Treatment with human placental extracts inhibits allergic rhinitis by modulating AMPK/SHP1/SHP2/STING signaling

Abstract

The present study aimed to investigate the regulatory effects and mechanisms of human placental extracts (HPE) on rats and cell models of ovalbumin (OVA)‑induced allergic rhinitis (AR). IFN‑y and LPS induced AR in vitro. A total of 32 male Sprague‑Dawley (SD) rats were randomly divided into the following four groups: Sham group, model group, model + HPE group and model + HPE + AMPK inhibitor group (n=8 rats/group). With the exception of the sham group, the remaining three groups were sensitized with OVA to establish an AR model, followed by various treatments. Hematoxylin and eosin staining was utilized to observe morphological changes in the nasal mucosa, ELISA was employed to measure serum levels of IL‑1β, interferon (IFN)β, immunoglobulin (Ig)E, IgG1 and IgG2a, and western blotting was conducted to assess protein expression across the groups. The sham group exhibited intact tissue structure with no notable pathological alterations. The model group demonstrated pronounced pathological features, including extensive infiltration of inflammatory cells, tissue shedding and edema. The model + HPE group revealed a gradual restoration of tissue architecture, characterized by reduced edema and inflammatory infiltration, whereas the model + HPE + AMPK inhibitor group again exhibited significant inflammatory cell infiltration and other pathological manifestations. Compared with the sham operation group, the levels of IL‑1β, IFNβ, IgE, IgG1 and IgG2a in the serum of the model group were elevated. The levels of IL‑1β, IFNβ, IgE, IgG1 and IgG2a in the model + HPE group were lower than those in the model group. In addition, the levels of IL‑1β, IFNβ, IgE, IgG1 and IgG2a in the model + HPE + AMPK inhibitor group were higher than those in the model + HPE group. Relative to the sham group, the expression levels of phosphorylated (p)‑AMPK/total (t)‑AMPK, p‑Src homology 2‑containing phosphatase (SHP)1/t‑SHP1 and p‑SHP2/t‑SHP2 were diminished, whereas the expression levels of p‑STING/t‑STING and p‑TBK1/t‑TBK1 were heightened in the model group. In comparison to the model group, the expression levels of p‑AMPK/t‑AMPK, p‑SHP1/t‑SHP1 and p‑SHP2/t‑SHP2 were enhanced, whereas the expression levels of p‑STING/t‑STING and p‑TBK1/t‑TBK1 were reduced in the model + HPE group. Conversely, when compared with the model + HPE group, the expression levels of p‑AMPK/t‑AMPK, p‑SHP1/t‑SHP1 and p‑SHP2/t‑SHP2 were decreased, whereas those of p‑STING/t‑STING and p‑TBK1/t‑TBK1 were increased in the model + HPE + AMPK inhibitor group. In conclusion, HPE may inhibit AR by modulating the AMPK/SHP1/SHP2/STING signaling pathway.

Keywords: AMPK/SHP1/SHP2/STING; allergic rhinitis; human placental extracts; ovalbumin.

Figure 1.

Bioinformatics analysis. (A) Sample normalization boxplot. (B) Principal component analysis plot showing the differences between samples. (C) Volcano plot of DEGs. (D) Heatmap of DEGs. (E) Bar chart of Gene Ontology enrichment analysis. (F) Chord diagram of Kyoto of Encyclopedia of Genes and Genomes enrichment analysis. Scatter plots of (G) AMPK and NF-κB correlation, (H) AMPK and PU.1 correlation, (I) PU.1 and SHP1 correlation, (J) NF-κB and SHP2 correlation, (K) SHP2 and TBK1 correlation and (L) TBK1 and IRF3 correlation. DEGs, differentially expressed genes; SHP, Src homology 2-containing phosphatase.

Figure 2.

HPE can alleviate allergic rhinitis damage, with AMPK serving a notable role. (A) Hematoxylin and eosin staining of nasal mucosal tissues. (B) ELISA was used to detect IL-1β, IFNβ, IgE, IgG1 and IgG2 levels in the serum of rats. (C) Immunofluorescence was used to detect the expression levels of CD68 in the rats. **P<0.01, *P<0.05. HPE, human placental extracts; IFN, interferon; Ig, immunoglobulin. **P<0.01, *P<0.05.

Figure 3.

HPE acts on AMPK and promotes SHP1/SHP2 protein expression while inhibiting STING/TBK1 expression. (A) Protein banding maps of p-AMPK, t-AMPK, p-STING, t-STING, p-TBK1, t-TBK1, p-SHP1, t-SHP1, and p-SHP2, t-SHP2 were detected in nasal mucosal tissues by Western blot analysis. (B) Western blot analysis was performed to detect the protein expression levels of p-AMPK/t-AMPK, p-STING/t-STING, p-TBK1/t-TBK1, p-SHP1/t-SHP1 and p-SHP2/t-SHP2 in nasal mucosal tissues. **P<0.01, *P<0.05. HPE, human placental extracts; p-, phosphorylated; SHP, Src homology 2-containing phosphatase; t-t, total.

Figure 4.

HPE alleviates the inflammatory response. (A) Immunofluorescence staining was used to detect extracted alveolar macrophages. (B) Western blot analysis was conducted to detect the protein expression levels of NOX4, p-AMPK/t-AMPK, p-SHP1/t-SHP1 and p-SHP2/t-SHP2 in macrophages. (C) Western blot analysis was conducted to detect the protein expression levels of p-STING/t-STING, p-TBK1/t-TBK1, NLRP3 and IFNβ in macrophages. ELISA was used to measure the levels of (D) MDA, SOD and GSH, and (E) IL-1β, IFNβ and IFNα in macrophage supernatants. **P<0.01, *P<0.05, ^nsP>0.05. GSH, glutathione; HPE, human placental extracts; IFN, interferon; LPS, lipopolysaccharide; MDA, malondialdehyde; NC, negative control; NOX4, NADPH oxidase 4; p-, phosphorylated; SHP, Src homology 2-containing phosphatase; SOD, superoxide dismutase; t-t, total.

Figure 5.

HPE reduces cell migration. (A) Transwell experiment assessed the migration of macrophages. (B) Statistical analysis of the migration assay. **P<0.01, *P<0.05. ns; not significant (P>0.05); HPE, human placental extracts; IFN, interferon; LPS, lipopolysaccharide; NC, negative control.

Figure 6.

HPE inhibits allergic rhinitis by modulating the AMPK/SHP1/SHP2/STING signaling pathway. HPE promotes the NF-κB signaling pathway through activation of AMPK, and NF-κB directly activates SHP2 on the one hand, and SHP1 on the other hand via C/EBPα/PU.1. SHP1 inhibits STING, and SHP2 inhibits TBK1, thereby inhibiting the STING/TBK1 signaling pathway, which mediates inflammatory responses and promotes mitochondrial via IRF3 Oxidative Stress. Mitochondrial oxidative stress upregulates the expression of cGAMP and P53: cGAMP binds to STING and stabilizes its dimers and oligomers; P53 inhibits SHP1 and SHP2, and promotes the expression of both SYK and EGFR. STING interacts with EGFR, leading to the autophosphorylation of EGFR and activation of SYK, which then phosphorylates STING and EGFR at Y240 and Y245 sites, respectively. SYK and EGFR phosphorylate STING at the Y240 and Y245 sites, respectively, promoting its translocation to ERGIC for signaling. cGAMP, cyclic GMP-AMP; EGFR, epidermal growth factor; HPE, human placental extracts; IFN, interferon; IRF3, IFN regulatory factor 3; OVA, ovalbumin; SHP, Src homology 2-containing phosphatase; SYK, spleen tyrosine kinase.