Asthma attack is developed by two essential factors, inflammation and bronchoconstriction, that is mediated by smooth muscle contraction. Inflammation is beginning slowly in airway but, quick attack is initiated by contraction of the ASM and inhibition of the bronchoconstriction help to decrease morbidity and mortality from the asthma attack. Initiated bronchoconstriction from ASM contraction in asthma attack reduced air flow and decreasing responsiveness of the ASM in airways will attenuate the bronchoconstrictor response in asthma and can be achieved effective pharmacotherapy (3, 8). In asthma, ASMs are key of bronchoconstriction, which have muscarinic receptors while nicotinic acetylcholine receptors (nAChRs) are only on airway neurons. It was showed that α7 subunit of nAChR is expressed in ASM cells in asthma and by exposure to pro-inflammatory cytokines TNFα and IL-13. Classically, nicotine acts via nAChRs. In ASMs, contractility involves muscarinic receptors. Allergy in asthma increases ASMs Ca2 + and response to bronchoconstrictor agonists [such as acetylcholine (Ach)]. Effect of ACh on ASM is usually associated with muscarinic receptors, while nAChR is generally considered to acts as Ca2 + channels in pre-ganglionic neurons (9–11). On the other hand, TNF-α and IL-13 attribute to the increased CD38-mediated calcium release (12). Also, TNF-a induced muscarinic receptor density, increases the activity, as well as the amount, of G-proteins in ASM, and may also modulate b2-adrenergic function (13). On the day 24, Treatment with BTX for all dosages via IN and N had no significant effect on AHR, but on day 26 and 28, could significantly decrease AHR for concentrations of MCh. AHR decreasing was continued on day 30 for all treated groups and has significant decreasing compared to non-treat group for concentrations of MCh. Also, decreasing of AHR on day 30, had significant difference compared to BTX-treat groups on day 26 and 28.
In normal airways, ASMs regulate the airway caliber, bronchomotor tone and assist to the mucus and in airways, β-2-adrenoceptor agonists or muscarinic antagonists can modulate the contractile function of the ASM (13). AHR as the airways predisposition to narrow excessively in response to stimuli, is considered an asthma cardinal feature. The AHR presence is associated with lung function decline and increases risk for the asthma development and persistence of wheeze. However, increased contractility of the ASM is a principal cause of AHR and the β2-adrenergic receptor genotype influences AHR. On the other hand, in asthma, ASM contraction is formed by actin-myosin cross-bridges and the activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Hypertrophy of the constructed ASM and changes in extracellular matrix composition lead to airway remodeling and therefore, AHR has correlation with airway wall thickening and bronchoconstriction (14–16). Prevention of bronchoconstriction potentially reduces the asthma attacks severity and BTX allows chemical denervation transient of the ASM. BTX acts to weaken muscle by preventing the acetylcholine vesicle docking of the presynaptic membrane (on the inner surface), which causes chemical denervation and paresis of smooth or skeletal muscle. Duration of BTX effect was estimated at 3–5 months for skeletal muscle. Use of BTX to treat asthma would be worth exploring. BTX can be administrated to treat asthma with three ways: one, intranasally, trans-tracheally or intratracheally via bronchoscopy, two, injection to the smooth muscle and three, aerosolized as a nebulized solution (8). After the first dose of BTX, AHR (on day 24) had no significant changes, because 1 day passed and in the measurement time, BTX had not effected to the ASMs, therefore, AHR was not significantly decreased. On the day 30, the most doses had effect on the AMSc, and therefore, the best effect of BTX on AHR was observed on day 30. In all treated groups, when BTX`s doses were increased, the AHR was decreased more than lower dose.
Vocal Cord Dysfunction is a respiratory problem in which vocal cord restricts airflow by closing during inspiration that leads to coughing, shortness of breath, wheezing and chest tightness. This problem is often misdiagnosed as asthma and treated as such. It was shown that steroids used to asthma therapy are not beneficial in the treatment of Vocal Cord Dysfunction, and are therefore unnecessary. It is suggested that BTX can relax the thyroarythenoid muscles surrounding vocal cords resulting in improves airflow (17, 18). BTX is a neurotoxin that has a heavy and a light chain. The heavy chain binds to specific neuronal ecto-acceptor, the light chain cleaves SNAP-25, inhibits synaptic exocytosis, disables neural transmission and blocks the acetylcholine releasing (19). BTX has therapeutic effect on allergic rhinitis symptoms and is useful in patients whose symptoms are non-control with standard treatment. The BTX has the greatest change in rhinorrhea compared with other symptoms. The BTX effects on the nasal cavity occurs through; acetylcholine release inhibition from the cholinergic nerve in the nasal mucosa, from preganglionic cholinergic nerve in the sphenopalatine ganglion, and apoptosis induction in the nasal gland. It acts by cleaving the synaptosomal-associated protein with a molecular weight of 25 kD (SNAP-25) and after damages SNAP-5, regulates exocytosis (20, 21). We administrated BTX via intra-nasal and nebulizing form. It was observed that in the same dose of BTX with different routs of administration, when BTX was used via nebulizing form, had strong effect on control of AHR compared to intra-nasal using form for all concentration.
Mild dose of the purified BTX attenuates chronic pneumonia, dyspnea, cough, acute respiratory failure, and neurological deficits that were recognized as the clinical symptoms of COVID-19. Therapeutic BTX improves oxygen supply thereby improving the survival rate, which can also be considered as a potent treatment for COVID-19 patients (22). In our study, all asthmatic mice that were received the first dose of BTX (10 mg/ml) via IN and N, and some groups after receiving 1 mg/ml were died. It may be happen for the effect of BTX on respiratory system and breathing related muscles that leads to paralysis and relaxation of muscles. Therefore, using of BTX for treatment of asthma and controlling of AHR, should be used carefully and the safe dose of BTX should be determined. It is a strong treatment to control of AHR and asthma attack, but can lead to respiratory paresis and death of asthmatic patients.
The nAChRs are muscle and neuronal types (23, 24) and the ERK as effector kinase is involved signaling pathway of the multiple essential cell processes such as survival, proliferation and differentiation (25, 26). TrkA is a receptor tyrosine kinase, and activated by Nerve growth factor (NGF) and auto-phosphorylation. Since TrkA receptor is expressed on the ASMs, it may be importance in asthma. However, NGF expression is increased in various allergic diseases, and TrkA receptor activation by NGF causes its internalization mediated by clathrin-dependent pathways, followed by lysosomal degradation. TrkA activation in the ASMs leads to increased TrkA expression and increased pathophysiological consequences. Therefore the TrkA receptor as an interesting target in the development of new therapeutic approaches for allergic asthma (27). Treatment with BTX via IN and N in asthma mice had no significant effect on the expressions of TrkA, TrkB, M1, M2, M3, M4, M5, α7nAChR, TNF-α and genes, perivascular inflammation, peribronchial inflammation, hyperplasia of the goblet cell and production of mucus compared to the non-treated asthma group.