Fig 2: Working model detailing the role of IP3R/Ca2+ in VILI. Mechanical overstretch induces cell inflammation through an accelerated ER-to-cytosolic and ER-to-mitochondria efflux of Ca2+ through IP3R, followed by ER stress and mitochondrial dysfunction.

Fig 3: MV with HTV induces IP3R1 activation in lungs. Immunofluorescence photomicrographs (A) and quantification analysis (B) of IP3R1 in lung tissues of mice with spontaneous breathing (CON group) or mechanical ventilation at high tidal volume (HTV group). Dapi (blue) was used to stain the nuclei. Scale bar: 200µm. An inset picture was employed to show the indicated area at 4X magnification. Western blot (C) and quantification analysis (D) of IP3R1 protein expression in lung extracts. (E) Western blot analysis of the indicated subcellular fractions for IP3R1, calnexin (CNX; ER and MAM marker), FACL-4 (MAM marker) and tubulin (cytosolic marker). (F) Quantification of IP3R1 protein expression in the ER and MAM was performed by normalizing to CNX. Data are expressed as means ± SD (n = 6 animals per group). *P < 0.05 vs. CON group.

Fig 4: Effects of carbachol and 2-APB on HTV-induced Ca2+ homeostasis in lungs. Single-cell suspensions from lung tissues were preloaded with Fluo-4 AM, Rhod-2 and Mag-Fluo-AM, respectively. (A) Fluo-4 AM labeling the cytoplasmic Ca2+ in lung tissues from CON group, HTV group, IP3R agonist carbachol pretreatment upon HTV stimulation group (HTV+Carbachol group) and IP3R inhibitor 2-APB pretreatment upon HTV stimulation group (HTV+2-APB group). An inset picture was employed to show the indicated area at 4X magnification. (B) Fluorescence quantification analysis of Fluo-4 AM. (C) Rhod-2 and Mag-Fluo-AM co-dyeing to label Ca2+ in the mitochondria and ER in different groups. An inset picture was employed to show the indicated area at 4X magnification. (D) Fluorescence quantification analysis of Rhod-2. (E) Fluorescence quantification analysis of Mag-Fluo-AM. Data are expressed as means ± SD (n = 6 per group). *P < 0.05 vs. CON group; #P < 0.05 vs. HTV group.