COL-3 prevents intracellular oxidative stress generated in microglial cells in response to inflammatory signals
To test the hypothesis of a ROS-dependent NADPH production, we used a quantitative cell-based fluorescence assay to evaluate ROS in microglial cells exposed to 10 ng/ml LPS or 70 µg/ml αSa (Figure 5). As expected, a 24-h treatment with LPS (10 ng/ml) or αSa (70 µg/ml) induced a significant increase of intracellular ROS levels in microglial cells. In contrast, αSm does not induce significant effects on ROS levels. Of interest, ROS production returned to control values when cultures exposed to either LPS or αSa were previously treated with 20 µM COL-3 (Figure 5A,B), confirming the hypothesis that the non-antibiotic tetracycline may exert anti-inflammatory effects by counteracting oxidative stress-dependent mechanisms. As expected, the inhibitory effect of COL-3 on ROS production was mimicked by DOX at 50 µM. Noticeably, the NADPH oxidase inhibitor APO (300 µM) and the antioxidant TROL (10µM) reduced ROS production with an efficacy similar to that demonstrated by COL-3.