Alleviating Asthma-Induced Mitochondrial Dysfunction and Oxidative Stress: Lactobacillus rhamnosus Intervention via Notch1 Signaling Pathway

doi:10.21203/rs.3.rs-4188427/v1

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Alleviating Asthma-Induced Mitochondrial Dysfunction and Oxidative Stress: Lactobacillus rhamnosus Intervention via Notch1 Signaling Pathway

https://doi.org/10.21203/rs.3.rs-4188427/v1

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Immune system disorders play a crucial role in asthma. Probiotics can be beneficial in maintaining a healthy balance of gut bacteria and improving metabolism. Lactobacillus rhamnosus (L. rhamnosus) is a gram-positive probiotic found primarily in the intestines of humans and animals. However, the mechanism by which L. rhamnosus treats asthma is unknown. Here, we confirmed that L. rhamnosus had a protective effect on the lung tissues of asthmatic mice. In addition, L. rhamnosus significantly modulated the balance of T cell subpopulations and promoted the expression of Treg cells, increasing the proportion of inhibited Th17 and restoring immune function. Mechanistically, L. rhamnosus inhibited the activation of NOD-like receptor heat protein domain-related protein 3 (NLRP3) inflammatory vesicles caused by asthma-induced oxidative stress and mitochondrial dysfunction. In conclusion, L. rhamnosus ameliorated asthma by mediating Notch1 signaling pathway-induced NLRP3 inflammasome activation and Dynamin-related protein 1 (Drp1) translational activation. In words, our study provides a theoretical basis for the use of probiotics, represented by L. rhamnosus, as effective agents for the prevention and treatment of asthma.

L. rhamnosus

Asthma

oxidative stress

mitochondrial dysfunction

immunity

Notch1 signaling pathway

Asthma, a chronic respiratory ailment, is distinguished by airway hyperresponsiveness, inflammation, and reversible airway remodeling ¹. Conventional asthma treatment primarily relies on medications, aiming to alleviate bronchospasm and reduce inflammation through anti-inflammatory effects ². Implementing environmental control measures, including humidity regulation, thorough vacuuming, and pest control, can effectively decrease asthma exacerbations triggered by common allergens like tobacco smoke, pollen, mold, or dust mites ³. The pathogenesis of asthma remains incompletely understood, but current research indicates that targeting the imbalance in immunoregulatory mechanisms triggered by airway inflammation could hold promise as a therapeutic approach ⁴. Hence, addressing the inflammatory response resulting from exposure to external substances has emerged as a pivotal focus in preventing and managing the onset and advancement of asthma. With approximately 100 trillion microorganisms inhabiting the human body, their crucial role in sustaining health is undeniable ⁵. Among their functions are the elimination or inhibition of pathogens, the reinforcement of epithelial barrier function through modulation of signaling pathways, and the regulation of host immunity—both locally and systemically—by targeting specific strains ⁶. Probiotics are microorganisms that confer health benefits to the host ⁷ and their therapeutic effects have been demonstrated in a large number of studies on gastrointestinal disorders ^8,9. Recent studies have highlighted promising prospects for the application of probiotics in the prevention and treatment of chronic respiratory diseases ^10,11. L. rhamnosus, a gram-positive probiotic predominantly residing in the intestines of humans and animals, demonstrates the ability to generate a biofilm. This biofilm serves as a mechanical shield, offering protection to the mucosa. Moreover, L. rhamnosus plays a pivotal role in intestinal health by enhancing intestinal crypt survival, decreasing intestinal epithelial apoptosis, and preserving cytoskeletal integrity ¹². Moreover, L. rhamnosus suppresses monocyte activation and the expression of inflammatory markers. It also fosters a type I immune response by augmenting the production of Interleukin-10 (IL-10), Interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) in macrophages ¹³. Studies have shown that probiotics colonizing in the intestine are able to affect the microbiota of the lungs through the lung-gut axis, thereby treating asthma ¹⁴. At present, L. rhamnosus has become one of the key probiotics for the treatment of asthma. Oral administration of L. rhamnosus sensitized mice reduced airway inflammation and airway hyperresponsiveness ¹⁵. Moreover, L. rhamnosus demonstrates the ability to suppress lung inflammation triggered by allergens by promoting the activity of CD4+CD25+Foxp3+Treg cells ¹⁶. In conclusion, L. rhamnosus emerges as a promising candidate for asthma prevention and treatment. Delving into the mechanisms underlying its therapeutic effects can lay a solid theoretical foundation for its extensive application.

Excessive accumulation of reactive oxygen species (ROS) and oxidative stress severely exacerbate the pathogenesis of asthma ¹⁷. Strong oxidizing substances such as ROS induce airway remodeling by damaging the structure of epithelial cells, further releasing inflammatory mediators and aggravating asthma to form a vicious circle ¹⁸. Healthy mitochondria serve to synthesize ATP, maintain calcium homeostasis and regulate programmed cell death. ¹⁹. However, oxidative phosphorylation of cellular mitochondria leads to ROS production, thus defective mitochondrial function may also be an underlying mechanism in asthma ²⁰. Clinical studies have shown that the levels of endogenous antioxidant enzymes in the peripheral tissues of asthmatic patients are significantly lower than those of normal patients, suggesting that asthma leads to impaired mitochondrial function and intense oxidative stress ¹⁸. Increased oxidative stress can further cause mitochondrial damage, eventually leading to oxidative inflammation ²¹. Further, mitochondrial dysfunction can be involved in the activation of inflammatory vesicles by interacting with components of the NLRP3 inflammatory vesicle ²². The NLRP3 receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and cysteinyl aspartate specific proteinase-1 (caspase-1) as an effector protein together NLRP3 inflammatory vesicles ²³. Among these, ASC is enzymatically active by promoting the attachment of the pro-caspase-1 precursor to NLRP3 and thus the activation of pro-caspase-1 ²⁴. caspase-1 acts as an effector protein of inflammasomes, activating the cytokines IL-1β and IL-18 and thereby activating downstream inflammatory pathways ²⁵. Activation of NLRP3 inflammatory vesicles is thought to be involved in the initiation and spread of allergen-driven airway inflammatory responses ²⁶. A significant reduction in eosinophil counts and airway inflammation have been reported in a chicken ovalbumin (OVA)-induced asthma mouse model deficient in NLRP3 and ASC ²⁷. Recently, Liu et al. found that L. rhamnosus ameliorated multidrug-resistant Bacillus cereus-induced cellular damage by inhibiting apoptosis in NLRP3 inflammasome and bovine endometritis cells ²⁸. Inhibition of production of ROS and mitochondrial dysfunction-induced NLRP3 inflammasome may be the mechanism of L. rhamnosus improves asthma.

In our investigation, we established a mouse model of asthma to delve into the intricate biological role and specific mechanisms orchestrated by L. rhamnosus in asthma pathology. Through meticulous experimentation, we meticulously examined the effects of L. rhamnosus on key aspects such as NLRP3 inflammasome activation, oxidative stress modulation, and mitochondrial function within the lung tissues of asthmatic mice. Subsequently, our rigorous in vitro studies further elucidated how L. rhamnosus exerts its influence on oxidative stress and mitochondrial function by orchestrating the modulation of NLRP3 inflammasome activation. By piecing together these intricate puzzle pieces, our study lays a robust theoretical foundation, suggesting the potential of L. rhamnosus as a preventive and therapeutic intervention for asthma. Through a comprehensive understanding of its mechanisms, L. rhamnosus emerges as a promising candidate for alleviating the burden of asthma, paving the way for future therapeutic exploration and clinical application.

Antibodies and Reagents

All antibodies and reagents used in this study are listed in detail in Tables S1 and S2.

Animal experiments

SPF-rated female BALB/C mice (6-8 weeks old) were purchased from Beijing Huafukang Laboratory Animal Technology Co., Ltd. The animal experiments were completed in the Laboratory Animal Department of the R&D Center of Shengjing Hospital affiliated to China Medical University. This study was approved by the Laboratory Animal Welfare and Ethics Committee of China Medical University (IACUC-2022PS264K). The mice were randomly divided into three groups: the control group, the asthma group, and the L. rhamnosus gavage group. Apart from the control group, mice in the other groups received intraperitoneal injections of 0.2 mL of sensitizing solution containing 50 μg of chicken egg albumin and 0.8 mg of aluminum hydroxide on the 8th, 15th, and 22nd days. Subsequently, they were exposed to ultrasonic atomization of 5% chicken egg albumin solution for 30 minutes each time, once a day, for 1 week. The control group received injections of the same volume of normal saline at the same intervals, and ultrasonic atomization was carried out using a normal saline solution. Furthermore, the L. rhamnosus suspension was administered via gavage at concentrations of either 10^8 or 10^9 CFU/mL, with a volume of 200 μL once daily for the initial 7 days.

Culture of L. rhamnosus

L. rhamnosus was purchased from the American Standard Bacteria Library (ATCC), and cultured at 37 °C in freshly prepared MRS agar medium according to ATCC culture methods, then collected bacteria, and calculated the bacterial concentration using the Turbidimetry method.

Airway reactivity Penh value detection

Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (70 mg/kg) 24 h after the last excitation, and the trachea was separated and intubated. An animal spirometer was connected and an intravenous indwelling needle was inserted into the tail vein of the mice. The mice were placed in a closed box and mechanically ventilated with a tidal volume of 6 ml/kg and a respiratory rate of 90 breaths/min. After stabilization of airway pressure, basal lung function values were measured (2 min), followed by different concentrations of acetylcholine (0, 3.125, 6.25, 12.5, and 25 and 50 g/l). Each application of acetylcholine was followed by a 3-minute nebulization period, a 2-minute rest period, and a total recording time of 5 minutes. Mouse Penh values were then collected.

HE staining of lung tissue

Twenty-four hours after the last excitation, the left upper lung tissue was taken from mice, fixed with 4% paraformaldehyde, embedded, and then cut into paraffin sections with a thickness of 3 μm, and then stained with HE staining in accordance with the instructions of the reagent manufacturer, and photographed under a light microscope.

Bronchoalveolar lavage fluid cell isolation and counting

After mice were euthanized, the trachea was secured to the cricoid cartilage with vascular clamps, the trachea was fully exposed, and a transverse incision was made under the trachea. Silicone tubing attached to a syringe was preinserted, and 0.5 mL of pre-cooled phosphate buffer was slowly injected in three fractions. The lavage fluid was recovered and centrifuged at 1000 rpm for 10 minutes, the cell mass was resuspended with PBS, and the total number of cells was counted by hemocytometer, and differential counting of cells was performed by Rachel's staining.

ELISA assay

Mouse lung tissues were collected 24 h after the last stimulation, and after centrifugation at 3000 rpm for 10 min at 4 ℃, the total IgE level in serum was detected by an IgE ELISA kit. The alveolar lavage fluid and lung tissue were collected as described above, and the levels of HMGB1, IFN-γ, IL-4, IL-17A, SOD, MDA, 8-OHdG and nitrotyrosine in the alveolar lavage fluid were detected according to the instructions of the kit.

ROS measurement

Frozen slices of mouse upper left lung tissue were prepared to a thickness of 10 μm, and the slices were rinsed with PBS for 5 minutes at room temperature and then blotted dry with PBS. Then, the tissue was titrated with DHE probe, and the slices were placed in a wet box and incubated at 37 ℃ with light protection for 1 hour, and the slices were rinsed with PBS for 3 times, sealed with glycerol, and then observed and photographed under a fluorescence microscope. For cell samples, after replacing the cells with fresh culture medium, add the DHE probe, incubate at 37 °C for 1 hour under light, then rinse with PBS, observe and image under a fluorescence microscope.

Flow cytometry

Mouse spleen wax was taken and ground through a sieve to collect the cell suspension. The collected cell suspension is centrifuged at 1,500 rpm for 10 minutes and the cell pellet is collected. Add 3 volumes of erythrocyte lysate to the pellet and lysed at 4 °C for 5 minutes. The pellet is then washed with PBS and centrifuged at 1,500 rpm for 10 minutes to collect the pellet. The cells were screened for Th1, Th2, Treg and T17 positivity and detected by flow cytometry.

Western blotting

The total protein of tissues and cells was extracted by RIPA lysate, the protein concentration was detected by BCA method, and the protein samples were configured to a final concentration of 2 μg/μL, and the protein was fully denatured by boiling. Proteins were isolated by SDS-PAGE electrophoretic, and transferred to PVDF membrane, then blocked by 5% skim milk at room temperature for 1h, incubated using primary antibody at 4 °C overnight. The membrane was washed using PBST for 3 times, and incubated with secondary antibody at room temperature for 1 h. Then washed by PBST for 3 times, and imaged using ECL luminescence method.

ATP content detection

The collected tissue samples were washed with pre-chilled PBS to prepare tissue homogenate. After full lysis, the samples were centrifuged at 4 °C at 10,000 g for 5 min, and collected the supernatant. Spectrophotometrically detect ATP content in tissues according to kit instructions.

Cell experiments

Human airway epithelial cell line BEAS-2B was purchased from the Typical Culture Collection Center of the Chinese Academy of Sciences, and cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37 ℃ in a constant temperature incubator with 5% CO2. The cells were divided into control group, model group, L. rhamnosus treatment group, Drp1 overexpression group and Notch1 agonist group. The control group was cultured routinely. The model group was treated with 20 ng/mL IL-4 for 8 hr on top of the control group; the L. rhamnosus-treated group was treated with 20 ng/mL IL-4 and 10⁹ or 10¹⁰ CFU/mL L. rhamnosus for 6 hours on top of the control group; the Drp1 overexpression group was transfected with Drp1 overexpression plasmid 48 hours prior to treatment in the L. rhamnosus-treated group; the Notch1 agonist group was transfected with Drp1 overexpression plasmid 48 hours before treatment in the L. rhamnosus -treated group. The Notch1 agonist group was incubated with 2 μg/mL Jagged1-Fc fusion protein for 8 hours on top of the L. rhamnosus treatment group.

Statistical analysis

Results were expressed as mean ± standard deviation (SD) of three independent experiments unless otherwise specified. Data were analyzed using SPSS 22.0 software (IBM, USA). One-way analysis of variance (ANOVA) with Tukey's multiple comparison post hoc test were used to compare the data among groups, and p<0.05 was considered as significant difference.

L. rhamnosus alleviates airway inflammation in asthmatic mice

Airway reactivity Penh values were assessed to validate the therapeutic efficacy of L. rhamnosus in asthmatic mice. Our findings indicate a significant deceleration in the increase of airway reactivity Penh value following L. rhamnosus administration (Figure 1a). Furthermore, histological examination via HE staining demonstrated the effectiveness of L. rhamnosus in alleviating asthma-induced airway smooth muscle thickening and reducing inflammatory cell infiltration (Figure 1b). Subsequent investigations revealed notable reductions in the number of neutrophils, eosinophils, and lymphocytes in the alveolar lavage fluid upon administration of L. rhamnosus to asthmatic mice (Figure 1c). Moreover, there was a significant decrease observed in total serum IgE levels (Figure 1d), as well as in the levels of IL-17, HMGB1, and IL-4 in the alveolar lavage fluid (Figure 1e-g). Asthma inflammation levels are often correlated with T cell activation in the airway wall, with previous studies implicating them in the initiation and development of the asthma inflammatory response ²⁹. Flow cytometry analysis was employed to assess the proportion of Th17 and Treg cells in the spleens of mice, revealing that L. rhamnosus administration led to decreased proportions of Th17 and Treg cells in the spleens of asthmatic mice (Figure 1h-j).

These comprehensive results collectively demonstrated the ability of L. rhamnosus to ameliorate the imbalance of T-cell subpopulations and attenuate airway inflammation in asthmatic mice.

L. rhamnosus inhibits oxidative stress and mitochondrial dysfunction in asthmatic mice

The accumulation of reactive oxygen species (ROS) and subsequent oxidative stress play pivotal roles in the initiation of asthma ¹⁸. To delve into the mechanism underlying its alleviating effects on asthma, we employed varying concentrations of L. rhamnosus (10^9 or 10^10 CFU/mL, denoted as LGG 1 and LGG 2) to ensure the precision of our findings. Measurement of ROS levels in lung tissue revealed a dose-dependent reduction upon administration of L. rhamnosus in asthmatic mice (refer to Figure 2a-b). These findings suggest that L. rhamnosus possesses the capability to attenuate ROS levels in asthmatic mice, thus mitigating oxidative stress imbalance. It is well-established that disturbances in mitochondrial homeostasis can lead to heightened ROS production, consequently triggering airway inflammatory responses (Figure 2a-b). These results suggested that L. rhamnosus could downregulate ROS levels in asthmatic mice and inhibit oxidative stress imbalance. Disturbances in mitochondrial homeostasis can induce a large production of ROS and induce an airway inflammatory response ³⁰. Peroxisome proliIerators-activated receptor γ coactivator lalpha (PGC-1α) interacts with its transcription factor mitochondrial transcription factor A (TFAM) to stimulate mitochondrial biogenesis and maintain the structure, transcription, and replication of mitochondrial DNA ³¹. We measured the protein expression levels of PGC-1α and TFAM in mouse lung tissue by western blotting (Figure 2d), and found that nasal drops and gavage administration of L. rhamnosus increased PGC-1α and TFAM protein expression levels in lung tissue of asthmatic mice (Figure 2e). Mitochondria have the function of synthesizing ATP to provide energy to cells, and the function of mitochondria can be directly reflected by measuring the level of ATP. We used the Mitochondrial ATP Assay Kit to detect ATP levels in mouse lung tissue and found that both methods given to L. rhamnosus were effective in increasing mitochondrial ATP levels in lung tissues of asthmatic mice (Figure 2c). Maintenance of mitochondrial division/fusion is critical to mitochondrial function ³². We measured the expression level of mitochondrial fusion and division markers Optic Atrophy 1(OPA1), mitochondrial fusion protein (Mfn2), dynamin-related protein 1(Drp1) and Fission 1 homologue (Fis1) proteins in mouse lung tissues by western blotting (Figure 2f), and found that the gavage administration of L. rhamnosus reduced the expression levels of OPA1, Mfn2, Drp1 and Fis1 proteins in lung tissue of asthmatic mice (Figure 2g). These results showed that L. rhamnosus had the functions of promoting the biosynthesis of mitochondria in lung tissue, maintaining mitochondrial stability, increasing mitochondrial synthesis of ATP and inhibiting mitochondrial division/fusion.

L. rhamnosus inhibits NLRP3 inflammasome activation in lung tissue of asthmatic mice by downregulating Drp1

The onset of asthma is closely related to cellular inflammation ³³. NLRP3 inflammasome is an important molecule involved in cellular inflammatory response, and previous clinical studies have confirmed that NLRP3 expression in peripheral blood of asthmatic patients is up-regulated, and animal experiments have also confirmed NLRP3 inflammasome activation in lung tissue of asthma model mice ^34,35. We measured the expression levels of NLRP3 inflammasome-related protein in mouse lung tissue through western blotting (Figure 3a), and found that the administration of L. rhamnosus reduced the expression levels of NLRP3, pro-caspase1, pro-IL-1β, GSDMD, and GSDMD-N proteins in lung tissue of asthmatic mice in two ways (Figure 3b). In addition, we also measured the content of IL-1β and IL-18 in the alveolar lavage solution of mice by ELISA, and found that the administration of L. rhamnosus reduced the content of IL-1β and IL-18 in the alveolar lavage solution of asthmatic mice (Figure 3c-d). Drp1/NLRP3 signaling axis can serve as a potential key point for slowing asthma damage. To verify that L. rhamnosus inhibited asthma-induced NLRP3 inflammasome activation by downregulating Drp1, we mimicked asthma by IL-4 treatment of human airway epithelial BEAS-2B cells in vitro. We found that the expression levels of NLRP3, pro-caspase1, pro-IL-1β, Gasdermin domain containin (GSDMD) and GSDMD-N proteins in BEAS-2B cells treated with IL-4 were increased, and the expression of NLRP3, pro-caspase1, pro-IL-1β, GSDMD and GSDMD-N proteins in BEAS-2B cells induced by IL-4 treatment was inhibited. Overexpression of Drp1 reversed the downregulation of the expression of NLRP3, pro-caspase1, pro-IL-1β, GSDMD and GSDMD-N proteins in IL-4-treated BEAS-2B cells (Figure 3e-f). Imbalances in the oxidative stress response often occur in asthmatic patients ³⁶. Oxidative stress factors SOD, Malondialdehyde (MDA), 8-hydroxy-2' -deoxyguanosine (8-OHdG) and nitrotyrosine were measured in mouse alveolar lavage fluids by ELISA. We found that nasal drops and gavage administration of L. rhamnosus increased the SOD content of alveolar lavage solution in asthmatic mice (Figure 3g) and decreased the content of MDA, 8-OHdG and nitrotyrosine in mouse alveolar lavage solution (Figure 3h-j). These results suggested that L. rhamnosus inhibited NLRP3 inflammasome activation in lung tissue of asthmatic mice, possibly by inhibiting the Drp1/NLRP3 signaling axis.

L. rhamnosus inhibits oxidative stress-induced mitochondrial damage in airway epithelial cells in vitro by inhibiting the Notch1/Jagged1 signaling pathway

The Notch1 signaling pathway plays an important role in asthma-induced oxidative stress ³⁷. Inhibition of the Notch1/Jagged1 pathway facilitates the migration of rat bone marrow mesenchymal stem cells (BM-MSCs) to targeted sites and effectively ameliorates asthma symptoms. ³⁸. We examined the effect of L. rhamnosus on the Notch1/Jagged1 signaling pathway in the lung tissue of asthmatic mice (Figure 4a), and found that the administration of L. rhamnosus reduced the expression levels of Notch1 and Jagged1 proteins in lung tissue of asthmatic mice in two ways (Figure 4b). Next, to replicate asthma conditions in vitro, we subjected BEAS-2B cells to IL-4 treatment. Subsequently, we aimed to counteract the inhibitory effects of L. rhamnosus on the Notch1/Jagged1 signaling pathway by administering Jagged1-Fc fusion protein. Surprisingly, our results demonstrated that the administration of Jagged1-Fc fusion protein led to a notable increase in ROS levels within the cellular environment. This unexpected outcome sheds new light on the intricate interplay between the Notch1/Jagged1 pathway and oxidative stress in the context of asthma pathogenesis. Further exploration of these mechanisms was warranted to elucidate their potential implications for asthma management strategies. (Figure 4c), SOD, MDA, 8-OhdG, and nitrotyrosine levels (Figure 4d-g) in L. rhamnosus-treated BEAS-2B cellsand decreased SOD content. The administration of Jagged1-Fc fusion protein also reduced PGC-1α and TFMA protein expression in BEAS-2B cells treated with L. rhamnosus (Figure 5a-b), and increased the expression of NLRP3, GSDMD-N, GSDMD-C, NLRP3, pro-caspase1, pro-IL-1β, OPA1, Mfn2, Drp1 and Fis1 proteins (Figure 5c-d). These in vitro findings provide compelling evidence demonstrating the activation of NLRP3 inflammatory vesicles consequent to the inhibition of the Notch1 signaling pathway. This intricate process involves multiple mechanisms, including the downregulation of oxidative stress, the inhibition of mitochondrial damage, and the over-activation of Drp1. Understanding these complex interactions sheds light on the underlying pathophysiological mechanisms of asthma and may pave the way for the development of targeted therapeutic interventions aimed at mitigating airway inflammation and improving asthma management.

In our study, L. rhamnosus had a protective effect on mice in an asthma model by significantly attenuating the hyperreactivity of the airways, ameliorating the histopathological damage in the lungs, and reducing the number of inflammatory cells. In addition, levels of key inflammatory factors in alveolar lavage fluid were significantly reduced. Notably, IFN-γ has been reported to play a critical role in ameliorating and modulating airway inflammation in asthma patients³⁹. In addition, IFN-γ also reduces IgE levels in patients by inhibiting IL-4 expression⁴⁰. Studies have shown that IL-4 enhances the expression of vascular endothelial cell adhesion molecules, leading to a selective recruitment of eosinophils and their aggregation at sites of inflammation in patients ⁴¹.IL-17A is a member of the IL-17 family, which is mainly derived from CD4⁺ T cells but is also secreted by CD8⁺ T cells, γδ T cells, and NKT cells ⁴². Studies have shown that the expression levels of Th17 cells and IL-17A are significantly elevated in the peripheral blood of asthmatic patients. This upregulation highlights the critical role of Th17-mediated immune responses in fueling and amplifying the inflammatory cascade within the airways leading to the onset and progression of asthma ⁴³. HMGB1, a highly conserved non-histone nuclear protein, is significantly upregulated in alveolar lavage fluid and lung tissue of asthmatics ⁴⁴. Furthermore, the HMGB1/TLR4/NF-κB signaling pathway is implicated in the pathogenesis of asthma and has important implications for developing airway inflammation and subsequent airway remodeling ⁴⁵. Our results showed that rhamnose treatment reduced the expression levels of IL-4, IL-17A, and HMGB1 while promoting the expression of IFN-γ.

These results strongly suggested that L. rhamnosus had anti-inflammatory effects in reducing inflammatory responses. Several cytokines produced by allergen-specific Th cells induce characteristic asthmatic responses, including airway hyperresponsiveness, inflammatory response, mucus hypersecretion, and airway asthma. Studies have shown that IL-12 and IFN-γ secreted by Th1 cells inhibit antigen-induced airway hyperresponsiveness in mice ⁴⁶. Furthermore, the development of asthma has been linked to an imbalance in the Th17/Treg subpopulation. Studies have shown that mechanisms leading to airway hyperresponsiveness and increased airway inflammation in T-bet knockout mice include IL-17A-mediated activation of neutrophils derived from Th17 cells and impaired regulation of the Th2 response ⁴⁷. Treg cells mediate immunosuppression through intercellular contacts and secretion of the cytokine IL-10, which affects the immune imbalance in asthma ⁴⁸.IL-10 alleviates allergic responses by stimulating B cell proliferation and inhibiting IgE secretion, and plays a negative regulatory role in asthma immune inflammation ⁴⁹. We found that the proportion of Th1 and Treg cells was significantly higher in the spleen of L. rhamnosus -treated mice, suggesting that L. rhamnosus ameliorates the asthma-induced imbalance of T cell subsets. Oxidative stress results from an imbalance between ROS production and the body's antioxidant defenses caused by endogenous and exogenous factors. Studies have shown that in ovalbumin-sensitized asthmatic mice, the accumulation of free radicals in the lung tissues is common and leads to oxidative damage, especially to macromolecular components such as lipids, nucleic acids, and proteins ^50,51. Our study found that L. rhamnosus reduced ROS levels in the lung tissues of asthmatic mice.

In addition, we quantified the levels of superoxide and nitrotyrosine in the alveolar lavage fluid of asthmatic mice. In our study, we observed an increase in SOD levels in the alveolar lavage fluid of asthmatic mice after administration of L. rhamnosus, along with a decrease in MDA, 8-OHdG, and nitrotyrosine levels. These findings suggested that L. rhamnosus had a potential protective effect against asthma and can attenuate asthma-induced oxidative stress imbalance.

Furthermore, L. rhamnosus increased ATP levels and mitochondrial biosynthesis-related proteins in the lung tissue, suggesting that L. rhamnosus may have the ability to promote mitochondrial biosynthesis and maintain mitochondrial stability. In addition, our study also showed that L. rhamnosus treatment decreased the expression levels of mitochondrial division and fusion-related proteins in lung tissues of asthmatic mice, suggesting that L. rhamnosus could inhibit the process of mitochondrial division and fusion. During epithelial cell injury, we found that L. rhamnosus inhibited asthma-induced NLRP3 inflammasome activation. Furthermore, in vitro experiments showed that overexpression of L. rhamnosus reversed the inhibitory effect on NLRP3 inflammasome activation in airway epithelial cells, suggesting that L. rhamnosus may ameliorate oxidative stress and mitochondrial dysfunction by inhibiting NLRP3 inflammasome activation through downregulation of Drp1. In addition, the NLRP3 inflammasome is also associated with Th cell activation, and knockdown of NLRP3 inhibited Th2 cell activation ⁵². Therefore, we hypothesized that the amelioration of asthma-induced T cell subset imbalance by L. rhamnosus may also be related to inhibiting NLRP3 inflammasome activation by L. rhamnosus, which needs to be further verified. Lung tissue contains the most Notch ligands and receptors, and the Notch signaling pathway is involved in the pathophysiological processes of lung-related diseases, including asthma ⁵³. We found that the Notch1/Jagged1 pathway was activated in mouse and cellular models of asthma, which is consistent with previous reports ³⁸ . L. rhamnosus inhibited the asthma-induced activation of the Notch1/Jagged1 pathway. More importantly, L. rhamnosus inhibited the activation of the Notch1/Jagged1 pathway. While Notch1 agonists reversed the effects of L. rhamnosus on the amelioration of oxidative stress and mitochondrial dysfunction in BEAS-2B cells treated with IL-4, they also reversed the inhibitory effects of NLRP3 inflammation. In addition, the results showed that the Notch signaling pathway is closely related to the differentiation and activation of T cells, and Notch directly regulate the expression of Th2 transcription factor GATA-3, and blocking the Notch signaling pathway reduces the level of GATA3, which in turn reduces the level of Th2 cells ⁵⁴. Therefore, the inhibitory effect of L. rhamnosus on the Notch1 pathway may also be a mechanism to ameliorate the asthma.

L. rhamnosus Lactobacillus rhamnosus

NLRP3 NOD-like receptor thermal protein domain associated protein 3

Drp1 dynamin-related protein 1

IL-10 Interleukin-10

IL-12 Interleukin-12

TNF-α tumor necrosis factor-α

ROS reactive oxygen species

ASC apoptosis-associated speck-like protein containing a CARD

pro-caspase-1 pro-cysteinyl aspartate specific proteinase-1,

PGC-1α Peroxisome proliIerators-activated receptor γ coactivator lalpha

TFAM mitochondrial transcription factor A

OPA1 Optic Atrophy 1

Mfn2 mitochondrial fusion protein

Drp1 dynamin-relatedprotein 1

Fis1 Fission 1 homologue

GSDM Gasdermin domain containin

SOD Superoxide Dismutase

MDA Malondialdehyde

8-OHdG 8-hydroxy-2' -deoxyguanosine

Ethics approval and consent for participate statement

We acknowledged and appreciated the colleague for their valuable comments on this document.

Acknowledgements

N. Yang and S. Liu participated in the conception of the study. N. Yang, S. Liu and S.T. Zhang contributed to experimental work and/or manuscript writing and critical revision. S. Liu and X.T. Jiang and Y.X.X. Shang conducted experimental work and processed the data. S. Liu, S.T.Zhang, X.T.Jiang, and Y.X.Shang explained the data. All authors reviewed and corrected the manuscript.

Consent for publication

All data generated or analysed during this study are included in this published article.

Funding

This work was supported by grants from the Applied Basic Research Project of Liaoning province (2022JH2/101500004)

Data availability statement

The study has no data required.

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Alleviating Asthma-Induced Mitochondrial Dysfunction and Oxidative Stress: Lactobacillus rhamnosus Intervention via Notch1 Signaling Pathway

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