Liu Tian-Yi, Hao Yu, Mao Qin, Zhou Na, Liu Meng-Hua, Wu Jun, Wang Yi, Yang Ming-Rui
School of Life Sciences, Beijing University of Chinese Medicine, Beijing, 102488, China.
Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Chin J Integr Med. 2025 Jan;31(1):19-27. doi: 10.1007/s11655-024-3905-3. Epub 2024 Jun 24.
To investigate the inhibitory effect of Tanreqing Injection (TRQ) on the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in macrophages infected with influenza A virus and the underlying mechanism based on mitophagy pathway.
The inflammatory model of murine macrophage J774A.1 induced by influenza A virus [strain A/Puerto Rico/8/1934 (H1N1), PR8] was constructed and treated by TRQ, while the mitochondria-targeted antioxidant Mito-TEMPO and autophagy specific inhibitor 3-methyladenine (3-MA) were used as controls to intensively study the anti-inflammatory mechanism of TRQ based on mitophagy-mitochondrial reactive oxygen species (mtROS)-NLRP3 inflammasome pathway. The levels of NLRP3, Caspase-1 p20, microtubule-associated protein 1 light chain 3 II (LC3II) and P62 proteins were measured by Western blot. The release of interleukin-1β (IL-1β) was tested by enzyme linked immunosorbent assay, the mtROS level was detected by flow cytometry, and the immunofluorescence and co-localization of LC3 and mitochondria were observed under confocal laser scanning microscopy.
Similar to the effect of Mito-TEMPO and contrary to the results of 3-MA treatment, TRQ could significantly reduce the expressions of NLRP3, Caspase-1 p20, and autophagy adaptor P62, promote the expression of autophagy marker LC3II, enhance the mitochondrial fluorescence intensity, and inhibit the release of mtROS and IL-1β (all P<0.01). Moreover, LC3 was co-localized with mitochondria, confirming the type of mitophagy.
TRQ could reduce the level of mtROS by promoting mitophagy in macrophages infected with influenza A virus, thus inhibiting the activation of NLRP3 inflammasome and the release of IL-1β, and attenuating the inflammatory response.
探讨痰热清注射液（TRQ）对甲型流感病毒感染巨噬细胞中含NOD样受体蛋白3（NLRP3）炎性小体激活的抑制作用及其基于线粒体自噬途径的潜在机制。
构建甲型流感病毒[株A/波多黎各/8/1934（H1N1），PR8]诱导的小鼠巨噬细胞J774A.1炎性模型，并用TRQ处理，同时以线粒体靶向抗氧化剂Mito-TEMPO和自噬特异性抑制剂3-甲基腺嘌呤（3-MA）作为对照，深入研究TRQ基于线粒体自噬-线粒体活性氧（mtROS）-NLRP3炎性小体途径的抗炎机制。采用蛋白质免疫印迹法检测NLRP3、半胱天冬酶-1 p20、微管相关蛋白1轻链3 II（LC3II）和P62蛋白水平。采用酶联免疫吸附测定法检测白细胞介素-1β（IL-1β）释放，采用流式细胞术检测mtROS水平，并在共聚焦激光扫描显微镜下观察LC3与线粒体的免疫荧光及共定位情况。
与Mito-TEMPO的作用相似，与3-MA处理结果相反，TRQ可显著降低NLRP3、半胱天冬酶-1 p20和自噬衔接蛋白P62的表达，促进自噬标志物LC3II的表达，增强线粒体荧光强度，抑制mtROS和IL-1β的释放（均P<0.01）。此外，LC3与线粒体共定位，证实了线粒体自噬类型。
TRQ可通过促进甲型流感病毒感染巨噬细胞的线粒体自噬来降低mtROS水平，从而抑制NLRP3炎性小体的激活和IL-1β的释放，减轻炎症反应。
