Antioxidant Effect

In this study, two CS exposure experiments were conducted: (1) the prophylactic approach, in which SUL-151 (4 mg/kg), budesonide (500 μg/kg) [27 (link)], or vehicle (saline) was administered via the oropharyngeal route 30 min before the first CS exposure daily for 5 consecutive days to investigate the anti-inflammatory and antioxidative effects of SUL-151 (SUL-151 administration during the induction of the disease, Figure 9A) and (2) the therapeutic approach, in which mice were first exposed to CS for 5 days without treatment, followed by 5 days of CS exposure with the different therapies (once daily, 30 min before the first CS exposure) and a possible anti-inflammatory and antioxidative therapeutic approach of SUL-151 and associating potential mechanism were investigated (SUL-151 after induction of the disease, Figure 9B).
Mice were exposed in whole-body chambers to air or to mainstream CS for 5 or 10 consecutive days (twice a day) using a peristaltic pump (SCIQ 232, Watson-Marlow 323, USA) at speed 35 rpm [50 (link)]. Research cigarettes (3R4F) were obtained from the Tobacco Research Institute (University of Kentucky, Lexington, Kentucky), and filters were removed before use.
Mice were exposed to either CS or to ambient air twice daily with a minimum interval of 5 h using 4–6 cigarettes on day 1, 8–10 cigarettes on day 2, 12–14 cigarettes on day 3, and 14 cigarettes till the end of the smoke exposure period (two cigarettes/round). CS exposures were analyzed periodically for carbon monoxide (CO), ranging between 200 and 400 ppm. The mass concentration of cigarette smoke total particulate matter (TPM) was determined by gravimetric analysis of type A/E glass fiber filter (PALL life sciences, Mexico) [51 (link)]. The TPM concentration in the smoke exposure box generated by 14 cigarettes reached approximately 828 μg/L (828 ± 4.5 μg/L).
Mice were killed by an intraperitoneal overdose of pentobarbital approximately 18 h hours after the last air or smoke exposure. Blood was obtained by heart puncture and collected in Mini collect tubes (Greiner Bio-One, Alphen aan den Rijn, the Netherlands). Blood samples were centrifuged (14,000 rpm for 10 min) and serum was stored at −20 °C. Lungs were collected, snap-frozen in liquid nitrogen, and kept at −80 °C until further analyses.

In the first step, we studied the acute anti-inflammatory effects of cashew nuts and a possible dose-response in a classical acute model of inflammation. Rats were divided into different groups:
CAR + vehicle: rats were subjected to CAR-induced paw edema, as described above and administered vehicle (saline);
CAR + cashew nuts (30mg/kg): same as the CAR+vehicle group but cashew nuts at dose of 30 mg/kg was administered instead of vehicle orally 30 min before CAR injection;
CAR + cashew nuts (60mg/kg): same as the CAR+vehicle group but cashew nuts at dose of 60 mg/kg was administered instead of vehicle orally 30 min before CAR injection;
CAR + cashew nuts (100mg/kg): same as the CAR+vehicle group but cashew nuts at dose of 100 mg/kg was administered instead of vehicle orally 30 min before CAR injection;
Sham operated groups received saline instead of CAR and were treated orally with saline or nuts.
Doses were given on the basis of a dose-response study. We started with 30 mg/kg dose based on a previous work on nuts [32 (link)].
The administration of cashew nuts was done orally for 4 days based on our previous studies on the effects of antioxidants on mouse model of colitis induced by intrarectal DNBS injection [24 (link),33 (link),34 (link)].
After a preliminary data on dose response, we decided to carry on with the experiments by evaluating the chronic effect of cashew nuts at a dose of 100 mg/kg (higher dose which showed a significant reduction of paw volume increase) in an experimental model of colitis by instillation of DNBS in the colon. In particular, mice were divided into the following groups:
DNBS +saline: mice were subjected to DNBS administration described as above, and saline was administered orally every 24 h, for 4 days, starting from 1 h after the administration of DNBS.
DNBS+cashew nuts: mice were subjected to DNBS administration described as above, and cashew nuts (100 mg/kg) was administered orally every 24 h, for 4 days, starting from 1 h after the administration of DNBS.
Sham operated groups: vehicle solution (saline) or cashew nuts were orally administered for 4 days.
Since no significant change was found between sham groups, we present data of sham+vehicle groups only.

The effects on the antioxidant activity and the inhibition mechanism of antibacterial substances were investigated by measuring the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) in B. sorokiniana mycelium treated with different levels of lipopeptide crude extracts. Briefly, 0.2 g of B. sorokiniana mature mycelium was taken and added to 1% and 2% lipopeptide crude extract liquid as described before, respectively, and PBS was supplemented to 1 mL and incubated for 2 h at 28 °C. The treatment group with only 1 mL of PBS added was used as a control and the experiment was repeated three times for each treatment group. The mycelial sediment was collected by centrifugation at 4 °C and 10,000× g for 10 min and ground well in liquid nitrogen. The SOD and POD enzyme activities, as well as the MDA content, were determined by referring to the previous report by Yi et al. [23 (link)]. The CAT enzyme activity was measured by referring to Zhou et al. [52 (link)].

Caenorhabditis elegans strains N2, Bristol (wild type) was obtained from the Caenorhabditis Genetics Center at the University of Minnesota and maintained at 20 °C on Nematode Growth Medium (NGM) plates with E. coli strain OP50 as normal diet for nematodes. Overnight, B. clausii CSI08 culture was grown, as described in Section 2.1, and then centrifuged for 10 min at 4000× g. Supernatants were then removed, and bacterial pellets were washed once with saline solution. C. elegans wild-type strain (N2) cultured in NGM (control fed condition), or NGM supplemented with B. clausii CSI08 at two different doses (10⁸ and 10⁹ cells/plate). Vitamin C (10 µg/mL) was used as positive control. Worms were incubated in these conditions and after several days were submitted to an acute oxidative stress (2 mM H₂O₂) according to a previously published protocol [46 (link)]. Afterwards, viability of nematodes was determined in each fed condition. Experiments were performed in duplicate. Each experiment was conducted on 5 different plates, each containing 10 worms (50 worms/assay). The antioxidant activity (worm survival) of total population was calculated. Final survival data correspond to the average of two independent assays (total population of 100 worms/condition). The effect of B. clausii CSI08 antioxidant activity was studied by comparing the survival of treated nematodes versus the control-fed nematodes.