Pre-Treatment with Zirconia Nanoparticles Reduces Inflammation Induced by the Pathogenic H5N1 Influenza Virus

Huo, Caiyun; Xiao, Jin; Xiao, Kai; Zou, Shumei; Wang, Ming; Qi, Peng; Liu, Tianlong; Hu, Yanxin

Pathological changes were evaluated by a veterinary pathologist and scored from 0 to 4 in a blind study. Descriptions of the lung scores were as follows: 0 = no microscopic lesions; 1 = extremely mild, characterized by mild interstitial edema and desquamation of rare epithelial cells; 2 = mild, characterized by interstitial edema, thickening of alveolar walls, and occasional bronchial structural damage; 3 = moderate, characterized by hyperemia, hemorrhage, interstitial edema, thickening of the alveolar wall, bronchial structural damage, and slight infiltration of inflammatory cells; and 4 = severe, characterized by hyperemia, hemorrhage, interstitial edema, thickening of the alveolar wall, serious bronchial structural damage, and greater infiltration of Quantitative PCR (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) QPCR and ELISA analyses were conducted as previously described. 38 Briefly, total RNA was extracted from approximately 10 mg of lung or spleen tissue homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was reverse transcribed using the EasyScript First-Strand cDNA Synthesis Super Mix (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. Real-time PCR was performed in triplicate using the Power SYBR ® Green PCR Master Mix kit (Applied Biosystems, Warrington, UK) on an Applied Biosystems 7500 system. Expression of the hemagglutinin (HA) gene was measured using the absolute quantification method. Amplification was performed as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s. The copy number was calculated using an HA-containing plasmid of known concentration as a standard. The expression levels of interferon (IFN)-α, IFN-β, IFN-γ, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1β, IL-6, IL-12, interferon-induced protein (IP)-10, and tumor necrosis factor (TNF)-α were measured using the relative quantification method, and normalized to the results of the control or glucose group using the 2 −ΔΔCT method with β-actin (forward primer, 5ʹ-GAG ACC TTC AAC ACC CCG C-3ʹ; reverse primer, 5ʹ-ATG TCA CGC ACG ATT TCC C-3ʹ) as an internal standard. Amplification was performed as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 40 s. Cytokine primers are listed in Table S2 . In addition, detection of protein expression of cytokines was performed using ELISA kits according to the manufacturer's instructions.