Original Article
Pyroptosis mediated by mitochondrial DNA amplifies the inflammatory response of alveolar macrophage
Ning Zhao, Yong Li, Fen Liu, Rong Jiang, Qiang Shao, Shilin Hu, Jiaquan Chen, Kejian Qian
Published 2018-02-10
Cite as Chin Crit Care Med, 2018, 30(2): 97-100. DOI: 10.3760/cma.j.issn.2095-4352.2018.02.001
Abstract
ObjectiveTo observe the amplification effect of mitochondrial DNA (mtNDA) on the inflammatory response of rat alveolar macrophage induced by lipopolysaccharide (LPS), and to preliminarily explore its mechanism.
MethodsmtDNA of Sprague-Dawley (SD) rat liver tissue was harvested, ultra-micro spectrophotometer and 1% agarose gel electrophoresis were used to detect the concentration and quality of mtDNA. The alveolar macrophages of SD rat were isolated and cultured, the macrophages in logarithmic growth phase were divided into phosphatic buffer solution (PBS) group, LPS group, mtDNA group and LPS+mtDNA group. The first three groups were added equal volumes of PBS, LPS 1 mg/L or mtDNA 10 mg/L to the alveolar macrophages medium for 6 hours and 12 hours, respectively; the alveolar macrophage medium of LPS+mtDNA group was stimulated with 1 mg/L LPS for 6 hours and then stimulated with 10 mg/L mtDNA for 6 hours. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were detected by enzyme linked immunosorbent assay (ELISA); the expression of the key protein of Pyroptosis-Gasdermin D (GSDMD) was detected by Western Blot.
Results① The mtDNA A260/280 ratios were between 1.8-2.0, and agarose gel electrophoresis showed a single band, with a size of about 16 kb. ② After alveolar macrophages stimulated by LPS or mtDNA for 6 hours or 12 hours, respectively, the levels of IL-1β and TNF-α were higher than those in PBS group. When cells were treated with mtDNA for 6 hours after LPS stimulation 6 hours, the levels of IL-1β were higher than those in LPS 12 hours group (ng/L: 366.27±23.35 vs. 154.70±23.32, P < 0.01), but the levels of TNF-α had no significant difference compared with LPS 12 hours group (ng/L: 836.13±25.01 vs. 802.67±30.48, P > 0.05). ③ The protein expressions of GSDMD in LPS group, mtDNA group and LPS+mtDNA group were significantly higher than those in PBS group (GSDMD/β-actin: 1.77±0.05, 1.65±0.04, 2.40±0.05, 1.00±0.02, all P < 0.01), and the protein expression of GSDMD in LPS+mtDNA group was higher than that in LPS group (P < 0.01).
ConclusionmtDNA amplifies LPS-induced alveolar macrophage inflammatory responses, which mechanism may be related to the increase in pyroptosis mediated by mtDNA.
Key words:
Mitochondrial DNA; Pyroptosis; Alveolar macrophage; Inflammatory responses
Contributor Information
Ning Zhao
Department of Intensive Care Unit, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
Yong Li
Department of Oncology, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
Fen Liu
Department of Intensive Care Unit, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
Rong Jiang
Department of Intensive Care Unit, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
Qiang Shao
Department of Intensive Care Unit, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
Shilin Hu
Department of Intensive Care Unit, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
Jiaquan Chen
Department of Intensive Care Unit, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
Kejian Qian
Department of Intensive Care Unit, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
