Objective To investigate the changes in osteoprotegerin (OPG) / receptor activator of nuclear factor-κB ligand (RANKL) ratio in sepsis-associated acute lung injury (SA-ALI) and the role of regulation of this ratio on the inflammatory response in SA-ALI. Methods Eighteen C57BL/6 male mice were randomly divided into sham operation group, cecal ligation and perforation (CLP) group and RANKL group, with 6 mice in each group. Before the experiment, the RANKL group was intraperitoneally injected with 5 μg (0.2 mL) of recombinant RANKL antibody, whereas both the sham operation group and the CLP group were intraperitoneally injected with a volume-matched normal saline. One hour later, the sham operation group underwent only abdominal exploration and repositioning, while the other groups underwent the CLP surgery to induce the SA-ALI model. After 24 h of modelling, all mice were sacrificed and samples were collected. Pathological evaluation of lung tissues was performed by haematoxylin-eosin staining; enzyme-linked immunosorbent assay was used to detect serum concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β; while the mRNA and protein expression of OPG and RANKL, along with their ratio values, were detected by real-time polymerase chain reaction for quantitative analysis and protein immunoblotting. Results The SA-ALI mouse model was successfully established. Compared with the sham operation group, mice in the CLP group showed disturbed alveolar structure, obvious alveolar and interstitial haemorrhage and inflammatory cell infiltration, elevated serum levels of IL-6, TNF-α and IL-1β (P<0.05), significantly increased mRNA and protein expression of OPG and elevated OPG/RANKL ratio in lung tissue (P<0.05), whereas RANKL mRNA and protein expression was significantly decreased (P<0.05). Compared with the CLP group, the pathological damage of lung tissue in the RANKL group was reduced, the infiltration of alveolar and interstitial inflammatory cells was significantly improved, and the alveolar structure and morphology were more regular, with lower serum levels of IL-6, TNF-α and IL-1β (P<0.05), significantly lower mRNA and protein expression of OPG and OPG/RANKL ratio in lung tissue (P<0.05), and significantly higher mRNA and protein expression of RANKL in lung tissue (P<0.05). Conclusion The alteration of OPG/RANKL ratio may be related to the pathophysiological process of SA-ALI, and the decrease in its level may reflect the attenuation of the inflammatory response in SA-ALI.
Objective To investigate the inhibitory effects and related mechanisms of NOD like receptor protein 3 (NLRP-3) inflammasome inhibitor MCC950 on oxidative stress, inflammation, and pyroptosis in human esophageal epithelial cells (HEECs). MethodsHEECs cells were passaged and divided into blank control group, acid stimulation group (stimulated 3 times a day with pH 4 acidic medium for 15 minutes each time, cultured for 48 hours), bile salt stimulation group (stimulated 3 times a day with 400 μmol/L bile salt mixture for 15 minutes each time, cultured for 48 hours), lipopolysaccharide (LPS) group (stimulated with 10 μL of 100 ng/mL LPS for 48 hours), MCC950 group (stimulated with 10 μL of 7.5 ng/mL MCC950 for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS for 48 hours), and N-acetyl-L-cysteine (NAC) group (stimulated with 1 mmol/L NAC for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS 48 hours). Three culture dishes were used in each group to detect the mRNA and protein expression levels of oxidative protein/antioxidant protein [Nox-4 (NADPH oxidase 4), nuclearfactor erythroidderived 2-like 2 (Nrf-2), heme oxygenase-1 (HO-1)], NLRP-3 signaling pathway [NLRP-3/caspase-1/intereukin (IL)-1β/IL-18], and cell apoptosis pathway [caspase-4/caspase-5/GSDMD] using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting experiments. Cell apoptosis were observed through Hoechst33342 staining. ResultsMCC950 intervention (0.023) and NAC intervention (0.031) effectively inhibited HEECs apoptosis induced by acid (0.042), bile salt (0.047), and LPS (0.054). The results of RT-PCR experiments showed that MCC950 intervention and NAC intervention significantly inhibited the high expression of Nox-4 mRNA (MCC950: 1.68; NAC: 1.62) in HEECs cells induced by acid (2.40), bile salt (3.07), and LPS (3.52), and significantly upregulated the mRNA expression levels of antioxidant proteins Nrf-2 (MCC950: 0.72; NAC: 0.57) and HO-1 (MCC950: 0.74; NAC: 0.57). MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA expression levels of NLRP-3 (MCC950: 1.58; NAC: 1.47), ASC (MCC950: 1.56; NAC: 1.93), caspase-1 (MCC950: 1.64; NAC: 1.96), IL-1β (MCC950: 1.66; NAC: 1.82), IL-18 (MCC950: 1.58; NAC: 1.84) in HEECs cells induced by acid stimulated, bile salt stimulated, and LPS. MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA expression levels of apoptosis pathway markers such as caspase-4 (MCC950: 1.51; NAC: 1.61), caspase-5 (MCC950: 1.38; NAC: 1.64), and GSDMD (MCC950: 1.41; NAC: 1.54) induced by acid stimulation, bile salt stimulation, and LPS in HEECs cells. The electrophoresis results were similar with RT-PCR. ConclusionAcid, bile salt, and LPS can all induce the overexpression of oxidative stress markers in HEECs, reduce the expression of antioxidant proteins, and activate the NLRP-3 inflammasome signaling pathway and cell pyroptosis pathway, promoting cellular inflammatory damage, but MCC950 has a protective effect.