## 4 Discussion
These data strongly suggest that TQ-6 can act as a strong antioxidant in different applications. The antioxidative properties of TQ-6 could not be only due to XO inhibition but also a direct inhibition of superoxide generation. Under pathological conditions, macrophages induced ROS through NADPH oxidase (NOX) activation could be involved in the damages of cell and tissue, killing of pathogen, and inflammatory signaling [31]. NOX-mediated free radical production is triggered by the induced p38 MAPK and NF-\(\kappa\)B signaling [32]. Studies found that TQ-6 inhibits LPS-stimulated macrophage activation through inhibiting of NF-\(\kappa\)B signaling pathway, such as \(\mathrm{I}\kappa\mathrm{B}\alpha\) degradation, NF-\(\kappa\)B p65 phosphorylation and its nuclear translocation [9]. This also inhibits OH\({}^{\bullet}\) formation in activated platelets [10]. A study was found that Ru(II) complexes, such as [Ru(bpy)2(maip)]\({}^{2+}\) and [Ru(bpy)2(paip)]\({}^{2+}\) exhibit good antioxidant activity in terms of inhibiting OH\({}^{\bullet}\) formation [33]. Here, the free radical scavenging ability of TQ-6 was found in cell-free system and LPS-induced macrophages through ESR spectrometry, and this assay may have provided direct evidence indicating that TQ-6 has antioxidant properties. Thus, TQ-6 is capable of reducing the severity of inflammatory diseases via reduction of oxidative stress and related inflammatory signaling. Certain anti-inflammatory signaling pathways negatively regulate inflammatory responses to maintain homeostasis. Several lines of confirmation revealed that HO-1 inhibits the production of nitric oxide (NO), prostaglandin E2 (PGE\({}_{2}\)), interleukin-1\(\beta\) (IL-1\(\beta\)), IL-6, and tumor necrosis factor-\(\alpha\) (TNF-\(\alpha\)) in vitro and in vivo [34; 35; 36]. Normally, Nrf2 attaches to its negative regulator, Kelch-like ECH-associated protein 1 (Keap1), and localizes in the cytoplasm; however, various stimuli, including ROS, induce nuclear translocation of Nrf2 and enhance HO-1 production [34]. HO-1 exhibits cytoprotective, antioxidant, and anti-inflammatory activities [37; 38]. Previous study explained that TQ-6 exhibits anti-inflammatory effects via suppressing of p38 MAPK, NF-\(\kappa\)B, iNOS and NO in LPS-stimulated macrophages [9]. A clear evidence indicated that ROS could activate Nrf2, NF-\(\kappa\)B and MAPK [39]. Here, we found that TQ-6 significantly upregulated Nrf2 expression followed by HO-1 expression in LPS-stimulated RAW cells. Moreover, [PERSON] et al. [40] provide evidence that macrophage apoptosis induced by LPS is mediated by both NO and TNF-\(\alpha\) production. A previous our paper also showed that TQ-6 dampened NO production, and TNF-\(\alpha\) expression in LPS-induced macrophages; however, these are not increased when treating with TQ-6 alone [9]. From this finding, it assumed that TQ-6 could inhibit LPS-induced macrophage apoptosis. The absence of evaluating the direct evidence whether TQ-6 has effective in LPS-induced apoptosis in macrophages and the similar attributes between macrophages and neutrophils as new therapeutic anti-inflammatory strategies subjected to TQ-6 treatment is regarded as the limitation of this study. The animal paw edema model has been commonly used to evaluate the anti-edematous effect of drugs. Carrageenan-induced edema model has long been appreciated as an excellent model for quick in vivo screening of NSAIDs [41]. [PERSON] et al. [42] noticed a zinc complex reduced carrageenan-induced rat paw edema. In this study, as TQ-6 administered to rats acutely for 24 h to test its in vivo anti-inflammatory efficacy, we have chosen carrageenann injection rather than LPS, and resulted in a significant reduction of rat paw thickness, thus demonstrating its anti-inflammatory properties. Consistently, in this inflammation model, TQ-6 showed a significant decrease in rat paw thickness, thus demonstrating its anti-inflammatory response. Pharmacokine (PK) study is an extensive area to focus for the new drug development.