Engeletin alleviates depressive-like behaviors by modulating microglial polarization via the LCN2/CXCL10 signaling pathway
Authors: Zhang Jie , Song Zheng , Huo Yanchao , Li Guangqiang , Lu Liming , Gao Xinfu , Wei Chuanmei , Zhang Shuping , Jiang Xingyue , Xu Yangyang 
Published: November 2023

Background
Microglial polarization and associated inflammatory activity are key mediators of depression pathogenesis. The natural Smilax glabra rhizomilax derivative engeletin has been reported to exhibit robust anti-inflammatory activity, but no studies to date have examined the mechanisms through which it can treat depressive symptoms.
Purpose
This study was designed to assess the therapeutic efficacy of engeletin in a murine chronic stress social defeat stress (CSDS) model system and to clarify the underlying mechanisms, with a particular focus on microglial polarization.
Methods
CSDS model mice were used to test the potential antidepressant effects of engeletin. Following a 21-day engeletin treatment period, a range of assays including the sucrose preference test (SPT), social interaction test (SIT), tail suspension test (TST), forced swim test (FST), and open field test (OFT) were used to measure depressive-like behaviors in these mice. Following the completion of such behavioral testing, 3.0 T multifunctional magnetic resonance imaging brain scans including T1-weighted imaging (T1WI), T2-weighted imaging (T2WI), T2 mapping, and diffusion tensor imaging (DTI) were performed. In addition, quantitative real-time PCR (qRT-PCR), and western blotting were used to measure levels of inflammatory cytokines including interleukin (IL)-6, IL-10, IL-1β, and TNF-α. Microglia activation was further evaluated through western blotting and immunohistochemical staining for markers of M1 (CD86, iNOS) and M2 (Arg1, CD206) polarization. The lipocalin-2 (LCN2)/ C-X-C motif chemokine ligand 10 (CXCL10) signaling pathway was additionally assessed via whole transcriptomic sequencing, qRT-PCR, and western blotting. Adeno-associated virus (AAV) particles encoding LCN2-EGFP were then infused into CSDS model mice to evaluate the effects of LCN2 overexpression and engeletin treatment in greater detail.
Results
Treatment for 21 days with engeletin significantly alleviated depressive-like behaviors in CSDS model mice. T1WI and T2WI imaging revealed no significant differences between groups, but the bilateral prefrontal cortex of CSDS mice exhibited significant increases in apparent diffusion coefficient (ADC) and T2 values relative to normal control mice, with a corresponding reduction in fractional anisotropy (FA), while engeletin reversed all of these changes. CSDS resulted in higher levels of IL-1β, IL-6, and TNF-a production, enhanced microglial activation, and greater M1 polarization with a concomitant decrease in M2 polarization in the mPFC, whereas engeletin treatment effectively abrogated these CSDS-related pathological changes. Engeletin was further found to suppress the LCN2/CXCL10 signaling axis such that AAV-induced LCN2 overexpression ablated the antidepressant effects of engeletin and reversed its beneficial effects on the M1/M2 polarization of microglia. These data suggest that the antidepressant effects of engeletin are correlated with the polarization of microglia, highlighting a potential avenue for future design of antidepressant strategies that specifically target the microglia.
Conclusion
Engeletin can alleviate CSDS-induced depressive-like behaviors by regulating the LCN2/CXCL10 pathway and thereby altering the polarization of microglia.