[Effect of methylene blue on changes in inducible nitric oxide synthase in lung of rats with sepsis]

To study the time course of effect of methylene blue on inducible nitric oxide synthase (iNOS) mRNA transcription and protein expression in lung tissue of rats with sepsis, and its mechanism.

126 female Wistar rats were randomly divided into sham group, sepsis group and methylene blue group. Each group was subdivided into 0-, 6-, 12-, 18-, 24-, 30-, and 36-hour subgroups according to the time after operation, with 6 rats in each subgroup. A model of sepsis was reproduced by cecal ligation and puncture (CLP), and the rats in sham group were only opened the abdominal cavity and isolated the membrane of the appendix without CLP. Rats in methylene blue group were given injection of 15 mg/kg methylene blue at all time points after CLP, the remaining rats were given 0.9%NaCl solution in same amount. Six hours after the injection, the rats were sacrificed and the lung tissue was harvested immediately. The expression of iNOS mRNA and protein in lung tissues were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot respectively, and the changes in histopathology were observed using hematoxylin and eosin (HE) staining.

Compared with sham group, the expression of iNOS mRNA was significantly up-regulated at 6, 12, 18 and 24 hours after CLP in sepsis group (2(-ΔΔCt): 2.42±0.66 vs. 1.00±0.38 at 6 hours, P = 0.002; 2.54±0.76 vs. 1.00±0.27 at 12 hours, P = 0.000; 5.46±2.26 vs. 1.00±0.38 at 18 hours, P = 0.000; 3.03±0.62 vs. 1.00±0.33 at 24 hours, P = 0.001), and iNOS protein expression was significantly up-regulated at 12, 18 and 24 hours (gray value: 2.54±0.45 vs. 1.00±0.35 at 12 hours, P = 0.000; 2.65±0.64 vs. 1.00±0.33 at 18 hours, P = 0.000; 3.03±0.59 vs. 1.00±0.24 at 24 hours, P = 0.000). Compared with sepsis group, the expression of iNOS mRNA was significantly down-regulated at 6, 12, 18 and 24 hours in methylene blue group (2(-ΔΔCt): 1.55±0.82 vs. 2.42±0.66 at 6 hours, P = 0.034; 1.84±0.42 vs. 2.54±0.76 at 12 hours, P = 0.016; 2.66±1.09 vs. 5.46±2.26 at 18 hours, P = 0.003; 2.20±0.29 vs. 3.03±0.62 at 24 hours, P = 0.002), and iNOS protein expression was significantly lowered at 12, 18 and 24 hours (gray value: 1.84±0.18 vs. 2.54±0.45 at 12 hours, P = 0.003; 1.87±0.27 vs. 2.65±0.64 at 18 hours, P = 0.008; 2.20±0.50 vs. 3.03±0.59 at 24 hours, P = 0.008). Histopathological observation showed that the degree of lung injury at each time point, including red blood cells effusion, lung interstitial edema, inflammatory cell infiltration, alveolar collapse etc., in sepsis group and methylene blue group were significantly higher than that of sham group, and the degree of lung injury in rats with methylene blue was not significantly improved as compared with that of sepsis group.

Lung iNOS mRNA expression was significantly increased at 6-24 hours after CLP induced sepsis in rat, and protein expression was increased at 12-24 hours. Methylene blue could inhibit mRNA transcription and protein expression of iNOS in lung of septic rat, but failed to reduce the degree of lung injury in sepsis.