*3.5. Effects of Compound* **1**–**5** *on PGE2 Secretion*

Anti-inflammatory COX-related activities of compounds **1**–**5** were evaluated by quantifying PGE2 secretion in the established oxidative stress conditions in vitro (Figure 6).

Consistent with literature data [27], even a short exposure to H2O2 for 3 h increased PGE2 secretion up to 1014.2 pg (Figure 6). Notably, all the tested compounds lowered the amount of PGE2 compared to the H2O2 pre-incubation but were less effective than Meloxicam (Figure 6). Compared to our previous experiments, the obtained data revealed a different trend of activity: compounds **1**, **2** and **5** showed remarkable anti-inflammatory effects and considerably reduced PGE2 secretion already at 24 h and mainly at 25 μM (240.5 pg, 84.8 pg and 357.1 pg, respectively), whereas compounds **3** and **4** were less efficient. Moreover, the modulation of PGE2 is time-dependent, being that this cytokine decreased over the time. Actually, the amount of PGE2 was almost halved with all the tested compounds after 72 h of exposure compared to 24 h treatment (Figure 6). These findings support the hypothesis that the observed anti-inflammatory effects rely on a COX-2 mediated mechanism of action and that the organometallic complexes retain the ability to inhibit COX-2.

Collectively, HO-1 expression and enzymatic activity are confirmed to influence positively and negatively both innate and adaptive immune responses; this dual action seems to be related to the stage of the inflammatory response or disease. The therapeutic potential of HO-1 may rely on limiting early inflammation, hampering successive tissue damage and modulating key pathways in most cell types of the immune system, given the complexity of heme catabolism and the role of HO-1 as a critical mediator of innate immune response. Immunomodulation is mostly related to higher demolition of the pro-inflammatory heme group, macrophage activation towards an anti-inflammatory macrophage profile with reduced secretion of pro-inflammatory cytokines and iNOS and interferon production by macrophages and dendritic cells. Indeed, T cells constitutively express HO-1, and their expansion regulatory is positively influenced by a tolerogenic phenotype sustained through HO-1 induction in dendritic cells. HO-1 modulation or application of low concentrations of CO to LPS-challenged macrophages reduced TNF-α and IL-1β expression and

simultaneously stimulated the anti-inflammatory IL-10 production through p38-MAPK activity [44–46].

This context was particularly evident in models of tendon-related diseases. In this light, the role of the macrophage is an area of emerging interest in tendinopathies and in general in the healing of tendons. In fact, inflammation appears to be driven by a high number of infiltrating macrophages at the inflamed tendon site [47]. Furthermore, damaged tendons from patients with tendinopathy show an abundance of CD14+ and CD68+ activated macrophages [48]. We have already reported that CORM hybrids exert their biological effects both on inflamed macrophages and tenocytes [22,26], disclosing a challenging field of application for molecules active on the HO-1/CO molecular axis not strictly related to the immune system. Finally, although once considered cells not involved in the immune-regulation and only related to tendon remodeling, tenocytes have been disclosed as active cells, secreting cytokines and expressing inflammation-related proteins [27].
