紫草素通过MAPK1/MAPK通路对小鼠胶原诱导型关节炎中滑膜炎症的影响  

Effect of Shikonin on Synovitis in Mice with Collagen-induced Arthritis Through MAPK1/MAPK Pathway

作　　者：童文婷 何莲花 TONG Wenting;HE Lianhua(Gannan Healthcare Vocational College,Ganzhou 341000,China;Shenzhen Traditional Chinese Medicine Hospital/The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine,Shenzhen 518033,China;Shenzhen China Resources Sanjiu Medicine Trading Co.Ltd.,Shenzhen 518109,China)

机构地区：[1]赣南卫生健康职业学院,江西赣州341000 [2]深圳市中医院/广州中医药大学第四临床医学院,广东深圳518033 [3]深圳华润三九医药贸易有限公司,广东深圳518109

出　　处：《中国实验方剂学杂志》2023年第22期56-63,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82204712);江西省中医药管理局项目(SZYYB20223908)。

摘　　要：目的:探究紫草素对DBA/1小鼠胶原诱导型关节炎(CIA)中滑膜炎的影响。方法:将36只DBA/1小鼠按体质量随机分为6组,每组6只。6组分别为正常组、CIA组,紫草素1、2、4 mg·kg^(-1)组及甲氨蝶呤(MTX)0.5 mg·kg^(-1)组。CIA组、紫草素低、中、高剂量组及MTX组在第1天以等体积牛Ⅱ型胶原和完全弗氏佐剂免疫DBA/1小鼠,于第21天以等体积牛Ⅱ型胶原和不完全弗氏佐剂二次免疫DBA/1小鼠建立CIA模型,并于二次免疫当天开始灌胃给药,除MTX为每周3次外,其余每天1次,共给药28 d。第22天开始观察CIA小鼠的关节红肿等症状并进行关节炎评分,第49天取材后,采用苏木素-伊红(HE)染色进行组织病理学检查观察CIA小鼠关节滑膜炎症的情况;免疫荧光(IF)双标法检测CIA小鼠关节滑膜中波形蛋白(Vimentin)和丝裂原活化蛋白激酶1(MAPK1)的表达;网络药理学预测出紫草素作用于RA的靶标是MAPK1,采用分子对接方式进行验证;蛋白免疫印迹法(Western blot)检测小鼠关节滑膜中细胞外调节蛋白激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38、磷酸化(p)-ERK、p-JNK、p-p38蛋白表达。结果:与正常组比较,CIA组小鼠关节炎评分、发病率及滑膜炎症情况显著升高,与正常组比较,CIA组小鼠关节结构严重紊乱,关节软骨及骨破坏明显,骨侵蚀严重(P<0.01);小鼠关节滑膜中Vimentin及MAPK1的表达明显增多;小鼠关节滑膜中p-ERK/ERK,p-JNK/JNK及p-p38/p38的蛋白表达显著增高(P<0.01);与CIA组比较,紫草素各剂量组和MTX组小鼠关节结构均相对正常,骨侵蚀、骨破坏较轻,关节面相对完整光滑;分子对接结果证明紫草素的作用靶点为MAPK1;紫草素各剂量组和MTX组关节滑膜中Vimentin及MAPK1表达显著降低(P<0.01),小鼠关节滑膜中p-ERK/ERK,p-JNK/JNK及p-p38/p38的蛋白表达显著降低(P<0.01)。结论:紫草素可靶向MAPK1抑制CIA小鼠中p-ERK,p-JNK及p-p38的蛋白表达,进而治疗RA。Objective:To observe the effect of shikonin(SKN)on synovitis in DBA/1 mice with collagen-induced arthritis(CIA).Method:Thirty-six DBA/1 mice were randomly divided into a normal group,a CIA group,low-,medium-,and high-dose SKN groups(1,2,and 4 mg·kg^(-1)),and a methotrexate(MTX,0.5 mg·kg^(-1))group,with 6 mice in each group.Mice in the CIA group,the SKN groups,and the MTX group were immunized with an equal volume of bovine typeⅡcollagen and complete Freund's adjuvant on day 1.On day 21,those mice received a second immunization with an equal volume of bovine typeⅡcollagen and incomplete Freund's adjuvant to establish the CIA model.On the day of the second immunization,mice were treated with drugs by gavage.Mice in the MTX group received oral administration three times a week,while others received once per day,for 28 days.On day 22,the symptoms of arthritis,such as redness and swelling of joints,in CIA mice were observed,and arthritis scores were recorded.On day 49 after sample collation,histopathological examination of synovial inflammation in CIA mice was performed using hematoxylin-eosin(HE)staining.Immunofluorescence(IF)double labeling was used to detect the expression of vimentin and mitogen-activated protein kinase 1(MAPK1)in the synovium of CIA mice.Network pharmacology predicted that the target of SKN in rheumatoid arthritis(RA)was MAPK1,which was verified by molecular docking.Western blot was used to detect the expression of extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),p38,phosphorylated(p)-ERK,p-JNK,and p-p38 proteins in the synovial membrane of mice.Result:Compared with the normal group,the CIA group showed significantly higher arthritis scores,morbidity,and synovial inflammation,severely disrupted joint structure,evident articular cartilage and bone destruction,severe bone erosion(P<0.01),increased expression of vimentin and MAPK1 in the synovium of mice,and increased protein expression of p-ERK/ERK,p-JNK/JNK,and p-p38/p38 in the synovium of mice(P<0.01).Compared with the

关 键 词：紫草素 胶原诱导型关节炎 滑膜炎症 丝裂原活化蛋白激酶(MAPK) 

分 类 号：R2-0[医药卫生—中医学] R33R593.22R289