Role and mechanism of mitochondrial DNA mediated Toll-like receptor 9-myeloid differentiation factor 88 signaling pathway activation in rats with ventilator-induced lung injury / 中华危重病急救医学

ABSTRACT

Objective To investigate the role and mechanism of mitochondrial DNA (mtDNA) in rats with ventilator-induced lung injury (VILI) via Toll-like receptor 9 (TLR9)-myeloid differentiation factor 88 (MyD88) signaling pathway. Methods Thirty adult male Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 10) spontaneous breathing group, normal tidal volume (VT) group (NVT group, VT = 8 mL/kg), and high VT group (HVT group, VT = 40 mL/kg). Rats in the NVT group and HVT group were ventilated mechanically with a positive end-expiratory pressure (PEEP) of 0 and the fraction of inspired oxygen (FiO2) at 0.50. After 4 hours of ventilation, the blood from the rats' hearts was collected and the rats were sacrificed, the levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) in serum were determined with enzyme-linked immune sorbent assay (ELISA). The bronchoalveolar lavage fluid (BALF) was collected for a determination of total protein by using the bicinchoninic acid (BCA) assay. The lung tissues were harvested to determine the wet/dry (W/D) ratio. The changes in pathobiology of lung tissue were observed with hematoxylin and eosin (HE) staining. The protein expression levels of mtDNA-encoded cytochrome C oxidase subunit Ⅳ (COX-Ⅳ), TLR9, MyD88 and nuclear factor-κB p65 (NF-κB p65) in lung tissues were determined by immunohistochemistry. Results The histopathology of lung tissues indicated that lungs from animals ventilated with HVT developed marked lung inflammation changes, whereas no major histological change was observed in animals ventilated with NVT or spontaneously breathing. The pathological score in HVT group was significantly higher than that of spontaneous breathing group and NVT group (3.50±0.41 vs. 0.25±0.09, 0.33±0.10, both P < 0.05). Compared with spontaneous breathing group and NVT group, the ratio of W/D in the HVT group was significantly increased (6.42±0.41 vs. 4.14±0.04, 4.28±0.11, both P < 0.05), the contents of total proteins in BALF were significantly increased (g/L 0.43±0.04 vs. 0.13±0.01, 0.14±0.01, both P < 0.05), and serum IL-6, IL-1β and TNF-α levels were also increased [IL-6 (μg/L) 1.15±0.17 vs. 0.42±0.10, 0.46±0.04; IL-1β (μg/L) 6.73±0.38 vs. 2.08±0.90, 2.19±0.18; TNF-α (μg/L) 4.10±0.11 vs. 1.12±0.10, 1.14±0.04; all P < 0.05]. Immunohistochemistry results demonstrated that the proteins of COX-Ⅳ, TLR9, MyD88 and NF-κB p65 in HVT group were shown in brown, which meant strongly expressed. However, these proteins in spontaneous breathing group and NVT group were uncolored or shown in buff, which meant unexpressed or weakly expressed. The results of quantitative analysis indicated that the immunoreactive scores (IRS) of COX-Ⅳ, TLR9, MyD88 and NF-κB p65 in HVT group were significantly higher than those in spontaneous breathing group and NVT group (COX-Ⅳ IRS 8.80±2.17 vs. 0.80±0.45, 1.40±0.55;TLR9 IRS 8.40±2.51 vs. 1.00±0.71, 1.20±0.84; MyD88 IRS 9.40±1.52 vs. 1.40±0.55, 1.60±0.55; NF-κB p65 IRS 9.80±2.05 vs. 1.00±0.71, 1.20±0.84; all P < 0.05). There was no significant difference in all of the parameters between spontaneous breathing group and NVT group (all P > 0.05). Conclusion mtDNA contributes significantly to VILI by activating the TLR9-MyD88 signaling pathway, resulting in subsequent secretion of NF-κB p65 and the proinflammatory cytokines, which induce acute inflammatory injury of lung tissue.