Effect of solasodine on mast cell-mediated OVA (ovalbumin)- induced airway inflammation in asthma via the Fyn-Lyn-Syk pathway

doi:10.21203/rs.3.rs-4186806/v1

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Effect of solasodine on mast cell-mediated OVA (ovalbumin)- induced airway inflammation in asthma via the Fyn-Lyn-Syk pathway

https://doi.org/10.21203/rs.3.rs-4186806/v1

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Introduction: Current clinical asthma therapeutic drugs can produce a series of adverse effects when used for a long period of time or in high doses, so it is urgent to find new therapeutic strategies. Solasodine can inhibit ovalbumin (OVA)-induced type II inflammation in mice, but the mechanism is still unclear. Mast cell (MCs) degranulation is an important part of allergic reactions, and Src family kinases (SFKs) are involved in the activation of the initiation signal of MC degranulation, and Lyn, Fyn, and Syk among SFKs play important regulatory roles in MC degranulation. Regulation of SFKs can inhibit the MC degranulation process and suppress the occurrence of allergic reactions. Here, we explored the effects of solasodine on OVA-induced asthma in mice and MC-mediated allergic reactions.

Methods: Mouse bone marrow-derived mast cells (BMMCs) cells were added with different concentrations (0, 2, 4, 6, 8,10, 12 and 14 µM) of solasodine to select the appropriate concentration. In transforming growth factor (TGF)-β1 treatment of BMMCs, cells were treated with 10 µM solasodine or dexamethasone (Dex), respectively, to analyze the possible mechanisms of action. A mouse model of bronchial asthma was constructed, and the mice were divided into control, OVA, OVA + Dex, OVA + solasodine (1 mg/kg) and OVA + solasodine (10 mg/kg) groups. The histopathological changes in the lungs of the mice were observed by staining with HE, Masson, and Tunel staining. ELISA assay was used to detect the differences between bronchoalveolar lavage fluid (BALF) and serum. IL-4, IL-5, IL-1β, TNF-α and LTD-4 levels were detected by ELISA. BALF inflammatory cells were detected by Wright staining and P-Fyn, P-Lyn and P-Syk protein expression in lung tissues were detected by Western blot.

Results: In TGF-β1-induced BMMCs, solasodine significantly reduced the expression of P-Fyn, P-Lyn, and P-Syk, decreased the expression of cytokines IL-6, TNF-α, and LTC₄ and calcium uptake, as well as inhibited mast cell degranulation. In ova mice, solasodine inhibited OVA-induced airway remodeling, production of IL-4, IL-5, IL-1β, TNF-α, and LTD-4, and degranulation of mast cells in asthmatic mice. Solasodine inhibited the activation of Fyn, Lyn, and Sky and reduced the number of inflammatory cells such as eosinophils, lymphocytes, and neutrophils in lung tissues of asthmatic mice.

Conclusion: In this study, we found that solasodine could have a concentration-dependent therapeutic effect on OVA-induced allergic asthma, and solasodine could inhibit the release of mast cell degranulation signature mediator β-HEX and histamine, and the molecular mechanism of its efficacy might be related to its regulation of the intracellular calcium concentration and the IgE/FcεRI-mediated signaling pathways such as P-Fyn/Fyn, P-Lyn/Lyn, P-Syk/Syk, and etc.

Solasodine

Mast cell

Airway inflammation

Fyn-Lyn-Syk pathway

Asthma is a major non-communicable disease (NCD) that can affect people of all ages and is the most common chronic lung disease among children. It is caused by inflammation and muscle tightening around the airways, leading to breathing difficulties and in the most severe cases, asthma can lead to death.[1] Asthma is characterized by hyperresponsiveness, airway inflammation, and airway remodeling. [2–4] And airway remodeling is irreversible with current asthma medications and is a major cause of severe asthma. And airway smooth muscle cells (ASMCs) as the main effector cells of airway remodeling, proliferation and hypertrophy are associated with airway remodeling. [5–7] MCs are important immune cells involved in mediating allergic responses. Activated MCs can selectively secrete and release vasoactive substances or inflammatory mediators through degranulation to participate in the immune or inflammatory response of the body, which plays an important role in asthma, allergy, tumor, atherosclerosis and rheumatoid arthritis.[8] The pathogenesis of allergic asthma is closely related to MCs, which promote inflammatory and allergic responses by releasing intermediate inflammatory and allergic mediators. [8–10]

Signaling pathways associated with MC activation FcεRI-mediated have already been demonstrated. MC activation is mediated by IgE binding to high-affinity IgE receptors (FcεRI) expressed on the cell surface, followed by FcεRI aggregation, which triggers an intracellular signaling pathway that promotes the activation of receptor-associated tyrosine kinases, such as Fyn and Lyn, and subsequent activation of splenic tyrosine kinase (Syk). Syk then stimulates downstream signaling molecules, including T-cell activation tether (LAT), which mediates inflammatory allergic responses, mitogen-activated protein kinases (MAPKs), PI3K/Akt, phospholipase Cγ1 (PLCγ1), NF-κB, and intracellular Ca²⁺. Besides, the signaling molecules are able to activate AKT via phosphorylation and stimulate MC degranulation and cytokine and chemokine production. [11–14]

Solasodine is a glycoalkaloid consisting of a C27 cholane structure, a basic nitrogen and six sugar molecules. [10] This glycoside is considered to be representative of the alkaloids of the Solanaceae family and is found in various members of the Solanaceae family. Solasodine glycosides have been a target of interest for researchers because of their pharmacological effects such as anti-schistosomal, anti-inflammatory, antioxidant, anti-fungal, anti-atherosclerotic, hepatoprotective, anti-convulsant and neuroprotective properties. [15–19] Current findings suggest that solasodine holds therapeutic promise for allergic asthma by preventing MC degranulation and the resulting airway histamine and T-helper type 2 (Th2) related inflammatory mediators. [1] However, the mechanism of solasodine action on asthma is not completely clear.

Therefore, the current study is intended to study the role of solasodine on OVA induced allergic asthma and to investigate its association with MCs and the Fyn-Lyn-Syk pathway. In this study, mouse BMMCs and mice were successfully induced with OVA as a model of allergic asthma to investigate the efficacy, pharmacological effects, and its mechanism of action of solasodine in bronchial asthma, with a focus on the upstream signaling pathways, including Fyn, Lyn, and Syk. It was found that solasodine may inhibit MC-mediated inflammatory responses and allergic asthma by inhibiting the phosphorylation of upstream signaling molecules Fyn, Lyn, and Syk.

2.1. Drug and reagents

Solasodine (^#MFCD00037844), OVA (^#MFCD00889188), and TGF-β1 (^#91701ES08) were ordered from Sigma Aldrich (MO, USA) and the purity of solasodine is ≥ 98%. Other drugs such as penicillin and streptomycin were purchased from Gibco (Grand Island, NY/Carlsbad, CA, USA), which also provided us the RMPI-1640 and fetal bovine serum (FBS). Nitric oxide (NO) calorimetric kit was obtained from BioVision (Research Products, USA). Water purification system (Millipore, MA, USA) supplied the milli-Q water used in experiments. The goat anti-rabbit IgG combined with horseradish peroxidase (HRP) was bought from Invitrogen (Carlsbad, CA, USA) as well as Alum Adjuvant (Thermo Scientific, USA).

2.2. Cell culture

Mouse BMMCs were purchased from Procell (^#ORCM249, Wuhan, China). RPMI-1640 was used to culture the mouse BMMCs cells at 37°C, 5% CO₂. And the Cells were cultured in the medium with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

2.3. Toxicity of solasodine for cells

0.1% dimethyl sulfoxide (DMSO) was used to solve the solasodine in different concentrations. Then, different dose of solasodine were used to treat the BMMC cells (1×10⁷ cells/well) seeded in 96-well plates for 24h. CCK-8 kit was used to detect cell viability according to the kit’s instruction. The 450 nm absorbance was recorded using a microplate reader (Biotek Corporation, USA).

2.4. Cell groups and treatment

The BMMCs cells were divided into 4 groups, which were control group, TGF-β1 model group, TGF-β1 + Dex group, and TGF-β1 + solasodine group. The control group was cultured under normal conditions for 48 h. Cells in the TGF-β1 model, TGF-β1 + Dex, and TGF-β1 + solasodine groups were cultured in medium containing 10 ng/mL of TGF-β1 for 24 h, and then transferred to fresh medium containing 10 µM Dex or 10 µM solasodine for 24 h. Finally, cells were transferred into fresh medium containing 10 µM Dex or 10 µM solasodine.

2.5. Western blot

The proteins were extracted from BMMCs using RIPA buffer and then separated through 12% SDS-PAGE. Then, they were transferred to polyvinylidene fluoride (PVDF) membranes, and incubated with primary antibodies overnight at 4 ℃. The primary antibodies using in the study were as follows: p-Fyn (^#ab182661, abcam UK), Fyn (^#4023, Cell Signaling Technology), p-Lyn (^#70926, Cell Signaling Technology), Lyn (^#2796, Cell Signaling Technology), p-Syk (^#2712, Cell Signaling Technology), Syk (^#2710, Cell Signaling Technology), GAPDH (^#8884, Cell Signaling Technology), at a 1:1000 dilution. Subsequently, the membranes were cultured with the second antibody HRP-linked anti-rabbit IgG for 60 min at 37 ℃ at a 1:2500 dilution. Then, the membranes were observed using an enhanced chemiluminescence reagent kit.

2.6. ELISA

BMMCs were seeded at 1×10⁶ cells/mL and cultured overnight. After different treatments, the supernatant was collected and centrifuged under the conditions of 4°C for 5 min at 3000 rpm. Then, BMMCs cells were collected and cytokine levels were analyzed by ELISA kit. Concentrations of IL-6, LTC₄ and TNF-α (R&D Systems, Minneapolis, MN, USA) were analyzed.

2.7. Measurement of β-hexosaminidase

To measure the percentage of MC degranulation, BMMCs cells were divided into 4 groups: control group, TGF-β1 model group, TGF-β1 + Dex group and TGF-β1 + solasodine group. The control group was cultured under normal conditions for 48 h. Cells in the TGF-β1 model, TGF-β1 + Dex, and TGF-β1 + solasodine groups were cultured in medium containing 10 ng/mL of recombinant human TGF-β1 for 24 h, and then transferred to fresh medium containing 10 µM Dex or 10 µM solasodine for 24 h. Finally, the P-nitrophenyl-N-acetyl-β-D-glucosaminide and citric acid (pH 4.5) were added to the supernatant to detect the percentage of β-hexosaminidase (β-hex) released.

2.8. Ca²⁺ concentration measurement

BMMCs were inoculated into flat-bottomed 12-well plates with 5 × 10⁵ cells/well. The cell suspension was mixed continuously during the inoculation process to ensure that the number of cells in each well was approximately the same. Then, the 12-well plates were incubated in a thermostatic incubator. The cells in the experiment were divided into control, TGF-β1 model, TGF-β1 + Dex, and TGF-β1 + solasodine groups. No treatment for the control group. Cells in the TGF-β1 model, TGF-β1 + Dex, and TGF-β1 + solasodine groups were cultured under 10 ng/mL of recombinant human TGF-β1 for 24 h, and then transferred to fresh medium containing 10 µM Dex or 10 µM solasodine for 24 h. Aspirate away the culture medium of each group and rinse the cells with HBSS buffer 3 times. Immediately following this, 1 mL of pre-prepared Fluo3-AM working solution containing 0.05% Pluronic F127 at a ratio of 0.05% (W/V) was added per well and loaded for 40 minutes away from light. After incubation, cells were rinsed 3 times with HBSS buffer and then 1 mL of HBSS buffer was added to each well to cover the cells and incubated at 37°C for half an hour to ensure complete intracellular de-esterification of Fluo3-AM. Fluorescence inverted microscope (Leica, Germany) was used to observe and photograph the cells in 528nm.

2.9. Immunofluorescence staining

BMMCs were inoculated into flat-bottomed 12-well plates with 5 × 10⁵ cells per well. The cell suspension was mixed continuously during the inoculation process to ensure that the number of cells in each well was approximately the same. 12-well plates were incubated in a thermostatic incubator. The cells were distributed into control, TGF-β1 model, TGF-β1 + Dex, and TGF-β1 + solasodine groups. No treatment for the control group. Cells in the TGF-β1 model, TGF-β1 + Dex, and TGF-β1 + solasodine groups were cultivated in medium containing 10 ng/mL of TGF-β1 for 24 h, and then transferred to fresh medium containing 10 µM Dex or 10 µM solasodine for 24 h. Each group of cells was planted into 10-cm dishes containing 15 to 20 sterilized glass lids each. 20 µL of cell suspension was taken on each cover slip for 10 min and then fixed with 4% paraformaldehyde for 10 min. Cells, as indicated, were closed with BS-T containing 1% BSA for 30 min and incubated with different antibodies overnight at 4°C. After TBS washing, cells were incubated with cy3-coupled antibody for 60 min, and the nuclei and tissues were incubated with 4′,6-diamino − 2-phenylindole (DAPI) for 60 min. The tissues were rinsed and sealed and then observed under a laser confocal microscopy (FluoView FV1000 Olympus Corporation, Japan).

2.10. Animal and asthma model

In this study, forty male mice of C57BL/6 J in 6–8-weeks-old were ordered from Jinan Pengyue experimental animal breeding Co., Ltd and all of them were kept in a specific condition of pathogen-free. After one week of acclimatization, the asthma model was constructed according to the instructions of the report. Firstly, 20µg of OVA and 1 mg of aluminum hydroxide were dissolved in 200µL of saline, which was used for intraperitoneally injection of sensitized mice on day 1 and day 8, respectively. On days 15, 16 and 17 of initial sensitization with OVA, mice inhaled 5% OVA in a whole-body exposure chamber using an ultrasonic nebulizer (NE-U17, Omron) for 30 min. These mice were then used to perform different experiments.

2.11. Animal treatment and groups

Randomly, the mice used in this study were randomly divided into control group, OVA model group, OVA + Dex group, OVA + low dose solasodine group and OVA + high dose solasodine group. 200µL saline containing 20µL PBS was injected to the mice of the control group on day 1 and day 8, respectively. On days 15, 16 and 17 after sensitization, control mice were nebulized for 30min with 5% PBS. OVA was used to sensitize the mice in the Dex group, low dose solasodine group and high dose solasodine group and then intranasally treated with 2.5 mg/kg Dex, 1mg/kg solasodine or 10 mg/kg solasodine solution every day.

2.12. Bronchioalveolar lavage

The lung of the mice was injected with 1 mL PBS via the tracheal tube to obtain the bronchioalveolar lavage fluids (BLAFs). Trypan blue dye exclusion was used to calculate the total number of cells in the BALFs. Wright stain solution (^#G1040, Solarbio). And the number of eosinophils, neutrophils and lymphocytes were calculated by Wright stain solution (^#G1040, Solarbio).

2.13. Determination of cytokine levels in serum and BALF fluid

BALFs were centrifuged (× 800 g, 15 min) to take supernatants to detect the levels of TNF-α, IL-4, IL-5, IL-1β and LTD-4 in serum and BALF fluid were measured using ELISA kits.

2.14 Western blot for Fyn-Lyn-Syk pathway-related protein expression

Using RIPA lysis buffer, the total protein was extracted from lung tissues and then quantified the concentration using the BCA kit. 10% SDS PAGE gel electrophoresis was performed to separate proteins, which were transferred to PVDF membranes. The membranes were closed with PBS containing 5% skimmed milk powder and incubated with primary antibodies against p-Fyn (^#ab182661, abcam UK), Fyn (^#4023, Cell Signaling Technology), p-Lyn (^#70926, Cell Signaling Technology), Lyn (^#2796, Cell Signaling Technology), p-Syk(^#2712, Cell Signaling Technology), Syk(^#2710, Cell Signaling Technology), GAPDH (^#8884, Cell Signaling Technology) overnight at 4 ℃.After the incubation with the secondary antibody, the membranes were visualized through ECL kit and the protein gray values were analyzed by Image J software.

2.15. Airway hyperresponsiveness (AHR)

Pentobarbital sodium (45 mg/kg) was injected to the mice to anesthetize them. Then, a tracheostomy tube was inserted into the trachea of the mice ventilation, which was performed using a Sci-Req FlexiVent machine. The respiratory rate was set at 150 breaths/min, tidal volume at 2 mL and the resistance was measured at baseline and after increasing doses (0, 12.5, 25, 50, and 100mg/mL) of nebulized methacholine.

2.16. Determination of histamine in lung tissue

The above preserved lung tissue was homogenized with 2.5 mL of physiological saline, acidified with 0.8 mol/L perchloric acid, and centrifuged at 4,000 r/min for 10 min. The supernatant was neutralized with sodium hydroxide, added with sodium chloride and n-butanol, and centrifuged at 4,000 r/min for 10 min. The n-butanol fractions were collected, and washed with 0.1 mol/L sodium hydroxide saturated solution for three times, then centrifuged again. centrifugation. The non-aqueous layer was mixed with 0.1 mol/L hydrochloric acid and heptane and centrifuged at 4000 r/min for 10 min. Histamine was measured spectrophotometrically at 650 nm.

2.17. HE and Masson staining

All mice were sacrificed after the collection of BALFs. Lung tissues of the mice were injected with 4% paraformaldehyde to fix for 48h, which then were embedded in paraffin and cut into serial sections (3µm). Hematoxylin and eosin (HE) were used to stain the sections according to the modified HE staining method. Subsequently, the sections were stained using Masson trichrome and observed using a light microscope.

2.18. Tunel staining

Paraffin sections were washed twice in xylene×5min; gradient ethanol (100%, 95%, 90%, 80%, 70%) washed once×3min; the tissue samples were treated with Proteinase K working solution at 37 ℃ for 20min; Pbs washed twice×5min; the Tunnel reaction mixture was prepared (50µl tdt + 450µl fluorescein-labeled tdt); the slides were dried and 50µL Tunnel reaction mixture was added on the tissue samples and reacted at 37 ℃×1h under light protection; PBS washed three times×5min; the Tunnel reaction mixture was added on the tissue samples and reacted in light protection. Fluorescein-labeled tdt); shake dry the slide, add 50µL Tunnel reaction mixture on the tissue sample, react at 37 ℃×1h under light protection; PBS washed 3 times×5min under light protection; observe the slide under fluorescence microscope (FluoView FV1000 Olympus Corporation, Japan) and take pictures (excitation wavelength 450 nm-500 nm, detection wavelength 515 nm-565 nm).

2.19. Statistical analysis

The data in this study were analyzed using GraphPad Prism 6.0 software (GraphPad Software Inc., CA) and showed as mean ± standard deviation. One-way analysis of variance following the Turkey teat was used to test the significance among groups, which was considered as a value of *p < 0.05, **p < 0.01, ***p < 0.001.

3.1. Effect of solasodine on TGF-β1-induced asthma cell model in BMMCs

In order to select the optimal concentration of the test compounds, the cytotoxicity of solasodine was first determined using the CCK-8 kit. We cultured BMMCs in serum-free medium supplemented with solasodine at concentrations of 0, 2, 4, 6, 8, 10,12 and 14 µM. When the concentration was as high as 14 µM, the tested compounds still lacked significant cytotoxic effects on the BMMCs still lacked significant cytotoxic effects (Fig. 1a). Based on these results, we performed further experiments using 10 µM concentration of solasodine. Airway wall remodeling in asthma patients is associated with enhanced subepithelial fibrosis and activation of human bronchial fibroblasts (HBFs) during fibroblast-to-myofibroblast transformation (FMT), which is mainly induced by transforming growth factor-β (TGF-β). We therefore used TGF-β1-induced BMMCs cells as an in vitro cultured asthma cell model.

We examined the expression of Fyn, Lyn, Syk p-Fyn, p-Lyn, p-Syk proteins in different groups of control, TGF-β1 model, TGF-β1 + Dex, and TGF-β1 + solasodine groups by Western blot method, and the results are shown in Fig. 1b. In order to quantify the results accurately, we used Image J software to quantify the gray values of p-Fyn, p-Lyn, p-Syk proteins and statistically analyzed this. As can be seen from Fig. 1c, d, and e, the expression of Fyn, Lyn, and Syk phosphorylated proteins in the TGF-β1-induced model group was much higher than that in the normal group (p < 0.01). In contrast, the expression of these three proteins in the model group with the addition of Dex, a clinical drug for asthma, showed a significant decreasing trend (p < 0.01), suggesting that Dex can treat asthma by decreasing the expression level of Fyn, Lyn, and Syk phosphorylated proteins. Surprisingly, the expression of Fyn, Lyn and Syk phosphorylated proteins was also significantly decreased in the experimental group with the addition of solasodine compared to the TGF-β1-induced model group (p < 0.01), which suggests that the principle of solasodine in the treatment of asthma may be similar to that of Dex, i.e., the treatment of asthma by decreasing the expression of Fyn, Lyn and Syk phosphorylated proteins.

To reconfirm our experimental conclusions, we measured the protein content in the cells using immunofluorescence (Fig. 2a) and quantified the average fluorescence intensity in the graphs using Image J software (Fig. 2b, c, d). The results were consistent with the Western blot results. TGF-β1 significantly increased the expression of Fyn, Lyn and Syk phosphorylated proteins (p < 0.01), whereas the clinical drugs Dex and solasodine both decreased the expression of all three phosphorylated proteins (p < 0.01), and all of these results were statistically significant.

3.2. Effect of solasodine on MC degranulation

Generally, the presence of β-hexosaminidase is almost undetectable in human blood [20], and when the body undergoes an allergic reaction, MC degranulation releases β-hexosaminidase into the extracellular compartment, which causes a significant increase in plasma β-hexosaminidase concentration. Only MCs are known to have the ability to release β-hexosaminidase. β-hexosaminidase has become a marker for the strength of the degranulation response occurring in MCs. Next, we used the β-hex assay to detect degranulation in BMMCs. As we can see from Fig. 3d, TGF-β1 was able to significantly increase the percentage of β-hex release (p < 0.01), while both Dex and solasodine significantly decreased the percentage of β-hex release (p < 0.01).

Studies have shown that bioactive mediators released during degranulation are closely associated with a range of pathophysiological functions of MCs. ELISA analysis of cytokines in the present study showed (Fig. 3a, b, c) that serum IL-6, LTC₄ and TNF-α levels were significantly higher in the TGF-β1 model group compared with the control group (p < 0.001). The cytokines IL-6, LTC₄ and TNF-α were significantly lower in the standard dexamethasone-treated and solasodine-treated groups compared to the TGF-β1 model group (p < 0.01).

Changes in Ca²⁺ are very important in stimulating MC degranulation and the production of inflammatory mediators.[21] Because of the key role of Ca²⁺ in the activation and degranulation of MCs, the fluorescent probe Fluo3 was used as a carrier of Ca²⁺, and the effect of solasodine on the intracellular concentration of Ca²⁺ in sensitized and stimulated BMMCs was observed by inverted fluorescence microscope.Fluo3 specifically binds to free intracellular Ca²⁺ and produces fluorescence at 528 nm (Fig. 4a). And the fluorescence intensity of the graph was quantitatively analyzed. From the figure, we can see that the fluorescence intensity of the control group was very low, indicating that the concentration of intracellular Ca²⁺ was very low in the normal state, whereas the concentration of intracellular Ca²⁺ increased dramatically after sensitization of BMMCs (p < 0.05), resulting in a significant enhancement of fluorescence intensity. Fluorescence intensity was significantly reduced after treatment with Dex and solasodine (p < 0.05).

3.3. Solasodine inhibits OVA-induced asthma symptoms in mice

The pathogenesis of asthma has a significant relationship with the airway inflammatory response, and the two are causative of each other. Under the stimulation of allergens, a large number of inflammatory cells, such as neutrophils, eosinophils, and lymphocytes, accumulate toward the lung tissues, which cause airway inflammatory response and induce asthma. [22] In this study, we used an OVA-induced allergic asthma model, and to further investigate the mechanism of action of solasodine in allergic asthma, an OVA-induced allergic asthma mouse model was used. A daily intranasal injection of 10 mg/kg Dex or 1 mg/kg solasodine solution (low dose) or 10 mg/kg solasodine solution (high dose) was administered for 8–17 days prior to OVA tapping (Fig. 5a).

It was found that the lung tissues of mice in the model group showed the phenomenon of massive inflammatory cell infiltration, peribronchial eosinophilia, and enlarged and detached epithelial cells, which showed the characteristics of airway allergic disease, suggesting that the modeling was successful. We analyzed the counts of eosinophils, lymphocytes and neutrophils in BALF (Fig. 5b, c, d) and found that the number of eosinophils, lymphocytes and neutrophils was significantly increased in BALF of OVA-induced mice (p < 0.01). The number of eosinophils, lymphocytes and neutrophils was significantly lower in the Dex group and the low-dose solasodine group than that in the OVA group (p < 0.05). Eosinophil, lymphocyte, and neutrophil counts were significantly lower at high doses of solasodine compared to low doses of solasodine (p < 0.05). We also measured airway resistance in each group (Fig. 6f). Airway resistance was significantly up-regulated after tapping OVA compared to the control group (p < 0.01). Besides, airway resistance was significantly down-regulated after administration of Dex or low concentrations of solasodine to mice (p < 0.05). However, airway resistance was significantly higher in mice in the low-dose solasodine group than in the high-dose solasodine group (p < 0.05). This suggests that solasodine has a concentration-dependent ameliorating effect on OVA-induced allergic asthma, which is not as effective as the clinical drug Dex, but still has a significant therapeutic effect.

Histamine promotes MC-mediated allergic and inflammatory responses [23] LTD-4 is one of the most potent bronchoconstrictors and chemotactic agents for eosinophils. As shown in Fig. 6a, b, c, d, e, we found that BALF levels of IL-4, IL-5, IL-1β, TNF-α, and LTD-4, were significantly increased in Model group rats (p < 0.01), suggesting that inflammatory response and bronchoconstriction were aggravated in allergic asthmatic rats. [24, 25] After the treatment of low-dose solasodine, all the above indexes of asthmatic mice decreased significantly (p < 0.01), and compared with low-dose solasodine, high-dose solasodine had a more obvious improvement effect (p < 0.05). It suggests that solasodine has the effect of improving the immune response in allergic asthmatic rats.

3.4 Solasodine inhibits OVA-induced expression of Fyn, Lyn, Sky protein phosphorylation in mouse lung tissue

Syk is a member of the tyrosine protein kinase family, which can be expressed in a variety of immune cells, is closely related to inflammatory responses, and is involved in asthma-associated cytokine signaling. Lyn, as a member of the non-receptor-type tyrosine protein kinase Src family, activates downstream signaling molecules by phosphorylating STAT3 through the recruitment of Syk. Fyn is a member of the same receptor-associated tyrosine kinases as Lyn, which can also inhibit IgE-mediated allergic responses.

Activation of the Fyn-Lyn-Syk pathway is an important result of allergic asthma in mice. Fyn is a receptor-associated tyrosine kinase, and also inhibits IgE-mediated allergic responses. Activation of the Fyn-Lyn-Syk pathway is important for the induction of allergic asthma in mice. As shown in Fig. 7a, b, c and d, the phosphorylation levels of Fyn, Lyn, and Syk proteins were elevated in the Model group of rats (p < 0.01), consistent with previous results, whereas the phosphorylation levels of Fyn, Lyn, and Syk proteins were significantly down-regulated by high-dose solasodine treatment (p < 0.01), suggesting that solasodine may play a role in the treatment of allergic asthma mice by down-regulating the Fyn-Lyn-Syk pathway.

3.5 Effect of solasodine on histamine levels in lung tissues

Histamine-specific receptors can interact with granulocyte surface molecules to release pro-inflammatory factors in direct response to the degree of allergic reaction. MCs in all organisms contain histamine-specific receptors and are capable of releasing histamine. In human cells, histamine is found only in basophils and MCs, so the amount of histamine released can be used as an important marker of the strength of MC degranulation. In Fig. 7e, analysis of histamine levels in lung tissue homogenates of experimental mice showed that chronic exposure to OVA significantly increased lung tissue histamine levels compared to normal control mice (p < 0.01). Both standard dexamethasone and low-dose solasodine treatment significantly reduced histamine levels compared with asthma controls (p < 0.05), and high-dose solasodine treatment of OVA-sensitized animals also reduced histamine levels compared with the low-dose solasodine group (p < 0.01).

3.6 Effect of solasodine on total nitric oxide and nitrite concentration

Calorimetric analysis showed (Fig. 8a, b) that serum total nitric oxide and nitrite levels were elevated in the OVA-sensitized group of mice compared with the unsensitized control group (p < 0.01), and serum nitric oxide and nitrite levels were significantly reduced in the dexamethasone and solasodine (1 mg/kg) groups compared with the asthmatic group (p < 0.01). The reduction in serum nitric oxide and nitrite levels was more pronounced in high-dose solasodine (10 mg/kg) than in low-dose solasodine.

3.7 Solasodine ameliorates ova-induced airway inflammation and lung tissue fibrosis in mice

Lung histopathological changes were observed by HE, Masson and Tunnel staining (Fig. 8c). In this experiment, HE staining was used to observe the inflammation in the lung tissue of mice in each group: under high magnification microscope, no thickening of the basal membrane of bronchial epithelium was seen in the control group, and no inflammatory cell infiltration was seen around the bronchioles. The inflammation was aggravated in the OVA groups compared with that in the normal control group, and the OVA group could see the proliferation of mucus glands in the bronchial mucosa, and the bronchial wall could be seen to be thickened with thickening of the basal membrane. Inflammation was reduced in the OVA + Dex group, OVA + solasodine group (1 mg/kg), and OVA + solasodine group (10 mg/kg) compared to the OVA group. In the clinical drug group, inflammation symptoms were more significantly reduced. Although the low-dose solasodine group was not as effective as the high-dose group, it also had the effect of reducing inflammation.

In this experiment, Masson staining was used to observe the collagen deposition around the airways in the lung tissues of mice in each group: collagen deposition increased in the OVA group compared with the normal control group, suggesting that early airway remodeling occurred in the asthma model mice. Collagen deposition was reduced in the OVA + solasodine group compared with the OVA group in a dose-dependent manner, with high doses of solasodine significantly reducing collagen deposition. Tunnel staining showed that apoptosis was increased in the OVA group, while Dex and solasodine improved the apoptosis status, particularly high doses of solasodine. Taken together, the above experimental results showed that high dose of solasodine could significantly improve the asthma symptoms in mice induced by OVA.

Immunohistochemistry was performed to detect the levels of fibrosis-related proteins c-kit, α-SMA, and TGF-β1 in lung tissues (Fig. 8d). Their relative expression levels were significantly higher in ova-induced lung tissues compared with control lung tissues. In damaged lung tissues, the expression of these proteins was significantly inhibited in the Dex or solasodine group compared with the model group. Dex inhibited lung fibrosis-related proteins c-kit, α-SMA and TGF-β1 more effectively. Similarly, the high-dose solasodine group was more able to improve lung tissue fibrosis than the low-dose group.

Allergic asthma is a multifactorial disease in which various immune mechanisms involving IgE antibodies, MCs, basophils, eosinophils, and Th2 hypersensitivity mediators together play a role in the inflammatory and immune response in acute or chronic allergic airway disease. [26] Although there are established medications used as rescue or maintenance therapy for asthma, the available treatments are no longer able to control the disease to a satisfactory level. [27] Through macrophage activation of helper T cells in vivo, Th2 cells can produce various interleukins such as IL-4, prompting eosinophils to gather in the airways, amplifying the inflammatory response cascade and promoting the development of asthma. Th1 cells secrete IFN-γ factor, which can inhibit the inflammatory response of the asthmatic airways, and TNF-α is a common pro-inflammatory factor. In patients with asthma, stimulated by a variety of factors, immune in asthma patients, stimulated by various factors, immune cells produce a large number of inflammatory factors such as TNF-α, IL-1β, IL-4, etc., and the synthesis and secretion of IFN-γ are reduced. [28, 29] After antigenic stimulation, MCs release some chemicals such as histamine, β-aminohexosidase, LTB4, LTC4, LTE4, etc. Because their surface contains IgE high-affinity receptor FcεRI, BMMCs can be activated in vitro by corresponding antigen induction to simulate the sensitization and excitation phases of MC activation and degranulation in asthma, and can be used to study the various MC-related physiology, pathology, mechanism of drug action and other studies.In this study, we established an asthmatic cell model of BMMCs and detected the expression of Fyn, Lyn, Syk phosphorylated proteins and the levels of β-HEX, TNF-α, IL-6, LTC4 and Ca²⁺ in different subgroups. As can be seen from the results, solasodine inhibited the expression of Fyn, Lyn, Syk phosphorylated proteins and reduced the levels of β-HEX, TNF-α, IL-6, LTC₄ and Ca²⁺. Although the lowering effect was not as significant as the clinical drug Dex, it did relieve asthma symptoms. In this study, we established a mouse model of OVA asthma and found that the lung histopathology of the mice was altered with a large number of inflammatory cell infiltration, and the number of inflammatory cells in the BALF and the levels of IL-1β, IL-5, TNF-α, IL-4, and LTD-4 in the serum and the BALF were elevated, which were consistent with the characteristics of the mice model of bronchial asthma caused by OVA.

Histamine is currently the most widely studied class of MC-releasing mediators. It is involved in allergic and inflammatory responses and in mediating cellular interactions in the inflammatory response. Its ability to increase anti-CD3-induced IL-5 production in Th2 T cells via the histamine H2 receptor. β- hexosaminidase shows optimal activity at low pH, a condition typical of the inflammatory process. β- hexosaminidase is now generally recognized as a marker of MC degranulation. In this study, after the action of solasodine on MCs, the release of histamine and β- hexosaminidase in the supernatant fluid of MCs after MC activation was significantly reduced, and the strength of the action of solasodine showed a concentration-dependent correlation, suggesting that solasodine has a strong inhibitory effect on the release of cellular contents in MC degranulation. FcεR I-triggered exponential increase in cytoplasmic calcium ions is an important step in MC activation and the initiation of immune response, especially at the time of degranulation. FcεRI-triggered by IgE resulted in the activation of multiple signaling pathways. In this study, solasodine inhibited the release of histamine and β-HEX from MC degranulation and showed a certain inhibitory effect on the intracellular calcium concentration in BMMCs activated degranulation cells. It is suggested that solasodine has the function of regulating the mobilization of MC calcium ions and MC membrane stabilizer-like effect, and it may cut down the power of transporting histamine mediators by regulating the concentration of intracellular calcium ions in MCs, thus affecting the release of histamine and β-HEX.

IgE-bound FcεR I undergoes a series of intracellular signaling events after encountering and binding to the corresponding antigen. The initial signaling leads to the activation of Src family kinases via specialized lipid carrier microstructural domains in the cell membrane and subsequently to tyrosine phosphorylation of the receptor subunits. one of the Src family kinases that is involved in this early activation is Lyn. the phosphorylated immunoreceptor tyrosine activation of the β- and γ-chain tyrosine residues of the FcεR I receptor is achieved when the tyrosine residues of the β- and γ-chain of FcεRI receptor are phosphorylated by Lyn. The phosphorylated immune receptor tyrosine activation motifs (ITAMs) on the β and γ chains of the FcεRI receptor provide high-affinity binding sites for Lyn's SH2 and the structural domain of the zwitterionic protein kinase (ZAP70) to the tyrosine kinase Syk. Syk also plays an important role in the activation of downstream signaling molecules such as PI3K, Akt, PLCγ, and MAPKs. Also Fyn plays an important role in MC degranulation. Upon activation of FcεRI, Fyn regulates MC degranulation by interacting with Gab2 and activating PI3K, thereby affecting calcium mobilization. It is also these activated signaling cascades that regulate MC degranulation and the production of allergic and inflammatory cytokines. In our present study, solasodine showed some inhibitory effects on antigen-induced phosphorylation of Fyn, Lyn and Syk in MCs. This suggests that solasodine has an effect on the antigen-induced phosphorylation signaling of the Src family kinases Fyn, Syk, and Lyn in the FcεRI receptor pathway, which is responsible for inflammatory and allergic responses. In combination with solasodine's modulation of histamine and β-HEX release and MC activation of intracellular calcium concentration, it exerts a MC membrane stabilizing effect and inhibits the release of related inflammatory factors such as IL-1β, IL-5, TNF-α, IL-4, LTD-4, etc., as well as calcium ions. This may be achieved by regulating the early activation of Fyn, Lyn and Syk on the FcεR I signaling pathway, resulting in a reduction of Fyn, Lyn and Syk phosphorylation and a weakening of signaling to various downstream molecules.

Taken together, our data support the notion that solasodine has the potential to attenuate asthma symptoms by reducing MC degranulation, Th2-associated cytokine production, airway remodeling, and recruitment of inflammatory cells into the lungs. solasodine may ameliorate the immune response in allergic asthma mice by down-regulating the Fyn-Lyn-Syk signaling pathway, which may have an ameliorating effect. However, the optimal therapeutic dose of solasodine and whether it can modulate other pathways to achieve the same therapeutic effect need to be further explored.

Data Availability statement

Data will be mde avallable on request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was no funding supported.

Author Contribution

Z. G.: Conceptualization, Validation, Formal analysis, Investigation, Writing - original draft, C. Z.: Conceptualization, Software, Visualization, Writing - review & editing, Y. F.: Data curation, Methodology, Investigation, Software, X. Y.：Data curation, Methodology, Investigation, Software, Q. W.: Data curation, Methodology, Investigation, C. Q.: Conceptualization, Funding acquisition, Methodology, Writing - review & editing, J. C.: Funding acquisition, Conceptualization, Methodology, Writing - review & editing

P. Arora, L.M. Nainwal, G. Gupta, S.K. Singh, D.K. Chellappan, B.G. Oliver, et al., Orally administered solasodine, a steroidal glycoalkaloid, suppresses ovalbumin-induced exaggerated Th2-immune response in rat model of bronchial asthma, Chem-Biol Interact, 366(2022) 110138-110154.
E.Y. Kim, H. Park, E.J. Kim, S.H. Lee, J.W. Choi, J. Kim, et al., Efficacy of Trigonella foenum-graecum Linne in an animal model of particulate matter-induced asthma exacerbation, J Ethnopharmacol, 319(2024).
L.J.D. Morel, F. Carmona, C.C. Guimaraes, L.G.Q. Moreira, P.D. Leao, E.J. Crevelin, et al., A methanolic extract of Eclipta prostrata (L.) L. decreases inflammation in a murine model of chronic allergic asthma via inhibition of the NF-kappa-B pathway, J Ethnopharmacol, 318(2024) 116930-116961.
S.I. Bukhari, B. Pattnaik, S. Rayees, S. Kaul, M.K. Dhar, Safranal of L. Inhibits Inducible Nitric Oxide Synthase and Attenuates Asthma in a Mouse Model of Asthma, Phytother Res, 29(2015) 617-627.
S.Y. He, M. Chen, X.L. Lin, Z.Q. Lv, R.Y. Liang, L.J. Huang, Triptolide inhibits PDGF-induced proliferation of ASMCs through G0/G1 cell cycle arrest and suppression of the AKT/NF-κB/cyclinDl signaling pathway, Eur J Pharmacol, 867(2020) 172811-172822.
Y.Y. Wei, B.F. Han, W.J. Dai, S.F. Guo, C.P. Zhang, L.X. Zhao, et al., Exposure to ozone impacted Th1/Th2 imbalance of CDT cells and apoptosis of ASMCs underlying asthmatic progression by activating lncRNA PVT1-miR-15a-5p/miR-29c-3p signaling, Aging-Us, 12(2020) 25229-25284.
X. Liang, J.J. Wang, W.W. Chen, X.Y. Ma, Y.Q. Wang, N. Nagao, et al., Inhibition of airway remodeling and inflammation by isoforskolin in PDGF-induced rat ASMCs and OVA-induced rat asthma model, Biomedicine & Pharmacotherapy, 95(2017) 275-286.
K. Amin, The role of mast cells in allergic inflammation, Resp Med, 106(2012) 9-14.
J. Kalesnikoff, S.J. Galli, New developments in mast cell biology, Nat Immunol, 9(2008) 1215-1223.
S.J. Galli, M. Tsai, IgE and mast cells in allergic disease, Nature Medicine, 18(2012) 693-704.
A.M. Gilfillan, C. Tkaczyk, Integrated signalling pathways for mast-cell activation, Nat Rev Immunol, 6(2006) 218-230.
J. Rivera, A.M. Gilfillan, Molecular regulation of mast cell activation, J Allergy Clin Immun, 117(2006) 1214-1225.
R. Sibilano, B. Frossi, C.E. Pucillo, Mast cell activation: A complex interplay of positive and negative signaling pathways, Eur J Immunol, 44(2014) 2558-2566.
R.P. Siraganian, R.O. de Castro, E.A. Barbu, J.A. Zhang, Mast cell signaling: The role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants, Febs Lett, 584(2010) 4933-4940.
M.A. Miranda, L.G. Magalhaes, R.F.J. Tiossi, C.C. Kuehn, L.G.R. Oliveira, V. Rodrigues, et al., Evaluation of the schistosomicidal activity of the steroidal alkaloids from fruits, Parasitol Res, 111(2012) 257-262.
Y.O. Son, J. Kim, J.C. Lim, Y. Chung, G.H. Chung, J.C. Lee, Ripe fruits of L. inhibits cell growth and induces apoptosis in MCF-7 cells, Food Chem Toxicol, 41(2003) 1421-1428.
A. Malik, M. Arooj, T.T. Butt, S. Zahid, F. Zahid, T.H. Jafar, et al., In silico and in vivo characterization of cabralealactone, solasodin and salvadorin in a rat model: potential anti-inflammatory agents, Drug Des Dev Ther, 12(2018) 1431-1443.
K. Chauhan, N. Sheth, V. Ranpariya, S. Parmar, Anticonvulsant activity of solasodine isolated from fruits in rodents, Pharm Biol, 49(2011) 194-199.
I. Margaill, M. Plotkine, D. Lerouet, Antioxidant strategies in the treatment of stroke, Free Radical Bio Med, 39(2005) 429-443.
S. Hendrix, P. Kramer, D. Pehl, K. Warnke, F. Boato, S. Nelissen, et al., Mast cells protect from post-traumatic brain inflammation by the mast cell-specific chymase, Faseb J, 27(2013) 920-929.
Y. Baba, K. Nishida, Y. Fujii, T. Hirano, M. Hikida, T. Kurosaki, Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses, Nat Immunol, 9(2008) 81-88.
T. Al-Shaikhly, R.C. Murphy, A. Parker, Y. Lai, M.C. Altman, M. Larmore, et al., Location of eosinophils in the airway wall is critical for specific features of airway hyperresponsiveness and T2 inflammation in asthma, Eur Respir J, 60(2022) 210865-210878.
E.B. Thangam, E.A. Jemima, H. Singh, M.S. Baig, M. Khan, C.B. Mathias, et al., The Role of Histamine and Histamine Receptors in Mast Cell-Mediated Allergy and Inflammation: The Hunt for New Therapeutic Targets, Front Immunol, 9(2018) 1873-1882.
N. Dholia, G.S. Sethi, A.S. Naura, U.C.S. Yadav, Cysteinyl leukotriene D(LTD) promotes airway epithelial cell inflammation and remodelling, Inflamm Res, 70(2021) 109-126.
K. Asayama, T. Kobayashi, C.N. D'Alessandro-Gabazza, M. Toda, T. Yasuma, H. Fujimoto, et al., Protein S protects against allergic bronchial asthma by modulating Th1/Th2 balance, Allergy, 76(2021) 1946-1953.
M. Cellat, M. Kuzu, C.T. Isler, M. Etyemez, N. Dikmen, A. Uyar, et al., Tyrosol improves ovalbumin (OVA)-induced asthma in rat model through prevention of airway inflammation, N-S Arch Pharmacol, 394(2021) 2061-2075.
S. Azman, M. Sekar, S. Wahidin, S.H. Gan, J. Vaijanathappa, S.R. Bonam, et al., Embelin Alleviates Severe Airway Inflammation in OVA-LPS-Induced Rat Model of Allergic Asthma, J Asthma Allergy, 14(2021) 1511-1525.
H. Zhang, L.L. Chen, X.P. Sun, Q.J. Yang, L.L. Wan, C. Guo, Matrine: A Promising Natural Product With Various Pharmacological Activities, Front Pharmacol, 11(2020) 588-606.
Y.A. Sun, L. Xu, Q.H. Cai, M.M. Wang, X.L. Wang, S.M. Wang, et al., Research progress on the pharmacological effects of matrine, Front Neurosci-Switz, 16(2022) 977374-977383.

No competing interests reported.

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Effect of solasodine on mast cell-mediated OVA (ovalbumin)- induced airway inflammation in asthma via the Fyn-Lyn-Syk pathway

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Abstract

Figures

1. Introduction

2. Experimental Methods

2.1. Drug and reagents

2.2. Cell culture

2.3. Toxicity of solasodine for cells

2.4. Cell groups and treatment

2.5. Western blot

2.6. ELISA

2.7. Measurement of β-hexosaminidase

2.8. Ca2+ concentration measurement

2.9. Immunofluorescence staining

2.10. Animal and asthma model

2.11. Animal treatment and groups

2.12. Bronchioalveolar lavage

2.13. Determination of cytokine levels in serum and BALF fluid

2.14 Western blot for Fyn-Lyn-Syk pathway-related protein expression

2.15. Airway hyperresponsiveness (AHR)

2.16. Determination of histamine in lung tissue

2.17. HE and Masson staining

2.18. Tunel staining

2.19. Statistical analysis

3. Results and Discussion

3.1. Effect of solasodine on TGF-β1-induced asthma cell model in BMMCs

3.2. Effect of solasodine on MC degranulation

3.3. Solasodine inhibits OVA-induced asthma symptoms in mice

3.4 Solasodine inhibits OVA-induced expression of Fyn, Lyn, Sky protein phosphorylation in mouse lung tissue

3.5 Effect of solasodine on histamine levels in lung tissues

3.6 Effect of solasodine on total nitric oxide and nitrite concentration

3.7 Solasodine ameliorates ova-induced airway inflammation and lung tissue fibrosis in mice

4. Discussion

5. Conclusion

Declarations

Data Availability statement

Declaration of competing interest

Funding

Author Contribution

References

Additional Declarations

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2.8. Ca²⁺ concentration measurement