Rosmarinic Acid Attenuates the Activation of Murine Microglial N9 Cells through the Downregulation of Inflammatory Cytokines and Cleaved Caspase-3.

Objective: The present study evaluated the ability of rosmarinic acid (RA) to inhibit microglia activation induced by lipopolysaccharide (LPS) in the N9 murine microglial cell line, and investigated the putative mechanisms involved in this process.

Methods: In all tests, N9 murine microglial cells were pretreated with RA (0.1, 1.0, and 10 μM) for 20 h and exposed to LPS (1 μM/mL) for 4 h. Cell viability was measured by Trypan blue exclusion assay. Flow cytometry was used to detect reactive oxygen species (ROS), quantify cleaved caspase-3, and analyze the mitochondrial electrochemical potential. iNOS, Arg-1, TNF-α, IL-1β, and IL-6 proteins were analyzed by Western blotting, and their antigens were detected using the chemiluminescence technique. The effect of RA on DNA was evaluated by the Comet assay.

Results: RA attenuated the expression of the M1 marker iNOS and the levels of proinflammatory factors, including TNF-α, IL-1β, and IL-6; it increased the expression of the M2 marker Arg-1, and inhibited, at least in part, ROS generation and loss of mitochondrial outer membrane permeabilization through the inhibition of cleaved caspase-3 activation. RA also inhibited DNA damage, reassuring cell protection.

Conclusions: The results suggested a protective effect of RA through downregulation of inflammatory cytokines and cleaved caspase-3.