Effect of Qingre Huatan Jiedu Formula on TXNIP/NLRP3 Inflammatory Pathway in Lung Tissue of Rats with Cerebral Ischemia-reperfusion

WANG Tao; LIU Hong-xiang; WANG Ying; QI Jiao; CHEN Xiao-jing; YAN Li-jing; SHI Xiao-pan; LUO Jin-hua

doi:10.13422/j.cnki.syfjx.20181040

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Effect of Qingre Huatan Jiedu Formula on TXNIP/NLRP3 Inflammatory Pathway in Lung Tissue of Rats with Cerebral Ischemia-reperfusion

更新时间：2024-01-04

- Effect of Qingre Huatan Jiedu Formula on TXNIP/NLRP3 Inflammatory Pathway in Lung Tissue of Rats with Cerebral Ischemia-reperfusion
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 24, Issue 12, Pages: 114-122(2018)
- 作者机构：
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- DOI：10.13422/j.cnki.syfjx.20181040
  CLC： R285.5
- Published：2018
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WANG Tao, LIU Hong-xiang, WANG Ying, et al. Effect of Qingre Huatan Jiedu Formula on TXNIP/NLRP3 Inflammatory Pathway in Lung Tissue of Rats with Cerebral Ischemia-reperfusion[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(12): 114-122.
DOI：

WANG Tao, LIU Hong-xiang, WANG Ying, et al. Effect of Qingre Huatan Jiedu Formula on TXNIP/NLRP3 Inflammatory Pathway in Lung Tissue of Rats with Cerebral Ischemia-reperfusion[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(12): 114-122. DOI： 10.13422/j.cnki.syfjx.20181040.

摘要

目的：探究清热化痰解毒方对脑缺血再灌注大鼠肺组织的影响，并初步探讨相关分子机制。方法：将SD大鼠分为假手术组、模型组、阳性药组（灌胃尼莫地平200 mg·kg-1）、清热化痰解毒方低、高剂量组（100，200 mg·kg-1），每组10只，除假手术组外，采用改良4动脉阻断法制备大鼠全脑缺血再灌注损伤模型，造模后，给药3 d。肺湿/干重（W/D）分析，采用苏木素-伊红（HE）染色进行组织病理学分析；酶联免疫吸附法检测支气管肺泡灌洗液（BALF）中白细胞介素（interleukin，IL）-1β和IL-18水平；肺髓过氧化物酶试剂盒检测髓过氧化物酶（myeloperoxidase，MPO）活性；免疫组化分析核苷酸结合域样受体蛋白3（nucleotide-binding domain-like receptor 3，NLRP3）阳性细胞数；半胱氨酸天冬氨酸酶-1（Caspase-1）比色测定试剂盒检测Caspase-1活性；实时荧光定量聚合酶链式反应（Real-time PCR）和蛋白免疫印迹法（Western blot）检测NLRP3，ASC，Caspase-1 mRNA和蛋白表达，免疫共沉淀检测硫氧还蛋白相互作用蛋白（TXNIP）-NLRP3蛋白表达。结果：与假手术组比较，模型组肺损伤评分，肺W/D，BALF中IL-1β和IL-18水平、总细胞和多形核白细胞数量，MPO活性均明显增加（P<0.05）；与模型组比较，尼莫地平组、清热化痰解毒方低、高剂量组肺损伤评分，肺W/D，BALF中IL-1β和IL-18水平、总细胞和多形核白细胞数量及MPO活性均明显降低（P<0.05）。与假手术组比较，模型组肺组织中NLRP3阳性细胞数，Caspase-1活性，NLRP3，ASC，Caspase-1蛋白和mRNA相对表达明显增加（P<0.05）；与模型组比较，尼莫地平组、清热化痰解毒方低、高剂量组肺组织中NLRP3阳性细胞数，Caspase-1活性，NLRP3，ASC，Caspase-1蛋白和mRNA相对表达均明显降低（P<0.05）。与假手术组比较，模型组肺组织中ROS产生，TXNIP蛋白表达均明显增加（P<0.05）；与模型组比较，尼莫地平组、清热化痰解毒方低、高剂量组肺组织中ROS产生，TXNIP蛋白表达均明显降低（P<0.05）。与假手术组比较，模型组肺组织中TXNIP-NLRP3蛋白共沉淀增加。与模型组比较，尼莫地平组、清热化痰解毒方低、高剂量组肺组织中TXNIP-NLRP3蛋白共沉淀减少。结论：清热化痰解毒方对全脑缺血再灌注大鼠的炎症性肺损伤具有保护作用，且这种作用可能通过抑制ROS产生，从而抑制TXNIP-NLRP3炎症通路介导的促炎介质IL-1β和IL-18的分泌，最终抑制肺组织中炎性细胞浸润，缓解肺组织损伤。

Abstract

Objective: To investigate the effect of Qingre Huatan Jiedu formula on lung tissue in rats with cerebral ischemia-reperfusion injury and explore the underlying molecular mechanisms. Method: SD rats were divided into shame group

model group

positive control group (PC group

nimodipine 200 mg·kg-1)

low-dose Qingre Huatan Jiedu formula group (LT group

100 mg·kg-1) and high-dose Qingre Huatan Jiedu formula group (HT group

200 mg·kg-1)

with 10 rats in each group. Except for sham group

modified 4 artery occlusion method was used to prepare the global cerebral ischemia reperfusion injury model in rats. After modeling

the drug was given for 3 d. Lung wet/dry weight (W/D) analysis was made. htoxylin easin(HE) staining was performed for histopathological analysis; the levels of interleukin(IL)-1β and IL-18 in bronchoalveolar lavage fluid (BALF) were detected by enzyme linked immunosorbent assay (ELISA); pulmonary myeloperoxidase kit assay was used to detect myeloperoxidase(MPO) activity;immunohistochemical analysis was performed for nucleotide-binding domain-like receptor 3(NLRP3) positive cells; Caspase-1 colorimetric assay kit was applied in detecting Caspase-1 activity; Real-time PCR and Western blot were used to detect the mRNA and protein expressions of NLRP3

ASC and Caspase-1.Co-immunoprecipitation was used to detect thioredoxon interaction protein(TXNIP)-NLRP3 protein expression. Result: Compared with shame group

lung injury score

W/D ratio of lung

IL-1β and IL-18 levels in BALF

total number of cells and polymorphonuclear leukocytes and MPO activity in model group were significantly increased (P<0.05). Compared with model group

lung injury score

W/D ratio of lung

IL-1β and IL-18 levels in BALF

total cell and polymorphonuclear leukocyte and MPO activity in PC group

LT group and HT group were significantly decreased (P<0.05). Compared with shame group

the relative expression of NLRP3 positive cells

Caspase-1 activity and NLRP3/Asc/Caspase-1 in model group lung tissue were significantly increased (P<0.05). Compared with model group

the expression of NLRP3 positive cells

Caspase-1 activity and NLRP3/Asc/Caspase-1 in PC group

LT group and HT group were significantly decreased (P<0.05). Compared with the shame group

the ROS production and TXNIP protein expression in model group were significantly increased (P<0.05). Compared with model group

ROS production and TXNIP protein expression in PC group

LT group and HT group were significantly decreased (P<0.05). TXNIP-NLRP3 co-precipitation was increased in lungs of the model group compared with the shame group. Compared with model group

the TXNIP-NLRP3 co-precipitation in PC group

LT group and HT group decreased. Conclusion: Qingre Huatanjiedu formula can inhibit the production of ROS

block the TXNIP-NLRP3 inflammatory pathway

reduce the infiltration of inflammatory cells in lung tissue

and alleviate the inflammatory lung injury induced by global cerebral ischemia-reperfusion in rats.

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