The key active ingredients identification and pharmacological mechanism investigation of extract of ethyl acetate from Er Miao San aganist rheumatoid arthritis

Background

Er Miao San (EMS) is a basic formula for clearing heat and drying dampness in traditional Chinese medicine (TCM), which is mainly used in the treatment of rheumatoid arthritis (RA). Previous studies have found that the ethyl acetate extract of EMS (EMS-EA) has the best anti-inflammatory effect, but its specific pharmacological material basis is still unclear.

Purpose

The aim of the study was to investigate the active components of the EMS-EA against RA and its mechanism of action using a combination of serum pharmacochemistry and network pharmacology.

Methods

The anti-RA efficacy of EMS-EA was evaluated by establishing a rat model of adjuvant arthritis (AA). The chemical constituents of the EMS-EA and the blood components in the serum of rats after the administration of EMS-EA were detected by the ultra-high liquid chromatography-quadrupole Extractive Orbitrap Mass spectrometry (UPLC-QE-Orbitrap-MS). Network pharmacological analysis was utilized to predict the potential mechanism of action of key blood-entry components against RA, molecular docking, molecular dynamics simulations and in vitro experiments were performed to preliminarily validate the results of network drug prediction. The anti-proliferative activity and pro-apoptotic ability of the key blood-entry components against TNF-α (10 ng/mL)-induced inflammatory injury model of MH7A were detected by MTT assay and TUNEL staining, the levels of IL-6 and IL-1β in the supernatant of the cells were detected by ELISA, and pathway proteins by WB assay.

Results

Compared with the model group, EMS-EA treatment significantly attenuated the ankle joint injury condition in AA rats, reduced foot volume, arthritis index, organ index and serum levels of TNF-α, IL-6 and IL-1β in rats, and alleviated the pathologies such as formation of vascular opacities and synovial hyperplasia of knee joints to different degrees. In positive and negative ion mode, 51 compounds including 19 alkaloids, 8 terpenoids, Subsequently, berberine (BER) and atractylenolide I (AT-I) were detected in the serum collected from rats after EMS-EA administration. Phellodendrine (PHE) found in rat abdominal aorta serum. Network pharmacology,molecular docking and molecular dynamics simulations results revealed that BER、AT-I and PHE may exert anti-RA effects by modulating the MAPK signaling pathway, whose core targets are mainly CASP3, MAPK1 and MAPK8. Finally, we performed in vitro experiments to investigate the anti-RA activity of the three blood entry components mentioned above. The results showed that all three compounds were able to significantly reduce the TNF-α-induced proliferation level of MH7A cells and increase their apoptotic ability, while inhibiting the release of IL-1β and IL-6. WB experiments showed that all three compounds significantly elevated the level of Cleaved-caspase 3 in TNF-α-induced MH7A cells and down-regulated the phosphorylation levels of JNK and ERK.

Conclusion

EMS-EA has excellent therapeutic effects on AA rats, and its chemical components are mainly alkaloids, organic acids and terpenoids. Among them, BER、AT-I and PHE may be its direct acting substances in vivo, and the mechanism of action may be related to the inhibition of MAPK signaling pathway.