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凉血活血方通过抑制NLRP3炎性小体活化保护脓毒症急性肺损伤小鼠的实验研究
卓玉珍,杨磊,鹿燕敏,李棣华,刘俊红,李彩霞,崔立华,钟成梁
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天津市南开医院中西医结合急腹症研究所天津 300100;天津中医药大学第一附属医院临床试验中心天津300193
摘要:
目的:观察凉血活血方对脓毒症急性肺损伤(ALI)小鼠的保护作用并探索其作用机制。方法:将72只雄性C57BL/6小鼠72只,采用盲肠结扎穿刺术(CLP)制备ALI小鼠模型,将小鼠随机分为3组:假手术组、模型组和凉血活血方组。凉血活血方组灌胃给予凉血活血方水煎剂(6 g/kg)连续2 d(1次/d),假手术组和模型组给予同等剂量生理盐水灌胃。术后24 h麻醉取材。HE染色观察肺组织病理变化;ELISA法检测支气管肺泡灌洗液(BALF)中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、IL-1β水平;Western blotting检测肺组织核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体、核因子(NF)-κB、信号传导与转录激活因子3(STAT3)信号通路相关蛋白表达;检测肺组织髓过氧化物酶(MPO)活性;检测肺组织湿干重比(W/D)。结果:HE染色发现模型组肺组织损伤严重,肺组织结构紊乱,肺泡壁增厚,有明显炎性细胞浸润或出血,经凉血活血方治疗能明显减轻肺组织损伤程度,炎性细胞浸润减少;与假手术组比较,模型组肺组织MPO活性增加,水肿明显,给予中药治疗后可以明显抑制MPO增加,并且减轻肺组织水肿;ELISA检测发现凉血活血方能够降低ALI小鼠BALF中TNF-α、IL-6、IL-1β等炎性因子的水平(P<0.05)。Western blotting检测证实凉血活血方能够降低ALI小鼠肺组织NLRP3、ASC、Caspase 1 P20、IL-18、IL-1β等NLRP3炎性小体相关蛋白表达水平(P<0.05),也能降低NF-κB、STAT3蛋白磷酸化水平(P<0.05)。结论:凉血活血方对脓毒症所致ALI小鼠具有明显的保护作用,其作用机制可能是通过抑制NLRP3炎性小体活化和NF-κB、STAT3通路激活,从而减少下游相关炎性因子的释放,减轻肺组织损伤。
关键词:  凉血活血方  脓毒症  急性肺损伤  NLRP3炎性小体  核因子-κB  信号传导与转录激活因子3
DOI:10.3969/j.issn.1007-6948.2022.02.005
投稿时间:2021-06-03
基金项目:天津市卫健委中西医结合课题(2019038、2019050)
Experimental Study of Liangxue Huoxue Decoction Ameliorates Sepsis-induced ALI in Mice by Inhibiting NLRP3 Inflammasome
ZHUO Yu-zhen,YANG Lei,LU Yan-min
Abstract:
Objective The aim of the present study is to assess the effects of Liangxue Huoxue Decoction on CLP-induced ALI and explore the underlying mechanism. Methods The cecal ligation and puncture (CLP) was used to induce ALI mouse model. 72 male C57BL/6 mice were randomly divided into three groups: shamoperated group (SO), CLP model group (CLP) and Liangxue Huoxue Decoction group. The SO group underwent the sham operation. The Liangxue Huoxue Decoction and CLP groups underwent the CLP operation. The Liangxue Huoxue Decoction groups were administered Liangxue Huoxue Decoction (6 g/kg) by intragastric administration for two consecutive days (once a day), and the SO and CLP groups were administered the same volume of vehicle NS after CLP. After 24h, mice were sacrificed, bronchoalveolar lavage fluids (BALF) were collected for proinflammatory cytokines assay, and the lung tissues were harvested for histologic analysis, W/ D, myeloperoxidase (MPO) activity and the expression of NLRP3, ASC, Caspase 1 P20, ASC, IL-18, IL1 β, STAT 3 ,and NF-κB. Results The results showed that in septic mice, treatment with Liangxue Huoxue Decoction significantly reduced histological damage in the lung, inhibited the MPO activity and W/D. The levels of in.ammatory cytokines, including tumor TNF-α, IL-1β and IL-6 in BALF were signi.cantly decreased by Liangxue Huoxue Decoction. Compared with CLP group, the protein expressions of NLRP3, ASC, Caspase 1 P20, ASC, IL-18, IL-1β, STAT3 and NF-κB in lung tissue were also decreased signi.cantly. Conclusion Liangxue Huoxue Decoction attenuate the degree of lung in.ammation reaction in mice with CLP by suppressing NLRP3 in.ammasome, STAT3 and NF-κB.
Key words:  Liangxue Huoxue Decoction  sepsis  acute lung injury  NLRP3 in.ammasome  NF-κB  STAT3

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