EVALUATION OF POSSIBLE ANTI-INFLAMMATORY AND NEUROPROTECTIVE EFFECTS OF CHLOROGENIC ACID: AN INNOVATIVE APPROACH FOR THE MANAGEMENT OF NEUROINFLAMMATION

Neuroinflammation and the kynurenine-immune pathway are known to play a key role in inflammation-induced psychiatric disorders. There is evidence in the literature on the anti-inflammatory and antioxidant properties of naturally occurring polyphenols. The SHSY5Y (human neuroblastoma cells) and HMC3 (human microglia) cell lines are used to reproduce an in vitro model of neuroinflammation. The evaluation of the expression profile of inflammatory molecules (IL6, IL 1 beta and TNF alpha) was performed with Real Time PCR, in SHSY5Y cells, after stimulation for 24 hours with LPS at a dose of 5 μg/ml and subsequent treatment with the polyphenolic extract titrated in chlorogenic acid, at doses of 50 μM, for another 24 hours. The same treatment was performed for HMC3 after stimulation with LPS at a dose of 0.1 μg/ml and 50 μM of CGA. Real Time PCR was also performed on SHS5Y, treated only with chlorogenic acid at doses of 12.5-25-50 μM to evaluate the dose-response curve. Oxidative stress was assessed using a specific probe (Invitrogen, Thermo Fisher Scientific CM-H2DCFDA) for the detection of ROS. Changes in cell viability induced by stimulation with LPS (5 μg/ml in SHSY5Y and 0.1 μg/ml in hMC3) and by the possible protective effect of the titrated extract of chlorogenic acid were also monitored using the combined FDA-PI assay capable of differentially marking live cells and dead cells. Further enhancements of the expression of specific molecular targets associated with oxidative stress (NRF2, HO-1) were conducted on both cell lines using immunofluorescence on stimulated and treated cells as previously described. At the end of the experimental activity, interesting implications emerged regarding the antioxidant, anti-inflammatory and anti-apoptotic effect of the polyphenolic extract which offer potential translational applications also for the continuation of further studies.