In vitro and in vivo anti-inflammatory active copper(II)-lawsone complexes

We report in vitro and in vivo anti-inflammatory activities of a series of copper(II)-lawsone complexes of the general composition [Cu(Law)2(LN)x(H2O)(2-x)]·yH2O; where HLaw = 2-hydroxy-1,4-naphthoquinone, x = 1 when LN = pyridine (1) and 2-aminopyridine (3) and x = 2 when LN = imidazole (2), 3-aminopyridine (4), 4-aminopyridine (5), 3-hydroxypyridine (6), and 3,5-dimethylpyrazole (7). The compounds were thoroughly characterized by physical techniques, including single crystal X-ray analysis of complex 2. Some of the complexes showed the ability to suppress significantly the activation of nuclear factor κB (NF-κB) both by lipopolysaccharide (LPS) and TNF-alpha (complexes 3–7 at 100 nM level) in the similar manner as the reference drug prednisone (at 1 μM level). On the other hand, all the complexes 1–7 decreased significantly the levels of the secreted TNF-alpha after the LPS activation of THP-1 cells, thus showing the anti-inflammatory potential via both NF-κB moderation and by other mechanisms, such as influence on TNF-alpha transcription and/or translation and/or secretion. In addition, a strong intracellular pro-oxidative effect of all the complexes has been found at 100 nM dose in vitro. The ability to suppress the inflammatory response, caused by the subcutaneous application of λ-carrageenan, has been determined by in vivo testing in hind-paw edema model on rats. The most active complexes 1–3 (applied in a dose corresponding to 40 μmol Cu/kg), diminished the formation of edema simalarly as the reference drug indomethacine (applied in 10 mg/kg dose). The overall effect of the complexes, dominantly 1–3, shows similarity to anti-inflammatory drug benoxaprofen, known to induce intracellular pro-oxidative effects.

On the other hand, there are no known reports about the anti-inflammatory activity of transition metal complexes containing lawsone derivatives in contrast to the 1,4-naphthoquinone derivatives alone, and therefore we focused our attention towards the in vitro and in vivo studies of anti-inflammatory activity of the copper(II) compounds bearing the above-mentioned ligands. Moreover, our motivation is also connected with the fact that the present complexes were shown (based on the results of electrochemical studies) to possess the ability to take place in the production of reactive oxygen species (ROS) and to interact with DNA, as published in the previous paper [12], and thus, we wished to extend biological screening on these complexes with the aim to reveal any positive biological feature of these bioinorganic systems.
The chemicals, media and methods used for the evaluation of biological activities were as follows: RPMI 1640 medium, phosphate-buffered saline (PBS) and a penicillin-streptomycin mixture were purchased from Biosera (Boussens, France). Fetal bovine serum (FBS) was obtained from HyClone (GE Healthcare, Logan, UT, USA). Phorbol myristate acetate (PMA), erythrosin B, Escherichia coli 0111:B4 lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF) for molecular biology, prednisone, dichlorofluorescein diacetate (DCFH-DA), and urethane were obtained from Sigma-Aldrich (Steinheim, Germany). The cytotoxicity against the THP-1 cell line, used for the in vitro evaluation of antiinflammatory activities, was tested using a Cell Proliferation Reagent WST-1 kit from Roche Applied Science (Mannheim, Germany). The production of TNF-α was evaluated using a Human TNF-α Instant ELISA from eBioscience (Vienna, Austria). Quanti-Blue medium (Invivogen, San Diego, CA, USA) was used to detect reporter alkaline phosphatase. FLUOstar Omega microplate reader (BMG, Ortenberg, Germany) was employed to measure absorbance in the 96-well plates.
All the prepared complexes were characterized by elemental analysis, UV-Visible, FT-IR, electrospray-ionization mass spectrometry (ESI+ MS). Selected complexes were also analyzed by TG/DSC analyses, SQUID magnetometry and single crystal X-ray analysis.
For comparative purposes, the aqua-complex of the composition [Cu(Law) 2 (H 2 O) 2 ]Á 0.5H 2 O was prepared by a modified method of S. Salunke-Gawali et al. [13]. The description of the preparative procedure and identification of the product is provided in S1 Text.

General methods for characterization
Elemental analysis (C, H, N) was performed on a Flash 2000 CHNS Elemental Analyser (Thermo Fisher Scientific, Waltham, USA). Infrared spectra were measured using a Nexus 670 FT-IR (Thermo Fisher Scientific, Waltham, USA) spectrometer using an ATR technique in the region of 650-4000 cm -1 . The intensities of bands are defined as s = strong, m = medium and w = weak. Electrospray ionization mass spectra (ESI+MS) of methanol solutions of complexes 1-7 were acquired using an LCQ Fleet Ion Trap mass spectrometer (Thermo Fisher Scientific, Waltham, USA) in the positive ionization mode. Electronic spectra of complexes in methanol solutions were recorded on an HP 8453 UV-Visible spectrometer (Agilent Technologies, Santa Clara, USA). The selected complexes (7 and the aqua-complex) were studied by thermal TG/ DSC analysis in the range of 20-600˚C with the linear thermal gradient of 5˚C/min. The temperature dependence of magnetization was studied for the selected complexes 1-5 and the aqua-complex using a SQUID XL-7 magnetometer (Quantum Design, San Diego, USA) in the temperature range of 300-1.9 K.

X-ray crystallography
Single crystal X-ray diffraction data of complex 2 were obtained on a Bruker D8 Quest diffractometer equipped with a Photon 100 CMOS detector, using the Mo-Kα radiation at 120(2) K. Data collection, data reduction, and cell parameters refinements were performed using the Bruker Apex III software package [14]. The molecular structure was solved by direct methods (SHELXS) and all non-hydrogen atoms were refined anisotropically on F 2 using full-matrix least-squares procedure in SHELXL-2014 [15]. Hydrogen atoms were found in differential Fourier maps and their parameters were refined using a riding model. Molecular graphics were prepared and some structural features were evaluated and interpreted using Mercury, ver. 3.9. [16] The crystal data and structure refinements are given in Table 1.

Anti-inflammatory activity testing in vitro
Maintenance and preparation of macrophages. The THP-1 human monocytic leukemia cell line was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and THP1-XBlue™-MD2-CD14 cells were purchased from Invivogen (San Diego, USA). The cells were cultivated at 37˚C in the RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO 2 . The culture was split twice a week when the cells had reached the concentration of 5-7×10 5 cells/mL. The cell count and viability were determined following staining with erythrosin B. The cells were counted manually using a hemocytometer and a light microscope.
One hundred microliters of the stabilized THP-1 cells (5 th -20 th passage) were split into 96-well plates to afford the concentration of 5×10 5 cells/mL, and differentiation into macrophages was induced by the addition of phorbol myristate acetate (PMA), as described previously. [17] THP1-XBlue™-MD2-CD14 cells were used without any differentiation due to their stabile expression of MD2 and CD14 co-receptors.
Cytotoxicity assay. The tested compounds 1-7, aqua-complex [Cu(Law) 2 (H 2 O) 2 ]Á 0.5H 2 O, and lawsone were dissolved in DMF or PBS (due to low solubility, the concentration limit for most of the tested compounds in the culture media was 10 μM) and added to the THP-1 monocyte suspension in a culture medium in at least 5 concentration levels. The final concentration of the solvent in the culture medium was 0.1% (v/v). The cells were incubated at 37˚C with 5% CO 2 for 24 h. After the incubation, the cytotoxicity was determined by the Cell With respect to the results of cytotoxicity assay, the concentration of 100 nM should not affect the viability of the target cells in any way. For comparative purposes, a conventional drug prednisone was used at the standard dose of 1 μM dissolved in DMSO. Then, the DMSO was added to the rest of the wells. The final concentration of DMSO reached the level of 0.1% in each well. Vehicle-treated cells contained a vehicle (0.1% DMSO) only.
The TNF-α secretion in the pretreated differentiated THP-1 macrophages was induced by the addition of 1 μg/mL LPS dissolved in sterile water. The basal cytokine expression level was determined in the control cells without the LPS stimulation. LPS triggers an inflammatory response through binding to toll-like receptor 4 (TLR-4) and subsequently activates the NF-κB signaling pathway. The cultivation medium was aspirated 24 h after the LPS addition and the cell residue was removed by centrifugation. The concentration of the secreted TNF-α was determined using the Human TNF-α Instant ELISA kit.
Determination of NF-κB activity. THP1-XBlue™-MD2-CD14 cells were transferred into a serum-free medium at the concentration of 5×10 5 cells/mL. The floated cells were pretreated with the tested compounds (i.e. complexes 1-7, [Cu(Law) 2 (H 2 O) 2 ]Á0.5H 2 O, and Hlaw, respectively) and stimulated either by LPS (1 μg/mL), or by the addition of human recombinant TNF-α (10 ng/mL; Invivogen), or by Pam3CSK4 (100 ng/mL; Invivogen), a synthetic triacylated lipoprotein and selective Toll-like receptor 1/2 agonist. After 24 h of the incubation, 20 μL of the cultivation medium was mixed with 180 μL of Quanti-Blue medium and incubated according to the manufacturer's instructions at 37˚C for 2 h. The activity of released alkaline phosphatase, directly proportional to the NF-κB activity, was quantified from the difference in absorbance at 655 nm against the untreated control.
Evaluation of intracellular ROS production. To elucidate the possible pro/anti-oxidative effect of the tested complexes, a dichlorofluorescein method was used. [18][19] THP1-XBlue™-MD2-CD14 cells were seeded into a black plate in the concentration of 5×10 5 cell/mL in serum-free medium and were incubated for 2 hours. In the following step, the solutions of the tested complexes, or a standard antioxidant Trolox 1 were added at the final concentrations of 100 nM. The production of ROS was determined 1 and 24 h after the application of the compounds. 30 minutes before the determination of ROS production, DCFH-DA (5 μg/mL) dissolved in DMF was introduced into the cell medium. The intracellular fluorescence of the dichlorofluorescein product was measured by Fluostar Omega Microplate Reader (BMG Labtech) using λ(ex./em.) = 480/530 nm.

Anti-inflammatory activity testing in vivo
This study was carried out in a strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health [20]. In addition, all the tests were conducted under the guidelines of the International Association for the Study of Pain. [21] The protocol was approved by the Expert Committee on the Protection of Animals Against Cruelty at the University of Veterinary and Pharmaceuticals Science in Brno (Permit Number: . To minimize the suffering of laboratory animals, all pharmacological interventions were done under urethane anesthesia (applied 30 min prior the start of the experiment at the dose of 1.2 g/kg i.p.). The animals remained anesthetized during the whole duration of the experiment, until they were sacrificed by cervical dislocation. The anesthetized animals were placed onto the thermostated heating pad for preventing the loss of thermoregulation and their breathing and heart rate were checked regularly in about 10 min intervals. The animal tissues for ex vivo experiments were taken post mortem, immediately after all animals were sacrificed by cervical dislocation.
Animals. Wistar-SPF (6-8 weeks male) rats were obtained from the AnLab, Ltd., Prague. The animals were kept in plexiglass cages at the constant temperature of 22±1˚C, and relative humidity of 55±5% for at least 1 week before the experiment. They were given food and water ad libitum. After a one-week adaptation period, male Wistar-SPF rats (200-250 g) were randomly assigned into ten groups (n = 7) of animals in the study. The first, control group, received 10% DMF (v/v in water, intraperitoneal; i.p.). The next eight groups were pretreated with complexes 1-7 and [Cu(Law) 2 (H 2 O) 2 ]Á0.5H 2 O (at the dose corresponding to 40 mmol Cu/kg; ca. 20 mg/kg) and involved into the carrageenan-treatment. The last group was treated with a non-steroidal anti-inflammatory drug Indomethacin (5 mg/kg), which served as a positive control (Indomethacin + carrageenan).
Carrageenan-induced hind paw edema evaluation and ex vivo histological evaluation. The carrageenan-induced hind paw edema model was used for the determination of the antiinflammatory activity. [22] The animals were i.p. pretreated with the complexes 1-7, or [Cu (Law) 2 (H 2 O) 2 ]Á0.5H 2 O or indomethacin (5 mg/kg; positive control) or 10% DMF (v/v in water for injections PhEur), 30 min prior to the injection of 1% λ-carrageenan (50 μL) into the plantar side of right hind paws of the rats. The paw volume was measured immediately after the carrageenan injection and during the next 6 h after the administration of the edematogenic agent using a plethysmometer (model 7159, Ugo Basile, Varese, Italy). The degree of swelling induced was evaluated as the percentage of change in the volume of the right hind paw after the carrageenan treatment from the volume of the right hind paw before the carrageenan treatment. These data were combined afterwards for all 7 animals within each experimental group and subjected to statistical evaluation by the one-way ANOVA with Bonferroni's multiple comparisons post-hoc test.
All the animals were sacrificed by cervical dislocation, and immediately after that, the affected hind paws were separated and underwent the process of dehydration and fixation, and were embedded into paraffin blocks by means of standard protocols. The histopathological changes, like infiltration of different skin elements and deeper laying tissues by the polymorphonuclear white blood cells (dominantly neutrophils and lymphocytes) stained by the standard hematoxylin/eosin staining, were evaluated.

Statistical analysis
All the experiments were performed in triplicate, and the results are presented as mean values, with the error bars representing the standard error of the mean (SEM). A one-way ANOVA test was used for statistical analysis, followed by Bonferroni's multiple comparisons post-hoc test. A value of p < 0.05 was considered as statistically significant. GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA) was used to perform the analysis.

Infrared spectra
The IR spectra of complexes 1-7 (selected spectra for complexes 1 and 7 are depicted in S6 and S7 Figs) showed several characteristic vibrations belonging to the organic ligands [23]. Very intensive bands assignable to the stretching vibrations of the carbonyl groups ν(C . . . O) were detected at ca 1260 cm -1 , while the bands observed in the region between 1640 cm -1 and 1450 cm -1 may be associated with the ν(C . . . C) ring , and ν(C . . . N) ring vibrations. The bands observed at ca. 3139-2968 cm -1 can be associated with the ν(C-H) arom vibrations. The complexes containing water molecules in the coordination sphere, or the water molecules of crystallization, or heterocyclic ligands containing hydroxyl, or amino groups (i.e. aminopyridines, and 3-hydroxypyridine) showed also the bands corresponding to ν(O-H) in the region of 3339-3207 cm -1 , and to the ν(N-H) vibrations at 3454-3407 cm -1 .

UV-Visible spectra
The diffusion-reflection spectra showed intensive maxima (or intensive shoulder) in the region of 465-502 nm owing to the ligand-to-matal charge transfer (LMCT) transitions. Further, either one absorption band at 727-746 nm or two bands at 628-673 nm and 791-800 nm, owing to the d-d transitions of differently axially distorted octahedral polyhedron of Cu(II), have been identified. [24] The representative spectra of complexes 1 and 2 are shown in S8 and S9 Figs.
The solution spectra of the complexes, measured in methanol at 5×10 −4 and 5×10 −5 M concentrations (see S10 Fig), revealed intensive absorption bands at 276-280 nm and shoulders at ca. 300-330 nm, assignable to intraligand electron transitions within the 1,4-naphthoquinone and/or heterocyclic N-donor ligands, and one band assignable to CT transitions at higher wavelengths (at ca. 460-472 nm). The maxima of the first band appeared at 280 nm with ε max % 26 000-34 300 M -1 cm -1 , except for complex 6, with the maxima shifted to 276 nm with ε max = 39 300 M -1 cm -1 and this is assignable to π-π Ã transitions of the aromatic systems (either naphthoquinone system, so heterocyclic N-donor ligands). The maxima of the intensive absorption bands, located in the region of 460-472 with ε max = 4 000-4 800 M -1 cm -1 , can be most likely associated with the n-π Ã transitions and they are probably overlapped with the LMCT chargetransfer transitions. Even the spectral measurements performed for the solutions of a higher concentration (at the concentration of 10 −3 M) did not reveal the positions of the d-d transitions.

SQUID magnetometry
The solid-state temperature dependence of magnetic susceptibility was measured over the temperature range of 300-1.9 K for the selected complexes (1-3, and 5). As a representative result of the temperature dependence of magnetic susceptibility for complex 5 is shown below (Fig 3).
In all the cases, the complexes behaved as paramagnets and followed the Curie-Weiss law (for the best fit parameters, see Table 2) in the wide range of temperatures. All the complexes showed a week antiferromagnetic exchange, probably mediated through the non-covalent interactions stabilizing the crystal structures of the complexes. In the case of aqua-complex, the participation of hydrogen bonds in the more intense antiferromagnetic exchange was evident at low temperature (1.9-3.0 K).

In vitro cytotoxicity
The in vitro cytotoxicity of complexes 1-7 was evaluated as a first step preceding the further in vitro analyses of anti-inflammatory activity. Due to low solubility of the complexes in the cultivation medium, we dissolved the complexes either in DMF or PBS to produce the master solution, which was successively diluted by culture medium to produce the final concentrations of the complex for cytotoxicity testing. The maximum content of the solvent in the final solutions was lower than 0.1% (v/v). All the complexes showed cytotoxic effect on THP-1 cell line with the IC 50 values higher than 10 μM. Lawsone alone demonstrated only negligible cytotoxic effect at 10 μM concentration as the viability of the THP-1 cells was lowered to ca. 80% (see the dose-viability curves, S1 Fig).

Anti-inflammatory activity in vitro
Effect of complexes on NF-κB activation. Lawsone is one of the main secondary metabolites isolated from the henna tree (Lawsonia inermis L., Lythraceae), which is used in folk medicine for treatment of inflammatory diseases [27][28]. Moreover, it is a well-known fact, that several copper(II) complexes have shown anti-inflammatory potential and ability to act by different mechanism of action [29][30]. In this study, we decided to evaluate the anti-inflammatory potential of mixed-ligand Cu(II) complexes involving lawsone and selected N-donor ligands, chosen from azine and azole groups of heterocyclic compounds, both by in vitro and in vivo methods.
The in vitro anti-inflammatory potential of the complexes was evaluated by their ability to modulate the activity of one of the key pro-inflammatory transcription factors, the nuclear factor-κB (NF-κB) after induction by three different activators (TNF-α, LPS, and Pam3CSK4, respectively), thus modelling the different signaling pathways of NF-κB activation (see Fig 5).
The first activator was bacterial lipopolysaccharide (LPS, 1 μg/mL), a Toll-like receptor-4 (TLR-4) agonist, which simulates the immune response of human body against the bacteria [31]. Complex 1 did not affect the activity of NF-κB stimulated by LPS, complexes 2 and 6 non-significantly decreased its activity by 10%, and 12%, respectively. All the remaining complexes (3, 4, and 7 applied at the 100 nM concentration), including lawsone (applied at the 100 nM concentration) were able to significantly attenuate the NF-κB activation by 19-24% ( Fig  5A). In comparison, the synthetic corticoid steroid prednisone, clinically used for the treatment of inflammatory diseases (applied at the 1 μM concentration), significantly decreased the activity of NF-κB by 26%.
The activation of NF-κB by the second activator, a cytokine tumor necrosis factor alpha (TNF-α), represents an aseptic inflammatory response model, proceeds via the activation of the appropriate receptor (TNFR). Complexes 3-7 were able to significantly reduce the activity of NF-κB by 18-28%. In this case, however, the pure lawsone reached similar effect as prednisone as these two compounds decreased the NF-κB by 32%, and 37%, respectively (Fig 5B).
The third pathway of the NF-κB activation was induced by the synthetic triacylated lipoprotein Pam3CSK4. Recognition of Pam3CSK4, which mimics the cell wall components found in both Gram positive and Gram negative bacteria, is mediated by TLR2 which cooperates with TLR1 through their cytoplasmic domain to induce the signaling cascade leading to the activation of NF-κB [32][33]. The effect of complexes 1-7 on the above-mentioned activation  pathway was only marginal. The complexes 3-7 decreased the activation of NF-κB by 15-29%, while the complex 3 was the most efficient ( Fig 5C). Also in this case, similar to other routes of the NF-κB activation, the complexes 1 and 2 showed no noticeable effect. Effect of complexes on the induction of intracellular oxidative stress. The ability of complexes 1-7 to induce the intracellular oxidative stress was studied by the dichlorofluorescein method, 1h and 24 h after the addition of the complexes into cell medium. By comparing the relative fluorescence intensities obtained after the addition of DCFH-DA in these two time-frames, it is evident that all the complexes caused the burst of ROS production at the beginning and the production of ROS was nearly diminished 24 h after the addition (see Fig  6). The combination of pro-oxidant action and the anti-inflammatory activity is not very common; however, it has been described previously for some non-steroidal anti-inflammatory drugs and their reactive metabolites [34][35], and specifically for two new anti-inflammatory drugs flunoxaprofen and benoxaprofen [36][37].
Effect of complexes on TNF-α secretion. Cytokine TNF-α is a prominent pro-inflammatory signaling peptide, which is under transcription control of NF-κB [38]. All the tested complexes decreased significantly its expression in LPS-stimulated cells by 31-48% (see Fig 7). The greatest potential showed the complex 3. Interestingly, pure lawsone reduced the TNF-α level non-significantly only by 28%.
It is a known fact, that naphthoquinones can attenuate the NF-κB activity and thus reduce the expression of pro-inflammatory genes. [39] On the other hand, they could also involve in dissolved in DMSO) for 1 h. Subsequently, LPS (1 μg/mL) (A) or TNF-α (10 ng/mL) (B) or Pam3CSK4 (100 ng/mL) (C) was added [except for the control cells (basal)] to trigger the activation of NF-κB. After 24 h, the activity of NF-κB was evaluated based on the amount of the secreted alkaline phosphatase measured spectrophotometrically. The results are expressed as the mean ± S.E.M. for six independent experiments. ** Indicates a significant difference in comparison with the vehicle-treated cells p < 0.01, and **** indicates a significant difference in comparison with the vehicle-treated cells p < 0.0001.
https://doi.org/10.1371/journal.pone.0181822.g005 the processes leading to the induction of the oxidative stress, for example in the presence of the redox-active transition metals. [40] As a consequence of the oxidation of some intermediates (e.g. IKKβ or p50), involved in the NF-κB activation pathway, the activity of NF-κB can, in fact, be decreased. [41] In the case of the tested complexes, we can observe both effects. Moreover, we can hypothesize that also other molecular mechanisms could influence the processes of TNF-α transcription/translation, or secretion, for example the oxidative stress can impede the processes of exocytosis [42] or proteolytic activation of membrane-bound TNF-α molecules. The tested complexes could also affect TNF-α transcription and/or translation which could explain the discrepancy between NF-κB activation and TNF-α secretion.
From this point of view, the Cu(II)-lawsone complexes represent promising group of new potential anti-inflammatory drugs.

Anti-inflammatory activity in vivo
In continuation of the in vitro testing of anti-inflammatory activities of complexes 1-7, all the copper(II) complexes were included to in vivo tests of anti-inflammatory activity using the carrageenan-induced hind paw edema model. The effect of the tested complexes on the acute inflammatory process (leading to swelling of the hind paw), caused by the carrageenan injection, was evaluated plethysmometrically. The clinically used non-steroidal anti-inflammatory drug indomethacin was used as a primary standard for anti-inflammatory activity. The overview of time-resolved antiedematous activity profiles of the tested compounds is summarized in Fig 8.  The results of antiedematous activity showed very similar pharmacological profiles of complexes 1-4 and aqua-complex [Cu(Law) 2 (H 2 O) 2 ]Á0.5H 2 O with the reference drug indomethacin. The most active complex 1 was able to diminish completely the swelling of the hind paw at the end of the experiment (after 6 h). With respect to the structural similarity of all the tested complexes, the similar mechanism of action can be expected, while the N-donor ligands (L N ) might play some additional role in these processes. For example, the 2-aminopyridine [43] and 4-aminopyridine [44] were found to be anti-inflammatory active as metabolites of anti-inflammatory drugs [43] or as a complementary mechanism in the treatment of neurodegenerative diseases. [44] On the other hand, their metabolism relates to the formation of several reactive intermediates and free radicals [45].
The results obtained by plethysmometric method were further confirmed by the histopathological analysis of tissue samples isolated from the plantar area of hind paws. The histopathological changes in tissues, stained by the standard hematoxylin/eosin staining for the most active complex 1, the least active complex 7, indomethacin and control group (see Fig 9), were evaluated on basis of the the presence of the inflammation infiltrate, which contained mainly neutrophils (polymorphonuclear cells-PMN). These changes provided evidence of the acute inflammation, which were manifested by the massive presence of PMN cells, in the samples from the control group (see Fig 9D) and the group pretreated with complex 7 (see Fig 9B). On the other hand, the PMN infiltration was mainly scarce and diffuse in samples obtained from indomethacin (see Fig 9C) and complex 1 (see Fig 9A) treated groups. Both these substances significantly decreased the inflammatory reaction.

Conclusions
A series of mixed-ligand copper(II) complexes (1-7), involving the lawsone and heterocyclic N-donor ligands, has been studied for anti-inflammatory activity on in vitro and in vivo levels.  Complexes 3-7 showed the ability to suppress significantly the activation of nuclear factor-κB (NF-κB) both by lipopolysaccharide (LPS) and by TNF-α in the similar manner as the reference drug prednisone, even at the 100 nM level. Moreover, all the studied complexes 1-7 decreased significantly the levels of the secreted TNF-α after the LPS activation of the THP-1 cells. The complexes also strongly induced the intracellular production of ROS. The in vivo testing on hind-paw edema model on rats revealed the significant anti-edematous effect of complexes 1-3, which diminished the formation of edema in the similar way as the reference drug indomethacine. The pharmacological profile of complexes 1-3 resembles that of antiinflammatory drug benoxaprofen, also known to cause intracellular production of ROS. To conclude, we may state that the obtained results clearly enrich the knowledge about the biological activities of copper(II) complexes and thus, they might serve as a clue for the development of new anti-inflammatory active complexes involving the 1,4-naphthoquinone ligands.