乌头汤通过下调HIF-1α/VEGFA/Ang信号通路抑制AIA风寒湿痹证大鼠血管翳形成  

Inhibition of Pannus Formation by Wutoutang in AIA Rats with Wind-cold-dampness Bi Syndrome Via Down-regulating HIF-1α/VEGFA/Ang Signaling Pathway

作　　者：欧阳丹 李鑫 湛港香 陈正 马远志 邹婕 曹建中 OUYANG Dan;LI Xin;ZHAN Gangxiang;CHEN Zheng;MA Yuanzhi;ZOU Jie;CAO Jianzhong(School of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学中医学院,长沙410208

出　　处：《中国实验方剂学杂志》2024年第9期10-18,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81803993)。

摘　　要：目的:探讨乌头汤对佐剂型关节炎(AIA)风寒湿痹证大鼠血管翳形成的抑制作用及其潜在作用机制。方法:将无特定病原体(SPF)雄性SD大鼠40只,按随机数字表法分为空白组、AIA风寒湿痹证组[完全弗氏佐剂(CFA),200μg]、乌头汤组(15 g·kg^(-1)·d^(-1))、吲哚美辛组(10 mg·kg^(-1)),除空白组外,其余各组注射CFA前给予风寒湿刺激。连续给药30 d,期间观察AIA风寒湿痹证大鼠的基本情况、发病时间、关节炎评分、足容积。最终取外周动脉血、踝关节和滑膜组织。酶联免疫吸附测定法(ELISA)检测血清缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子A(VEGFA)蛋白含量和风湿3项,包括抗O(ASO)、C反应蛋白(CRP)和类风湿因子(RF);苏木素-伊红(HE)染色观察关节组织形态学改变;免疫组化检测踝关节通路关键蛋白HIF-1α和VEGFA;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠滑膜组织中HIF-1α、VEGFA、血管生成素-1(Ang-1)、血管生成素-2(Ang-2)mRNA表达。结果:与空白组比较,AIA风寒湿痹证大鼠足肿容积和关节炎评分显著升高(P<0.01);血清CRP、RF、ASO阳性表达显著(P<0.01);HE染色可见踝关节滑膜炎性增生,血管新生、血管翳形成,骨破坏程度加重,提示模型构建成功。乌头汤干预后,发病时间显著延迟(P<0.01);足容积和关节炎评分显著降低(P<0.01);血清CRP、RF、ASO含量显著下降(P<0.01);滑膜组织炎性增生明显缓解,血管新生及血管翳减少,骨破坏减轻;滑膜HIF-1α、VEGFA、Ang-1、Ang-2mRNA明显降低(P<0.05,P<0.01);血清与踝关节HIF-1α和VEGFA蛋白表达显著下降(P<0.01)。在吲哚美辛组中,大鼠发病时间显著延迟(P<0.01);足容积和关节炎评分显著降低(P<0.01);血清CRP、RF、和ASO含量显著下降(P<0.01);HIF-1α/VEGFA/Ang信号通路激活,病理组织损伤改善。结论:乌头汤可延缓AIA风寒湿痹证大鼠发病时间,降低足容积、关节炎评分、风湿活动度,改善关节组Objective:This study aims to investigate the inhibitory effect of Wutoutang on pannus formation in adjuvant-induced arthritis(AIA)rats with wind-cold-dampness Bi syndrome and its potential mechanism.M ethod:A total of 40 male SD specific pathogen-free(SPF)rats were selected and divided into blank group,wind-cold-dampness Bi syndrome group[Complete Freund's Adjuvant(CFA),200μg],Wutoutang group(15 g·kg^(-1)·d^(-1)),and indometacin group(10 mg·kg^(-1))according to random number table method.Except for the blank group,the other groups were given wind-cold-dampness stimulation before the CFA injection.After the rats were administered for 30 days,the basic conditions,onset time,arthritis index score,and foot swelling volume of AIA rats with wind-cold-dampness Bi syndrome were observed.Finally,peripheral arterial blood,ankle joint,and synovial tissue were taken.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor A(VEGFA)protein content,and rheumatism,including anti-O(ASO),C-reactive protein(CRP),and rheumatoid factor(RF).Hematoxylin-eosin(HE)staining revealed the changes in joint histomorphology.Immunohistochemistry was used to detect the expression of HIF-1αand VEGFA,two important proteins in the ankle pathway.Quantitative real-time polymerase chain reaction(Real-time PCR)was used to reveal mRNA levels of HIF-1α,VEGFA,angiopoietin-1(Ang-1),and angiopoietin-2(Ang-2)in rat synovial tissue.Result:The foot swelling volume and arthritis score of AIA rats with wind-cold-dampness Bi syndrome were substantially higher(P<0.01)compared with the blank group.Serum CRP,RF,and ASO levels were considerably elevated(P<0.01).HE staining showed obvious hyperplasia of ankle synovium and synovial inflammation,angiogenesis and pannus formation,and aggravated bone destruction,indicating successful modeling.After the intervention of Wutoutang,the onset time was delayed(P<0.01).Foot swelling volume and arthritis score were decreased(P<0.01).Serum CRP,R

关 键 词：类风湿关节炎 乌头汤 血管翳 缺氧诱导因子-1α(HIF-1α)/血管内皮生长因子A(VEGFA)/血管生成素(Ang)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33