Ma Lan, Han Zhengpu, Yin Haoyu, Tian Jiaqi, Zhang Jing, Li Ning, Ding Chunjie, Zhang Lin
School of Public Health, Weifang Medical University, Weifang, 261053, People's Republic of China.
Clinical Medical Research Center for Women and Children Diseases, Maternal and Child Health Care Hospital of Shandong Province, Shandong University, Jinan, 250001, People's Republic of China.
J Inflamm Res. 2022 Aug 8;15:4537-4545. doi: 10.2147/JIR.S371536. eCollection 2022.
Silica nanoparticles (SiNPs) are one of the most widely used inorganic nanomaterials, and exposure to SiNP has been demonstrated to induce pulmonary inflammation, primarily promoted by the NLRP3-mediated macrophage pyroptosis. However, mechanisms underlying the activation of NLRP3 signaling are complex, and whether cathepsin B (CTSB), an enzyme released by the ruptured lysosome, could trigger NLRP3 assembly is controversial.
To further characterize the role of CTSB in silica-induced pyroptosis, we conducted this study by establishing SiNP exposure models in vitro. The morphological features of SiNPs were exhibited by the SEM and TEM, and the effects of SiNPs' internalization on macrophages were examined by the TEM and immunofluorescent staining. Moreover, Western blot was performed to detect the expression of proteins related to pyroptosis and CTSB after blocking the expression of NLRP3 and CTSB.
We found that SiNPs internalization caused the rupture of macrophage membrane and promoted the aging of cells with increased intracellular vacuoles. Also, the expression of NLRP3, ASC, Caspase-1, GSDMD, Pro-IL-1β, IL-1β, and CTSB increased under the stimulation of SiNP, which could be suppressed by additional treatment with MCC950, an NLRP3-specific inhibitor. Besides, we found SiNP joint treatment with leupeptin, a CTSB inhibitor, could inhibit the expression of CTSB, but it had no effect on the expression of NLRP3, ASC, and Caspase-1, and the process of macrophage pyroptosis was also not affected.
SiNP exposure induces rupture of macrophages and the release of lysosomal CTSB, but CTSB fails to specifically act on the NLRP3 inflammasome to induce pyroptosis which is causally linked to lung inflammation and fibrosis.
二氧化硅纳米颗粒（SiNPs）是应用最为广泛的无机纳米材料之一，已证实暴露于SiNP会引发肺部炎症，主要由NLRP3介导的巨噬细胞焦亡所推动。然而，NLRP3信号激活的潜在机制十分复杂，并且溶酶体破裂释放的组织蛋白酶B（CTSB）是否能够触发NLRP3组装存在争议。
为了进一步明确CTSB在二氧化硅诱导的焦亡中的作用，我们通过建立体外SiNP暴露模型开展了本研究。利用扫描电子显微镜（SEM）和透射电子显微镜（TEM）展示SiNPs的形态特征，并通过TEM和免疫荧光染色检测SiNPs内化对巨噬细胞的影响。此外，在阻断NLRP3和CTSB的表达后，采用蛋白质免疫印迹法检测与焦亡及CTSB相关的蛋白质表达。
我们发现SiNPs内化导致巨噬细胞膜破裂，并促进细胞老化，细胞内空泡增多。此外，在SiNP刺激下，NLRP3、ASC、半胱天冬酶-1、Gasdermin D、前白细胞介素-1β、白细胞介素-1β和CTSB的表达增加，而NLRP3特异性抑制剂MCC950的额外处理可抑制上述增加。此外，我们发现SiNP与CTSB抑制剂亮抑酶肽联合处理可抑制CTSB的表达，但对NLRP3、ASC和半胱天冬酶-1的表达无影响，且巨噬细胞焦亡过程也未受影响。
暴露于SiNP会导致巨噬细胞破裂和溶酶体CTSB释放，但CTSB无法特异性作用于NLRP3炎性小体以诱导与肺部炎症和纤维化有因果关系的焦亡。
