Penehyclidine hydrochloride alleviates LPS-induced inflammatory responses and oxidative stress via ROS/Nrf2/HO-1 activation in RAW264.7 cells.

ABSTRACT

BACKGROUND: Inflammation is a biological response of the immune system to harmful stimuli. Penehyclidine hydrochloride (PCH) can alleviate inflammation and oxidative stress by activating reactive oxygen species (ROS), nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase 1 (HO-1) in animal models, but there is a lack of cellular evidence. OBJECTIVES: This study investigated the effects of PHC on lipopolysaccharide (LPS)-induced inflammation response and oxidative stress in RAW264.7 cells. MATERIAL AND METHODS: RAW264.7 cells were treated with 1 µg/mL or 5 µg/mL of PHC, with interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-1ß, and prostaglandin E2 (PGE2) levels measured with enzyme-linked immunosorbent assay (ELISA) and nitric oxide (NO) measured using the Griess test. Reactive oxygen species were examined with flow cytometry and immunofluorescence, and b-related factor 2 (BRF-2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) using western blot. RESULTS: Penehyclidine hydrochloride partly, but substantially, reversed LPS-related NO and PGE2 production by RAW264.7 cells in a dose-dependent manner and suppressed LPS-induced expression of IL-6, TNF-α and IL-1ß messenger ribonucleic acid (mRNA), secretion of IL-6, TNF-α and IL-1ß, and ROS production. Lipopolysaccharide stimulation did not affect Nrf2, heme oxygenase 1 (HO-1) or NQO1 protein expression in RWA264.7 cells not treated with PHC. However, PHC treatment significantly elevated Nrf2, HO-1 and NQO1 protein in LPS-treated RWA264.7 cells, an effect that was dose-dependent. The ROS scavenging using N-acetyl-L-cysteine abolished the PHC-induced upregulation of Nrf2 and HO-1. CONCLUSIONS: Penehyclidine hydrochloride may alleviate LPS-induced inflammation and oxidative stress by activating Nrf2 signaling in RAW264.7 macrophages. These findings suggest that PHC could alleviate inflammation by targeting activated macrophages.