目的:通过蛋白质组学技术及组织病理学观察并验证灵猫方调控HBV相关免疫应答的作用及机制.方法:将 30 只SPF级雄性C57BL/6N小鼠随机分为正常组、模型组、灵猫方组,每组10 只.除正常组外,其余小鼠采用水动力转染技术建立HBV体内复制模型,灵猫方组小鼠给予灵猫方灌胃,2 次/d,模型组和正常组小鼠给予等体积饮用水,连续灌胃3 d.Elisa检测分析小鼠血清HBsAg、HBeAg、IFNα含量,IHC染色观察小鼠肝组织F4/80 表达,实时荧光定量PCR检测小鼠肝组织TNFα、IL-1β、IL-6 等促炎因子的含量,高通量蛋白质组学技术检测小鼠肝脏免疫细胞蛋白质表达,筛选差异表达的信号通路.结果:与模型组比较,灵猫方组小鼠血清HBsAg、HBeAg滴度明显下降(P<0.05,P<0.01),IFNα水平显著上升(P<0.000 1),肝组织F4/80 蛋白表达量明显降低(P<0.01).GO分析显示,灵猫方组小鼠差异蛋白显著富集于代谢相关功能;KEGG分析显示,差异蛋白显著富集于PPAR通路;差异蛋白表达定量分析显示,与模型组相较,灵猫方组小鼠免疫细胞的代谢能力下降,天然免疫应答上升.结论:灵猫方可能通过作用于PPAR信号通路,参与免疫细胞脂质代谢重编程,进而增强HBV相关免疫应答,同时抑制促炎因子的释放.
Objective:By employing proteomic techniques and histopathological observations,we aim to corroborate the role and mechanisms by which the Lingmao formula regulates the immune response associated with HBV.Methods:Thirty SPF male C57BL/6N mice were randomly divided into normal group,model group and Lingmao formula group.In addition to 10 in the normal group,the other 20 were established by hydrodynamic transfection technology.Lingmao formula was gavaged for 3 days,twice a day.The model group and normal group were gavaged with drinking water.The contents and expression of HBsAg,HBeAg and IFNα in serum were detected by Elisa.The expression of F4/80 in liver tissue was observed by IHC staining.The contents of TNFα,IL-1β,IL-6 and other proinflammatory factors in liver of mice were detected by PCR.High-throughput proteomics technique was used to detect the changes of protein expression in liver immune cells and screen the differential pathways.Results:Compared with model group,the titers of HBsAg and HBeAg in Lingmao Formula group decreased significantly(P<0.05,P<0.01).The level of IFNα in lingmao Formula group was significantly higher than that in normal group and model group(P<0.0001);Compared with model group,F4/80 protein expression level of Lingmao Formula was significantly decreased(P<0.01).GO analysis showed that differential proteins were significantly enriched in metabolism-related functions.KEGG analysis showed that differential proteins were significantly enriched in PPAR pathway.Quantitative analysis of differential protein expression showed that compared with model group,the metabolic capacity of immune cells in Lingmao Formula group decreased,and the natural immune response increased.Conclusion:Lingmao Formula may be involved in lipid metabolism reprogramming of immune cells by acting on PPAR signaling pathway,thereby enhancing HBV-related immune response and inhibiting the release of pro-inflammatory factors.