南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (10): 1232-.doi: 10.12122/j.issn.1673-4254.2019.10.16

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乌司他丁减轻失血性休克大鼠肺炎性损伤的机制

陈硬，徐志鹏，宋琦，王振杰，纪忠，邱兆磊，程峰，姜海   

  • 出版日期:2019-10-20 发布日期:2019-10-20

Mechanism of ulinastatin in reducing lung inflammatory injury in rats with hemorrhagic shock

  • Online:2019-10-20 Published:2019-10-20

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摘要： 目的探讨乌司他丁对创伤失血性休克大鼠肺炎性介质及其信号通路miR-146a/TLR4/NF-κB的影响。方法72只SD大 鼠，随机分为休克不复苏组（SR，n=24），醋酸钠林格液组（AR，n=24）及乌司他丁组（UR，n=24）。通过股动脉放血将3组制成休 克模型（平均动脉压维持在30~40 mmHg），SR组休克制备成功后不予复苏，并分别于休克后1、4、6 h（若大鼠死亡则大鼠死亡即 刻）取大鼠肺组织；AR组、UR组分别系维持休克60 min后应用醋酸钠林格液、醋酸钠林格液联合乌司他丁进行30 min液体复 苏，并分别于复苏后1、4、6 h取大鼠肺组织。利用实时荧光定量PCR法检测各组肺组织中miR-146a、TNF-α、IL-1、IL-4、IL-6、 IL-10的mRNA含量以及通过Western blot检测TLR4、MyD88、IκB-α、p-IκB-α、NF-κB p65、IRAK4、p-IRAK4（Thr345、Ser346）、 p-IRAK4（Thr342）、TRAF6的蛋白相对表达量。在光镜下观察3组肺组织病理学变化。结果与休克组相比，醋酸钠林格液组 肺组织炎症浸润程度稍减轻，其中miR-146a、IL-4 和IL-10 的mRNA水平升高（P<0.05），IκB-α、p-IRAK4（Thr342）及p-IRAK4 (Thr345、ser346)的蛋白表达增加（P<0.05）；TNF-α、IL-1和IL-6的水平降低（P<0.05），TLR4、MyD88、NF-κB p65、p-IκB-α、IRAK- 4及TRAF6的蛋白表达减少（P<0.05）。与醋酸钠林格液组比较，乌司他丁组的肺组织炎性损伤得到了进一步改善，升高了miR- 146a、IL-4 和IL-10 的mRNA水平（P<0.01）及IκB-α、p-IRAK4（Thr342）及p-IRAK4(Thr345、ser346)的蛋白表达（P<0.01），降低 了TNF-α、IL-1和IL-6的水平（P<0.01）和TLR4、MyD88、NF-κB p65、p-IκB-α、IRAK-4及TRAF6的蛋白表达（P<0.01）。结论乌 司他丁联合醋酸钠林格液复苏创伤失血性休克大鼠可能是通过增强miR-146a的表达，负反馈调控TLR4/NF-kB信号通路，进而 调节体内促炎因子和抗炎因子平衡，以达到保护肺组织的作用。

Abstract: Objective To investigate the effect of ulinastatin on the inflammatory mediators and their signaling pathways miR-146a/TLR4/NF-κB in rats with hemorrhagic shock. Methods Seventy-two SD rats were randomly assigned into shock without resuscitation group (SR group, n=24), acetated Ringer’s solution resuscitation group (AR group, n=24) and ulinastatin treatment group (n=24). In all the 3 groups hemorrhagic shock models were established by femoral artery bleeding (with the mean arterial pressure maintained at 30-40 mmHg) without resuscitation (in SR group) or with resuscitation (in AR and ulinastatin groups) using acetated Ringer’s solution for 30 min at 60 min after the onset of shock. At 1, 4, and 6 h after the shock onset or immediately after shock if the rats died, the lung tissues were taken for measurement of mRNA expressions of miR-146a, tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-4, IL-6 and IL-10 using real-time quantitative PCR and the protein expressions of TLR4, MyD88, IκB-α, p-IκB-α, NF-κB p65, IRAK4, p-IRAK4 (Thr345, Ser346), p-IRAK4 (Thr342) and TRAF6 using Western blotting. The lung histopathology of the rats was examined under optical microscope with HE staining. Results Compared with the SR group, the rats in the AR group showed slightly alleviated inflammatory infiltration in the lung tissues with significantly increased mRNA levels of miR-146a, IL-4 and IL-10 (P<0.05) and protein expressions of IκB-α, p-IRAK4 (Thr342) and p-IRAK4 (Thr345, ser346) (P<0.05), and decreased mRNA levels of TNF-α, IL-1 and IL-6 (P<0.05) and protein expressions of TLR4, MyD88, NF-κB p65, p-IκB-α, IRAK-4 and TRAF6 (P<0.05). Compared with those in AR group, the rats in ulinastatin group showed further alleviation of inflammatory lung tissue injury, with increased mRNA levels of miR-146a, IL-4 and IL-10 (P<0.01) and protein expressions of IκB-α, p-IRAK4 and p-IRAK4 (P<0.01) and decreased mRNA levels of TNF-α, IL-1 and IL-6 (P<0.01) and protein expressions of TLR4, MyD88, NF-κB p65, p-IκB-α, IRAK-4 and TRAF6 (P< 0.01). Conclusion Ulinastatin combined with acetated Ringer’s solution resuscitation alleviates lung inflammations in rats with hemorrhagic shock possibly by enhancing miR-146a expression to regulate TLR4/NF-κB signaling pathway through a negative feedback mechanism and thus modulate the balance of pro-inflammatory and anti-inflammatory factors.