孟河医派特色炮制辅料猪心血和猪血的蛋白质组学分析及其对丹参抗小胶质细胞炎症损伤的影响  

Proteomic Research on Characteristic Processing Materials of Porcine Cardiac Blood and Porcine Blood from Menghe Medical School and Their Effect on Anti-microglial Inflammatory Damage of Salviae Miltiorrhizae Radix et Rhizoma

作　　者：刘楠 廉源沛 尹春燕 蔡佳丽 王地均 朱力 刘产明[1] 黄玮[1] 颜晓静[1] LIU Nan;LIAN Yuanpei;YIN Chunyan;CAI Jiali;WANG Dijun;ZHU Li;LIU Chanming;HUANG Wei;YAN Xiaojing(Changzhou Key Laboratory of Human Use Experience Research&Transformation of Menghe Medical School,Changzhou Hospital of Traditional Chinese Medicine,Nanjing University of Chinese Medicine,Changzhou 213003,China)

机构地区：[1]南京中医药大学附属常州市中医医院、常州市孟河医派人用经验研究与转化重点实验室,江苏常州213003

出　　处：《中国实验方剂学杂志》2023年第18期137-145,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82074016,81603296);江苏省中医药管理局项目(2020ZX18);常州市卫生健康委员会重大项目(ZD202028);江苏省科协青年科技人才托举工程项目(2021-098);常州市科协青年科技人才托举工程项目(常科协[2020]86);常州市卫生健康青苗人才培养工程项目(CZQM2020087);常州市科技计划项目(CM20223006,CJ20219008);江苏省研究生科研创新计划项目(SJCX22_0866)。

摘　　要：目的:采用蛋白质组学考察猪心血(PCB)和猪血(PB)的蛋白差异,并比较二者对丹参(DS)抗小胶质细胞炎症损伤的影响。方法:采用纳升液相色谱-四极杆轨道阱质谱法(nLC-MS/MS)联合生物信息学比较PCB和PB模拟胃肠道消化的蛋白质组学差异,应用蛋白质免疫印迹法(Western blot)验证二者部分共有蛋白和差异蛋白含量;应用皮质酮(CORT)联合脂多糖(LPS)构建BV2神经炎症模型,考察PCB和PB对DS抗炎症细胞中肿瘤坏死因子(TNF-α)和白细胞介素-6(IL-6)水平的影响。结果:蛋白质组学结果显示,PCB和PB中共有蛋白69种、差异蛋白68种,其中共有蛋白中包括具有脑靶向作用的转铁蛋白(Tf),二者差异蛋白分别为41、27种。Western blot验证结果显示,二者共有Tf含量差异无统计学意义,差异蛋白肌酸激酶M及心脏型脂肪酸结合蛋白(H-FABP)含量差异则具有统计学意义(P<0.05)。进一步体外实验研究发现,与同浓度DS组比较,除100 mg·L^(-1) PB拌DS(PB-DS)组外,PCB拌DS(PCB-DS)组和PB-DS组均能明显抑制BV2炎症细胞的TNF-α和IL-6水平(P<0.05,P<0.01);与同浓度PB-DS组比较,PCB-DS组均能明显抑制BV2炎症细胞TNF-α和IL-6水平(P<0.05,P<0.01)。结论:孟河医派临方特色炮制辅料PCB和PB均能促进DS抑制神经炎症作用,且PCB促进DS抑制作用更显著,这可能是由于二者存在参与能量代谢相关差异蛋白所致,可为揭示PCB-DS及PB-DS的炮制科学内涵奠定基础。Objective:Proteomics was used to investigate the protein differences between porcine cardiac blood(PCB)and porcine blood(PB)from Menghe medical school and to compare the effects of both on the microglial inflammation of Salviae Miltiorrhizae Radix et Rhizoma(DS).Method:Nanoliquid chromatography-quadrupole orbitrap mass spectrometry(nLC-MS/MS)and bioinformatics were utilized to compare the proteomic differences of PCB and PB in simulated gastrointestinal digestion.Furthermore,Western blot was used to verify the contents of some shared proteins and differential proteins identified in PCB and PB.In addition,BV2 neuroinflammation model constructed by corticosterone(CORT)and lipopolysaccharide(LPS)was applied to detect the intervention effects of PCB and PB on the levels of tumor necrosis factor(TNF)-αand interleukin(IL)-6 in BV2 inflammatory cells of DS.Result:A total of 69 common proteins and 68 differential proteins were identified in PCB and PB,among which the common proteins included transferrin(Tf)with braintargeting effect,and the differential proteins in the two were 41 and 27,respectively.Western blot validation showed that the difference in the content of the same protein Tf between PCB and PB was not statistically significant,while the difference in the contents of the specific proteins of creatine kinase M and heart-type fatty acid binding protein(H-FABP)were statistically significant(P<0.05).Moreover,in vitro experimental studies revealed that compared with the same concentration of DS group,in addition to the 100 mg·L^(-1) PB-DS group,PCB-DS and PB-DS groups could significantly inhibit the levels of TNF-αand IL-6 in BV2 inflammatory cells(P<0.05,P<0.01),and PCB-DS group had more significant anti-inflammatory effect than PB-DS group with the same concentration(P<0.05,P<0.01).Conclusion:Both of PCB and PB can enhance the inhibitory effect of DS on the release of inflammatory factors,thus playing a neuroprotective role,and PCB promotes DS inhibition more significantly,which may be due to the existence of

关 键 词：孟河医派 特色炮制辅料 猪心血 猪血 液质联用技术 蛋白质组学 BV2细胞 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R943.1R96