Melatonin

Animals were randomly assigned to either the Melatonin administration (n = 5) or the control group (vehicle treated, n = 8). Vehicle solution was prepared with 8 ml of phosphate buffered saline (PBS), 1 ml of dimethyl sulfoxide (DMSO) and 1 ml of Cremophor (Sigma, St. Louise, MO, USA). The animals of the control group received 100 µl of vehicle solution by intraperitoneal injection (IP).
Melatonin (Sigma, St. Louise, MO, USA) was diluted in vehicle and the animals from the Melatonin group received IP of 100 µl of Melatonin treatment (at dose of 40 mg/kg of body weight) for five days a week. Melatonin was administered 1 hr before room lighting was switched off. Administration of Melatonin prior to the nocturnal increase in endogenous Melatonin may be most effective because tissues are most sensitive to the hormone at this time [38] (link), [39] (link).
Melatonin or vehicle administration started on the day of tumor implantation (soon after implantation) and continued for five days a week for 21 days. On the 22nd day, all animals underwent SPECT scanning with Tc-99m-HYNIC-VEGF-c followed by euthanasia and collection of tumors for immunohistochemistry and membrane antibody array to determine the expression of different pro-angiogenic and growth factors in tumor extracts.

In some experiments, HUVEC were co-cultured with Mf in medium supplemented with different concentrations and types of serum or other additional additives. These included heat-inactivated or non-heat inactivated human serum, C3-depleted human serum, 250μg/ml mannan, 2.5μM DEC or 1nM melatonin (Sigma-Aldrich Ltd. The melatonin concentration used in this experiment corresponded approximately to the concentration of melatonin observed in human plasma at early night [29 (link)]. In some experiments HUVEC were stimulated with 10ng/ml human IFN-γ (Immuno-Contact, USA) for 24-48h or 20ng/ml human TNF-α (Insight Biotechnology Ltd., Wembley, UK) for 18h prior to co-culture with Mf. In other experiments, cell culture plates containing endothelial cells were placed inside a sealed plastic container that also contained a GasPak TM EZ Campy Container System Sachet (BENEX Ltd., Maryland, USA) to achieve a hypoxic environment. (Each sachet contains inorganic carbonate, activated carbon, ascorbic acid and water, which reduces the oxygen concentration within the container to 6–16%). The number of Mf adhering after 24 h co-culture was counted. Both cell and Mf viability, and Mf motility was unaltered after 24 hours in the hypoxic environment.

To determine the effect of melatonin on BMMSCs, cells were subjected to a 24-h pretreatment with 5 µM melatonin (Sigma, USA). melatonin-pretreated BMMSCs (MT-BMMSCs) were then washed three times with PBS (Sigma, USA) for complete removal of the hormone from cell suspension[9 (link),12 (link)].

The murine model of allergic airway inflammation was established according to previous study (27 (link)). Briefly, the mice (6–8 weeks old) were sensitized on day 0 with an intra-peritoneal injection of OVA (Sigma, St. Louis, MO, USA) emulsified with 1 mg potassium aluminum sulfate (Sangon Biotech, Shanghai, China) in 500 μl of saline. Airway inflammation was induced by inhalation of 1% aerosolized OVA 30 min per day, for consecutive 7 days. Control mice received saline-sensitization and inhalation of nebulized saline solution. The mice from WT-OVA-Melatonin group were administered with Melatonin (10 mg/kg, Sigma), and the mice from TLR2^−/−-OVA-Luzindole group were administered with Luzindole (30 mg/kg, Sigma) 1 h before allergen-challenge through intra-peritoneal injection, respectively, the dose of Melatonin and Luzindole was used according to previous studies (19 (link), 28 (link)). Experiments were performed with six mice per group. Mice were harvested 24 h following the last challenge.