滋补脾阴方药对糖尿病认知功能障碍大鼠海马乳酸及乳酸化修饰的影响

Effects of Zibu Piyin Formula(滋补脾阴方)on Lactate and Lactylation in Hippocampus of Rats with Diabetes-Associated Cognitive Decline

目的:探讨滋补脾阴方药对糖尿病认知功能障碍(DACD)大鼠学习记忆能力及海马中糖酵解产物乳酸及乳酸化修饰(Kla)的影响。方法:18只雄性SD大鼠随机分为正常对照组、模型对照组、滋补脾阴方药34.6 g/kg组,每组6只。除正常对照组外,其余大鼠以高脂喂养联合链脲佐菌素(STZ)建立DACD模型,大鼠灌胃给予相应药物。行为学实验分析大鼠学习记忆能力;全自动生化分析仪测定血脂含量;ELISA法测定海马中β-淀粉样多肽(Aβ)1-40、Aβ_(1-42)含量;比色法测定海马乳酸含量;Western blot法测定海马糖酵解过程中的关键酶己糖激酶2(HK2)、M2型丙酮酸激酶(PKM2)、乳酸脱氢酶A(LDHA)、丙酮酸脱氢酶Α(PDHA1)、组蛋白赖氨酸乳酸酰化(Kla)的蛋白表达;通过免疫荧光分析大鼠海马Kla的水平。结果:与正常对照组比较,模型对照组的大鼠空腹血糖、糖化血红蛋白、逃避潜伏期明显增加(P<0.05或P<0.01),血清TG和LDL-c明显升高(P<0.05或P<0.01),海马Aβ_(1-40)、Aβ_(1-42)含量显著升高(P<0.01);跨越平台次数显著减少(P<0.01),HDL-c含量明显降低(P<0.05),海马HK2、PKM2、LDHA、PDHA1、Kla蛋白表达显著下调(P<0.01),乳酸含量明显降低(P<0.05);与模型对照组比较,滋补脾阴方药34.6 g/kg组能够显著增加大鼠跨越平台次数(P<0.01),明显升高血清HDL-c含量(P<0.05),上调海马HK2、PKM2、LDHA、PDHA1、Kla蛋白表达,明显升高乳酸含量(P<0.05或P<0.01),明显降低血糖、血清TG、TC和LDL-c含量(P<0.05);显著降低海马Aβ_(1-40)、Aβ_(1-42)含量(P<0.01)。结论:滋补脾阴方药有利于改善DACD大鼠的糖脂代谢紊乱及学习记忆能力。其机制可能是通过激活糖酵解通路,上调乳酸水平调控蛋白乳酸化,改善大鼠的记忆功能。

Objective:To investigate the effects of Zibu Piyin Formula(滋补脾阴方)on learning and memory abilities and the levels of lactate and lactylation(Kla)in the hippocampus of rats with diabetes-associated cognitive decline(DACD).Methods:Eighteen male SD rats were randomLy divided into a normal control group,a model control group,and a Zibu Piyin Formula group(34.6 g/kg),with six rats in each group.The DACD model was induced in rats except for those in the normal control group by feeding on a high-fat diet combined with streptozotocin(STZ),and the rats were orally administered with corresponding drugs.Behavioral experiments were conducted to analyze the learning and memory abilities of the rats.Blood lipid levels were determined using an automated biochemical analyzer.The levels ofβ-amyloid peptides(Aβ)1-40 and Aβ_(1-42) in the hippocampus were measured by ELISA.Lactate content in the hippocampus was determined using a colorimetric method.The protein expression of key enzymes in the glycolysis,including hexokinase 2(HK2),pyruvate kinase M2(PKM2),lactate dehydrogenase A(LDHA),Pyruvate dehydrogenaseα(PDHA1),and Kla in the hippocampus,was assessed by Western blot.The level of Kla in the hippocampal was analyzed through immunofluorescence.Results:Compared with the normal control group,the model control group showed significantly increased fasting blood glucose,glycated hemoglobin,and escape latency(P<0.05 or P<0.01),elevated serum triglyceride(TG)and low-density lipoprotein cholesterol(LDL-c)levels(P<0.05 or P<0.01),increased levels of hippocampal Aβ_(1-40) and Aβ_(1-42)(P<0.01),decreased number of platform crossings(P<0.01),decreased high-density lipoprotein cholesterol(HDL-c)(P<0.05),downregulated protein expression of HK2,PKM2,LDHA,PDHA1,and Kla in the hippocampus(P<0.01),and reduced lactate content(P<0.05).Compared with the model control group,the Zibu Piyin Formula group(34.6 g/kg)significantly increased the number of platform crossings(P<0.01),elevated serum HDL-c levels(P<0.05),upregulated the protein expression of HK2,PKM2,LDHA,PDHA1,and Kla in the hippocampus,increased lactate content(P<0.05 or P<0.01),significantly reduced blood glucose,serum TG,total cholesterol(TC),and LDL-c levels(P<0.05),and decreased hippocampal levels of Aβ_(1-40) and Aβ_(1-42)(P<0.01).Conclusion:Zibu Piyin Formula is beneficial in improving the disordered glucose and lipid metabolism and learning and memory abilities in rats with DACD.Its mechanism may involve the activation of the glycolysis pathway,upregulation of lactate levels,modulation of protein lactylation,and improvement of memory function in rats.