基于海马小胶质细胞P2X7/NLRP3 信号探讨左归降糖解郁方对糖尿病并发抑郁症模型大鼠海马神经突触可塑性作用及机制

Based on the P2X7/NLRP3 signaling of hippocampal microglia, this study explores the effect and mechanism of the Zuo Gui Jiang Tang Jie Yu Formula on hippocampal synaptic plasticity in a rat model of diabetes complicated with depression

关键词:  左归降糖解郁方  糖尿病并发抑郁症  突触可塑性  成体海马神经发生  P2X7/NLRP3信号通路

Key words:  Zuogui Jiangtang Jieyu Formula  diabetes mellitus complicated with depression  synaptic plasticity  adult hippocampal neurogenesis  P2X7/NLRP3 pathway

摘要:

目的 基于P2X7-NLRP3轴,探讨左归降糖解郁方改善糖尿病并发抑郁症(diabetes mellitus complicated with depression, DD)模型大鼠神经炎症及增强神经保护作用的相关机制。方法 通过4周高脂饲料饲养联合链脲佐菌素(streptozotocin, STZ)腹腔注射及4周慢性温和不可预知应激(chronic unpredictable mild stress, CUMS)联合孤笼饲养建立DD大鼠模型,实验分为对照组、模型组、阳性药组(二甲双胍0.18 g/kg+氟西汀1.8 mg/kg)、左归降糖解郁方低、中、高剂量组和左归降糖解郁方中剂量+BBG抑制剂组。采用旷场实验、强迫游泳实验和Morris水迷宫检测大鼠抑郁样行为和记忆认知功能；酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测血清中去甲肾上腺素(Norepinephrine, NE)、5-羟色胺(5-hydroxyteyptamine,5-HT)、多巴胺(dopamine,DA)含量；免疫荧光检测海马P2X7/Iba-1、NLRP3/Iba-1、突触后致密蛋白-95(postsynaptic density protein-95,PSD95)、突触蛋白-1(synapsin1,SYN1)；苏木素-伊红(hematoxylin-eosin,HE)染色和尼氏(Nissl)染色检测海马神经元损伤情况；免疫印迹法(Western blot)检测P2X7-NLRP3轴相关蛋白P2X7、NLRP3、ASC、Caspase-1、IL-1β和脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)的表达情况。结果 与模型组比较,左归降糖解郁方可以改善DD大鼠抑郁行为,增强神经保护作用；显著升高PSD95、SYN1、BDNF表达(P<0.01),降低P2X7/Iba-1、NLRP3/Iba-1、ASC、Caspase-1和IL-1β表达(P<0.01)。结论 左归降糖解郁方通过抑制P2X7-NLRP3轴和海马小胶质细胞(microglia, MG)激活,进一步改善神经炎症与NLRP3炎性小体的异常激活,从而提高海马中突触相关蛋白PSD95、SYN和BDNF的表达来发挥神经保护作用。

Abstract:

Objective: To explore the mechanism by which the Zuo Gui Jiang Tang Jie Yu Formula improves neuroinflammation and enhances neuroprotection in a rat model of diabetes mellitus complicated with depression (DD) based on the P2X7-NLRP3 axis. Methods: A DD rat model was established by feeding a high-fat diet for 4 weeks combined with intraperitoneal injection of streptozotocin (STZ) and 4 weeks of chronic unpredictable mild stress (CUMS) combined with solitary housing. The experiment was divided into the control group, model group, positive drug group (metformin 0.18 g/kg + fluoxetine 1.8 mg/kg), low-, medium-, and high-dose Zuo Gui Jiang Tang Jie Yu Formula groups, and the medium-dose Zuo Gui Jiang Tang Jie Yu Formula + BBG inhibitor group. Open field test, forced swimming test, and Morris water maze were used to detect depressive-like behaviors and memory and cognitive functions in rats. Enzyme-linked immunosorbent assay (ELISA) was used to detect the contents of norepinephrine (NE), 5-hydroxytryptamine (5-HT), and dopamine (DA) in serum. Immunofluorescence was used to detect P2X7/Iba-1, NLRP3/Iba-1, postsynaptic density protein-95 (PSD95), and synapsin1 (SYN1) in the hippocampus. Hematoxylin-eosin (HE) staining and Nissl staining were used to detect hippocampal neuronal injury. Western blot was used to detect the expression of P2X7-NLRP3 axis-related proteins P2X7, NLRP3, ASC, Caspase-1, IL-1β, and brain-derived neurotrophic factor (BDNF). Results: Compared with the model group, the Zuo Gui Jiang Tang Jie Yu Formula could improve depressive behaviors in DD rats and enhance neuroprotection; significantly increase the expression of PSD95, SYN1, and BDNF (P < 0.01), and decrease the expression of P2X7/Iba-1, NLRP3/Iba-1, ASC, Caspase-1, and IL-1β (P < 0.01). Conclusion: The Zuo Gui Jiang Tang Jie Yu Formula exerts neuroprotective effects by inhibiting the P2X7-NLRP3 axis and microglial (MG) activation, further improving neuroinflammation and abnormal activation of NLRP3 inflammasomes, and increasing the expression of synaptic-related proteins PSD95, SYN, and BDNF in the hippocampus.