Determination of Fibrinogen C Domain-Containing Protein 1(FIBCD1) as A Novel Target for Treatment of Post-Traumatic Stress Disorder(PTSD) and Discovery of A Novel FIBCD1 Inhibitor

摘要

Objective: Post-traumatic stress disorder(PTSD) is a complex mental health condition marked by persistent psychological and physiological responses following traumatic events. Research indicates that PTSD patients often exhibit immune system imbalances and inflammatory states, which may be closely linked to their pathophysiological mechanisms. In terms of treatment, in addition to traditional psychotherapy and medication, new approaches like immunotherapy are being explored to provide more effective treatments for this disorder. As a chitin-binding receptor in mammals, FIBCD1(Fibrinogen C domain-containing protein 1) acts as a pattern recognition receptor, identifying chitin on microbial surfaces and thereby initiating host immune responses. FIBCD1 is detected both peripherally and within the brain glial cells, demonstrating regulatory functions in immune processes. However, the potential role of FIBCD1 in neuroinflammation and PTSD remains unclear. Methods: In this study, a modified single prolonged stress(mSPS) model was employed to induce PTSD in C57 BL/6J mice. The expression of FIBCD1 in various brain regions was assessed via immunofluorescence staining and western blot analysis. To evaluate the effect of FIBCD1 in PTSD-like behaviors, male FIBCD1-deficient mice underwent behavioral test including the elevated plus maze(EPM), open field test(OFT), fear conditioning response, and fear generalization assays. The underlying mechanism was explored by immunofluorescence and western blot. Additionally, the involvement of FIBCD1 in neuroinflammatory processes was investigated using lipopolysaccharide(LPS)-activated BV-2 cells. To target FIBCD1 therapeutically, virtual screening was conducted to identify potentical small-molecule inhibitors, followed by validation using SPR, DARTs, and western blotting. The anti-neuroinflammatory efficacy of the identified inhibitor was subsequently examined in LPS-stimulated BV-2 cells. Finally, the blood-brain barrier(BBB) permeability of the candidated inhibitor was assessed in an in vitro BBB model utilizing BEND3 cells. Results: First, we observed a marked upregulation of microglial FIBCD1 expression in amygdala of mSPS mice. Western blot analysis confirmed that adeno-associated virus(AAV)-mediated delivery of shRNA effectively knocked down FIBCD1 expression in amygdala. In the OFT, FIBCD1 knockdown significantly increased both the number of entries into and time spent in the center zone, without affecting locomotor activity. Furthermore, in the EPM, mSPS-induced AAV-shNC group exhibited anxietylike behavior characterized by reduced time in the open arms, this phenotype was ameliorated by FIBCD1 knockdown. During cued fear conditioning, FIBCD1 knockdown reulted in reduced freezing behavior. However, no significant difference in contextual fear memory was observed mSPS-induced AAV-shNC and AAV-shFIBCD1 group. FIBCD1 knockdown also attenuated fear generalization to the conditioned tone. Mechanistically, FIBCD1 knockdown suppressed neuroinflammation in mSPS mice via inhibition of the TLR2-NF-κB signaling pathway. In vitro, LPS stimulation increased FIBCD1 protein levels in BV-2 cells. Knockdown of FIBCD1 reduced the expression pro-inflammatory factors including iNOS, IL-1β, and IL-6, and decrease the proportion of M1 polarized microglia following LPS challenge. Further experiments indicated that FIBCD1 knockdown attenuates LPS-induced inflammatory cytokines production by inhibiting NF-κB activation. Subsequently, Virtual screening was performed to identify potential FIBCD1 inhibitors. The top 200 compounds ranked by docking score were subjected to strucural clustering, from which 8 representative candidates were selected for experimental validation. DARTs assays confirmed direct binding between compound 1 and FIBCD1 protein. SPR measurements revealed a binding affinity of 585 μM. Western blot analysis demonstrated that compound 1 significantly suppressed FIBCD1 expression at concentrations from 40 μM. Thus, compound 1 was identified as a novel small-molecule inhibitor of FIBCD1. Finally, compound 1 exhibited blood-brain barrier permeability and anti-inflammatory effects in the in vitro model. Conclusion: In summary, this study identifies FIBCD1 as a potential therapeutic target for PTSD and reports the development of a first small-molecule FIBCD1 inhibitor. These findings provide a mechanistic and pharmacological foundation for targeting FIBCD1 in the treatment of PTSD.

关键词

post-traumatic stress disorder / fibrinogen C domain containing 1 / neuroinflammation / amygdala / inhibitor