摘要
目的:基于免疫浸润特征,采用细胞实验与动物实验探讨冬虫夏草蛋白缓解狼疮性肾炎(Lupus nephritis,LN)的作用及机制。方法:从GEO数据库中获取LN患者肾脏组织数据集,采用R软件、Import和CIBERSORT数据库对肾脏中免疫细胞浸润情况进行分析,并通过随机森林法评估LN患者肾脏中的免疫浸润特征。提取冬虫夏草总蛋白并通过LC-MS/MS鉴定其组成;构建M1型巨噬细胞模型,通过实时荧光定量PCR和流式细胞术评价冬虫夏草蛋白对M1型巨噬细胞极化的影响;利用MRL/lpr小鼠建立LN模型,通过ELISA、HE染色、PAS染色、流式细胞术和免疫荧光等技术分析冬虫夏草总蛋白对LN小鼠肾脏及对肾脏M1型巨噬细胞极化的影响;通过Western blot法检测冬虫夏草总蛋白对M1型巨噬细胞p-p38、p38、p-ERK、ERK、p-JNK和JNK蛋白表达的影响。结果:免疫浸润分析结果表明,M1型巨噬细胞在LN患者肾脏中浸润增加,抑制肾脏M1型巨噬细胞的浸润可能是治疗LN的有效干预方案。LC-MS/MS从冬虫夏草蛋白中,共鉴定出422个非冗余蛋白。细胞实验结果表明,与模型对照组相比,冬虫夏草蛋白100、200、400μg/mL均可降低巨噬细胞中一氧化氮合酶(Inos)和白细胞介素1β(Ilb)的基因表达(P<0.01),抑制M1型巨噬细胞(F4/80^(+)CD86^(+)细胞)的极化(P<0.05)。动物实验结果表明,与模型对照组相比,冬虫夏草蛋白1.5 g/kg组小鼠尿蛋白含量显著降低(P<0.01),尿肌酐含量显著增加(P<0.01),血清中dsDNA抗体和血清ANA含量明显降低(P<0.05),HE和PAS染色的结果均表明冬虫夏草蛋白1.5 g/kg小鼠肾组织损伤减轻,LN小鼠肾脏M1型巨噬细胞的浸润减轻,p-p38和p-JNK蛋白表达下调(P<0.05)。结论:冬虫夏草总蛋白可能通过JNK/p38 MAPK信号通路抑制M1型巨噬细胞活化缓解狼疮性肾炎。
Objective:To investigate the role and mechanism of Dongchongxiacao(冬虫夏草)protein in alleviating lupus nephritis(LN)by cell and animal experiments based on the immune infiltration characteristics.Methods:A data set of the kidney tissue in LN patients was obtained from the Gene Expression Omnibus(GEO).Immune cell infiltration in the kidneys was analyzed by R,Import,and CIBERSORT.The immune infiltration characteristics in the kidneys of LN patients were assessed by the random forest method.The total protein of Dongchongxiacao was extracted and its composition was identified by LC-MS/MS.M1-type macrophage model was established,and real-time fluorescence quantitative PCR and flow cytometry were employed to evaluate the effect of Dongchongxiacao protein on M1-type macrophage polarization.An LN model was established in MRL/lpr mice,and the effects of Dongchongxiacao total protein on M1-type macrophage polarization were examined by ELISA,hematoxylin-eosin(HE)staining,periodic acid-Schiff(PAS)staining,flow cytometry,and immunofluorescence.Furthermore,the protein levels of p38,phosphorylated p38(p-p38),extracellular signal-regulated kinase(ERK),phosphorylated ERK(p-ERK),c-Jun N-terminal kinase(JNK),and phosphorylated JNK(p-JNK)in the M1-type macrophages treated with Dongchongxiacao total protein were determined by Western blot.Results:Immune infiltration analysis revealed M1-type macrophages in the kidneys of LN patients increased,suggesting that targeting renal M1-type macrophage infiltration might be a promising treatment approach for LN.A total of 422 non-redundant proteins were identified in Dongchongxiacao.In the cell experiment,compared with the model group,Dongchongxiacao protein(100,200,and 400μg/mL)down-regulated the mRNA levels of inducible nitric oxide synthase(iNOS)and interleukin(IL)-1βin macrophages(P<0.01)and inhibited the polarization of M1-type macrophages(F4/80+CD86+)(P<0.05).In the animal experiment,compared with the model group,Dongchongxiacao protein led to a reduction of urinary protein content(P<0.01),an increase of urinary creatinine level(P<0.01),and decreases of levels of dsDNA antibody and anti-nuclear antibody(ANA)in the serum(P<0.05).HE and PAS staining results indicated that Dongchongxiacao protein attenuated the renal tissue damage in mice after treatment.The results of real-time PCR,immunofluorescence,and flow cytometry suggested that Dongchongxiacao protein reduced the infiltration of Ml-type macrophages in the kidneys of the mouse model of LN(P<0.05).Western blot results suggested that Dongchongxiacao protein down-regulated the expressions of p-p38 and p-JNK(P<0.05).Conclusion:Dongchongxiacao protein may alleviate LN by inhibiting M1-type macrophage activation via the p38/JNK MAPK signaling pathway.
作者
周小仝
何丽英
廖郑悦
杨兴茂
李勇
白静
牛舒琪
刘思静
国锦琳
ZHOU Xiaotong;HE Liying;LIAO Zhengyue;YANG Xingmao;LI Yong;BAI Jing;NIU Shuqi;LIU Sijing;GUO Jinlin(School of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611100;State Key Laboratory of Characteristic Chinese Drug Resources in Southwest China,Chengdu 611100;College of Medical Technology,Chengdu University of Traditional Chinese Medicine,Chengdu 611100)
出处
《中药药理与临床》
北大核心
2025年第7期45-51,共7页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然科学基金(编号:81872959、81373920)
四川省科技厅自然科学基金(编号:2023NSFSC1757)。
