Ginsenoside Rh1 Alleviates Allergic Rhinitis by Mediating Mitochondrial Autophagy via Activation of the AMPK/ULK1/FUNDC1 Pathway

Abstract

Ginsenoside Rh1, a bioactive compound derived from ginseng, exhibits notable anti-inflammatory and antioxidant effects and has shown promising therapeutic potential in the treatment of allergic diseases. However, its exact role in allergic rhinitis (AR) and the underlying molecular mechanisms remain inadequately understood. This study investigates whether Rh1 alleviates AR through AMPK/ULK1/FUNDC1-mediated mitochondrial autophagy. In this study, human nasal epithelial cells (HNEpCs) were stimulated with house dust mite (HDM) and treated with mitochondrial autophagy inhibitors or siRNA transfection techniques to assess the effects of Rh1. Network pharmacology and molecular docking (MD) were used to explore the interactions between Rh1 and AMPK, ULK1, and FUNDC1. To explore the effects of Rh1, enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC) were employed to measure IgE levels and various inflammatory mediators. Western blot (WB) analysis was conducted to assess protein expression related to mitochondrial autophagy, inflammation, and apoptosis in nasal tissues and HNEpCs. Immunofluorescence (IF) and transmission electron microscopy (TEM) provided further verification. The experimental data reveal that Rh1 effectively alleviates HDM-induced nasal mucosal epithelial thickening and eosinophil infiltration by modulating mitochondrial autophagy via the AMPK/ULK1/FUNDC1 signaling pathway. Additionally, Rh1 inhibits IL-4 secretion in nasal airway lavage fluid (NALF) and helps restore the Th1/Th2 immune balance. It also reduces mtROS production, inhibits NLRP3 inflammasome activation, and prevents apoptosis, thereby mitigating tissue damage associated with AR. Knockdown of AMPK or treatment with 3-Methyladenine (3-MA) further confirmed Rh1's inducing effect on mitophagy. In summary, Rh1 modulates mitophagy through the AMPK/ULK1/FUNDC1 pathway, reducing inflammatory responses and inhibiting apoptosis, thereby offering significant protection against AR.

Keywords: AMPK/ULK1/FUNDC1 pathway; Ginsenoside Rh1; allergic rhinitis; apoptosis; mitochondrial autophagy.

FIGURE 1

Inflammation and restoration of Th1/Th2 imbalance in AR mice. (A) Experimental protocol outlining treatment with HDM and PBS. (B) Frequency of sneezing and nasal rubbing observed in the mice. (C) HE staining of nasal mucosa tissues. (D–F) FC analysis depicting the ratio of CD4⁺ IL‐4⁺ cells to CD4⁺ IFN‐γ⁺ cells in the spleen, lymph nodes, and supernatant of NALF. (G) ELISA measurements of HDM‐specific IgE levels in serum, eosinophil count, and cytokine levels (IL‐4, IL‐5, and IL‐13) in the NALF supernatant. Data are presented as mean ± SD (n = 8). *p < 0.05, **p < 0.01 compared with control group. ^# p < 0.05,^## p < 0.01 compared with HDM‐challenged group.

FIGURE 2

Ginsenoside Rh1 inhibits apoptosis and inflammation in nasal mucosal epithelial cells of AR mice. (A) WB analysis showing the expression of apoptosis‐related proteins in nasal mucosal cells of mice. (B) WB analysis revealing the levels of inflammation‐related proteins in the nasal mucosa of mice. (C) Histological analysis indicating the expression of Cleaved‐Caspase‐3. (D) TUNEL assay results for detection of cell apoptosis in nasal tissue sections. Data are presented as mean ± SD (n = 8). *p < 0.05, compared with control group. ^# p < 0.05 compared with HDM‐challenged group.

FIGURE 3

Ginsenoside Rh1 activates the AMPK/ULK1/FUNDC1 pathway to enhance mitochondrial autophagy in AR mice. (A) Venn diagram illustrating the overlap between AR and the targets of Ginsenoside Rh1. (B) PPI network highlighting interactions among key targets, including AMPK (PRKAG1), ULK1, and FUNDC1. (C) GO analysis of the three biological ontologies. (D) The chemical structure of Ginsenoside Rh1. (E) MD of Ginsenoside Rh1 and AMPK. (F) WB analysis demonstrating protein levels of AMPK, ULK1, and FUNDC1, as well as their phosphorylated forms. (G) WB results showing the protein levels of PINK1 and Parkin. (H, I) IF detection of PINK1 and Parkin in the nasal mucosa. Data are presented as mean ± SD (n = 8). *p < 0.05, compared with control group. ^# p < 0.05 compared with HDM‐challenged group.

FIGURE 4

si‐AMPK treatment of HDM‐stimulated HNEpCs cells affects mitophagy. (A) WB analysis assessing the expression of AMPK and its phosphorylated form. (B) WB analysis revealing protein levels of AMPK, ULK1, and FUNDC1, along with their phosphorylated forms. (C) WB analysis of PINK1 and Parkin protein expression. (D, E) Colocalization of MitoTracker Red with PINK1 and Parkin in HNEpCs. (F) TEM analysis of mitochondrial morphology. (G) Detection of ROS using DCFH‐DA. (H) Representative microscopy images and quantification of mitochondrial ROS (MitoSOX) in HNEpCs. (I) JC‐1 dye analysis of mitochondrial membrane potential in HNEpCs post‐HDM treatment. Data are presented as mean ± SD (n = 8). *p < 0.05, compared with control group. ^# p < 0.05compared with HDM‐challenged group.

FIGURE 5

Pretreatment with 3‐MA enhances MitoROS production, NLRP3 inflammasome activation, and apoptosis in HDM‐stimulated HNEpCs by inhibiting mitochondrial autophagy (A) ROS detection using DCFH‐DA. (B) Mitochondrial membrane potential analysis in HNEpCs treated with HDM using JC‐1 dye. (C) Representative micrographs and quantification of mitochondrial ROS (MitoSOX) in HNEpCs. (D) WB analysis of PINK1 and Parkin protein levels. (E, F) Co‐localization of MitoTracker Red with PINK1 and Parkin in HNEpCs. (G) Assessment of mitochondrial morphology in HNEpCs using TOM20. Data are presented as mean ± SD (n = 8).*p < 0.05 compared with control group. ^# p < 0.05 compared with HDM‐challenged group.

FIGURE 6

Pretreatment with 3‐MA enhances NLRP3 inflammasome activation, product formation, and apoptosis in HDM‐stimulated HNEpCs. (A, B) WB analysis of apoptosis‐ and inflammation‐related proteins in mouse nasal mucosal cells. (C) Apoptosis detection using the TUNEL method. (D, E) WB analysis of apoptosis and inflammation‐related proteins in nasal mucosal cells. Data are presented as mean ± SD (n = 8). *p < 0.05 compared with control group. ^# p < 0.05 compared with HDM‐challenged group.

FIGURE 7

Mechanism diagram. Schematic representation of mitophagy, mitochondrial ROS, NLRP3 inflammasome activation, and apoptosis mechanisms in AR. HDM stimulation induces mitochondrial damage in HNEpCs, resulting in mitochondrial ROS production and NLRP3 inflammasome activation. Ginsenoside Rh1 promotes autophagy via the AMPK/ULK1/FUNDC1 pathway, facilitating mitochondrial repair, reducing mitochondrial ROS, and attenuating NLRP3 inflammasome activation, ultimately preventing AR. Rh1 enhances AMPK activation, modulates AMPK phosphorylation, inhibits mitochondrial fission, and consequently suppresses the TXNIP/NLRP3 pathway.