Ginsenoside Rb1 Alleviates Asthma Inflammation by Regulating Mitochondrial Dysfunction through SIRT1/PGC-1α and PI3K/AKT Pathways

Abstract

The aim of this study was to investigate whether ginsenoside Rb1 attenuates cockroach extract (CRE)-induced asthma by interfering with mitochondrial dysfunction. After induction with CRE, mice were administered different doses of Rb1. Hematoxylin–eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and flow cytometry analysis revealed that inflammatory cell infiltration, total immunoglobulin E (IgE) and CRE-specific IgE in serum, and inflammatory cytokines in bronchoalveolar lavage fluid were effectively inhibited by Rb1. Through Western blot, TUNEL, and immunofluorescence colocalization assays, we observed Rb1 also inhibited endogenous reactive oxygen species (ROS), tightly associated with increased superoxide dismutase, catalase levels, and decreased malondialdehyde levels. Subsequently, the silent information regulator sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) pathway was activated, whereas the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was inhibited. Additionally, Rb1 could rescue mitochondrial dysfunction by promoting the mitochondrial fusion protein mitofusion 1 (MFN1) and inhibiting dynamin-related protein 1 (DRP1) expression and apoptosis in the lungs. In BEAS-2B cells, Rb1 plays a role similar to that of a SIRT1 agonist (SRT1720), including enhancing mitochondrial membrane potential and decreasing mitochondrial ROS and DRP1 translocation to mitochondria. Our findings suggest that Rb1 maintains mitochondrial integrity by activating SIRT1/PGC-1α and inhibiting PI3K/AKT, thereby ameliorating asthmatic airway inflammation.

INTRODUCTION

Bronchial asthma is characterized by chronic airway epithelial inflammation that seriously endangers human health.¹⁾ Cockroaches are insects that are often found in modern urban environments and are also a common class of allergens that can cause allergic asthma.^2,3) Reactive oxygen species (ROS) are mainly generated from mitochondria, and excessive ROS mediate mitochondrial dysfunction, including mitochondrial fission/fusion imbalance, decreased mitochondrial membrane potential (MMP), and apoptosis, which are important factors leading to airway epithelial injury in asthma.^4,5) Therefore, it is of great clinical significance to investigate new therapeutic agents targeting cockroach extract (CRE)-induced mitochondrial dysfunction in asthmatic airway inflammation.

Sirtuin family proteins (SIRT) are class III histone deacetylases that play an anti-inflammatory role in asthma. Aili⁶⁾ et al. demonstrated that lipid lysophosphatidylglycerol (LPG) 18 : 0 compromises regulatory T-cell (Treg) function through the NAD⁺/SIRT1/FOXP3 pathway, thereby aggravating asthma. The use of SIRT1 agonists in ovalbumin (OVA)-induced asthmatic mice reduced both peri-airway inflammatory cell infiltration and inflammatory cytokine production.⁷⁾ Diminished activation of the SIRT1 pathway results in the acetylation and extracellular release of high-mobility group box 1 (HMGB1), as well as impaired ROS scavenging, contributing to the exacerbation of asthma.⁸⁾ Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is a transcriptional coactivator that can be activated by the upstream protein SIRT1, whose functions include oxidative phosphorylation and catalytic elimination of ROS and whose mechanism of action has been widely studied in mitochondrial research.⁹⁾ Augmenting the activation of the SIRT1/AMPK/PGC-1α pathway effectively mitigates hydrogen peroxide (H₂O₂)-induced proliferation in mouse airway smooth muscle cells (ASMCs) and reduces asthmatic airway inflammation.¹⁰⁾ Consequently, the SIRT1/PGC-1α signaling axis emerges as a promising therapeutic target for asthma treatment.

The ginsenoside Rb1 belongs to the protopanaxadiol class of compounds and is derived from the rhizome of the perennial herb ginseng in the Araliaceae family, which has a variety of pharmacological activities such as anti-inflammatory and antiapoptotic effects. Nam et al.¹¹⁾ indicated that Rb1 inhibits that Rb1 suppresses the upregulation of cell adhesion molecules and inflammatory cytokines triggered by lipopolysaccharide (LPS), as well as the phosphorylation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), nuclear factor-κB (NF-κB) transcription factors, and the mitogen-activated protein kinase (MAPK) signaling pathways including extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) in dental pulp cells. Yin et al.¹²⁾ reported that Rb1 treatment boosts cell proliferation, reduces apoptosis, and decreases the protein expression of collagen II, fibronectin (FN), and α-smooth muscle actin (α-SMA) in muscle tissue. In recent years, studies on the regulation of mitochondrial dysfunction by Rb1 have been widely reported. Pu et al.¹³⁾ demonstrated that Rb1 enhances cardiac function in heart failure through the modulation of inflammation, mitochondrial function, and gut microbiota. Rb1 has been implicated in regulating mitochondrial autophagy and the NF-κB pathway to inhibit astrocyte pyroptosis, thereby preserving neural homeostasis by dampening inflammation and promoting synaptic plasticity.¹⁴⁾ In addition, the induction of mouse embryonic fibroblasts with Rb1 resulted in a significant dose-dependent increase in both SIRT1 and PGC-1α.¹⁵⁾ In OVA-induced asthma, Rb1 can suppress inflammatory changes and hyperresponsiveness in airways.¹⁶⁾ Nevertheless, the underlying mechanisms by which Rb1 modulates the SIRT1/PGC-1α signaling pathway in asthma remain unclear.

The objective of our study was to confirm that Rb1 mitigates mitochondrial dysfunction via the SIRT1/PGC-1α and PI3K/AKT pathways, thereby repressing asthmatic airway inflammation. These insights are poised to advance the pursuit of strategies for asthma prevention and therapeutic intervention.

MATERIALS AND METHODS

Mice

Forty-five BALB/c female mice (free of specific pathogens, 6- to 8-week-old, weighing 20–22g) were obtained from the animal feeding center of Yanbian University. The breeding conditions were 50–60% relative humidity, room temperature at 22 ± 2°C, and 12-h light-dark cycle. All animal experimental procedures were performed in accordance with the Regulations for the Administration of Laboratory Animals and approved by the Ethics Committee of the College of Medicine of Yanbian University (approval number YD20230710003). All methods were reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org).

Animal Model and Grouping

Mice were randomly divided into 5 groups (n = 9): the control group, the CRE group, the low-dose ginsenoside Rb1-treated group, high-dose ginsenoside Rb1-treated group, and the dexamethasone group (positive control group, Dex). The mice in the CRE group were treated with CRE (50µg, XP46D3A4, Greerlabs, Lenoir, NC, U.S.A.) dissolved in 50 µL of saline from Day 1 to 5 via intranasal (i.n.) administration for sensitization. Then, 50 µL of saline was applied to the control group mice. On Days 11 to 15, the mice were continuously stimulated with an equivalent volume and concentration of CRE (i.n.) daily under light anesthesia.¹⁷⁾ Mice in the Rb1-treated groups were gavaged with Rb1 (10 or 20 mg/kg, S26692, Yuanye Bio-Technology Co., Shanghai, China) for 5 d, once daily, 1 h before CRE stimulation.¹⁸⁾ Mice in the Dex group were intraperitoneally injected with Dex (1 mg/kg, D1756, Sigma, St. Louis, MO, U.S.A.) for 5 d, 1 time/d before CRE stimulation. The structural formula is shown in Supplementary Fig. S1. The control group was treated with 50 µL of saline. Mice were sacrificed by cervical dislocation under deep anesthesia using halothane 2 h after the last challenge for further analysis.

Sample Collection

Bronchoalveolar lavage fluid (BALF) was obtained from 3 randomly selected mice in each group and then stained with Diff-Quick (G1541, Solarbio, Beijing, China) to calculate the proportion of eosinophils. Serum was extracted from the peripheral blood, mediastinal lymph nodes (mLNs), and right lung of the other 6 mice for molecular biology analysis. The left lungs were subjected to histochemical staining. The remaining lungs were frozen for further analysis.

ELISA

The levels of cytokines, including interleukin-4 (IL-4), IL-5, IL-13, and interferon-γ (IFN-γ) (M4000B, M5000, M1300CB, and MIF00), in the BALF supernatant were measured using ELISA (enzyme-linked immunosorbent assay) kits (R&D Systems, Minneapolis, MN, U.S.A.). All had a sensitivity of 2.0 pg/mL. Serum CRE-specific and total immunoglobulin E (IgE) (SEKR-0019, Solarbio) levels were also measured. An ELISA for malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) (A003-1-2, A007-1-1, A001-3-2; Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) was performed.

Histological Analysis

Paraffin-embedded lung sections were stained with a Hematoxylin and Eosin Kit (G1120, Solarbio) to observe airway inflammation. Modified Masson’s trichrome staining (G1346, Solarbio) was performed to observe collagen deposition.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide (MTT) Assay

After treatment, BEAS-2B cells were treated with MTT solution as previously described.¹⁹⁾

Cell Culture and Treatment

BEAS-2B, bronchial epithelium transformed with Ad12-SV40 2B, are derived from the bronchial epithelial cells of non-tumorigenic individuals and immortalized by infection with an adenovirus 12 (Ad12) and SV40 hybrid virus, followed by cloning. BEAS-2B cells were purchased from the Cell Resource Centre, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). The cell culture medium was Dulbecco’s modified Eagle’s medium (DMEM) (Vivacell) supplemented with 10% fetal bovine serum (Vivacell, Shanghai, China), streptomycin (100 g/mL), and penicillin (100 U/mL), and the cells were incubated at 37°C in 5% CO₂. For stimulation, 1 × 10⁵ cells were treated with 60 µM of Rb1 for 24 h,¹⁸⁾ MitoTempo (10 µM, HY-112879, MCE, Monmouth Junction, NJ, U.S.A.) for 30 min,²⁰⁾ or the SIRT1 agonist SRT1720 (HY-15145, 1 µM, MCE) for 6 h,²¹⁾ or the SIRT1 inhibitor Ex527 (HY-15452, 10 µM, MCE) for 24 h, and then exposed to 50 µg/mL CRE for 24 h.

ROS, Mitochondrial Morphology, MMP Assay, and TUNEL

Total cellular ROS was detected by incubation with a 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (10 µM, S0033S, Beyotime, Shanghai, China) fluorescent probe at 37°C for 10 min. Tissue ROS were detected by Dihydroethidium (DHE) (10 µM, S0063, Beyotime) at 37°C for 5 min. For mitochondrial morphology, intact tubular networks of mitochondria were characterized as not being injured; in contrast, the ruptured and spherical morphology of mitochondria was defined as fission mitochondria. Mitochondrial length and dynamin-related protein 1 (DRP1) density were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.). Cellular mitochondrial ROS (mtROS) were detected by MitoSOX (dye, 5 µM, M36008, Thermo Fisher Scientific, Waltham, MA, U.S.A.) for 10 min. For MMP detection, the cells were stained with JC-1 (5 µM, C2006, Beyotime) at 37°C for 15 min, and the red/green fluorescence intensity ratio was calculated. Apoptosis was detected using a Dead End fluorescent TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) kit (C1089, Beyotime). All images were photographed using the Cytation5 Cell Imaging Microplate Detection System (BioTek Inc., Winooski, VT, U.S.A.).

Western Blotting

After extraction, proteins (20 µg) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was incubated with antibodies against SIRT1 (110304, Abcam, U.S.A.), cleaved caspase-3 (32042, Abcam), PGC-1α (77210, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 5174, CST, U.S.A.), Bax (182734, Abcam), DRP1 (ab184247, Abcam), Bcl-2 (182858, Abcam), (MFN1) (221661, Abcam), and phosphorylated-DRP1 (p-DRP1) (Ser616) (3455, CST), PI3K (4292, CST), p-PI3K (17366, CST), nuclear factor erythroid 2-related factor 2 (Nrf2) (SAB4501984, Sigma), heme oxygenase 1 (HO-1, ab189491, Abcam), AKT (8805, Abcam), and p-AKT(38449, Abcam) at 4°C overnight. Then, the membranes were incubated with an antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (5151, CST) or an HRP-conjugated anti-mouse antibody (5257, CST). Grayscale values were measured using Quantity One software (Bio-Rad, Hercules, CA, U.S.A.). For phosphorylated proteins, the ratio of phosphorylated protein to total protein was calculated; for other proteins, the ratio of protein to GAPDH was determined.

Flow Cytometry

BAL cells and single-cell suspensions of mLNs were incubated with the surface marker fluorescein isothiocyanate (FITC)-conjugated anti-CD4 antibody (11-0041-82, Invitrogen, U.S.A.). The cells were then treated with an Intracellular Fixation and Permeabilization Kit (88-8824-00, Invitrogen), followed by staining with an allophycocyanin (APC)-conjugated anti-IL-4 antibody (554436, BD, U.S.A.) and PE-CY7-conjugated anti-IFN-γ antibody (25-7311-82, Invitrogen). Samples were finally collected using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, U.S.A.), and the ratios of IL-4⁺ CD4⁺ and IFN-γ⁺ CD4⁺ cells were calculated using CytoExpert 2.4: Beckman Coulter, Brea, CA, U.S.A.

Immunofluorescence Assay

For mitochondrial imaging, treated cells were incubated with the mitochondrial marker MitoTracker Red (M7512, Thermo Fisher Scientific) at 37°C for 30 min. For costaining of MFN1 and mitochondria, lung sections were first incubated with rabbit anti-MFN1 antibody at 4°C overnight, then washed, and finally incubated with MitoTracker Red for 30 min. For the DRP1 translocation assay, the cells were stained with MitoTracker Red and then incubated with rabbit anti-DRP1 antibody (8570S, CST). Finally, the tissue sections were treated with 4′,6-diamidino-2-phenylindole (DAPI) (P0131, Beyotime). The slides were photographed by Cytation5. The fluorescence density was analyzed by ImageJ.

Statistical Analysis

The data are presented as the mean ± standard deviation of three independent experiments. The significance of differences between two groups was determined by Student’s t-test with SPSS 19.0 software (IBM Co., Armonk, NY, U.S.A.). Multiple comparisons were performed using ANOVA or the Wilcoxon rank-sum test. A p-value of < 0.05 was considered statistically significant.

RESULTS

Rb1 Attenuates CRE-Induced Airway Inflammation in Asthma

To investigate the modulatory effect of ginsenoside Rb1 on airway inflammation in CRE-induced asthmatic mice, we observed histopathological changes around the airway and the T helper 1 cell/T helper 2 cell (Th1)/Th2 balance in BALF and mLNs. A mouse model of CRE-induced asthmatic inflammation was established (Fig. 1A). Rb1 treatment inhibited eosinophil occupancy (Figs. 1B and 1C) and Th2 cytokine (IL-4, IL-5, and IL-13) levels in the BALF and promoted Th1 cytokine (IFN-γ) levels compared to those in the CRE model group (Fig. 1D). Rb1 also reduced the serum total IgE and CRE-specific IgE levels (Fig. 1E), reduced the percentage of IL-4⁺ CD4⁺ cells, and increased the percentage of IFN-γ⁺ CD4⁺ cells in the mLNs (Figs. 1F and 1G). Pathological staining revealed that Rb1 reduced inflammatory cell infiltration and collagen deposition (Fig. 1H). Taken together, Rb1 inhibited airway inflammation, downregulated serum IgE levels, reversed Th1/Th2 imbalance, and alleviated CRE-induced airway inflammation in asthma.

Fig. 1. The Effect of Rb1 on CRE-Induced Airway Inflammation in Asthmatic Mice

(A) Diagrammatic representation of the CRE-triggered mouse model of asthma with or without Rb1 (n = 9). (B, C) Quantitative analysis of eosinophils in BAL fluid by Diff–Quick staining. The arrow indicates eosinophils. Scale bar = 200 µm. (D) Cytokine (IL-4, IL-5, IL-13, and IFN-γ) levels in the supernatants of mouse BALF. (E) Total IgE and CRE-specific IgE levels. The data are presented as the means ± S.D.s. (F, G) Flow cytometric detection of IL-4⁺ and IFN-γ⁺ CD4⁺ cells in the mediastinal lymph nodes (mLNs). (H) H&E and Masson staining were applied to lung tissue sections. Arrows represent peri-airway inflammatory cells and collagen deposition, respectively. Scale bar = 200 µm. The data are presented as the means ± S.D.s. n = 9.

Rb1 Attenuates Oxidative Stress

To determine the effects of Rb1 under CRE induction, we measured the levels of oxidative stress-related agents and the antioxidant enzymes in lung tissues. Rb1 inhibited ROS and MDA production compared to that in the CRE model group (Figs. 2A and 2B) and significantly upregulated both SOD and CAT levels in tissues (Figs. 2C and 2D). Additionally, Rb1 markedly upregulated antioxidant enzymes Nrf2 and HO-1 (Fig. 2E). Therefore, Rb1 reduced oxidative damage through the counteraction of ROS and, at the same time, the upregulation of antioxidant enzymes.

Fig. 2. The Effect of Rb1 on CRE-Induced Oxidative Stress

(A) Detection of ROS production in lung sections by fluorescence assay. Scale bar = 200 µm. (B, D) ELISA for MDA, CAT, and SOD in BALF supernatants. (E) Immunoblotting of Nrf2 and HO-1. Relative densities were calculated. The data are presented as the means ± S.D.s. n = 9.

Rb1 Inhibits Mitochondrial Fission by Regulating SIRT1/PGC-1α and PI3K/AKT Axis

We evaluated the modulatory effect of Rb1 on SIRT1/PGC-1α and PI3K/AKT signaling, followed by downstream mitochondrial dynamics analysis by immunoblotting. As shown in Figs. 3A and 3B, Rb1 activated SIRT1/PGC-1α protein expression compared to that in the CRE group, but inhibited p-PI3K and p-AKT protein expression. Moreover, Rb1 inhibited DRP1 and p-DRP1 (ser616) expression but promoted MFN1 expression (Figs. 3C and 3D). Consistent with these findings, immunofluorescence costaining analysis revealed that the deficiency of MFN1 in mitochondria caused by CRE-induced asthma was reversed by treatment with both Rb1 and Dex (Fig. 3E). Considering the effect of Rb1 on airway inflammation and oxidative stress, the alleviation of asthmatic inflammation by Rb1 may be associated with the suppression of the SIRT1/PGC-1α and PI3K/AKT pathways.

Fig. 3. Rb1 Affects Mitochondrial Fission by Activating the SIRT1/PGC-1α and PI3K/AKT Axes

(A) Immunoblotting of SIRT1, PGC-1α, p-PI3K, p-AKT, and GAPDH. (B) Relative densities were calculated using Quantity One software. (C) Immunoblots showing the levels of p-DRP1 (Ser616), DRP1, MFN1, and GAPDH. (D) Relative densities were calculated. (E) Colocalization of MitoTracker Red and MFN1 in lung tissues. Scale bar = 200 µm. The data are presented as the means ± S.D.s. n = 9.

Rb1 Alleviates Asthmatic Apoptosis in Airway Epithelial

Western blotting indicated that the levels of apoptotic proteins, such as cleaved caspase-3 and Bax, were significantly reduced in lung tissues after Rb1 treatment, while the level of Bcl-2 increased (Figs. 4A and 4B). Similarly, the TUNEL assay of the lungs revealed that the most TUNEL-positive cells were airway epithelial cells, where oxidative and mitochondrial damage had occurred (Fig. 4C), representing the pivotal role of Rb1 in modulating mitochondrial apoptosis in the airway epithelium of asthmatic mice.

Fig. 4. The Effect of Rb1 on CRE-Induced Mitochondrial Apoptosis

(A) Immunoblotting of Bax, Bcl-2, cleaved caspase-3, and GAPDH. (B) Calculation of relative densities. (C) TUNEL assay for apoptosis in lung tissues. Scale bar = 200 µm. n = 9.

Rb1 Inhibits CRE Triggered mtROS and Mitochondrial Membrane Potential in BEAS-2B

In vitro, we treated BEAS-2B cells with Rb1 in the presence of CRE to simulate in vivo experiments. Figure 5A shows that compared with the control, Rb1 had no significant toxic effect on cell viability (at 10, 30, or 60 µM). The morphology of the mitochondria was next observed by the fluorescent dye MitoTracker Red (Figs. 5B and 5C). Rb1 reversed the extent of CRE-stimulated mitochondrial breakage and fragmentation. A similar antioxidative effect of Rb1, which can decrease total ROS and mtROS, was also shown in CRE-induced BEAS-2B cells (Figs. 5D–5G). Analysis of the MMP with JC-1 dye showed that the red/green fluorescence ratio decreased after CRE treatment and was significantly improved by Rb1, signifying the recovery of the membrane potential (Figs. 5H and 5I). Taken together, these findings suggested that Rb1 can reduce CRE-triggered ROS/mtROS accumulation, thereby enhancing the MMP and preventing CRE-induced mitochondrial dysfunction.

Fig. 5. The Effect of Rb1 on CRE-Induced Mitochondrial Dysfunction in BEAS-2B Cells

A total of 1 × 10⁵ cells were treated with different concentrations of Rb1 (10, 30, 60, and 100 µm) for 1 h, followed by stimulation with CRE (50 µg/mL) for 24 h. (A) MTT assay to evaluate the effect of Rb1 at different concentrations on cell viability. (B, C) Mitochondrial morphology using MitoTracker Red staining and mitochondrial length calculations. Scale bar = 25 µm. (D–G) Fluorescence assay and quantitative analysis of total intracellular ROS and mtROS (mitochondrial superoxide indicator) production. Scale bar = 200 µm. (H, I) MMP detection by JC-1 dye and its quantitative analysis. Scale bar = 200 µm. The data are presented as the means ± S.D.s. n = 3.

Rb1 Inhibits CRE-Induced Mitochondrial Fission and Apoptosis in BEAS-2B Cells by Increasing SIRT1/PGC-1α and Decreasing PI3K/AKT Expression

Consistent with the above results, Rb1 promoted SIRT1/PGC-1α protein expression, inhibited p-PI3K and p-AKT expression (Figs. 6A and 6B), and further modulated the mitochondrial fission/fusion imbalance (Figs. 6C and 6D). These effects were similar to those of the SIRT1 agonist SRT1720 and superoxide scavenger MitoTEMPO. Immunofluorescence colocalization assays revealed that DRP1 and MitoTracker Red significantly colocalized in cells exposed to CRE, whereas this effect was notably reversed by Rb1, SRT1720, and MitoTEMPO treatment (Fig. 6E). Furthermore, Rb1 was observed to mitigate the SIRT1 inhibitor Ex527-induced downregulation of SIRT1 expression and upregulation of p-PI3K expression (Supplementary Fig. 2). Immunoblot analysis revealed that Rb1 inhibited cleaved caspase-3 and Bax expression while promoting Bcl-2 expression (Figs. 7A and 7B). According to the TUNEL assay, the proportion of TUNEL⁺ cells was evidently decreased after Rb1 treatment (Figs. 7C and 7D). These factors have a tremendous regulatory effect on mitochondrial homeostasis. Collectively, our data demonstrated that Rb1 attenuates mitochondrial dysfunction by modulating the SIRT1/PGC-1α and PI3K/AKT signaling pathways, thereby suppressing asthmatic inflammation.

Fig. 6. The Effect of Rb1 on CRE-Induced Mitochondrial Fission in BEAS-2B Cells through the Enhancement of the SIRT1/PGC-1α and PI3K/AKT Axes

(A) Immunoblotting of PGC-1α, SIRT1, p-AKT, p-PI3K, and GAPDH. (B) Relative densities were calculated using Quantity One software. (C) Immunoblotting of p-DRP1 (ser616), DRP1, MFN1, and GAPDH. (D) Calculated relative band densities. (E) Colocalization of DRP1 and MitoTracker Red in the CRE-induced BEAS-2B cells. Scale bar = 100 µm. The data are presented as the means ± S.D.s. n = 3.

Fig. 7. The Effect of Rb1 on CRE-Induced Mitochondrial Apoptosis in BEAS-2B Cells

(A) Immunoblotting of Bax, Bcl-2, cleaved caspase-3, and GAPDH. (B) Calculation of relative densities. (C) TUNEL assay for apoptosis and (D) the proportion of TUNEL-positive cells (%). Scale bar = 200 µm. The data are presented as the means ± S.D.s. n = 3.

DISCUSSION

Here, we validated the molecular mechanisms by which Rb1 attenuates CRE-induced mitochondrial fission and apoptosis by regulating the SIRT1/PGC-1α and PI3K/AKT signaling pathways, thereby alleviating asthmatic airway inflammation. Rb1 prevents CRE-induced inflammatory cell infiltration around the airway, decreases ROS generation in lung tissues, upregulates SIRT1/PGC-1α expression, and downregulates PI3K/AKT expression. Rb1 further reduced DRP1, BAX, and cleaved caspase-3 expression and increased MFN1 and Bcl-2 expression. In vitro, Rb1 effectively increased CRE-induced SOD and CAT levels and decreased MDA levels in BEAS-2B cells, while inhibiting ROS/mtROS production and DRP1 translocation to mitochondria. Our supplementary studies further clarified Rb1’s enhancement of endogenous antioxidant defenses: in the CRE-induced asthma model, Rb1 significantly upregulated the key antioxidant transcription factor Nrf2 and its effector HO-1 in lung tissue. Since Nrf2 controls antioxidant enzymes like SOD and CAT^22,23) (which Rb1 increased in this study), Rb1’s activation of the Nrf2/HO-1 pathway explains this induction and its mitigation of oxidative damage by enhancing host defenses. This Nrf2-mediated mechanism, consistent with other natural antioxidants, underscores Rb1’s role in modulating redox homeostasis in asthma. Notably, these protective mechanisms and therapeutic outcomes share similarities with the effects of Dex, which was used as a positive control and effectively regulated the relevant signaling pathways and provided therapeutic benefits in both the in vivo asthma model and in vitro cellular studies.²⁴⁾ This finding suggests that Rb1 has a protective effect on CRE-induced asthmatic inflammation.

PGC-1α has been shown to have a potential therapeutic effect on bronchial asthma.^10,24) When BEAS-2B cells were treated with montelukast, which has anti-inflammatory and antioxidant properties, the expression of PGC-1α significantly increased.²⁵⁾ According to Han et al.,²⁶⁾ 6-gingerol reversed arsenic trioxide (As₂O₃)-induced inhibition of the AMPK/SIRT1/PGC-1α pathway, thereby inhibiting myocardial oxidative stress, inflammation, and apoptosis and attenuating the myocardial toxicity of As₂O₃. On this basis, we further explored and validated that the activation of SIRT1/PGC-1α is effective in alleviating CRE-induced inflammatory lesions in asthma. Moreover, herein, we found that the SIRT1/PGC-1α axis could additionally mediate the protective effects of altered mitochondrial dynamics and apoptosis. This finding is supported by a review of the literature showing that PGC-1α is a major agent for preserving the dynamic homeostasis of mitochondria.^27,28) PGC-1α directly upregulates MFN1 gene transcription by coactivating estrogen-related receptor-α on conserved DNA elements,²⁹⁾ and immunoprecipitation analysis revealed that PGC-1α also binds to the DRP1 promoter and downregulates DRP1 expression.³⁰⁾ Following stimulation by PM2.5 in BEAS-2B cells, rhodopsin increased the mitochondrial membrane potential through activation of the SIRT1/PGC-1α signaling axis and inhibited mitochondrial cytochrome c release into the cytoplasm to mediate apoptosis.³¹⁾ It has also been reported that the neuroprotective drug BMS-470539 protects against early brain injury by activating the AMPK/SIRT1/PGC-1α pathway to reduce neuronal ROS, cleaved caspase-3, and DRP1 expression.³²⁾ Thus, SIRT1/PGC-1α is an important target for attenuating dysfunction caused by mitochondrial oxidation, fusion–fission imbalance, and apoptosis.

Our study demonstrated that Rb1 could activate the SIRT1/PGC-1α axis to inhibit the fission/fusion imbalance of mitochondria caused by oxidative stress and reduce airway inflammation in asthma. Rb1 is closely associated with mitochondrial dysfunction, playing a pivotal role in modulating the integrity and functionality of these organelles, which is crucial for maintaining cellular energy metabolism and reducing oxidative stress. Rb1 significantly increases mitochondrial content in the myocardium and enhances aerobic cellular respiration by activating the SIRT1 pathway to promote mitochondrial energy metabolism and ATP synthesis, thereby protecting cardiomyocytes.³³⁾ Rb1 induces browning of 3T3-L1 cells and primary white adipocytes, enhances PGC-1α expression in brown adipocytes, and thus increases mitochondrial density.³⁴⁾ Although there is no direct evidence for the role of Rb1 in mitochondrial fission and fusion, ginsenoside Rd inhibits the oxygen–glucose deprivation/reoxygenation-induced overexpression of DRP1 and the activation of NLRP3 inflammatory vesicles in BV-2 cells³⁵⁾; thus, we hypothesize that ginsenoside Rb1 also has a modulatory effect on mitochondrial fission and fusion. In the present study, Rb1 effectively inhibited CRE-induced ROS in lung tissues and mtROS in cells. Hence, it can be inferred that Rb1 may regulate mitochondrial fission–fusion in an oxidative stress-mediated manner. Our previous research showed that elevated ROS/mtROS levels could trigger HDM (house dust mite)-induced DRP1 protein overexpression and apoptosis in BEAS-2B cells.³⁶⁾ It is therefore concluded that Rb1 activates the SIRT1/PGC-1α pathway through the downregulation of CRE-induced ROS/mtROS and inhibits DRP1-mediated mitochondrial damage, ultimately achieving a therapeutic effect on asthma-induced airway inflammation.

Many studies have shown that inhibition of the PI3K/AKT pathway is an effective way to alleviate mitochondrial fusion imbalance and suppress asthma airway inflammation. Cigarettes exacerbate asthma airway inflammation by activating the PI3K/AKT signaling axis, increasing DRP1 and decreasing MFN2-mediated mitochondrial dysfunction in ASM cells.³⁷⁾ In the present study, we also obtained a similar conclusion that Rb1 treatment alleviated asthmatic inflammation through PI3K/AKT pathway deactivation. There is also a reciprocal regulatory relationship between SIRT1/PGC-1α and PI3K/AKT expression. When resveratrol was applied to diabetic mice, it increased AMPK/SIRT1/PGC-1α and decreased PI3K-AKT protein expression, thereby improving diabetic nephropathy.³⁸⁾ The combined application of minocycline and botulinum toxin promotes SIRT1 expression, inactivates the PI3K/AKT signaling pathway, reduces inflammation and oxidative stress in LPS-treated primary microglia, and alleviates neuropathic pain due to spinal cord injury.³⁹⁾ The PI3K/AKT pathway and apoptosis were reported to be inhibited following Rb1 treatment in a rabbit model of osteoarthritis.⁴⁰⁾ We therefore hypothesized that the upregulation of SIRT1/PGC-1α expression by Rb1, along with the inhibition of PI3K/AKT expression, could effectively alleviate the oxidative stress-induced imbalance in mitochondrial fission and fusion.

However, it is noteworthy that ginsenoside Rb1, as a natural product with a wide range of biological activities, may exert protective effects, including anti-inflammatory, antioxidant, modulation of Th1/Th2 imbalance, and antiapoptotic activities, that are not exclusively limited to the SIRT1/PGC-1α and PI3K/AKT pathways. Literature reports indicate that Rb1 can also modulate other key inflammatory signaling pathways, such as the NF-κB and MAPK pathways, which also play significant roles in the pathogenesis of asthma.¹¹⁾ SIRT1 not only regulates mitochondrial function and antioxidation via PGC-1α but is also known to suppress the activity of key inflammatory transcription factors like NF-κB through deacetylation.⁴¹⁾ NF-κB is essential for the expression of many pro-inflammatory cytokines, including some that promote Th2 differentiation or function. Because the activation of NF-κB results in the production of the Th2-associated cytokines IL-4, IL-5, and IL-13,⁴²⁾ the ameliorating effect of Rb1 on asthma is likely a consequence of its concerted actions on multiple targets and pathways. The findings of this study highlight an important mechanism by which Rb1 contributes to asthma treatment through the regulation of mitochondrial homeostasis, but its complete pharmacological network still warrants further exploration.

CONCLUSION

We found that Rb1 could improve asthmatic inflammation by decreasing CRE-induced oxidative stress, followed by mitochondrial dysfunction and apoptosis. The enhancement of SIRT1/PGC-1α protein expression and the suppression of PI3K/AKT expression may be the underlying mechanisms. Thus, Rb1 plays a critical role in ameliorating the development of asthma and may become a promising pharmaceutical preparation for treating asthma.

Funding

This study was funded by the National Natural Science Foundation of China (NSFC: 82160004); Jilin Provincial Department of Education Project (JJKH20220547KJ, JJKH20230635KJ, and JJKH2 0240690KJ); Natural Science Research Foundation of Jilin Province for Sciences and Technology (20240404025ZP); and the 2024 Jilin Province Health Science and Technology Capability Enhancement Plan (2024A058).

Author Contributions

Huiwen Li: Writing—original draft; conceptualization; methodology; data curation. Ying Piao: Data curation; methodology. Qiaoyun Bai: Methodology; formal analysis. Xue Han: Formal analysis. Lin Shen: Resources; methodology. Xu Yinling: Visualization. Guanghai Yan: Investigation. Yihua Piao: Writing—review and editing; funding acquisition. Yilan Song: Project administration; conceptualization; funding acquisition.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All data and materials used in this study are available upon request from the corresponding author.

Supplementary Materials

This article contains supplementary materials.

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