电针通过调节CD39-NLRP3通路减轻CpG1826攻击小鼠的肺损伤

Electroacupuncture Alleviates Lung Injury in CpG1826-Challenged Mice via Modulating CD39-NLRP3 Pathway.

作者信息

Wu Jiasi, Xiong Xin, Hu Xiumin

机构信息

Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, People's Republic of China.

出版信息

J Inflamm Res. 2023 Aug 3;16:3245-3258. doi: 10.2147/JIR.S413892. eCollection 2023.

PURPOSE

Cytokine storm secondary lung injury (CSSLI) is the leading death cause in COVID-19 virus infection, and CD39-dominated purinergic brake drives NLRP3 inflammasome activation and pyroptosis, which plays a crucial role in the pathogenesis of CSSLI. Though electroacupuncture (EA) can alleviate lung injury caused by a variety of inducers, its effect on CSSLI and the underlying mechanism needs further investigation.

METHODS

We established a widely recognized CSSLI mice model with CpG1826 (CpG), a TLR-9 agonist agent. Luminex liquid chip was employed to detect serum levels of 12 cytokines/chemokines to evaluate cytokine storm formation. H+E staining and transmission electron microscope were applied to examine pulmonary pathological injury and alveolar macrophage structure, respectively. IL-1β, IL-18, IL-1α, and HMGB-1 in BAL fluid were determined by ELISA kits. mRNA and protein levels of lung CD39 and NLRP3 were assessed by qRT-PCR and Western blotting. An in vitro model was also established by incubating PMA-differentiated THP-1 cells with serum samples obtained from relevant group of mice.

RESULTS

Repeated CpG induced CSSLI together with the elevation of 11 cytokines/chemokines including GM-CSF, IL-16, IL-1α, MCP-1, IL-2, IL-10, CCL3, IL-1β, TNF-α, IL-6, and IL-17A, though not IFN-γ, which was reduced by EA pretreatment to a different extent. EA also alleviated lung injury and recovered lung macrophage structure. Moreover, CpG enhanced IL-1β and IL-18 level in BAL fluid, promoted NLRP3, while suppressing CD39 expression in lung, all of which were reversed by EA pretreatment. Of note, EA failed to further decrease BAL fluid IL-1β, IL-18, IL-1α, and HMGB-1 levels when A438079, a selective inhibitor of P2X7, was administered. However, both CD39 and NLRP3 are dispensable for EA decreasing multi-cytokine secretion in serum-incubated and CpG-stimulated THP-1 cells. Taken together, EA alleviated CSSLI in CpG-challenged mice by regulating the CD39-NLRP3 pathway in a P2X7-dependent way.

CONCLUSION

EA demonstrated potential to be applied in COVID-19 treatment.

目的

细胞因子风暴继发的肺损伤（CSSLI）是新型冠状病毒肺炎（COVID-19）病毒感染的主要死亡原因，以CD39为主导的嘌呤能制动驱动NLRP3炎性小体激活和细胞焦亡，这在CSSLI的发病机制中起关键作用。虽然电针（EA）可减轻多种诱导剂所致的肺损伤，但其对CSSLI的作用及潜在机制仍需进一步研究。

方法

我们用TLR-9激动剂CpG1826（CpG）建立了一个广泛认可的CSSLI小鼠模型。采用Luminex液相芯片检测12种细胞因子/趋化因子的血清水平，以评估细胞因子风暴的形成。分别应用苏木精-伊红（H+E）染色和透射电子显微镜观察肺组织病理损伤和肺泡巨噬细胞结构。采用酶联免疫吸附测定（ELISA）试剂盒测定支气管肺泡灌洗液（BALF）中白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）、白细胞介素-1α（IL-1α）和高迁移率族蛋白B1（HMGB-1）的水平。通过实时荧光定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测肺组织中CD39和NLRP3的mRNA和蛋白水平。还用从小鼠相关组获得的血清样本孵育经佛波酯（PMA）分化的人单核细胞白血病细胞株（THP-1）细胞，建立了体外模型。

结果

重复给予CpG可诱导CSSLI，并使包括粒细胞-巨噬细胞集落刺激因子（GM-CSF）、白细胞介素-16（IL-16）、白细胞介素-1α、单核细胞趋化蛋白-1（MCP-1）、白细胞介素-2、白细胞介素-10、趋化因子配体3（CCL3）、白细胞介素-1β、肿瘤坏死因子-α（TNF-α）、白细胞介素-6和白细胞介素-17A在内的11种细胞因子/趋化因子水平升高，而干扰素-γ（IFN-γ）水平降低，EA预处理可在不同程度上降低上述细胞因子/趋化因子水平。EA还减轻了肺损伤并恢复了肺巨噬细胞结构。此外，CpG可提高BALF中IL-1β和IL-18水平，促进NLRP3表达，同时抑制肺组织中CD39表达，而EA预处理可逆转上述变化。值得注意的是，当给予P2X7选择性抑制剂A438079时，EA未能进一步降低BALF中IL-1β、IL-18、IL-1α和HMGB-1水平。然而，在血清孵育和CpG刺激的THP-1细胞中，EA降低多种细胞因子分泌的作用并不依赖于CD39和NLRP3。综上所述，EA通过以P2X7依赖的方式调节CD39-NLRP3通路减轻了CpG攻击小鼠的CSSLI。