Sun Qian, Li Ling, Li Jing, Li Si-Yu, Zhang Yan, Chen Xin-Si, Liu Sha-Sha, Hua Zi-Yu
Department of Neonatology, Children's Hospital of Chongqing Medical University, Chongqing, China; Key Laboratory of Child Neurodevelopment and Cognitive Disorders, Chongqing, China; Key Laboratory of Child Rare Diseases in Infection and Immunity, Chongqing, China; International Science and Technology Cooperation base of Child development and Critical Disorders, Chongqing, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
Department of Neonatology, Children's Hospital of Chongqing Medical University, Chongqing, China; Key Laboratory of Child Neurodevelopment and Cognitive Disorders, Chongqing, China; Key Laboratory of Child Rare Diseases in Infection and Immunity, Chongqing, China; International Science and Technology Cooperation base of Child development and Critical Disorders, Chongqing, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
Int Immunopharmacol. 2025 Mar 26;150:114294. doi: 10.1016/j.intimp.2025.114294. Epub 2025 Feb 18.
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.
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.
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.
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.
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.
坏死性小肠结肠炎（NEC）引起的全身炎症会对发育中的中枢神经系统产生不利影响。有证据表明，肠道来源的高迁移率族蛋白B1（HMGB1）可迁移至大脑并激活小胶质细胞。
确定HMGB1抑制剂甘草酸是否可通过调节HMGB1/ Toll样受体4（TLR4）途径减轻NEC中的小胶质细胞焦亡和神经炎症损伤。
分析临床NEC样本中的HMGB1水平。通过缺氧、冷暴露和过度喂养诱导建立NEC模型。用脂多糖（LPS）刺激BV2小胶质细胞以模拟NEC诱导的炎症。对肠道和大脑进行组织学评估。采用细胞计数试剂盒-8（CCK-8）法评估细胞增殖。实时定量聚合酶链反应（RT-qPCR）定量检测HMGB1、TLR4、核因子κB p65（NF-κB p65）、肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-6和IL-18的mRNA水平。使用蛋白质印迹法、酶联免疫吸附测定（ELISA）、免疫组织化学和免疫荧光分析关键标志物的蛋白表达。针对HMGB1和TLR4设计小干扰RNA（siRNA）。
在临床NEC样本和NEC小鼠模型中均观察到HMGB1水平升高，在粪便、脑脊液和脑组织中检测到更高浓度。在NEC患者中，Spearman分析显示脑脊液和粪便上清液中升高的HMGB1水平之间存在显著相关性。甘草酸治疗似乎可减轻NEC小鼠的脑损伤，并似乎可减轻BV2小胶质细胞中LPS诱导的炎症。此外，甘草酸可增强紧密连接蛋白闭合蛋白和髓鞘碱性蛋白（MBP）的表达，这可能与NEC小鼠行为表现的改善有关。此外，甘草酸治疗可导致脑组织和BV2小胶质细胞中关键炎症介质减少，包括NF-κB p65、NOD样受体蛋白3（NLRP3）、半胱天冬酶-1 p20、gasdermin D（GSDMD）、IL-1β和IL-18。这些结果表明，甘草酸可能至少部分通过调节HMGB1/TLR4信号通路发挥作用。
甘草酸通过抑制HMGB1/TLR4途径、保护MBP、保护血脑屏障并减少BV2小胶质细胞焦亡，有效减轻NEC中的神经炎症损伤。这些发现表明，甘草酸可能为改善NEC的神经学预后提供一种新的治疗方法。
