Evaluating Macrophage Polarization and Inflammation in Bitongqing-Treated Collagen-Induced Arthritis Rats

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation and immune dysregulation. Traditional Chinese medicine formulas such as Bitongqing Decoction (BTQ) are used clinically, but reproducible workflows are needed to characterize their absorbed constituents and associated immunological effects. This article presents an integrated protocol comprising herbal preparation, collagen-induced arthritis (CIA) induction, 21-day oral treatment, serum ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS), arthritis scoring, micro-computed tomography (micro-CT), hematoxylin and eosin staining, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and western blotting. The workflow evaluates macroscopic disease manifestations, joint pathology, inflammatory mediators, macrophage-associated M1-like and M2-like markers, splenic T helper 1 (Th1)- and regulatory T cell (Treg)-associated markers, and NF-κB/MAPK signaling proteins. Representative data showed that BTQ treatment was associated with improved arthritis-related outcomes, lower levels of inflammatory mediators, reduced CD80, iNOS, MHCII, and STAT1 signaling, increased CD163 and ARG-1 signaling, altered CXCR3 and FOXP3 signaling, and reduced phosphorylation of P65, ERK, and JNK. Because marker intensities, rather than cell-number ratios, were measured, these findings are described as changes in marker profiles rather than as direct measurements of M1/M2 or Th1/Treg balance. This protocol provides a multimodal framework for pharmacological studies of complex herbal formulas by linking the characterization of circulating constituents with structural, histological, and immunological readouts while preserving physiological interactions among synovial macrophages, lymphocytes, fibroblast-like synoviocytes, and other joint-resident cells.