Effect of methylene blue on liver inducible nitric oxide synthase in rats with different stages of sepsis / 中国中西医结合急救杂志

ABSTRACT

Objective To observe the effect of methylene blue on the expression of liver inducible nitric oxide synthase (iNOS) in rats with different stages of sepsis.Methods One hundred and twenty-six adult female Wistar rats were randomly divided into three groups sham operation group, sepsis group and methylene blue group, each group was again subdivided into 0, 6, 12, 18, 24, 30, 36 hours subgroups, each subgroup6 rats. The model of sepsis was established by cecal ligation and puncture (CLP) method, and in the sham operation group, the abdominal incision was performed and the intestinal mesentery was separated only, without ligation and perforation. In methylene blue group, 15 mg/kg methylene blue was injected into a caudal vein at 0, 6, 12, 18, 24, 30, 36 hours after CLP in the rats in corresponding subgroups, respectively; in the sepsis and sham operation subgroups, the same amount of 0.9% normal saline was given. After administration for 6 hours in various groups, the rats were sacrificed and the liver tissue was harvested immediately. The expression of iNOS mRNA of liver tissues was determined by the real-time fluorescent quantitative reverse transcription-polymerase chain reaction (qRT-PCR),and the protein expression of iNOS was determined by Western Blot.Results Compared with sham operation group, the liver tissue expression of iNOS mRNA was significantly up-regulated in sub-sepsis groups at 0, 6, 12 and 18 hours after CLP (2-ΔΔCt 16.66±2.81 vs. 1.00±0.36, 12.26±5.78 vs. 1.00±0.30, 6.08±1.33 vs. 1.00±0.18, 2.42±0.64 vs. 1.00±0.12, allP < 0.01), after 24 hours the expression of iNOS mRNA had no significant change; the liver tissue expression of iNOS protein was obviously up-regulated in sub-sepsis groups at 6, 12 ,18 and 36 hours after CLP (gray value 0.350±0.011 vs. 0.210±0.005, 1.460±0.085 vs. 0.090±0.005, 0.230±0.012 vs. 0.18±0.008, 0.310±0.017 vs. 0.200±0.010, allP < 0.01). Compared with sepsis group, the expression of the liver tissue iNOS mRNA was down-regulated in methylene blue subgroups at 0, 12 and 18 hours after CLP (2-ΔΔCt 9.90±3.06 vs. 16.66±2.81, 1.56±0.58 vs. 6.08±1.33, 1.11±0.15 vs. 2.42±0.64, allP < 0.05), and the expression of iNOS protein was down regulated in methylene blue subgroups at 6, 12, 18 and 36 hours after CLP (gray value 0.150±0.008 vs. 0.350±0.011, 0.950±0.009 vs. 1.460±0.085, 0.150±0.007 vs. 0.230±0.012, 0.170±0.009 vs. 0.310±0.017, allP < 0.05).Conclusion Zero-24 hours after CLP, the expressions of mRNA iNOS and protein in liver of septic rats are significantly increased; methylene blue can markedly inhibit the expressions of iNOS mRNA and protein in the liver of rats with sepsis.