Glycyrrhizin alleviates brain injury in necrotizing enterocolitis model mice by suppressing HMGB1/TLR4 pathway

Abstract

Background: Systemic inflammation from necrotizing enterocolitis (NEC) can adversely affect the developing central nervous system. Evidence indicates that gut-derived high mobility group box 1 (HMGB1) can migrate to the brain and activate microglia.

Objective: To determine if glycyrrhizin, an HMGB1 inhibitor, can reduce microglial pyroptosis and neuroinflammatory injury in NEC by modulating the HMGB1/ Toll-like receptor 4 (TLR4) pathway.

Methods: HMGB1 levels were analyzed in clinical NEC samples. NEC models were induced through hypoxia, cold exposure, and overfeeding. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) to mimic NEC-induced inflammation. Histological assessments were performed on the intestines and brain. Cell proliferation was evaluated employing the cell counting kit-8 (CCK-8) assay. Real-time quantitative polymerase chain reaction (RT-qPCR) quantified mRNA levels of HMGB1, TLR4, nuclear factor kappa B p65 (NF-κB p65), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-18. Protein expression was analyzed using western blotting, ELISA, immunohistochemistry, and immunofluorescence for key markers. Small interfering RNAs (siRNAs) targeted HMGB1 and TLR4.

Results: Elevated levels of HMGB1 were observed in both clinical NEC samples and the NEC mouse model, with higher concentrations detected in feces, cerebrospinal fluid, and brain tissue. In the NEC patients, Spearman analysis revealed a significant correlation between elevated HMGB1 levels in CSF and fecal supernatant. Treatment with glycyrrhizin appeared to mitigate brain damage in the NEC mice and seemed to reduce LPS-induced inflammation in BV2 microglial cells. Additionally, glycyrrhizin enhanced the expression of tight junction protein occludin and myelin basic protein (MBP), which may be associated with improvements in behavioral performance observed in the NEC mice. Furthermore, glycyrrhizin treatment resulted in a reduction of key inflammatory mediators, including NF-κB p65, NOD-like receptor protein 3 (NLRP3), Caspase-1 p20, gasdermin D (GSDMD), IL-1β, and IL-18 in brain tissue and BV2 microglial cells. These results suggest that glycyrrhizin may exert its effects, at least in part, through modulation of the HMGB1/TLR4 signaling pathway.

Conclusion: Glycyrrhizin effectively mitigates neuroinflammatory injury in NEC by inhibiting the HMGB1/TLR4 pathway, preserving MBP, protecting the blood-brain barrier, and reducing pyroptosis in BV2 microglial cells. These findings suggest that glycyrrhizin may provide a novel therapeutic approach for improving neurological outcomes in NEC.

Keywords: Glycyrrhizin; High mobility group box 1; Microglia; Necrotizing enterocolitis; Pyroptosis; Toll-like receptor 4.