Therapeutic Effects of Fengshi Feibi Decoction on Rheumatoid Arthritis-Associated Interstitial Lung Disease in TNF-Tg Mice postprint

Abstract: Background　Rheumatoid arthritis-associated interstitial lung disease （RA-ILD） is a common complication of rheumatoid arthritis （RA）， with 20% to 50% of RA patients developing ILD， which increases the risk of mortality. Currently， there is a lack of highly targeted and safe therapeutic agents for this condition. Traditional Chinese medicine has certain advantages in the treatment of RA-ILD， Fengshi Feibi Decoction （FSFBF）， as a commonly used prescription，has proven efficacy， but its mechanism of action remains unclear and urgently requires validation through experimental research. Objective　To investigate the therapeutic efficacy and underlying mechanisms of FSFBF in TNF-α transgenic （TNF-Tg） mice with RA-ILD. Methods　From March 2024 to January 2025， female TNF-Tg mice were used as an RA-ILD model. A total of 24 TNF-Tg mice （2.5 months old） were randomly divided into four groups: model group （TNF-Tg）， low-dose FSFBF group（Low），medium-dose FSFBF group （Middle）， and high-dose FSFBF group （High）. Additionally， six age-matched wild-type（WT）littermates were randomly selected as controls. The TNF-Tg group received normal saline， while the treatment groups received FSFBF via oral gavage at doses of 5.825， 13.65， and 27.3 g·kg-1·day-1， respectively， for 9 weeks. Body weight， ankle joint clinical scores， and forelimb grip strength were recorded. Ankle joint and lung tissues were collected for histological analysis using H&E， Masson's trichrome， and Safranin O-fast green staining. Serum levels of inflammatory cytokines were measured by enzyme linked immunosorbent assay （ELISA）. Immunofluorescence staining was performed to assess macrophage polarization in lung tissues. Quantitative real-time polymerase chain reaction （RT-qPCR） was used to evaluate the relative mRNA expression of inflammatory cytokines in ankle joints and lungs. Results　At 4.5 months of age， body weight in the TNF-Tg group was lower than that in the WT group， while the Low and High groups had higher body weights than the TNF-Tg group; ankle joint clinical scores in the TNF-Tg group were higher than those in the WT group， while the Middle and High groups had lower scores than the TNF-Tg group; forelimb grip strength in the TNF-Tg group was lower than that in the WT group， while the High group had higher grip strength than the TNF-Tg group （P<0.05）. Pathological experimental results showed: The TNF-Tg group had larger ankle joint inflammation areas， lung inflammation areas， and lung type I collagen fibers areas than the WT group; the Middle and High groups had smaller ankle joint inflammation areas than the TNF-Tg group; the Middle group had larger joint cartilage areas than the TNF-Tg group; and the Low， Middle， and High groups had smaller lung inflammation and lung type I collagen fibre areas than the TNF-Tg group （P<0.05）. Immunofluorescence staining results showed that the number of M1 and M2 macrophages in the lungs of the TNF-Tg group was greater than that in the WT group. The number of M1 macrophages in the Low， Middle， and High groups was lower than that in the TNF-Tg group， while the number of M2 macrophages was higher than that in the TNF-Tg group （P<0.05）. ELISA results showed that serum TNF-α， IL-1β and IL-6 levels in the TNF-Tg group were higher than those in the WT group（P<0.05）. The serum TNF-α and IL-1β levels in the Low， Middle， and High groups were lower than those in the TNF-Tg group， and the serum IL-6 levels in the High group was lower than that in the TNF-Tg Group （P<0.05）. RT-qPCR results showed that the relative expression levels of TNF-α and IL-1β in the lungs of mice in the TNF-Tg group were higher than those in the WT group， while the High group was lower than the TNF-Tg group（P<0.05）；The Middle group and High group had lower relative expression levels of IL-1β in the lungs than the TNF-Tg group（P<0.05）；The relative expression levels of TNF-α and IL-1β in the ankle joints of the TNF-Tg group were higher than those in the WT group， while the Low， Middle and High groups were lower than the TNF-Tg group （P<0.05）. Conclusion　FSFBF ameliorates RA-ILD symptoms in TNF-Tg mice by modulating macrophage polarization in the lungs. It alleviates joint inflammation， cartilage degradation， pulmonary inflammation， and alveolar fibrosis. These findings suggest that FSFBF may represent a promising novel therapeutic strategy for the clinical treatment of RA-ILD.