Ferroptosis in Rat Lung Tissue during Severe Acute Pancreatitis-Associated Acute Lung Injury: Protection of Qingyi Decoction

Abstract

Qingyi decoction (QYD) has anti-inflammatory pharmacological properties and substantial therapeutic benefits on severe acute pancreatitis (SAP) in clinical practice. However, its protective mechanism against SAP-associated acute lung injury (ALI) remains unclear. In this study, we screened the active ingredients of QYD from the perspective of network pharmacology to identify its core targets and signaling pathways against SAP-associated ALI. Rescue experiments were used to determine the relationship between QYD and ferroptosis. Then, metabolomics and 16s rDNA sequencing were used to identify differential metabolites and microbes in lung tissue. Correlation analysis was utilized to explore the relationship between core targets, signaling pathways, metabolic phenotypes, and microbial flora, sorting out the potential molecular network of QYD against SAP-associated lung ALI. Inflammatory damage was caused by SAP in the rat lung. QYD could effectively alleviate lung injury, improve respiratory function, and significantly reduce serum inflammatory factor levels in SAP rats. Network pharmacology and molecular docking identified three key targets: ALDH2, AnxA1, and ICAM-1. Mechanistically, QYD may inhibit ferroptosis by promoting the ALDH2 expression and suppress neutrophil infiltration by blocking the cleavage of intact AnxA1 and downregulating ICAM-1 expression. Ferroptosis activator counteracts the pulmonary protective effect of QYD in SAP rats. In addition, seven significant differential metabolites were identified in lung tissues. QYD relatively improved the lung microbiome's abundance in SAP rats. Further correlation analysis determined the correlation between ferroptosis, differential metabolites, and differential microbes. In this work, the network pharmacology, metabolomics, and 16s rDNA sequencing were integrated to uncover the mechanism of QYD against SAP-associated ALI. This novel integrated method may play an important role in future research on traditional Chinese medicine.

Figure 1

Protective effect of QYD on pancreatic and pulmonary tissue injury in rats with SAP. (a) Representative images of HE staining of the pancreas (upper) and lung tissues (lower) of rats in each group (scale bar, 100 μm). (b, c) Histopathological scores of pancreas and lung tissues of rats in each group. (d) Serum amylase levels of rats in each group. (e) The lung W/D ratio was used to evaluate pulmonary edema in each group of rats. (f, g) Serum levels of inflammatory factors TNF-α and IL-6 of rats in each group. Data are presented as representative images or as the mean ± SD of each group of rats (n = 6 per group) from at least three separate experiments. ^∗∗∗P < 0.001.

Figure 2

Determination of core targets of QYD against SAP-associated ALI. (a) Volcano plot showing DEPs between the SAP and CON groups. (b) Volcano plot showing DEPs between the QYD and SAP groups. (c) GO enrichment analysis of 202 overlapped DEGs in the term of biological process. (d) Pathway annotation of 202 overlapped DEGs by KEGG terms. (e) Venn diagram between the 202 overlapped DEGs and the 514 drug targets of QYD. The integrated analysis of proteomic data and network pharmacology results yielded three core targets of QYD for SAP-associated ALI: ALDH2, AnxA1, and ICAM-1. (f) Construction of a drug-active ingredient-target network based on the three core targets obtained. Yellow dots represented each herb of QYD; purple dots represented the active ingredients of QYD; red dots represented the target proteins.

Figure 3

Interactions between ingredients and targets. (a) Molecular docking interaction of pulmatin, chrysophanein, and 8-methoxy-5-O-glucoside flavone with ALDH2. (b) Molecular docking interaction of palbinone and gardenolic acid B with AnxA1. (c) Molecular docking interaction of quercetin and kaempferol with ICAM-1. The left part of each molecular docking result showed the structure of the ingredient-target complex after docking; the middle part showed the interaction space of interpolated charge and hydrogen bonds (acceptor in green and donor in pink) between the ingredient and the target; and the right part showed the two-dimensional diagram of interaction sites between the ingredient and the target.

Figure 4

QYD enhancing ALDH2 activity protects against ferroptosis in SAP-associated ALI. (a) qRT-PCR was performed to assess the mRNA expression level of ALDH2 in the rat lung tissue of each group. GAPDH was used as the reference gene. (b, c) Western blotting was conducted to evaluate the protein expression levels of ALDH2, GPX4, p65, and p-p65 in the lung tissue of rats. β-Actin was used as a loading control. (d–f) Semiquantification of protein expression of ALDH2, GPX4, and p-p65 using histograms. (g) Measurement of Fe²⁺ in rat lung tissue of each group served as an iron metabolism indicator. (h) MDA concentration in the rat lung tissue of each group was measured as an indicator of lipid peroxidation. (i) GSH levels in rat lung tissue of each group were monitored to reflect antioxidant capacity. Data are presented as representative images or as the mean ± SD of each group of rats (n = 6 per group) from at least three separate experiments. ^∗∗∗P < 0.001.

Figure 5

QYD decreases neutrophil infiltration via upregulating active AnxA1. (a) Analysis of the relative levels of AnxA1 mRNA in lung tissues using quantitative RT-PCR. (b, c) Western blotting analysis of the protein expression levels of AnxA1 in the lung tissues. (d, e) Analysis of the relative levels of ICAM-1 mRNA and protein in lung tissues. (f) MPO activity in the lung tissues. Data are presented as representative images or as the mean ± SD of each group of rats (n = 6 per group) from at least three separate experiments. ^∗P < 0.05 and ^∗∗∗P < 0.001.

Figure 6

Effect of QYD on the lung metabolites of SAP rats. (a) PLS-DA score plots of the CON group, SAP group, SAP+QYD group, and CON+QYD group. (b) OPLS-DA score plots of the SAP vs. CON groups. (c) OPLS-DA score plots of the SAP+QYD vs. SAP groups. (d) Venn diagram of differential metabolites between SAP vs. CON groups and SAP+QYD vs. SAP groups. Effects of QYD on 7 differential metabolites levels in rats. (e) Heat map of 7 metabolites in each sample. (f) Analysis of metabolic pathway of differential metabolites between the QYD and SAP groups. Data are presented as the mean ± SD. n = 10 per group. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 7

Effect of QYD on the lung microbiota composition of SAP rats. (a) Linear discriminant analysis between the SAP and CON groups (LDA score ≥ 3, P < 0.05). (b) Linear discriminant analysis between the QYD and SAP groups (LDA score ≥ 3, P < 0.05). (c) Metabolic pathways predicted by PICRUSt analysis that were statistically different between the QYD and SAP groups. Data are presented as the mean ± SD. n = 10 per group.

Figure 8

Correlation analysis of ferroptosis, metabolites, and microbiota. (a) Ferroptosis and metabolites. (b) Ferroptosis and microbiota. (c) Microbiota and metabolites. Each point in the RDA plot represents a sample, and the connecting arrows' length indicates the correlation's magnitude. The angle between the connecting arrows indicates the correlation, with acute angles indicating positive correlations and obtuse angles indicating negative correlations. The smaller the angle, the higher the correlation. The red color in the heat map indicates a positive correlation, while the blue indicates a negative one. ^∗P < 0.05 and ^∗∗P < 0.01.