Application of Gelatin Zymography in Nanotoxicity Research

Zhang, Yue; Wan, Rong; Zhang, Qunwei; Mo, Yiqun

doi:10.1007/978-1-4939-8916-4_8

Cited by 5 publications

(2 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The enzymatic activities of MMP-2 and MMP-9 were determined by gelatin zymography assay as described in our previous studies [ 19 , 22 ]. Briefly, the cells were cultured in FBS-free media for 24 h prior to PM 2.5 exposure.…”

Section: Methodsmentioning

confidence: 99%

High glucose enhances the activation of NLRP3 inflammasome by ambient fine particulate matter in alveolar macrophages

Mo,

Zhang

et al. 2023

Part Fibre Toxicol

Self Cite

View full text Add to dashboard Cite

Background Epidemiological studies have demonstrated that individuals with preexisting conditions, including diabetes mellitus (DM), are more susceptible to air pollution. However, the underlying mechanisms remain unclear. In this study, we proposed that a high glucose setting enhances ambient fine particulate matter (PM2.5)-induced macrophage activation and secretion of the proinflammatory cytokine, IL-1β, through activation of the NLRP3 inflammasome, altering the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Results Exposure of mouse alveolar macrophages to non-cytotoxic doses of PM2.5 led to upregulation of IL-1β, activation of the NLRP3 inflammasome, increased nuclear translocation of the transcription factor NF-κB, increased generation of reactive oxygen species (ROS), and increased expression and enzymatic activity of MMP-9; these effects were enhanced when cells were pretreated with high glucose. However, pretreatment in a high glucose setting alone did not induce significant changes. ROS generation following PM2.5 exposure was abolished when cells were pretreated with ROS scavengers such as Trolox and superoxide dismutase (SOD), or with an NADPH oxidase inhibitor, DPI. Pretreatment of cells with DPI attenuated the effects of a high glucose setting on PM2.5-induced upregulation of IL-1β, activation of the NLRP3 inflammasome, and nuclear translocation of NF-κB. In addition, enhancement of PM2.5-induced expression and enzymatic activity of MMP-9 following high glucose pretreatment was not observed in primary alveolar macrophages obtained from NLRP3 or IL-1R1 knockout (KO) mice, where pro-IL-1β cannot be cleaved to IL-1β or cells are insensitive to IL-1β, respectively. Conclusions This study demonstrated that exposure of mouse alveolar macrophages to PM2.5 in a high glucose setting enhanced PM2.5-induced production of IL-1β through activation of the NLRP3 inflammasome and nuclear translocation of NF-κB due to PM2.5-induced oxidative stress, leading to MMP-9 upregulation. The key role of NADPH oxidase in PM2.5-induced ROS generation and activation of the IL-1β secretion pathway and the importance of IL-1β secretion and signaling in PM2.5-induced increases in MMP-9 enzymatic activity were also demonstrated. This study provides a further understanding of the potential mechanisms underlying the susceptibility of individuals with DM to air pollution and suggests potential therapeutic targets.

show abstract

Section: Methodsmentioning

confidence: 99%

High glucose enhances the activation of NLRP3 inflammasome by ambient fine particulate matter in alveolar macrophages

Mo,

Zhang

et al. 2023

Part Fibre Toxicol

Self Cite

View full text Add to dashboard Cite

show abstract

“…MMP-2 and MMP-9 activities in the BALF and lung tissues were determined by a gelatin zymography assay as described previously (Mo et al 2009b;Mo et al 2019;Wan et al 2008;Wan et al 2011;Zhang et al 2016;Zhang et al 2019). A total of 60 μL of BALF mixture (48 μL BALF + 12 μL 5x loading buffer) was loaded into each lane of 10% SDS-PAGE copolymerized with 0.5 mg/mL gelatin, which was used as the substrate under nonreducing conditions.…”

Section: Gelatin Zymography Assaymentioning

confidence: 99%

miR-21 mediates nickel nanoparticle-induced pulmonary injury and fibrosis

Zhang

Wan

et al. 2020

Nanotoxicology

Self Cite

View full text Add to dashboard Cite

We and other groups have demonstrated that exposure to nickel nanoparticles (Nano-Ni) results in severe and persistent lung inflammation and fibrosis, but the underlying mechanisms remain unclear. Here, we propose that miR-21 may play an important role in Nano-Ni-induced lung inflammation, injury, and fibrosis. Our dose-and time-response studies demonstrated that exposure of C57BL/6J (WT) mice to Nano-Ni resulted in upregulation of miR-21, proinflammatory cytokines, and profibrotic mediators. Histologically, exposure to Nano-Ni caused severe pulmonary inflammation and fibrosis. Based on the dose-and time-response studies, we chose a dose of 50 μg of Nano-Ni per mouse to compare the effects of Nano-Ni on WT with those on miR-21 KO mouse lungs. At day 3 post-exposure, Nano-Ni caused severe acute lung inflammation and injury that were reflected by increased neutrophil count, CXCL1/KC level, LDH activity, total protein concentration, MMP-2/9 protein levels and activities, and proinflammatory cytokines in the BALF or lung tissues from WT mice, which were confirmed histologically. Although Nano-Ni had similar effects on miR-21 KO mice, the above mentioned levels were significantly lower than those in WT mice. Histologically, lungs from WT mice exposed to Nano-Ni had infiltration of a large number of polymorphonuclear (PMN) cells and macrophages in the alveolar space and interstitial tissues. However, exposure of miR-21 KO mice to Nano-Ni only caused mild acute lung inflammation and injury. At day 42 post-exposure, Nano-Ni caused extensive pulmonary fibrosis and chronic inflammation in the WT mouse lungs. However, exposure of miR-21 KO mice to Nano-Ni only caused mild lung fibrosis and chronic lung

show abstract