摘要
目的 从快速加速纤维肉瘤激酶(RAF)/丝裂原活化蛋白激酶(MEK)/细胞外信号调节激酶(ERK)通路调控中性粒细胞胞外诱捕网(NETs)失衡探讨凉血退紫方治疗过敏性紫癜(HSP)血热证的可能作用机制。方法 将70只Wistar大鼠随机分为空白组14只和造模组56只。造模组大鼠采用改良的卵白蛋白(OVA)致敏联合热性中药灌胃构建HSP血热证大鼠模型,造模周期共8周。将造模成功的50只大鼠随机分为模型组、复方甘草酸苷组、凉血退紫方组、RAF抑制组、凉血退紫方+RAF激动组,每组10只,另取空白组10只。造模第11周开始,空白组、模型组大鼠在0.9%氯化钠溶液0.5 ml/(kg·d)腹腔注射基础上给予1 ml/(100 g·d)超纯水灌胃;凉血退紫方组、复方甘草酸苷组在0.9%氯化钠溶液腹腔注射基础上分别给予7.5 g/(kg·d)凉血退紫颗粒混悬液灌胃、13.5 mg/(kg·d)复方甘草酸苷混悬液灌胃;RAF抑制组予1 mg/(kg·d)GW5074混悬液腹腔注射及超纯水灌胃;凉血退紫方+RAF激动组予7.5 g/(kg·d)凉血退紫颗粒混悬液灌胃及1 mg/(kg·d)紫杉醇混悬液腹腔注射。各组干预均每天1次,持续4周。干预结束后,HE染色法观察大鼠皮肤组织病理形态,免疫荧光法检测皮肤组织免疫球蛋白A(IgA)沉积情况,ELISA法检测大鼠血清中性粒细胞弹性蛋白酶(NE)、肿瘤坏死因子α(TNF-α)、血管细胞黏附分子1(VCAM-1)含量,比色法检测大鼠血清髓过氧化物酶(MPO)水平,RT-qPCR法检测大鼠皮肤组织RAF、MEK、ERK mRNA表达水平,Western Blot法检测大鼠皮肤组织RAF、MEK、ERK、磷酸化丝裂原活化蛋白激酶(p-MEK)、磷酸化细胞外信号调节激酶(p-ERK)水平。结果 空白组大鼠皮肤组织正常,模型组大鼠皮肤组织出现中性粒细胞浸润、红细胞破裂出血;各药物干预组大鼠皮肤组织中性粒细胞浸润、出血渗出现象明显减轻,且以凉血退紫方组大鼠改善效果显著。与空白组比较,模型组大鼠皮肤组织IgA荧光强度增强,血清NE、MPO、TNF-α及VCAM-1含量升高,RAF、MEK、ERK1、ERK2 mRNA表达升高,RAF蛋白水平及p-MEK/MEK、p-ERK/ERK比值升高(P<0.05)。与模型组比较,各药物干预组大鼠皮肤组织IgA荧光强度减弱,血清NE、MPO、TNF-α及VCAM-1水平降低(P<0.05);凉血退紫方组及RAF抑制组大鼠皮肤组织RAF、MEK、ERK1、ERK2mRNA表达降低,RAF蛋白水平及p-MEK/MEK、p-ERK/ERK比值降低(P<0.05)。与凉血退紫方+RAF激动组比较,复方甘草酸苷组、凉血退紫方组及RAF抑制组大鼠皮肤组织IgA荧光强度减弱,血清NE、MPO、TNF-α及VCAM-1含量降低,MEK mRNA表达及p-MEK/MEK比值降低(P<0.05)。结论 凉血退紫方治疗过敏性紫癜血热证的可能机制是抑制皮肤组织RAF/MEK/ERK通路,减少NETs过度形成,从而降低皮下小血管IgA沉积及机体炎症反应。
Objective To investigate the potential mechanism of Liangxue Tuizi Formula(凉血退紫方,LXTZF)in treating Henoch-Schönlein Purpura(HSP)by examining its regulatory effect on neutrophil extracellular trap(NETs)dysregulation via the rapidly accelerated fibrosarcoma kinase(RAF)/mitogen-activated protein kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling pathway.Methods Seventy Wistar rats were randomly allocated into a blank control group(n=14)and a modeling group(n=56).Rats in the modelling group underwent an eight-week modelling period to establish HSP rat models with blood-heat syndrome via modified ovalbumin(OVA)induction method combined with oral administration of heat-property Chinese herbal medicine.Fifty successfully modeled rats were subsequently randomly divided into five groups(n=10 per group),model group,compound glycyrrhizin group,LXTZF group,RAF inhibitor group,and LXTZF+RAF agonist group.Additionally,10 rats were selected from the original blank control group for the final experiment.From the 11th week of modelling,rats in the blank control group and the model group received 1 ml/(100 g·d)ultrapure water via oral administration,in addition to 0.5 ml/(kg·d)0.9%sodium chloride solution via intraperitoneal injection.The LXTZF group and the compound glycyrrhizin group received 7.5 g/(kg·d)LXTZF granule suspension via gavage,13.5 mg/(kg·d)compound glycyrrhizin suspension via gavage,respectively.The RAF inhibitor group received 1 mg/(kg·d)GW5074 suspension via intraperitoneal injection and ultrapure water via oral administration;the LXTZF+RAF agonist group received 7.5 g/(kg·d)LXTZF granule suspension via gavage and 1 mg/(kg·d)paclitaxel suspension via intraperitoneal injection.All administrations were performed once daily for 4 weeks.After intervention,skin tissue histopathology was examined by hematoxylin and eosin(H&E)staining,immunoglobulin A(IgA)deposition was assessed via immunofluorescence,serum levels of neutrophil elastase(NE),tumor necrosis factor-α(TNF-α),and vascular cell adhesion molecule-1(VCAM-1)were measured using enzyme-linked immunosorbent assay(ELISA),serum myeloperoxidase(MPO)level was determined by a colorimetric assay;the mRNA expression levels of RAF,MEK,and ERK in skin tissue were detected by real-time quantitative polymerase chain reaction(RT-qPCR);and the protein expression of RAF,MEK,ERK,as well as phosphorylated MEK(p-MEK)and phosphorylated ERK(p-ERK),were analyzed by Western Blot.Results Skin tissue in the blank control group rats remained normal,whereas the model group exhibited neutrophil infiltration and haemorrhage with red blood cell rupture.In all drug intervention groups,neutrophil infiltration and haemorrhagic exudation reduced markedly,with LXTZF group demonstrating the most pronounced improvement.Compared with the blank control group,rats in the model group exhibited enhanced IgA fluorescence intensity in skin tissue,elevated serum levels of NE,MPO,TNF-αand VCAM-1,increased mRNA expression of RAF,MEK,ERK1 and ERK2,as well as heightened RAF protein levels and p-MEK/MEK and p-ERK/ERK ratios(P<0.05).Compared with the model group,the drug intervention groups exhibited reduced IgA fluorescence intensity in skin tissue,along with decreased serum levels of NE,MPO,TNF-α,and VCAM-1(P<0.05).In LXTZF group and RAF inhibition groups,reduced mRNA expression of RAF,MEK,ERK1,and ERK2 was observed in rat skin tissue,alongside decreased RAF protein levels and reduced p-MEK/MEK and p-ERK/ERK ratios(P<0.05).Compared with LXTZF+RAF agonist group,the compound glycyrrhizin group,LXTZF group,and RAF inhibitior group exhibited reduced IgA fluorescence intensity in skin tissue,decreased serum NE,MPO,TNF-α,and VCAM-1 levels,and decreased MEK mRNA expression and p-MEK/MEK ratio(P<0.05).Conclusion The potential mechanism by which LXTZF treats Henoch-Schönlein purpura with blood heat syndrome may involve blocking the RAF/MEK/ERK signaling pathway in skin tissue,and suppressing excessive formation of NETs,thereby reducing IgA deposition in dermal microvessels and attenuating systemic inflammatory responses.
作者
姜盈盈
杨满翔
袁振华
席乐迎
蔡明阳
马迪雅
李一凡
牛宇航
刘润泽
曹嘉文
陈熙麟
任献青
JIANG Yingying;YANG Manxiang;YUAN Zhenhua;XI Leying;CAI Mingyang;MA Diya;LI Yifan;NIU Yuhang;LIU Runze;CAO Jiawen;CHEN Xilin;REN Xianqing(Pediatric Hospital,First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou,450000;College of Pediatrics,Henan University of Chinese Medicine)
出处
《中医杂志》
北大核心
2025年第23期2475-2483,共9页
Journal of Traditional Chinese Medicine
基金
国家自然科学基金(82374519)
河南省省级科技研发计划联合基金(优势学科培育类)项目(222301420022)
河南省中原英才计划(育才系列)项目(豫组通[2021]44号)
中国博士后科学基金(2025M773948)
河南省卫生健康委员会国家中医药传承创新中心联合共建科研专项(2024ZXZX1056)
河南省中医学“双一流”创建科学研究专项(HSRP-DFCTCM-2023-8-07、HSRP-DFCTCM-2023-3-02)。
关键词
过敏性紫癜
血热证
免疫球蛋白A
中性粒细胞胞外诱捕网
快速加速纤维肉瘤激酶
丝裂原活化蛋白激酶
细胞外信号调节激酶
凉血退紫方
Henoch-Schönlein purpura
blood heat syndrome
immunoglobulin A
neutrophil extracellular traps
rapidly accelerated fibrosarcoma kinase
mitogen-activated protein kinase
extracellular signal-regulated kinase
Liangxue Tuizi Formula(凉血退紫方)
