Regulatory effect of integrated stress response inhibitors on endoplasmic reticulum stress signals in macrophages in silicotic mice

Background Silicosis is one of the most serious occupational diseases in China, requiring new treatment targets and therapies. The effects and mechanisms of integrated stress response inhibitors (ISRIB) on silicosis are still unknown.

Objective To observe the effects of ISRIB on silicosis fibrosis and its possible mechanisms.

Methods  The study was divided into two parts: in vivo and in vitro experiments. For the in vivo part, 40 SPF grade male C57BL/6J mice were randomly divided into four groups: control group, ISRIB group, silicotic model group, and ISRIB treatment group, with 10 mice in each group. A silicotic mouse model was established by using a single tracheal infusion of 50 μL 200 mg·mL⁻¹ SiO₂ suspension. After one week of perfusion with SiO₂ (the control group and the ISRIB group were perfused with an equal amount of sodium chloride solution), the ISRIB group and the ISRIB treatment group were intraperitoneally injected with 200 μL 2.5 mg·kg⁻¹ ISRIB for four weeks, and mice of other groups were injected with equal amounts of sodium chloride solution. A micro-CT instrument was used to observe the lung field clarity and lung texture of each group; hematoxylin eosin (HE) staining was used to observe the histopathology morphology of the lung and the formation of silicon nodules; Van Gieson (VG) staining was used to observe the deposition of collagen in silicotic nodules; immunofluorescence assay was used to detect the expression and localization of p-protein kinase RNA-like ER kinase (PERK) in lung tissue; immunoblotting was used to detect the expression of collagen I (Col I) and endoplasmic reticulum stress signal related proteins p-PERK, p-inositol-requiring enzyme-1α (p-IRE-1α), p-eukaryotic initiation factor 2α (p-eIF-2α), activating transcription factor 4 (ATF4), and NOD-like receptor thermal protein domain associated protein 3 (NLRP3). For the in vitro part, mouse alveolar macrophages MH-S cells were cultured in vitro and divided into a control group, an ISRIB (1 μg·mL⁻¹) group, a SiO₂ induction group (100 μg·mL⁻¹), and an ISRIB treatment group (1 μg·mL⁻¹ ISRIB treatment for 1 h, followed by 100 μg·mL⁻¹ SiO₂ induction). Immunofluorescence assay was used to detect the expression of p-PERK in MH-S cells; immunoblotting was used to determine expressions of endoplasmic reticulum stress signal related proteins p-PERK, p-IRE-1α, p-eIF-2α, and ATF4.

Results The CT images showed that the lung markings of the silicotic model group mice were thickened, and several high-density shadows of varying sizes were observed in the lung field, mainly distributed around the bronchi. Compared with the silicotic model group, the ISRIB treatment group showed a decrease in the number and volume of high-density shadows. The HE staining results showed that lung tissues in the silicotic model group lost their normal structure, with the formation of silicon nodules, around which were thickened alveoli around the silicon nodules, and infiltration of inflammatory cells; the area and number of silicon nodules in the ISRIB treatment group were significantly reduced, and the range of silicon nodules was limited. The results of VG staining showed that the proportion of collagen fiber area in the lung tissue of the silicotic model group was 21.47%±2.59%, and it decreased to 9.34%±1.06% in the ISRIB treatment group, with a statistically significant difference (P<0.05). The immunofluorescence results showed that p-PERK was strongly expressed in the silicotic nodules and localized in macrophages; the expression of p-PERK was also significantly increased in the SiO₂ induced MH-S cells, while the intensity of p-PERK was weakened in the ISRIB treatment groups both in vitro and in vivo. The results of immunoblotting showed that compared with the control group, the expressions of endoplasmic reticulum stress signal related proteins p-PERK, p-IRE-1α, p-eIF-2α, and ATF4 were upregulated after SiO₂ stimulation in vivo and in vitro; compared with the SiO₂ induction group, the expressions of p-PERK, p-IRE-1α, p-eIF-2α, and ATF4 were significantly downregulated in the ISRIB treatment group in vivo and in vitro, and the differences were statistically significant (P<0.05).

Conclusion ISRIB antagonizes silicosis fibrosis by inhibiting the activation of macrophage endoplasmic reticulum stress signals.