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脓毒症是指因感染引起的宿主反应失调导致的危及生命的器官功能障碍,病人常常需要入住重症医学科加强监护治疗,需要多种脏器支持措施,医疗耗费巨大,许多病人因缺乏有效救治而死亡[1-6]。在脓毒症综合治疗措施中,寻找经济实惠且具有确切治疗效果的药物成为临床追求的目标之一。已有的研究[6]表明静脉输注大剂量维生素C(Vit C)可以作为脓毒症的辅助治疗。Vit C作为一种常见的抗氧化剂,能清除氧自由基,刺激内皮细胞增殖,抑制凋亡,通过增加一氧化氮(NO)的生物利用度调节血流,具有一定程度的抗炎、抗氧化及增强免疫的作用[7]。相关研究[8]证实,补充Vit C可以预防、治疗呼吸道及全身感染。FISHER等通过雄性辛克莱猪的实验证明了Vit C对脓毒症具有抗炎作用,Vit C对脓毒症的抗炎作用机制与miRNA表达变化可能存在一定的关系[9-12];另有研究[13]显示Vit C没有显著改善脓毒症器官功能障碍评分和改善炎症和血管损伤标志物的作用。目前,Vit C对脓毒症的临床疗效尚不确切。应用Vit C后,脓毒症病人血浆miR126水平显著增高;而在脓毒症小鼠血浆miR155水平显著增高[11-12],其机制尚不完全清楚。本研究通过制作脓毒症小鼠模型,探讨Vit C对脓毒症肺脏组织的影响及其潜在的作用机制,为临床治疗脓毒症提供思路。
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与CON组相比,SEP组W/D明显增加,肺泡孔径明显减小;与SEP组相比,SEP+IR组W/D降低,肺泡孔径增大(P<0.05)(见表 1、2)。
分组 n W/mg D/mg W/D Con组 6 77.9±2.0 19.0±2.0 4.10±0.4 SEP组 6 142.2±1.0* 26.9±2.0 5.27±0.3* SEP+IR组 6 130.0±20* 25.0±10.0 5.23±0.2* F — 51.84 2.83 27.45 P — < 0.01 >0.05 < 0.01 MS组内 — 135.000 36.000 0.097 q检验:与Con组比较*P<0.05 表 1 3组小鼠左肺质量比较(x±s)
分组 n 肺泡直径/μm F P MS组内 Con组 6 60.800±8.000 SEP组 6 13.146±3.000* 60.48 < 0.01 57.667 SEP+IR组 6 30.592±10.000*# q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 表 2 3组肺组织的肺泡孔径(x±s)
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3组miR155-5p和miR126a-5p含量差异均有统计学意义(P<0.01);与SEP组相比,CON组和SEP+IR组miR155-5p和miR126a-5p含量均低,差异均有统计学意义(P<0.05)(见表 3)。
分组 n miR155-5p相对表达量 miR126a-5p相对表达量 Con组 6 1±0 1±0 SEP组 6 6.500±0.500* 7.200±0.500* SEP+IR 6 1.190±0.050# 1.137±0.030# F — 694.84 899.39 P — < 0.01 < 0.01 MS组内 — 0.084 0.084 q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 表 3 3组肺组织miR155-5p、miR126a-5p的表达情况(x±s)
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3组Caspase 1、Caspase 3蛋白含量差异均有统计学意义(P<0.05~P<0.01);与CON组相比,SEP组、SEP+IR组Caspase 1、Caspase 3蛋白含量均升高;与SEP组相比,SEP+IR组Caspase 1、Caspase 3蛋白含量减低,差异均有统计学意义(P<0.05)(见表 4)。
分组 n Caspase 1/β-actin Caspase 3/β-actin Con组 6 0.840 0±0.001 1.3090±0.000 3 SEP组 6 1.870 0±1.000* 1.991 0±0.000 4* SEP+IR组 6 1.269 0±0.090# 1.740 0±0.004 0*# F — 4.78 131 794.34 P — < 0.05 < 0.01 MS组内 — 0.336 0.000 q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 表 4 3组肺组织Caspase 1和Caspase 3蛋白的表达情况(x±s)
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与CON组相比,SEP组和SEP+IR组肺组织IL-6、TNF-α含量均升高;与SEP组相比,SEP+IR组肺组织IL-6、TNF-α含量均减低,差异均有统计学意义(P<0.05)(见表 5)。
分组 n IL-6因子表达量 TNF-α因子表达量 Con组 6 390±20 60±4 SEP组 6 830±30* 120±2* SEP+IR组 6 740±50*# 100±4*# F — 255.95 466.67 P — < 0.01 < 0.01 MS组内 — 1 266.667 12.000 q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 表 5 3组肺组织的IL-6及TNF-α表达(x±s; ×105pg/μg)
维生素C预处理对脓毒症小鼠肺组织miR126a-5p、miR155-5p表达的影响
Effect of vitamin C on the expression of miR126a-5p and miR155-5p in the lung tissue of sepsis mice
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摘要:
目的观察维生素C(vitamin C,Vit C)干预后脓毒症小鼠肺组织炎症表达情况及miR126a-5p和miR155-5p含量的变化,分析Vit C对脓毒症急性肺损伤的潜在保护机制。 方法选择4~6周雄性小鼠18只随机分为假手术组(对照组、CON组)、脓毒症+NS组(SEP组)、脓毒症组+Vit C干预组(SEP+IR组),予Vit C 1 000 mg/kg术前连续3 d尾静脉进行注射,采用盲肠穿刺结扎制作模型,术后12 h检测肺组织的湿重/干重(W/D);用HE染色的方法观察肺脏病理形态,测量肺泡的直径;用RT-qPCR检测miR126a-5p、miR155-5p的变化,Western blotting检测Caspase 1及Caspase-3的蛋白表达量,ELISA法检测白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α表达。 结果与CON组相比,SEP组中肺的W/D增加, 肺泡孔径明显减小;SEP组中的miR126a-5p和miR155-5p以及蛋白Caspase 1和Caspase 3表达量升高;同时可进一步促进炎症因子IL-6和TNF-α增加(P<0.05)。与SEP组相比,SEP+IR肺组织的W/D减低,肺泡孔径增大,miR126a-5p和miR155-5p以及蛋白Caspase 1和Caspase 3表达量降低,同时可进一步抑制炎症因子IL-6和TNF-α的增加(P<0.05)。 结论Vit C具有减轻肺水肿和稳定肺泡体积的作用,可能与miR126a-5p和miR155-5p诱导的Caspase 1、Caspase 3表达降低有关。 -
关键词:
- 脓毒症 /
- 维生素C /
- miR126a-5p /
- miR155-5p
Abstract:ObjectiveTo observe the inflammatory cytokines expression of lung tissue and the changes of miR126a-5p and miR155-5p in septic mice after vitamin C(Vit C) intervention, and to analyze the potential protective mechanism of Vit C in the acute lung injury in sepsis. MethodsA total of 18 male mice(4 to 6 weeks) were selected and randomly divided into the control group(CON), sepsis+NS group(SEP) and sepsis+Vit C intervention group(SEP+IR), before the injection of Vit C 1 000 mg/kg by tail vein for three consecutive days in the cecum puncture ligation model.Wet weight/dry weight(W/D) of lung tissue was measured 12 h after operation.HE staining was used to observe the pathological appearance of lung and the diameter of alveoli was measured.The changes of miR126a-5p and miR155-5p were detected by RT-qPCR, the protein expressions of Caspase 1 and Caspase 3 were detected by Western blot, and the expressions of IL-6 and TNF-α were detected by ELISA. ResultsCompared with CON group, the W/D of lung in SEP group was increased and the alveolar aperture was decreased significantly in SEP group.The expressions of miR126a-5p, miR155-5p, Caspase 1 and Caspase 3 were increased in SEP group.Meanwhile, the inflammatory cytokines IL-6 and TNF-α were further increased(P<0.05).Compared with SEP group, SEP+IR group had lower W/D, higher alveolar pore size, lower expression in miR126a-5p, miR155-5p, Caspase 1 and Caspase 3.Vit C further inhibited the increase of inflammatory cytokines IL-6 and TNF-α(P<0.05). ConclusionsVit C can reduce pulmonary edema and stabilize alveolar volume, which may be related to the decreased expressions of Caspase 1 and Caspase 3 induced by miR126a-5p and miR155-5p. -
Key words:
- sepsis /
- vitamin C /
- miR126a-5p /
- miR155-5p
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表 1 3组小鼠左肺质量比较(x±s)
分组 n W/mg D/mg W/D Con组 6 77.9±2.0 19.0±2.0 4.10±0.4 SEP组 6 142.2±1.0* 26.9±2.0 5.27±0.3* SEP+IR组 6 130.0±20* 25.0±10.0 5.23±0.2* F — 51.84 2.83 27.45 P — < 0.01 >0.05 < 0.01 MS组内 — 135.000 36.000 0.097 q检验:与Con组比较*P<0.05 表 2 3组肺组织的肺泡孔径(x±s)
分组 n 肺泡直径/μm F P MS组内 Con组 6 60.800±8.000 SEP组 6 13.146±3.000* 60.48 < 0.01 57.667 SEP+IR组 6 30.592±10.000*# q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 表 3 3组肺组织miR155-5p、miR126a-5p的表达情况(x±s)
分组 n miR155-5p相对表达量 miR126a-5p相对表达量 Con组 6 1±0 1±0 SEP组 6 6.500±0.500* 7.200±0.500* SEP+IR 6 1.190±0.050# 1.137±0.030# F — 694.84 899.39 P — < 0.01 < 0.01 MS组内 — 0.084 0.084 q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 表 4 3组肺组织Caspase 1和Caspase 3蛋白的表达情况(x±s)
分组 n Caspase 1/β-actin Caspase 3/β-actin Con组 6 0.840 0±0.001 1.3090±0.000 3 SEP组 6 1.870 0±1.000* 1.991 0±0.000 4* SEP+IR组 6 1.269 0±0.090# 1.740 0±0.004 0*# F — 4.78 131 794.34 P — < 0.05 < 0.01 MS组内 — 0.336 0.000 q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 表 5 3组肺组织的IL-6及TNF-α表达(x±s; ×105pg/μg)
分组 n IL-6因子表达量 TNF-α因子表达量 Con组 6 390±20 60±4 SEP组 6 830±30* 120±2* SEP+IR组 6 740±50*# 100±4*# F — 255.95 466.67 P — < 0.01 < 0.01 MS组内 — 1 266.667 12.000 q检验:与Con组比较*P<0.05;与SEP组比较#P<0.05 -
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