Caveolin-1 identified as a key mediator of acute lung injury using bioinformatics and functional research

Abstract

Acute lung injury (ALI) is a potentially life-threatening, devastating disease with an extremely high rate of mortality. The underlying mechanism of ALI is currently unclear. In this study, we aimed to confirm the hub genes associated with ALI and explore their functions and molecular mechanisms using bioinformatics methods. Five microarray datasets available in GEO were used to perform Robust Rank Aggregation (RRA) to identify differentially expressed genes (DEGs) and the key genes were identified via the protein-protein interaction (PPI) network. Lipopolysaccharide intraperitoneal injection was administered to establish an ALI model. Overall, 40 robust DEGs, which are mainly involved in the inflammatory response, protein catabolic process, and NF-κB signaling pathway were identified. Among these DEGs, we identified two genes associated with ALI, of which the CAV-1/NF-κB axis was significantly upregulated in ALI, and was identified as one of the most effective targets for ALI prevention. Subsequently, the expression of CAV-1 was knocked down using AAV-shCAV-1 or CAV-1-siRNA to study its effect on the pathogenesis of ALI in vivo and in vitro. The results of this study indicated that CAV-1/NF-κB axis levels were elevated in vivo and in vitro, accompanied by an increase in lung inflammation and autophagy. The knockdown of CAV-1 may improve ALI. Mechanistically, inflammation was reduced mainly by decreasing the expression levels of CD3 and F4/80, and activating autophagy by inhibiting AKT/mTOR and promoting the AMPK signaling pathway. Taken together, this study provides crucial evidence that CAV-1 knockdown inhibits the occurrence of ALI, suggesting that the CAV-1/NF-κB axis may be a promising therapeutic target for ALI treatment.

Fig. 1. The research scheme and RRA analysis of DEGs in ALI.

A Research scheme and a flow framework. We analyzed 5 ALI datasets using bioinformatics, screened out common differential genes, and performed GO, KEGG, and PPI analysis on these differential genes. The mRNA and protein expressions of the CAV-1 and RELA were verified in vitro and in vivo. The functions of CAV-1 and RELA were further verified to explore the potential mechanism in ALI. B Heatmap demonstrated the 40 robust DEGs between the ALI and control. Twenty genes were promoted and twenty genes were suppressed in ALI. Red shows upregulated gene expression and green shows downregulated gene expression in ALI. Numbers in the heatmap indicate log2 FC of genes in the five datasets compared with control groups. C The log2 FC of 40 DEGs was calculated from five datasets. The log2 FC sums of CAV-1 and RELA were 0.99 and 0.63, suggesting these two genes were significantly upregulated in ALI.

Fig. 2. Functional enrichment and PPI network construction of DEGs.

A, B Bubble plots show the results of GO functional enrichment and KEGG signaling pathway enrichment of DEGs. C The PPI network of the DEGs. Nodes represent proteins, edges represent interactions between proteins, and the intensity of the color indicates the degree of interaction of each protein. Proteins with more interaction were shown in the center of the network.

Fig. 3. Expression of CAV-1 and NF-κB in mouse model of ALI.

A–D The expression levels of CAV-1 and NF-κBp65 in lung tissues were analyzed by IHC. Scale bar = 50 μm. E, F qRT-PCR was performed to detect the levels of CAV-1 and NF-κBp65 in lung tissues. G The degree of pulmonary edema in each group was evaluated by lung weight coefficient. H, I H&E staining was used to detect the pathological changes in lung tissue and the score of morphological injury of lung tissue in each group. Scale bar = 50 μm. Results were represented as mean ± SEM (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4. Knocking down of CAV-1 improved autophagy and ameliorated ALI.

A, B qRT-PCR demonstrated the mRNA levels of LC3 and Beclin-1 in the lung tissues. C The protein levels of LC3II/I and Beclin-1 were examined by Western blotting in lung tissues. D, E Quantification of LC3 and Beclin-1 protein bands. Results were represented as mean ± SEM (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 5. Knockdown of CAV-1 could reduce the infiltration of CD3⁺ T cells and F4/80⁺ macrophages and inhibit proinflammatory cytokines.

A, C The expression of CD3 protein in T cells was detected by immunohistochemistry and the quantification of CD3⁺ T cells. B, D The protein levels of F4/80 was determined by IHC and quantification of F4/80⁺ cells. Scale bar = 50 μm. E–H The secretion of TNF-α, IL-1β, IL-6, and IL-18 in serum were determined by ELISA. I–L qRT-PCR analysis of TNF-α, IL-1β, IL-6, and IL-18 in lung tissues. Results were represented as mean ± SEM (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 6. Knockdown of CAV-1 activated autophagy in BMDMs.

A, B The mRNA levels of LC3 and Beclin-1 by qRT-PCR in BMDMs. C The protein expression of LC3II/I and Beclin-1 were assessed by western blotting in BMDMs. D, E Quantification of LC3 and beclin-1 expression. F The formation of autophagosomes in different groups of BMDMs was observed by TEM. Red arrows indicate autophagosomes. G Quantitative analysis of BMDMs autophagosomes number in each group. H Immunofluorescence analysis of LC3 (red) expression in BMDMs (magnification ×63). Results were represented as mean ± SEM (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 7. Knockdown of CAV-1 inhibited autophagy-related signaling pathways in BMDMs.

A GSEA analysis of NF-κB signaling pathway in ALI samples. B Western blotting analysis of CAV-1, p-IκBα, IκBα, p-NF-κBp65, and NF-κBp65 protein levels in BMDMs. C–E Quantification of these protein bands. F Confocal laser immunofluorescence showed NF-κBp65 nuclear translocation in BMDMs from different groups (magnification ×63). G GSEA analysis of mTOR signaling pathway in ALI sample. H, L The expression of p-AKT, AKT, p-mTOR, mTOR, p-AMPK, and AMPK were detected by western blotting in BMDMs. I, J, M Quantification of these protein bands. K GSEA analysis of AMPK signaling pathway in ALI samples. Results were represented as mean ± SEM (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 8. Molecular mechanism of CAV-1-mediated LPS-induced ALI development.

CAV-1 expression is upregulated in LPS-induced ALI, which recruits CD3⁺ T lymphocytes and F4/80⁺ macrophages to lung tissue, thereby triggering inflammatory response. Meanwhile, upregulated CAV-1 could promote AKT/mTOR and inhibit AMPK to downregulate autophagy, resulting in ALI. Conversely, knockdown of CAV-1 ameliorates ALI.