby suppressing lactate production and reducing hippocampal lactate content(P<0.05).Conclusion Zuogui Jiangtang Jieyu Formula can regulate the hippocampal SIRT1/c-Myc/LDHA signaling pathway,catalyze the conversion of pyruvate into lactic acid,and thus improve the metabolism of lactic acid in hippocampus of rats with diabetes-related depression.