Inhibition of PI3K/Akt signaling pathway is involved in the inhibitory effects of luteolin-plus-scoparone on the hyperproliferation of fibroblast-like synoviocytes

Rheumatoid Arthritis (RA) is a systematic autoimmune disease associated with chronic inflammation and synovial hyperplasia. Fibroblast-like synoviocytes (FLS) are characterized with tumor-like properties, such as abnormal proliferation and enhanced migration, and play a pathogenic role in RA. Current RA medications have toxicities and side-effects. Safe and highly effective drugs for treating RA are needed. Multi-target TCM (traditional Chinese medicine)- based herbal drugs are advantageous in treating RA. Our previous studies showed that a TCM formula comprising Lonicerae Japonicae Flos (LJF) and Rosae Multiflorae Fructus (RMF) has anti- RA effects without over toxicity in collagen-induced arthritis (CIA) rats. Luteolin, a flavone, is the main compound of LJF. Scoparone, a coumarin, is one of the main compounds of RMF. Luteolin has been shown to exert anti-RA effects by inhibiting MARK (ERKs) and PI3K/Akt signaling pathways. Scoparone is able to alleviate inflammation through inhibiting TLR4/NF-kB pathway. Activation of the pathways targeted by the two compounds is known to promote RA development. Since luteolin and scoparone target different pathogenic pathways of RA, we speculated that luteolin-plus-scoparone (Lu-plus-Sco for short) has synergistic anti-RA effects.

TNF-α-stimulated RA-FLS were used to evaluate the in vitro effects of Lu-plus-Sco. Synergism of the drug combination was evaluated using coefficient of drug interaction (CDI) values. Cell proliferation assay results showed that luteolin (10 μM) in combination with scoparone (50, 100, 200, 300 μM) treatments synergistically reduced the viability of TNF-α-stimulated RA-FLS (CDI<1). The CDI values of luteolin in combination with scoparone, at the ratio of 1:5, 1:10, 1:20 and 1:30, were 0.77, 0.83, 0.65, 0.73, respectively. At the 1:20 ratio, Lu-plus-Sco exhibited the optimal synergistic effect. Network pharmacology analysis predicted that PI3K/Akt signaling pathway is involved in the anti-RA effects of Lu-plus-Sco. Western blotting results showed that TNF-α stimulation increased the phosphorylation of Akt (Ser473) in RA-FLS, which was suppressed by Lu- plus-Sco.

In summary, we for the first time demonstrated that Lu-plus-Sco exerts synergistic effects in suppressing RA-FLS hyperproliferation, and that inhibition of the PI3K/Akt signaling pathway is involved in the anti-RA effects of Lu-plus-Sco. This study provided pharmacological data necessary for developing Lu-plus-Sco into a novel drug for treating RA.