Abstract
18β-Glycyrrhetinic acid (18β-GA), an active component from Glycyrrhiza glabra L. root (licorice), has been demonstrated to be able to protect against inflammatory response and reduce methotrexate (MTX)-derived toxicity. This study was therefore designed to test the therapeutic possibility of 18β-GA on rheumatoid arthritis (RA) and to explore the underlying mechanism. LPS or TNF-α-induced inflammatory cell models and collagen-induced arthritis (CIA) animal models were applied in this study. Real-time quantitative PCR (RT-qPCR) was used to measure the mRNA levels of various cytokines and FOXO family members. The protein levels of molecules in the MAPK/NF-κB signaling pathway were analyzed using western blot. The cell proliferation assay and colony-forming assay were used to test the influence of 18β-GA on cell viability. The cell apoptosis assay and cell cycle assay were performed to detect the effect of 18β-GA on cell proliferative capacity by using flow cytometry. Hematoxylin and eosin (H&E) staining was performed to evaluate pathological changes after drug administration. The enzyme-linked immunosorbent assay (ELISA) was carried out for the detection of cytokines in serum. In vitro, we found that 18β-GA decreased the mRNA levels of IL-1β, IL-6, and COX-2 by inhibiting the MAPK/NF-κB signaling pathway in MH7A and RAW264.7 cell lines. Moreover, 18β-GA was able to suppress cell viability, trigger cell apoptosis, and G1 phase cell cycle arrest in our in vitro studies. 18β-GA dramatically enhanced the mRNA level of FOXO3 in both TNF-α- and LPS-induced inflammation models in vitro. Interestingly, after analyzing GEO datasets, we found that the FOXO3 gene was significantly decreased in the RA synovial tissue as compared to healthy donors in multiple microarray studies. In vivo, 18β-GA exhibited a promising therapeutic effect in a collagen-induced arthritis mouse model by alleviating joint pathological changes and declining serum levels of TNF-α, IL-1β, and IL-6. Finally, we observed that 18β-GA administration could mitigate liver damage caused by collagen or MTX. Collectively, the current study demonstrates for the first time that 18β-GA can inhibit inflammation and proliferation of synovial cells, and the underlying mechanism may be associated with its inhibition of MAPK/NF-κB signaling and promotion of FOXO3 signaling. Therefore, 18β-GA is expected to be a new drug candidate for RA therapy.
Highlights
Rheumatoid arthritis (RA) is a common chronic autoimmune disease that affects 1% of the world’s population (Frisell et al, 2016)
Data are shown as mean ± SD. *, **, and *** mean p < 0.05, p < 0.01, and p < 0.001, n 3. (D–F) RAW264.7 cells were pretreated in Dulbecco’s modified Eagle’s medium (DMEM) without fetal bovine serum (FBS) overnight and the cells were exposed to LPS (1 μg/ml) and 18β-Glycyrrhetinic acid (18β-glycyrrhetinic acid (GA)) (100 and 200 μM) for 3 h
RA is recognized as a chronic autoimmune disease characterized by inflammation of joints and hyperplasia of fibroblast-like synoviocytes (FLSs) (Bottini and Firestein, 2013)
Summary
Rheumatoid arthritis (RA) is a common chronic autoimmune disease that affects 1% of the world’s population (Frisell et al, 2016). After long-term exposure to the pro-inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), interleukin 1β (IL1β), and interleukin 6 (IL-6), synovial cells will produce a set of chemokines that recruit circulating immune cells to migrate to the affected tissue, further exacerbating and perpetuating joint inflammation (Brzustewicz and Bryl, 2015; Kosmaczewska et al, 2011). In this inflammatory microenvironment, synoviocytes will shift to a hyperproliferative state, which will amplify the inflammatory response (Mcinnes and Schett, 2011). Our results provide a basis for the development of 18β-GA as a potential drug for the treatment of RA
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