亚甲蓝对不同时期脓毒症大鼠肝脏诱导型一氧化氮合酶的影响

Effect of methylene blue on liver inducible nitric oxide synthase in rats with different stages of sepsis

目的 探讨亚甲蓝对不同时期脓毒症大鼠肝脏诱导型一氧化氮合酶（iNOS）表达的影响.方法 按随机数字表法将126只成年雌性Wistar大鼠分为假手术组、脓毒症组和亚甲蓝组,每组再按术后第0、6、12、18、24、30、36 h给药分为7个亚组,每个亚组6只.采用盲肠结扎穿孔术（CLP）制备脓毒症动物模型;假手术组仅行开腹、分离盲肠系膜,不进行结扎和穿孔.亚甲蓝组术后0、6、12、18、24、30、36 h经尾静脉注射亚甲蓝15 mg/kg;脓毒症组和假手术组给予等量生理盐水.各组于给药后6 h处死大鼠取肝组织,用实时荧光定量反转录-聚合酶链反应（qRT-PCR）检测iNOS mRNA表达,用蛋白质免疫印迹试验（Western Blot）检测iNOS蛋白表达.结果 与假手术组比较,脓毒症组术后0、6、12、18 h肝组织iNOS mRNA表达明显升高（2-ΔΔCt：16.66±2.81比1.00±0.36,12.26±5.78比1.00±0.30,6.08±1.33比1.00±0.18,2.42±0.64比1.00±0.12,均P〈0.01）,24 h后则无明显改变;术后6、12、18、36 h肝组织iNOS蛋白表达明显升高（灰度值：0.350±0.011比0.210±0.005,1.460±0.085比0.090±0.005,0.230±0.012比0.180±0.008,0.310±0.017比0.200±0.010,均P〈0.01）.与脓毒症组比较,亚甲蓝组术后0、12、18 h肝组织iNOS mRNA表达明显降低（2-ΔΔCt：9.90±3.06比16.66±2.81,1.56±0.58比6.08±1.33,1.11±0.15比2.42±0.64,均P〈0.05）,术后6、12、18、36 h肝组织iNOS蛋白表达明显降低（灰度值：0.150±0.008比0.350±0.011,0.950±0.009比1.460±0.085,0.150±0.007比0.230±0.012,0.170±0.009比0.310±0.017,均P〈0.05）.结论 脓毒症大鼠24 h内肝脏iNOS mRNA和蛋白表达明显升高;亚甲蓝可明显抑制脓毒症时肝脏iNOS表达.

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.