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

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.

Keywords: High mobility group box 1, 5-Hydroxytryptamine receptor 7, Ferroptosis, Depression