目的：观察亚甲蓝对脓毒症大鼠肺组织诱导型一氧化氮合酶（iNOS）mRNA转录及蛋白表达影响的时间窗及作用机制。方法将126只雌性Wistar大鼠按随机数字表法分为假手术（Sham）组、脓毒症组和亚甲蓝组，每组再按术后时间分为0、6、12、18、24、30、36h7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，Sham组仅开腹和分离盲肠系膜但不结扎和穿孔；亚甲蓝组于CLP术后各时间点分别给予15mg/kg亚甲蓝，其余大鼠给予等量生理盐水。于给药后6h处死各组大鼠，收集肺脏组织标本，用实时荧光定量反转录-聚合酶链反应（RT-qPCR）检测肺组织iNOSmRNA表达，用蛋白质免疫印迹试验（WesternBlot）检测肺组织iNOS蛋白表达，苏木素-伊红（HE）染色后光镜下观察肺组织病理学改变。结果与Sham组比较，脓毒症组术后6、12、18、24h肺组织iNOSmRNA表达明显升高（2-ΔΔCt：6h为2.42±0.66比1.00±0.38，P＝0.002；12h为2.54±0.76比1.00±0.27，P＝0.000；18h为5.46±2.26比1.00±0.38，P＝0.000；24h为3.03±0.62比1.00±0.33，P＝0.001）；12、18、24hiNOS蛋白表达也明显升高（灰度值：12h为2.54±0.45比1.00±0.35，P＝0.000；18h 为2.65±0.64比1.00±0.33，P＝0.000；24h 为3.03±0.59比1.00±0.24，P＝0.000）。与脓毒症组比较，亚甲蓝组6、12、18、24h肺组织iNOSmRNA表达明显降低（2-ΔΔCt：6h为1.55±0.82比2.42±0.66，P＝0.034；12h为1.84±0.42比2.54±0.76，P＝0.016；18h为2.66±1.09比5.46±2.26，P＝0.003；24h为2.20±0.29比3.03±0.62，P＝0.002）；12、18、24hiNOS蛋白表达也明显降低（灰度值：12h为1.84±0.18比2.54±0.45，P＝0.003；18h为1.87±0.27比2.65±0.64，P＝0.008；24h为2.20±0.50比3.03±0.59，P＝0.008）。组织病理学观察显示，各时间点脓毒症组和亚甲蓝组大鼠肺损伤程度（包括红细胞渗出、肺间质水肿、炎性细胞浸润、肺泡塌陷等）均明显重于Sham组，而亚甲蓝组大鼠肺损伤程度较脓毒症组无明显改善。结论脓毒症大鼠肺组织iNOSmRNA表达于CLP术后6～24h上调，蛋白表达则于12～24h上调；亚甲蓝可明显抑制脓毒症大鼠肺组织iNOSmRNA和蛋白表达，但不能减轻脓毒症肺损伤的程度。%Objective 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. Methods 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. Results 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. Conclusions 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.
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