Supplementary Material for: Bacillus Calmette-Guérin Confers Neuroprotection in a Murine Model of Japanese Encephalitis

Objective: Japanese encephalitis (JE) is a debilitating disease caused by infection with the JE virus (JEV; family: Flaviviridae), which leaves neurological sequelae in survivors but more often leads to mortality. Neurodegeneration caused by inflammation is the primary pathology behind the clinical manifestation of encephalitis caused by JEV. Bacillus Calmette-Guérin (BCG) has been used in immunoprophylaxis for tuberculosis and in the adjuvant therapy of many malignancies, and has exhibited neuroprotective activities in experimental models of Parkinson and Alzheimer disease. This study aimed at assessing the neuroprotective role of BCG in a murine model of JE. Methods: Suckling mice were inoculated with 106 CFU of BCG and at 18 days postinoculation were challenged with 100 LD50 of JEV. PBS-inoculated mice were used as controls. Mice were sacrificed on days 2, 4, 6, and 8. Brain tissue was homogenized for RNA extraction. One-step real-time RT-PCR was performed to assess the relative gene expressions of TNF-α, IL-6, and iNOS. Results: The BCG-inoculated (BCG+JEV) group exhibited a significant delay in the presentation of neuropathological symptoms, longer survival, and a downregulation in the expression of TNF-α, IL-6, and iNOS on days 2, 4, and 6 post-JEV challenge compared to the JEV group. Conclusion: These findings indicate that the administration of BCG offers neuroprotection in the murine model of JE. BCG should therefore be further investigated as an adjuvant in the management of JE. BCG is an accepted vaccine for tuberculosis in many countries that are endemic for JEV. This approach may have a significant impact on the public health burden in these countries.研究目的：日本脑炎（Japanese encephalitis, JE）是由JE病毒（Japanese encephalitis virus, JEV; 黄病毒科Flaviviridae）感染引发的致残性疾病，幸存者常遗留神经后遗症，且病死率较高。炎症介导的神经退行性变是JEV所致脑炎临床症状的核心病理机制。卡介苗（Bacillus Calmette-Guérin, BCG）曾被用于结核病的免疫预防及多种恶性肿瘤的辅助治疗，且在帕金森病、阿尔茨海默病的实验模型中已展现出神经保护活性。本研究旨在评估卡介苗在日本脑炎小鼠模型中的神经保护作用。 研究方法：将乳鼠接种10⁶ CFU的卡介苗，于接种后18天以100 LD₅₀的JEV进行攻毒。以PBS（磷酸盐缓冲液）接种的小鼠作为对照组。分别于攻毒后第2、4、6、8天处死小鼠，取脑组织匀浆用于RNA提取。采用一步法实时荧光定量RT-PCR检测肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）及诱导型一氧化氮合酶（iNOS）的相对基因表达水平。 研究结果：与单纯JEV攻毒组相比，卡介苗预处理组（BCG+JEV）的神经病理症状出现时间显著延迟，存活时长更长，且在JEV攻毒后第2、4、6天的TNF-α、IL-6及iNOS表达水平均显著下调。 研究结论：本研究结果表明，卡介苗给药可在日本脑炎小鼠模型中发挥神经保护作用。因此，卡介苗作为日本脑炎治疗的辅助手段值得进一步深入研究。鉴于JEV流行区的多个国家已将卡介苗纳入结核病常规疫苗接种计划，该策略或可对上述地区的公共卫生负担产生显著改善作用。