Mechanism of isorhynchophylline in lipopolysaccharide-induced acute lung injury based on proteomic technology

Abstract

Isorhynchophylline (IRN), a tetracyclic indole alkaloid, has anti-inflammatory and antioxidant activities against cardiovascular diseases and central nervous system disorders. Acute lung injury (ALI) is a manifestation of inflammation concentrated in the lungs and has a high incidence rate and mortality The purpose of this study is to explain the mechanism of IRN in the treatment of acute lung injury and to provide a new scheme for clinical treatment. The experimental mice were divided into three groups: CTRL, LPS, LPS+IRN. The mouse model of ALI was established by inhaling LPS solution through nose. After continuous administration of IRN solution for 7 days, the mice in LPS+IRN group were killed and the lung tissue was collected for detection. Proteomic (Data are available via ProteomeXchange with identifier PXD050432) results showed that 5727 proteins were detected in mouse lung tissues, and 16 proteins were screened out. IRN could reverse the trend of these differential proteins. In addition, IRN can act on integrin αM to reduce neutrophil recruitment and thereby produce anti-inflammatory effects and may suppress neutrophil migration through the leukocyte transendothelial migration pathway. TUNEL and RT-PCR experiments revealed that LPS-induced ALI in mice increases the apoptosis of lung tissues, damage to alveolar epithelial cells and levels of inflammatory factors. Treatment with IRN can repair tissues, improve lung tissue pathology and reduce lung inflammation.

Keywords: Nano-LC-MS/MS; RT-PCR; TUNEL; acute lung injury; isorhynchophylline; proteomic technology.

FIGURE 1

(A) Experimental plan. (B) Pearson correlation coefficient chart: The horizontal and vertical axes represent each sample, and the colour depth (bottom left part) or numerical size (top right part) represents the size of the correlation coefficient between the two samples. The closer to the red portion (the closer the correlation coefficient is to 1), the greater the correlation. The closer to the middle colour yellow (the closer the correlation coefficient is to 0), the smaller the correlation.

FIGURE 2

(A,B) Heat map: The horizontal axis represents the sample and the vertical axis represents the screened differentially expressed proteins (default is to take the Top100 protein with the lowest p-value or all differentially expressed proteins for heat map display). (C,D) Volcano plot: Using log2 (FC) as the x-axis and −log10 (p-value) as the y-axis, plot a volcano plot for all proteins in differential expression analysis. Among them, red represents significantly upregulated differentially expressed proteins, blue represents significantly downregulated differentially expressed proteins and grey dots represent nonsignificant differentially expressed proteins. (E) Wayne plot: Counting the intersection of differentially expressed proteins in each comparison group. (F) Box plot: The horizontal axis represents the group name, the vertical axis represents the protein abundance, and the graphic title is protein ID (corresponding gene name).

FIGURE 3

(A,B) GO enrichment analysis; the first circle (from outside to inside) shows the enriched top (with the lowest p-value) GO entry, the outer circle shows the coordinate ruler of protein number, and different colours represent the classification of the three major categories of GO. The second circle represents the number of proteins annotated to the GO entry, and the colour represents the −log10 value of the enrichment analysis P or Q value. The third circle shows the statistics of the number of differentially expressed proteins in GO, where the number represents the number. The fourth circle represents the percentage of enrichment factors (Rich. Factor). (A) LPS group VS CTRL group, (B) LPS + IRN group VS LPS group. (C,D) The first circle (from outside to inside) is the KEGG pathway enriched in the Top (with the lowest p-value), and the outer circle is the coordinate ruler of the number of proteins. Different colours represent different KEGG first-level classifications (KEGG Level 1). The second circle represents the number of proteins annotated to the KEGG pathway, and the colour represents the −log10 value of the enrichment analysis P or Q value. The third circle shows the statistics of the number of differentially expressed proteins in the KEGG pathway, where the number represents the number. The fourth circle represents the percentage of enrichment factors (Rich. Factor), where (C) represents the LPS group VS CTRL group and (D) represents the LPS + IRN group VS LPS group.

FIGURE 4

(A) The size of bubbles is related to connectivity. The higher the connectivity, the larger the bubbles and the higher their importance in the network. The line represents the correlation, and the thickness of the line indicates the strength of the correlation. The thicker the line, the stronger the correlation. (B) TUNEL status of lung tissue in each group (×100). (C) The Relative Fluorescsncs Ratio of TUNEL. (D) The relative expression level of IL-1β in lung tissue of mice in each group. (E) The relative expression level of Arg-1 in lung tissue of mice in each group. (C,D,E) * Compared with CTRL, # Compared with LPS; *, #p < 0.05, **, ##p < 0.01, ***, ###p < 0.001.