高迁移率族蛋白B1通过调节5-羟色胺受体7信号通路介导小鼠抑郁样行为的作用及机制研究

杜欧; 段上上; 唐轮; 杜俊蓉

doi:10.12182/20251160101

高迁移率族蛋白B1通过调节5-羟色胺受体7信号通路介导小鼠抑郁样行为的作用及机制研究

Role and Mechanism of High Mobility Group Box 1 in Mediating Depression-Like Behaviors in Mice via Regulation of the 5-Hydroxytryptamine Receptor 7 Signaling Pathway

摘要

摘要:
目的探究高迁移率族蛋白B1（high mobility group box 1 protein, HMGB1）与5-羟色胺受体7（5-hydroxytryptamine receptor 7, 5-HT7R）对小鼠抑郁样行为的调控作用及潜在机制。
方法将5-HT7R敲除（5-HT7R^−/−）小鼠与同窝野生型（WT）小鼠分为以下4组：WT/空白病毒（AAV-Scramble）、5-HT7R^−/−/AAV-Scramble、WT/HMGB1过表达病毒（AAV-HMGB1）和5-HT7R^−/−/AAV-HMGB1组，对AAV-Scramble组与AAV-HMGB1组小鼠分别进行空白病毒与AAV-HMGB1海马区注射。3周后通过抑郁样行为学评价小鼠的抑郁状态，利用免疫荧光染色检测小鼠海马区的神经元损伤，使用商业试剂盒测定小鼠海马区还原性谷胱甘肽与氧化型谷胱甘肽比值（GSH/GSSG）、Fe²⁺与丙二醛（MDA）的水平以及超氧化物歧化酶（SOD）的活性。此外，通过Western blot和ELISA检测小鼠海马区5-HT7R/环磷酸腺苷（cAMP）/蛋白激酶A（PKA）与核因子E2相关因子2（Nrf2）/胱氨酸-谷氨酸交换体（xCT）/谷胱甘肽过氧化物酶4（GPX4）信号通路水平，采用免疫荧光双染检测小鼠海马区M2型小胶质细胞铁死亡。最后，利用qRT-PCR检测小鼠海马区促炎因子〔白细胞介素（IL）-1β、肿瘤坏死因子-α（TNF-α）〕与抗炎因子〔IL-10、精氨酸酶-1（Arg-1）〕的mRNA水平。
结果与AAV-Scramble组相比，海马区HMGB1过表达诱导小鼠抑郁样行为与神经元损伤（P<0.01），而5-HT7R敲除减轻上述病理表型（P<0.01）。与AAV-Scramble组相比，HMGB1过表达降低小鼠海马区GSH/GSSG比值与SOD活性（均P<0.01），同时上调Fe²⁺与MDA水平（均P<0.01），而5-HT7R敲除改善上述铁死亡相关指标（P<0.01）。与AAV-Scramble组相比，HMGB1过表达上调小鼠海马区5-HT7R表达（P<0.01），同时下调cAMP/PKA和Nrf2/xCT/GPX4信号通路（P<0.01），而5-HT7R敲除改善上述信号通路蛋白的降低（P<0.01）。与AAV-Scramble组相比，HMGB1过表达上调小鼠海马区小胶质细胞上5-HT7R与铁蛋白重链（FTH）的表达（P<0.01），下调Nrf2的表达（P<0.01），同时诱导FTH与CD206的共定位（P<0.01）；而5-HT7R敲除逆转上述小胶质细胞上FTH与Nrf2的表达变化（P<0.01），同时降低FTH与CD206的共定位（P<0.01）。此外，HMGB1过表达诱导小鼠海马区小胶质细胞极化为M1型与M2型（P<0.01），上调促炎因子（IL-1β、TNF-α）与抗炎因子（IL-10、Arg-1）mRNA水平（P<0.01），而5-HT7R敲除减轻上述小胶质细胞M1型极化以及降低上述促炎因子的mRNA水平（P<0.01），同时促进小胶质细胞M2型极化以及上调上述抗炎因子的mRNA水平（P<0.01）。
结论 HMGB1通过激活5-HT7R，下调cAMP/PKA/AKT/Nrf2/GPX4信号通路，进而介导M2型小胶质细胞铁死亡，最终促进小鼠抑郁样行为。

Abstract:
Objective To investigate the regulatory effects and underlying mechanisms of high mobility group box 1 (HMGB1) and 5-hydroxytryptamine receptor 7 (5-HT7R) on depressive-like behaviors in mice.
Methods 5-HT7R knockout (5-HT7R^−/−) mice and their wild-type (WT) littermates were assigned to 4 groups, including the WT/control virus (AAV-Scramble), 5-HT7R^−/−/AAV-Scramble, WT/HMGB1 overexpression virus (AAV-HMGB1), and 5-HT7R^−/−/AAV-HMGB1 groups. Mice in the AAV-Scramble and AAV-HMGB1 groups received hippocampal injections of control virus and AAV-HMGB1, respectively. Three weeks later, the depression state of the mice were assessed with depressive behavior tests, the neuronal damage in the hippocampus was evaluated using immunofluorescence staining, and the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), the levels of Fe²⁺ and malondialdehyde (MDA), and superoxide dismutase (SOD) activity in the hippocampus were measured using commercial kits. Additionally, Western blot and ELISA were used to determine the levels of the 5-HT7R/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway and the nuclear factor erythroid 2-related factor 2 (Nrf2)/cystine-glutamate exchanger (xCT)/glutathione peroxidase 4 (GPX4) signaling pathway in the hippocampus. Immunofluorescence double staining was performed to assess M2 microglial ferroptosis in the hippocampus. Finally, the mRNA levels of pro-inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor-α (TNF-α), and anti-inflammatory cytokines, IL-10 and arginase-1 (Arg-1), in the hippocampus was measuring using qRT-PCR.
Results Compared with the AAV-Scramble group, hippocampal HMGB1 overexpression induced depressive-like behaviors and neuronal damage (P < 0.01), while 5-HT7R knockout alleviated these pathological phenotypes (P < 0.01). Compared with the AAV-Scramble group, HMGB1 overexpression reduced the GSH/GSSG ratio and SOD activity (both P < 0.01) and increased Fe²⁺ and MDA levels (both P < 0.01) in the hippocampus, whereas 5-HT7R knockout improved these ferroptosis-related indicators (P < 0.01). Compared with the AAV-Scramble group, HMGB1 overexpression upregulated 5-HT7R expression (P < 0.01) and downregulated the cAMP/PKA and Nrf2/xCT/GPX4 signaling pathways (P < 0.01) in the hippocampus, whereas 5-HT7R knockout ameliorated the reduction in these signaling pathways (P < 0.01). Compared with the AAV-Scramble group, HMGB1 overexpression upregulated the expression of 5-HT7R and ferritin heavy chain (FTH) in microglia (P < 0.01), downregulated Nrf2 expression (P < 0.01), and induced colocalization of FTH and CD206 (P < 0.01). 5-HT7R knockout reversed the changes in FTH and Nrf2 expression in microglia (P < 0.01) and reduced the colocalization of FTH and CD206 (P < 0.01). Furthermore, HMGB1 overexpression promoted the polarization of microglia to both the M1 and M2 types (P < 0.01) and upregulated the mRNA levels of pro-inflammatory (IL-1β and TNF-α) and anti-inflammatory (IL-10 and Arg-1) cytokines (P < 0.01), whereas 5-HT7R knockout attenuated microglial M1-type polarization and reduced the mRNA levels of the aforementioned pro-inflammatory cytokine (P < 0.01) while promoting microglial M2-type polarization and increasing the mRNA levels of the aforementioned anti-inflammatory cytokines (P < 0.01).
Conclusion HMGB1 activates 5-HT7R and downregulates the cAMP/PKA/AKT/Nrf2/GPX4 signaling pathways, thereby mediating M2 microglial ferroptosis and neuroinflammation, ultimately promoting depressive-like behaviors in mice.

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