基于定量蛋白质组学研究当归拈痛汤对风湿热痹佐剂性关节炎大鼠的作用机制

Mechanism of Danggui Niantongtang on Adjuvant Arthritis Rats with Wind-dampness-heat Arthralgia Based on Quantitative Proteomics

目的:应用定量蛋白质组学研究当归拈痛汤对风湿热痹佐剂性关节炎(AA)大鼠的作用机制。方法:60只SD大鼠随机分为正常组、模型组、当归拈痛汤低、中、高剂量组及甲氨蝶呤(MTX)组,每组10只,大鼠尾根部皮下注射灭活结核分支杆菌佐剂(Mtb)进行AA的造模,人工气候箱干预16 d建立风湿热痹证模型,造模当天开始给药干预,持续干预28 d。提取大鼠滑膜组织蛋白质,使用4D非标记定量(4D-LFQ)蛋白质组学研究当归拈痛汤中剂量组与风湿热痹证模型组之间的差异蛋白情况,采用免疫组化及蛋白免疫印迹法(Western blot)验证与线粒体途径细胞凋亡相关的差异蛋白。结果:从滑膜组织中检测出4756个蛋白质,其中4234个蛋白质包含定量信息。当归拈痛汤与模型组差异蛋白为814个。基因本体(GO)和京都基因与基因组百科全书(KEGG)分析显示,当归拈痛汤对风湿热痹证AA大鼠的滑膜蛋白质组确有影响,且差异蛋白集中在对免疫系统的调控、急性炎症的反应和凋亡水平的调节。免疫组织化学与蛋白免疫印迹法验证发现,与模型组比较,当归拈痛汤各剂量组及MTX组滑膜组织中B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)及细胞色素C(Cyt C)蛋白表达水平明显升高(P<0.05,P<0.01),Bcl-2的表达水平明显下降(P<0.05,P<0.01),剪切型胱天蛋白酶-9(cleaved Caspase-9)/胱天蛋白酶-9(Caspase-9)显著升高(P<0.01),磷酸化蛋白激酶B(p-Akt)/蛋白激酶B(Akt)明显下降(P<0.05,P<0.01)。结论:当归拈痛汤对风湿热痹证类风湿关节炎的治疗涉及多靶点,其可能通过调节Akt/Bax/Bcl-2通路,促进线粒体途径细胞凋亡,从而发挥其对风湿热痹证类风湿关节炎的防治作用。

Objective:To explore the mechanism of Danggui Niantongtang(DGNT)against adjuvant arthritis(AA)rats with wind-dampness-heat arthralgia by quantitative proteomics.Method:Sixty SD rats were randomly divided into normal group,model group,angelica came pain soup low,medium and high dose group and methotrexate(MTX)group,each group of 10,only the rat tail root subcutaneously inactivated mycobacterium tuberculosis(Mtb)of adjuvant to build model of AA,artificial climate box intervention 16 d rheumatic fever bi syndrome model is set up,building the day began to drug intervention,The intervention lasted for 28 days.The proteins of synovial tissues in experimental rats were extracted.The differential proteins in the medium-dose DGNT group and the model group were detected and analyzed by 4D label-free quantification(4D-LFQ)proteomics.The differentially expressed proteins associated with mitochondrial pathway apoptosis were verified by immunohistochemistry and Western blot.Result:A total of 4756 proteins were identified from rat synovial tissues,of which 4234 proteins contained quantitative information.There were 814 differential proteins between the model group and the DGNT group.As revealed by Gene Ontology(GO)and Kyoto Encyclopedia of Gene and Genome(KEGG)enrichment analyses,DGNT had an effect on the synovial proteome of AA rats with wind-dampness-heat arthralgia,and the differential proteins were enriched in the regulation of the immune system,response to acute inflammation,and apoptosis regulation.As demonstrated by the results of immunohistochemistry and Western blot,compared with the model group,the DGNT groups and the MTX group showed increased protein expression of B-cell lymphoma 2(Bcl-2)-associated X protein(Bax)and cytochrome C(Cyt C)(P<0.05,P<0.01),reduced Bcl-2 level(P<0.05,P<0.01),elevated level of cleaved cysteinyl aspartate-specific protease 9(Caspase-9)/Caspase-9(P<0.01),and decreased level of phosphorylated protein kinase B(p-Akt)/Ak(t P<0.05,P<0.01).Conclusion:DGNT involved multiple targets in the treatment of AA with wind-dampness-heat arthralgia and it may exert its effect in the prevention and treatment by regulating the Akt/Bax/Bcl-2 pathway and promoting the cell apoptosis in the mitochondrial pathway.