Effect of Shikonin on Synovitis in Mice with Collagen-induced Arthritis Through MAPK1/MAPK Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo observe the effect of shikonin （SKN） on synovitis in DBA/1 mice with collagen-induced arthritis （CIA）. MethodThirty-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 type Ⅱ collagen and complete Freund's adjuvant on day 1. On day 21， those mice received a second immunization with an equal volume of bovine type Ⅱ collagen 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. ResultCompared 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 CIA group， the SKN groups and the MTX group showed relatively normal joint structure， with milder bone erosion and bone destruction， and smoother articular surfaces. Molecular docking results confirmed that the target of SKN was MAPK1. In the SKN groups and the MTX group， the expression of vimentin and MAPK1 in the synovial membrane was significantly reduced （P<0.01）， and the protein expression of p-ERK/ERK， p-JNK/JNK， and p-p38/p38 in the synovium of mice was significantly reduced （P<0.01）. ConclusionSKN can target MAPK1 to inhibit the protein expression of p-ERK， p-JNK， and p-p38 in CIA mice， thereby treating RA.