基于海马DG区小胶质细胞P2X7/NLRP3信号研究DD海马CA3神经突触可塑性机制

Research on the mechanism of hippocampal CA3 synaptic plasticity in diabetes mellitus with depression based on P2X7/NLRP3 signaling in microglia of the DG region

摘要

目的: 探讨嘌呤能受体P2X配体门控离子通道7(P2X purinoceptor 7,P2X7)-核苷酸结合寡聚化结构域样受体蛋白3(NOD like receptor family pyrin domain containing 3,NLRP3)通路在糖尿病并发抑郁症(diabetes mellitus with depression,DD)中的作用机制。方法: 将60只SD大鼠随机分为对照组(Con)、糖尿病组(DM)、抑郁组(Dep)、糖尿病并发抑郁组(DD)、阳性药[二甲双胍(0.18 g·kg^－1)联合氟西汀(1.8 mg·kg^－1)]组(Y)、P2X7抑制剂(BBG,30 mg·kg^－1)组,每组10只。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。抑制剂组大鼠于造模d 15,每天进行腹腔注射,连续14 d。在造模和药物干预完成后,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、凋亡相关斑点样蛋白、胱天蛋白酶-1(caspase-1)、白介素-1β和脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的表达情况。结果: BBG和阳性药可有效改善DD大鼠的体质量和空腹血糖。行为学结果表明,BBG和阳性药可减少强迫游泳不动时间,增加旷场实验活动总距离和活动总次数,缩短成功潜伏期时间,增加平台所在象限停留时间。病理结果显示,BBG和阳性药可改善DD大鼠海马脑区神经元的病理损伤。生化结果表明,BBG和阳性药可上调DD大鼠血清中5-HT、DA、NE水平。免疫荧光和Western blot结果表明,BBG可降低海马中P2X7、NLRP3等的表达,增加PSD-95、SYN1、BDNF的表达。结论: BBG可通过抑制P2X7/NLRP3轴和海马小胶质细胞激活,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 investigate the mechanism of the P2X purinoceptor 7 (P2X7)-NOD like receptor family pyrin domain containing 3 (NLRP3) pathway in diabetes mellitus with depression (DD). Methods: Sixty Sprague Dawley (SD) rats were randomly divided into six groups (n=10 per group): control (Con), diabetes mellitus (DM), depression (Dep), diabetes mellitus with depression (DD), positive drug [metformin (0.18 g·kg^－1)+fluoxetine (1.8 mg·kg^－1)] (Y), and P2X7 inhibitor [Brilliant Blue G (BBG), 30 mg·kg^－1]. DD rat models were established using 4 weeks of high-fat diet feeding combined with intraperitoneal streptozotocin (STZ) injection, followed by 4 weeks of chronic unpredictable mild stress (CUMS) combined with single caging. Rats in the inhibitor group received daily intraperitoneal injections starting from day 15 of modeling for 14 consecutive days. After modeling and drug intervention, behavioral tests (open field test, forced swim test, Morris water maze) were used to assess depression-like behaviors and cognitive function. Serum levels of norepinephrine (NE), 5-hydroxytryptamine (5-HT), and dopamine (DA) were measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was performed to detect hippocampal P2X7/Iba-1, NLRP3/Iba-1, postsynaptic density protein-95 (PSD95), and synapsin-1 (SYN1). Hippocampal neuronal damage was assessed using hematoxylin-eosin (HE) and Nissl staining. Western blotting was used to measure the expression of P2X7-NLRP3 axis-related proteins (P2X7, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, interleukin-1β (IL-1β) and brain-derived neurotrophic factor (BDNF). Results: Both BBG and the positive drug effectively attenuated the loss of body weight and elevated fasting blood glucose in DD rats. Behavioral results showed that BBG and the positive drug reduced immobility time in the forced swim test, increased total distance traveled and total activity in the open field test, shortened escape latency, and increased the time spent in the target quadrant in the Morris water maze. Pathological results demonstrated that BBG and the positive drug ameliorated neuronal damage in the hippocampal region of DD rats. Biochemical analysis results indicated that BBG and the positive drug upregulated serum levels of 5-HT, DA, and NE. Immunofluorescence and Western blot results showed that BBG could reduce the expression of P2X7 and NLRP3 in the hippocampus, while increasing the expression of PSD-95, SYN-1, and BDNF. Conclusion: BBG exerts neuroprotective effects by inhibiting the P2X7/NLRP3 axis and hippocampal microglial activation, thereby ameliorating neuroinflammation and abnormal NLRP3 inflammasome activation. This leads to increased expression of synaptic-related proteins (PSD95, SYN) and BDNF in the hippocampus.

关键词

P2X配体门控离子通道7/核苷酸结合寡聚化结构域样受体蛋白3信号通路 / 糖尿病并发抑郁症 / 突触可塑性 / 成体海马神经发生

Key words

P2X purinoceptor 7/NOD like receptor family pyrin domain containing 3 pathway / diabetes with depression / synaptic plasticity / adult hippocampal neurogenesis

[1] SUN H, SAEEDI P, KARURANGA S, et al. IDF Diabetes Atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045[J]. Diabetes Res Clin Pract, 2022, 183: 109119.
[2] FANELLI G, RASCHI E, HAFEZ G, et al. The interface of depression and diabetes: treatment considerations[J]. Transl Psychiatry, 2025, 15(1): 22.
[3] FERRARI AJ, CHARLSON FJ, NORMAN RE, et al. Burden of depressive disorders by country, sex, age, and year: findings from the global burden of disease study 2010[J]. PLoS Med, 2013, 10(11): e1001547.
[4] DIABETES CANADA CLINICAL PRACTICE GUIDELINES EXPERT COMMITTEE, PUNTHAKEE Z, GOLDENBERG R, et al. Definition, classification and diagnosis of diabetes, prediabetes and metabolic syndrome[J]. Can J Diabetes, 2018, 42(Suppl 1): S10-S15.
[5] ZANOVELI JM, DE MORAIS H, DA SILVA DIAS IC, et al. Depression associated with diabetes: from pathophysiology to treatment[J]. Curr Diabetes Rev, 2016, 12(3): 165-178.
[6] CHAMPANERI S, WAND GS, MALHOTRA SS, et al. Biological basis of depression in adults with diabetes[J]. Curr Diab Rep, 2010, 10(6): 396-405.
[7] KAUSHIK DK, GUPTA M, KUMAWAT KL, et al. NLRP3 inflammasome: key mediator of neuroinflammation in murine Japanese encephalitis[J]. PLoS One, 2012, 7(2): e32270.
[8] MATURANA CJ, AGUIRRE A, SÁEZ JC. High glucocorticoid levels during gestation activate the inflammasome in hippocampal oligodendrocytes of the offspring[J]. Dev Neurobiol, 2017, 77(5): 625-642.
[9] FREEMAN L, GUO HT, DAVID CN, et al. NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes[J]. J Exp Med, 2017, 214(5): 1351-1370.
[10] THUNDYIL J, LIM KL. DAMPs and neurodegeneration[J]. Ageing Res Rev, 2015, 24(pt a): 17-28.
[11] MANGAN MSJ, OLHAVA EJ, ROUSH WR, et al. Targeting the NLRP3 inflammasome in inflammatory diseases[J]. Nat Rev Drug Discov, 2018, 17(8): 588-606.
[12] GIULIANI AL, SARTI AC, FALZONI S, et al. The P2X7 receptor-interleukin-1 liaison[J]. Front Pharmacol, 2017, 8: 123.
[13] BHATTACHARYA A, JONES DNC. Emerging role of the P2X7-NLRP3-IL1β pathway in mood disorders[J]. Psychoneuroendocrinology, 2018, 98: 95-100.
[14] LI ZR, HAN YS, LIU Z, et al. GR/NF-κB signaling pathway regulates hippocampal inflammatory responses in diabetic rats with chronic unpredictable mild stress[J]. Eur J Pharmacol, 2021, 895: 173861.
[15] 刘洋, 李萍, 杨蕙, 等. 左归降糖解郁方改善糖尿病并发抑郁症大鼠N-甲基D-天冬氨酸受体过激致海马突触损伤的作用机制[J]. 中草药, 2023, 54(19): 6323-6335.
[16] YANG H, LING J, MENG P, et al. Activation of hippocampal IR/IRS-1 signaling contributes to the treatment with Zuogui Jiangtang Jieyu decoction on the diabetes-related depression[J]. Evid Based Complement Alternat Med, 2021, 2021: 6688723.
[17] LI P, WANG TT, GUO HP, et al. Pramipexole improves depression-like behavior in diabetes mellitus with depression rats by inhibiting NLRP3 inflammasome-mediated neuroinflammation and preventing impaired neuroplasticity[J]. J Affect Disord, 2024, 356: 586-596.
[18] BURNSTOCK G, KNIGHT GE. The potential of P2X7 receptors as a therapeutic target, including inflammation and tumour progression[J]. Purinergic Signal, 2018, 14(1): 1-18.
[19] BHATTACHARYA A, BIBER K. The microglial ATP-gated ion channel P2X7 as a CNS drug target[J]. Glia, 2016, 64(10): 1772-1787.
[20] BHATTACHARYA A. Recent advances in CNS P2X7 physiology and pharmacology: focus on neuropsychiatric disorders[J]. Front Pharmacol, 2018, 9: 30.
[21] DI VIRGILIO F, BEN DD, SARTI AC, et al. The P2X7 receptor in infection and inflammation[J]. Immunity, 2017, 47(1): 15-31.
[22] FAROOQ RK, TANTI A, AINOUCHE S, et al. A P2X7 receptor antagonist reverses behavioural alterations, microglial activation and neuroendocrine dysregulation in an unpredictable chronic mild stress (UCMS) model of depression in mice[J]. Psychoneuroendocrinology, 2018, 97: 120-130.
[23] ADINOLFI E, GIULIANI AL, DE MARCHI E, et al. The P2X7 receptor: a main player in inflammation[J]. Biochem Pharmacol, 2018, 151: 234-244.
[24] BASSO AM, BRATCHER NA, HARRIS RR, et al. Behavioral profile of P2X7 receptor knockout mice in animal models of depression and anxiety: relevance for neuropsychiatric disorders[J]. Behav Brain Res, 2009, 198(1): 83-90.
[25] CSÖLLE C, BARANYI M, ZSILLA G, et al. Neurochemical changes in the mouse hippocampus underlying the antidepressant effect of genetic deletion of P2X7 receptors[J]. PLoS One, 2013, 8(6): e66547.

基金资助

湖南省科校联合基金资助项目(2025JJ90061);教育厅优青项目(24B0342;23B0360);长沙市自然科学基金资助项目(kq2502215);湖南省卫健委一般项目(w20243097);中药粉体与创新药物研究省部共建国家重点实验室培育基地开放基金资助项目(24PTKF1013)