安宫牛黄丸对小儿严重脓毒症体外膜肺氧合脑损伤的脑保护作用

Cerebral Protective Effect of Angong Niuhuang Pill on the Brain Injury of Children Severe Sepsis with Extracorporeal Membrane Oxygenation

目的探讨安宫牛黄丸对小儿肺炎合并严重脓毒症体外膜肺氧合(ECMO)支持下的生化指标、危重程度、免疫指标及脑部并发症的作用。方法选择2017年2月1日—2020年4月30日河南省人民医院儿童重症监护病房(PICU)收住20例肺炎合并严重脓毒症需ECMO支持并脑部并发症患儿,通过信封编码法随机分为Ⅱ组和Ⅲ组,每组10例。同时收住10例小儿肺炎合并一般脓毒症为Ⅰ组,Ⅰ组给予常规治疗。Ⅱ、Ⅲ组均给予重症常规干预,Ⅲ组患儿入组初始即同时给予鼻饲安宫牛黄丸10天。观察外周血生化指标及危重程度,流式细胞法分析外周血中性粒细胞和巨噬细胞各种亚群、CD4^(+)、CD8^(+)T细胞的变化;ELISA、RT-PCR和ELISPOT检测外周血免疫因子和适应性免疫应答的变化。结果与Ⅰ组比较,Ⅱ组和Ⅲ组C反应蛋白(CRP)、降钙素原(PCT)、碱剩余(BE)绝对值、乳酸(Lac)、平均住院天数均升高(P<0.01),氧分压/氧浓度(PF值)降低(P<0.01),危重评分降低(P<0.01);外周血CD63^(+)单核细胞比例、CD4^(+)、CD8^(+)T细胞数目、CD8^(+)细胞毒性T细胞(CTL)功能、肿瘤坏死因子-α(TNF-α)和趋化因子CXCL9蛋白和m RNA、Toll样受体9(TLR9)、趋化因子CXCL10、IL-27 mRNA、内皮保护因子可溶性TEK酪氨酸激酶2(s TIE2)蛋白降低(P<0.01,P<0.05),弹性蛋白酶(elastase)^(+)、趋化因子受体CCR2^(high)CX3CR1^(low)/CCR2^(low)CX3CR1^(high)比例、IL-10和血管内皮生长因子(VEGF)蛋白、高迁移率族蛋白B1(HMGB1)和IL-17蛋白及m RNA、晚期糖基化终末产物受体(RAGE)和YTH结构域蛋白家族分子1(YTHDF1)m RNA、毛细血管渗漏指标促血管生成素2/1(Ang2/Ang1)蛋白及脑损伤分子神经元烯醇化酶(NSE)蛋白表达均升高(P<0.01,P<0.05)。与Ⅱ组比较,Ⅲ组危重评分略有好转,BE绝对值、Lac、平均住院天数均降低(P<0.01),PF值升高(P<0.01);外周血CD63^(+)单核细胞比例,脂多糖刺激后的CD8^(+)CTL适应性免疫应答,CXCL9蛋白和m RNA及s TIE2蛋白表达升高(P<0.01,P<0.05),elastase^(+)和CCR2^(high)CX3CR1^(low)/CCR2^(low)CX3CR1^(high)比例,HMGB1、IL-17蛋白和m RNA、NSE蛋白及RAGE、YTHDF1基因表达有一定程度下降(P<0.01,P<0.05)。结论安宫牛黄丸干预肺炎合并严重脓毒症ECMO脑部并发症的免疫机制与下调CD11b^(+)细胞的HMGB1/RAGE/YTHDF1通路、调节CD11b^(+)细胞功能和极性,改善Th1/Th17比例,减少内皮细胞损伤,抑制炎症细胞进入脑内的调节作用有关。

Objective To explore the effect of Angong Niuhuang Pill(AGNHP)on biochemical inators,criticality,immune mechanisms and brain complications in extracorporeal membrane oxygenation(ECMO)children with pneumonia and severe sepsis.Methods Totally 20 ECMO children patients with pneumonia and severe sepsis accompanied by brain complications admitted into Pediatric Intensive Care Unit of People’s Hospital of Henan Province from February 1,2017 to April 30,2020 were randomly assigned to groupⅡand groupⅢby envelope coding method,with 10 cases in each group.Ten children patients with pneumonia and general sepsis were also enrolled as the groupⅠ.GroupⅠwas given conventional treatment.GroupⅡand groupⅢwere given conventional intensive care intervention.AGNHP was given nasal feeding for 10days at the beginning of pediatric patients enrollment in groupⅢ.The research work included biochemical parameters of peripheral blood and criticality,the subgroups of macrophages and neutrophils,CD4^(+)and CD8^(+)T-lymphocytes in peripheral blood analyzed by flow cytometry,changes of peripheral blood immune factors and adaptive immune responses by ELISA,RT-PCR and ELISPOT.Results Compared with groupⅠ,C-reactive protein(CRP),procalcitonin(PCT),the absolute value of base excess(BE),lactic acid(LAC)and average length of hospital stay increased(P<0.01)in groupsⅡandⅢ,the value of oxygen partial pressure/oxygen concentration(PF)decreased(P<0.01),and the critical score decreased(P<0.01),while the ratio of peripheral blood CD63^(+)monocytes,the number of CD4^(+)and CD8^(+)T-lymphocytes,the function of CD8^(+)cytotoxic T-lymphocytes(CTL)stimulated by LPS,the protein and mRNA express level of tumor necrosis factor-α(TNF-α)and chemokin CXCL9,toll-like receptor 9(TLR9),chemokin CXCL10,interleukin-27(IL-27)mRNA,endothelial protective factor soluble TEK tyrosine kinase 2(sTIE2)protein in groupⅡand groupⅢdecreased(P<0.01,P<0.05),while the ratio of elastase^(+)and chemokin receptor CCR2^(high)CX3 CR1low/CCR2^(low)CX3CR1^(high)(P<0.01),the levels of IL-10 and vascular endothelial growth factor(VEGF)protein,High mobility group protein B1(HMGB1)and IL-17 protein and mRNA,receptor for advanced glycation end products(RAGE)and YTH domain family protein 1(YTHDF1)mRNA,capillary leaking indexes Angiopoietin2/1(Ang 2/1)and brain injury factor Neuron enolase(NSE)protein increased(P<0.01,P<0.05).Compared with groupⅡ,the critical score in groupⅢimproved slightly,the absolute value of BE,LAC and average length of hospital stay decreased(P<0.01),and the value of PF increased(P<0.01),while the ratio of CD63^(+)monocytes,the adaptive immune response of CD8^(+)CTL stimulated by LPS,and the expression levels of CXCL9 protein and mRNA and sTIE2 protein in groupⅢincreased(P<0.01,P<0.05),and the ratio of elastase^(+)and CCR2^(high)CX3CR1^(low)/CCR2^(low)CX3CR1^(high),HMGB1,IL-17 protein and mRNA,NSE protein,RAGE and YTHDF1 gene decreased to some extent(P<0.01,P<0.05).Conclusion The interference immune mechanism of AGNHP on brain complication in pneumonia accompanied by severe sepsis with ECMO support was related to the down-regulation of HMGB1/RAGE/YTHDF1,the regulation of the function and the polarity of CD11b^(+)cells,betterring Th1/Th17 ratio,decreasing endothelial cell injury and inhibiting inflamatory cells entering brain.