Knockout of GGPPS1 restrains rab37-mediated autophagy in response to ventilator-induced lung injury

Abstract

Mechanical ventilation may cause ventilator-induced lung injury (VILI) in patients requiring ventilator support. Inhibition of autophagy is an important approach to ameliorate VILI as it always enhances lung injury after exposure to various stress agents. This study aimed to further reveal the potential mechanisms underlying the effects of geranylgeranyl diphosphate synthase large subunit 1 (GGPPS1) knockout and autophagy in VILI using C57BL/6 mice with lung-specific GGPPS1 knockout that were subjected to mechanical ventilation. The results demonstrate that GGPPS1 knockout mice exhibit significantly attenuated VILI based on the histologic score, the lung wet-to-dry ratio, total protein levels, neutrophils in bronchoalveolar lavage fluid, and reduced levels of inflammatory cytokines. Importantly, the expression levels of autophagy markers were obviously decreased in GGPPS1 knockout mice compared with wild-type mice. The inhibitory effects of GGPPS1 knockout on autophagy were further confirmed by measuring the ultrastructural change of lung tissues under transmission electron microscopy. In addition, knockdown of GGPPS1 in RAW264.7 cells reduced cyclic stretch-induced inflammation and autophagy. The benefits of GGPPS1 knockout for VILI can be partially eliminated through treatment with rapamycin. Further analysis revealed that Rab37 was significantly downregulated in GGPPS1 knockout mice after mechanical ventilation, while it was highly expressed in the control group. Simultaneously, Rab37 overexpression significantly enhances autophagy in cells that are treated with cyclin stretch, including GGPPS1 knockout cells. Collectively, our results indicate that GGPPS1 knockout results in reduced expression of Rab37 proteins, further restraining autophagy and VILI.

Keywords: Autophagy; GGPPS1; Inflammation; Rab37; Ventilator-induced lung injury.

Fig. 1

GGPPS1 knockout ameliorated VILI in mice. C57BL/6 mice with lung-specific GGPPS1 knockout (GGPPS1^−/−) and wild-type (WT) mice were subjected to mechanical ventilation (VT) or not. a Lung injury was analyzed by HE staining and b lung injury was scored. c Mouse lungs were collected for calculation of the wet-to-dry ratio. d Total protein levels in BALF were analyzed using a BCA kit. e Neutrophils in BALF were analyzed by flow cytometry. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the indicated group

Fig. 2

GGPPS1 knockout ameliorated mechanical ventilation-induced inflammation in mice. C57BL/6 mice with lung-specific GGPPS1 knockout (GGPPS1^−/−) and wild-type (WT) mice were subjected to mechanical ventilation (VT) or not. a The contents of inflammatory cytokines, including IL-1β, IL-6, IL-18, and TNF-α, in BALF were measured by ELISA. b mRNA and c protein levels of the inflammatory cytokines were measured by qRT-PCR and western blot, respectively. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the indicated group

Fig. 3

GGPPS1 knockout inhibited autophagy in VILI mice. C57BL/6 mice with lung-specific GGPPS1 knockout (GGPPS1^−/−) and wild-type (WT) mice were subjected to mechanical ventilation (VT) or not. a The ultrastructural changes of lung tissues were observed by TEM. b Protein expression of autophagy markers, including LC3 I/II, ATG5, and Beclin1, was detected by western blot analysis. c, d LC3B-positive puncta in lung tissues were analyzed by immunofluorescence. ns, not significant; *P < 0.05; ***P < 0.001 vs. the indicated group

Fig. 4

Inhibition of autophagy ameliorated VILI in mice. Wild-type (WT) C57BL/6 mice were subjected to mechanical ventilation (VT) and intravenously injected with 3-MA (a key inhibitor of autophagy). a Lung injury was analyzed by HE staining and b lung injury was scored. c Mouse lungs were collected for calculation of the wet-to-dry ratio. d Total protein levels in BALF were analyzed using a BCA kit. e Neutrophils in BALF were analyzed by flow cytometry. f The contents of inflammatory cytokines, including IL-1β, IL-6, IL-18, and TNF-α, in BALF were measured by ELISA. g Protein expression of LC3 I/II and cytokines was analyzed by western blot. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the indicated group

Fig. 5

GGPPS1 knockout ameliorated VILI in mice via inhibition of autophagy. C57BL/6 mice with lung-specific GGPPS1 knockout (GGPPS1^−/−) and wild-type (WT) mice were subjected to mechanical ventilation (VT) or not. The GGPPS1^−/− mice in the VT group were then intravenously injected with RAPA (a key activator of autophagy). a Lung injury was analyzed by HE staining and b lung injury was scored. c Mouse lungs were collected for calculation of the wet-to-dry ratio. d Total protein levels in BALF were analyzed using a BCA kit. e Neutrophils in BALF were analyzed by flow cytometry. f The contents of inflammatory cytokines, including IL-1β, IL-6, IL-18, and TNF-α, in BALF were measured by ELISA. g Protein expression of LC3 I/II and cytokines was analyzed by western blot. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the indicated group

Fig. 6

GGPPS1 knockdown ameliorated cyclic stretch-induced cytokine release from RAW264.7 cells. RAW264.7 cells were transfected with GGPPS1-specific shRNA (shGGPPS1) or negative control shRNA (shNC). The transfected cells were then subjected to cyclic stretch (CS) or not. a The content of inflammatory cytokines in the cell supernatant was analyzed by ELISA. b Protein expression of cytokines was analyzed by western blot. c Autophagosomes and autolysosomes were observed by TEM. d Protein expression of autophagy markers was detected by western blot. e Protein expression of LC3 II and cytokines in chloroquine (CQ)-treated cells was analyzed by western blot. f, g Intracellular LC3B-positive puncta were analyzed by immunofluorescence. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the indicated group

Fig. 7

GGPPS1 knockdown ameliorated cytokine release from RAW264.7 cells via regulation of Rab37-mediated autophagy. a RAW264.7 cells transfected with GGPPS1-specific shRNA (shGGPPS1) or negative control shRNA (shNC) were subjected to cyclic stretch (CS) or not. Cells were treated with RAPA to induce autophagy. The content of inflammatory cytokines in the cell supernatant was analyzed by ELISA. b Protein expression of cytokines was analyzed by western blot. Rab37 protein expression levels in c GGPPS1^−/− mice subjected to mechanical ventilation (VT) and d GGPPS1 knockdown RAW264.7 cells subjected to CS were analyzed by western blot. e The Rab37 overexpression plasmid (pcRab37) was transfected into RAW264.7 cells. The content of inflammatory cytokines in the cell supernatant was analyzed by ELISA. f Protein expression of cytokines was analyzed by western blot. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the indicated group

Fig. 8

Schematic diagram of the effects of GGPPS1 on mechanical stretch-induced lung injury