摘要
目的利用label-free蛋白质组学技术探究扶正化瘀方(FZHY)抑制巨噬细胞向促炎表型(M1)极化的分子机制。方法脂多糖(LPS)和γ干扰素(IFN-γ)诱导小鼠巨噬细胞RAW264.7向M1极化。细胞分为空白对照组、M1组和FZHY低、高剂量组(100、200 mg/L),通过非标记定量蛋白质组学遴选差异蛋白(DEPs)及进行生物信息学分析,并通过实验验证差异蛋白及其信号通路。结果受LPS和IFN-γ诱导后,巨噬细胞向M1极化,与空白对照组比较,共有223个DEPs,其中120个上调,103个下调。经FZHY处理后,与M1组比较,共有103个DEPs,其中80个上调,23个下调。以上各组DEPs的交集为28个,其中脂肪分化相关蛋白2(Plin2)、溶酶体酸脂肪酶A(Lipa)、含犰狳重复序列蛋白6(Armc6)等25个DEPs在M1组中水平降低,在FZHY高剂量组中显著上调;IFN-γ诱导的GTP酶1(Iigp1)、三结构域蛋白质30a(Trim30a)和RAB11家族相互作用蛋白1(Rab11fip1)共3个蛋白在M1组中水平提高,在FZHY高剂量组中显著下调。差异蛋白采用基因本体(GO)数据库分析,发现其生物学功能包括定位修复等、细胞组成涉及细胞质部分等、分子功能包括催化活性等。采用京都基因与基因组百科全书(KEGG)数据库分析显示差异蛋白富集于代谢通路、溶酶体以及谷胱甘肽代谢等。与空白对照组比较,M1组的炎症表面标志物白细胞分化抗原40(CD40)、白细胞分化抗原86(CD86)和诱导型一氧化氮合酶(iNOS)及差异蛋白Iigp1的表达显著提高,而差异蛋白Plin2的表达降低。FZHY处理后可逆转上述炎症诱导的蛋白表达。M1巨噬细胞代谢以糖酵解为主,α-酮戊二酸脱氢酶(AKGDH)和谷氨酰胺连接酶(GLUL)在M1组中的表达降低,伴随着糖酵解速率提高,FZHY的处理可显著提高AKGDH和GLUL的基因表达并降低糖酵解速率。结论FZHY方可通过调控代谢中的关键代谢酶AKGDH、GLUL和差异蛋白Plin2、Iigp1及炎症标记物CD40、CD86、iNOS的表达,下调糖酵解速率而抑制巨噬细胞向M1的极化。
Objective To explore the molecular mechanisms behind the Fuzheng Huayu Formula's(FZHY)suppression of macrophage polarization towards a pro-inflammatory(M1)phenotype using label-free proteomics.Methods Lipopolysaccharides(LPS)and interferon-gamma(IFN-γ)induced the polarization of mouse macrophage RAW264.7 cells towards a M1 phenotype.The cells were divided into a blank control group,a M1 group,and low-and high-dose FZHY groups(100 mg/L and 200 mg/L).Differential expression proteins(DEPs)were selected through labelfree quantitative proteomics for bioinformatics analysis.The differences in DEPs and their signaling pathways were then experimentally validated.Results Following induction by LPS and IFN-γ,macrophages polarized towards the M1 phenotype.Compared with the blank control group,there were a total of 223 DEPs,with 120 upregulated and 103 downregulated in the M1 group.Compared with the M1 phenotype group,post-FZHY treatment resulted in the identification of 103 DEPs,with 80 upregulated and 23 downregulated.Among these groups,there were 28 common DEPs,and 25 DEPs including Perilipin-2(Plin2),lysosomal acid lipase A(Lipa),and armadillo repeatcontaining 6(Armc6)were downregulated in the M1 group but significantly upregulated in the high-dose FZHY group.Conversely,three proteins,namely IFN-γ-induced GTPase 1(Iigp1),tripartite motif-containing protein 30a(Trim30a),and RAB11 family-interacting protein 1(Rab11fip1),were upregulated in the M1 group but significantly downregulated in the high-dose FZHY group.Gene ontology(GO)analysis of the DEPs highlighted that their biological functions included localization repair,cellular components involved cytoplasmic parts,and their molecular functions included catalytic activity.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed that these DEPs were enriched in metabolic pathways,lysosomes,and glutathione metabolism.Compared with the blank control group,the expression of inflammatory surface markers CD40,CD86,inducible nitric oxide synthase(iNOS),and Iigp1 was significantly increased in the M1 group,whereas the expression of the protein Plin2 was decreased.FZHY treatment reversed the expression of these inflammation-induced proteins.The metabolism of M1 macrophages primarily relied on glycolysis,with the expression ofα‑ketoglutarate dehydrogenase(AKGDH)and glutamine synthetase(GLUL)decreased in the M1 group,accompanied by an increased glycolysis rate.FZHY treatment significantly increased the gene expression of AKGDH and GLUL and reduced the glycolysis rate.Conclusion FZHY suppressed macrophage polarization towards the M1 phenotype by regulating the expression of key metabolic enzymes(AKGDH and GLUL),differentially expressed proteins(Plin2 and Iigp1),and inflammatory markers(CD40,CD86,and iNOS)to downregulate the glycolysis rate.
作者
廖雅芳
陈龙
袁春露
林超颖
李荣胜
潘一峰
章丹丹
LIAO Yafang;CHEN Long;YUAN Chunlu;LIN Chaoying;LI Rongsheng;PAN Yifeng;ZHANG Dandan(Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Experiment Center for Science and Technology,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Research Department,Shanghai Huanghai Pharmaceutical Co.Ltd.,Shanghai 200942,China)
出处
《上海中医药杂志》
CSCD
2024年第8期72-79,共8页
Shanghai Journal of Traditional Chinese Medicine
基金
国家自然科学基金项目(82374116,81773946,81573673,81001666)
上海市国际科技合作项目(18400731400)
上海市战略性新兴产业重大项目(沪发改高技[2020]49号)。
关键词
肝纤维化
扶正化瘀方
代谢物
促炎表型
蛋白质组学
糖酵解
中药研究
hepatic fibrosis
Fuzheng Huayu Formula
metabolite
pro-inflammatory phenotype
proteomics
glycolysis
traditional Chinese herbal medicine research