Application of low-intensity focused ultrasound in preparation of noninvasive asthma treatment product

Abstract

The invention provides application of low-intensity focused ultrasound in preparation of a noninvasive asthma treatment product, and belongs to the technical field of asthma treatment. The low-intensity focused ultrasound treats the asthma by regulating and controlling the activity of the spleen nerves, the low-intensity focused ultrasound has high targeting, accuracy and durability on the asthma, and the spleen histological injury or dysfunction of adjacent organs (such as pancreas and kidneys) is not observed, so that the effectiveness and safety of the technology are proved.

Description

Technical Field

[0001] This invention belongs to the field of asthma treatment technology, specifically involving the application of low-intensity focused ultrasound in the preparation of non-invasive asthma treatment products. Background Technology

[0002] Bronchial asthma is a heterogeneous disease characterized by chronic airway inflammation and airway remodeling. Current clinical treatment primarily relies on inhaled corticosteroids, long-acting β2-receptor agonists, and targeted therapies. However, these treatments all have limitations: First, long-term use of inhaled corticosteroids may lead to local side effects such as oral candidiasis and hoarseness, while systemic corticosteroid use is closely associated with risks of osteoporosis and metabolic syndrome. Second, some patients with moderate to severe asthma do not respond well to existing medications and are classified as having "refractory asthma." While bronchial thermoplasty for severe patients can reduce airway smooth muscle mass through radiofrequency ablation, this technique is invasive and may cause complications such as bronchospasm and atelectasis, and its efficacy is limited in duration. These clinical challenges have created an urgent need for novel, non-pharmacological, non-invasive therapies that precisely regulate the immune microenvironment.

[0003] In recent years, groundbreaking advances in neuroimmunology have provided a novel perspective for asthma treatment. However, existing neuromodulation techniques face significant challenges: while vagal nerve stimulation can suppress systemic inflammatory responses by activating cholinergic anti-inflammatory pathways, it requires surgical electrode implantation, posing risks such as infection and nerve damage; optogenetics, although capable of cell-specific neuromodulation, relies on viral vector-mediated gene editing, facing ethical approval and safety hurdles in clinical application; and drug interventions (such as β-receptor agonists) lack tissue specificity and are prone to causing cardiovascular side effects. Therefore, developing a non-invasive, reversible, and spatially precise neuromodulation technique for asthma treatment has become crucial to overcoming current treatment bottlenecks. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of low-intensity focused ultrasound in the preparation of non-invasive asthma treatment products. Low-intensity focused ultrasound (LIFUS) has high targeting, accuracy and persistence for asthma, and no histological damage to the spleen or functional abnormalities of adjacent organs (such as pancreas and kidneys) were observed, which confirms the effectiveness and safety of the technology.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides the application of low-intensity focused ultrasound in the preparation of non-invasive asthma treatment products.

[0006] Preferably, the low-intensity focused ultrasound is used to target and modulate the activity of the spleen nerve to treat asthma.

[0007] Preferably, the power of the low-intensity focused ultrasound is 0.5-2.5W and the total time is 8-12min.

[0008] Preferably, the low-intensity focused ultrasound selects a pulse mode, in which the stimulation duration is 0.5-1.5s, the interval is 4-6s, and the frequency is 640-660KHz.

[0009] This invention provides a non-invasive treatment system for asthma, comprising a coupling agent filling module and an abdominal ultrasound treatment module; the coupling agent filling module is used to fill the gap between the probe and the skin with coupling agent, and the abdominal ultrasound treatment module is used to perform ultrasound stimulation on the spleen of the subject.

[0010] Preferably, the coupling agent includes sterile ultrasonic gel, electrolyte hydrogel, bioadhesive gel, ionic liquid gel, or biodegradable gel.

[0011] Preferably, the abdominal ultrasound treatment module includes an abdominal ultrasound generator module, an abdominal ultrasound probe module, and an abdominal ultrasound control module; the abdominal ultrasound generator module is used to generate mechanical vibration waves; the abdominal ultrasound probe module is used to transmit ultrasound waves into the subject's body to provide ultrasound stimulation to the subject's abdomen; and the abdominal ultrasound control module is used to control the ultrasound parameters output by the abdominal ultrasound generator module.

[0012] Preferably, the abdominal ultrasound probe module is in contact with the subject's skin to ensure that the sound beam axis passes through the center of the spleen's long axis.

[0013] Preferably, the ultrasound stimulation includes: stimulation at a power of 0.5-2.5W for 8-12 minutes; the ultrasound stimulation is performed in pulse mode, with a stimulation duration of 0.5-1.5s, an interval of 4-6s, and a frequency of 640-660KHz.

[0014] This invention provides the application of the non-invasive treatment system for asthma in the preparation of products for treating asthma.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to propose a non-invasive treatment of asthma using low-intensity focused ultrasound by modulating splenic nerve activity, which has the following significant advantages compared to existing technologies: Non-invasive and highly safe: Completely non-invasive, requiring no surgery or implantation devices, it uses low-intensity focused ultrasound as a physical stimulus, with an intensity below the tissue damage threshold, and uses non-thermal effects to dominate neural modulation, avoiding many risks associated with traditional invasive techniques.

[0016] High targeting and precise regulation: Combining ultrasound phased array sensors and real-time imaging technology, it can precisely target the spleen nerve and its associated immune regulation circuits, and can also flexibly control nerve excitation or inhibition by adjusting ultrasound parameters to optimize the immune regulation effect.

[0017] Bidirectional immune regulation and long-lasting therapeutic effect: It can bidirectionally regulate the immune response by modulating the activity of the spleen nerve, and provide fundamental intervention for asthma at the neuro-immune axis level.

[0018] Compatibility and clinical applicability: It can be used in conjunction with existing asthma treatments and is suitable for patients with drug-resistant asthma and those who are contraindicated for traditional therapies. The LIFUS treatment device can also be integrated with magnetic resonance imaging or ultrasound-guided systems to achieve real-time monitoring and dynamic parameter adjustment. Attached Figure Description

[0019] Figure 1 Flowchart for modeling asthmatic mice and ultrasound stimulation.

[0020] Figure 2 The results show the airway resistance test results in mice. P < 0.05.

[0021] Figure 3 The results show the serum liver, kidney, and cardiac function of mice. NS (Neutral Stroke Scale) showed no statistical significance.

[0022] Figure 4 The results of HE and PAS staining of lung tissue from each treatment group are shown. A. Representative HE staining images of lung tissue from each group; B. Representative PAS staining images of lung tissue from each group; C. Statistical results of HE staining inflammation scores for each group; D. Statistical results of PAS staining scores for each group. Scale bars are 2 mm, 200 μm, and 50 μm. Six mice were used in each group, and the results are expressed as mean ± standard error. P < 0.05.

[0023] Figure 5 HE staining results of mouse spleen. Detailed Implementation

[0024] This invention provides the application of low-intensity focused ultrasound (HIFU) in the preparation of non-invasive asthma treatment products. HIFU is a non-invasive neuromodulation tool that, through the interaction of mechanical waves with neuronal ion channels, modulates nerve excitability or inhibition with millisecond-level precision, and the energy intensity can be controlled within a safe threshold to avoid tissue thermal damage. Unless otherwise specified, the devices used for HIFU in this invention are all commercially available devices well-known in the art; the preferred device is a low-power focused ultrasound experimental device (UPG series), sourced from Shenzhen Huanying Medical Technology Co., Ltd.

[0025] In this invention, low-intensity focused ultrasound is used to target and modulate splenic nerve activity to treat asthma. This invention combines an ultrasound phased array sensor with real-time imaging technology to precisely target the splenic nerve and its associated immune regulatory circuits. Furthermore, it allows for flexible control of nerve excitation or inhibition by adjusting ultrasound parameters, optimizing the immunomodulatory effect. This invention can bidirectionally regulate the immune response by modulating splenic nerve activity, providing fundamental intervention for asthma at the neuro-immune axis level.

[0026] In this invention, the power of the low-intensity focused ultrasound (HIFU) is 0.5-2.5W and the total time is 8-12 minutes, preferably 0.8-2.2W and 9-11 minutes, more preferably 1W and 10 minutes, or 2W and 10 minutes. Excessive power or prolonged duration of the HIFU may cause spleen damage. Appropriate HIFU power and total time can effectively and non-invasively treat asthma without damaging the spleen.

[0027] In this invention, the low-intensity focused ultrasound uses a pulsed mode with a stimulation duration of 0.5-1.5 s, an interval of 4-6 s, and a frequency of 640-660 kHz; preferably, the stimulation duration is 0.8-1.3 s, the interval is 4.5-5.5 s, and the frequency is 645-655 kHz; more preferably, the stimulation duration is 1 s, the interval is 5 s, and the frequency is 650 kHz. The pulsed mode of this invention, by intermittently emitting ultrasound waves (duty cycle typically 10-50%), allows the tissue to dissipate heat during the pulse intervals, avoiding cumulative temperature rise (>1°C) caused by continuous waves and preventing tissue thermal damage.

[0028] This invention provides a non-invasive treatment system for asthma, comprising a coupling agent filling module and an abdominal ultrasound treatment module; the coupling agent filling module is used to fill the gap between the probe and the skin with coupling agent, and the abdominal ultrasound treatment module is used to perform ultrasound stimulation on the spleen of the subject. The coupling agent of this invention includes one or more of sterile ultrasound gel, electrolyte hydrogel, bioadhesive gel, ionic liquid gel, and biodegradable gel, preferably sterile ultrasound gel.

[0029] In this invention, the abdominal ultrasound treatment module includes an abdominal ultrasound generator module, an abdominal ultrasound probe module, and an abdominal ultrasound control module. The abdominal ultrasound generator module generates mechanical vibration waves, i.e., ultrasound waves. The abdominal ultrasound probe module transmits the ultrasound waves into the subject's body to provide ultrasound stimulation to the subject's abdomen. The abdominal ultrasound control module controls the ultrasound parameters output by the abdominal ultrasound generator module. The abdominal ultrasound probe module of this invention is placed in close contact with the subject's skin with gentle pressure to ensure that the sound beam axis passes through the center of the spleen's long axis.

[0030] In this invention, the ultrasound stimulation includes: stimulation at a power of 0.5-2.5W for 8-12 minutes, preferably at a power of 0.8-2.2W for 9-11 minutes, more preferably at a power of 1W for 10 minutes, or at a power of 2W for 10 minutes. The ultrasound stimulation of this invention selects a pulse mode, in which the stimulation duration is 0.5-1.5s, the interval is 4-6s, and the frequency is 640-660kHz; preferably, the stimulation duration is 0.8-1.3s, the interval is 4.5-5.5s, and the frequency is 645-655kHz; more preferably, the stimulation duration is 1s, the interval is 5s, and the frequency is 650kHz.

[0031] This invention provides the application of the non-invasive treatment system for asthma in the preparation of products for treating asthma.

[0032] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0033] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] In the following embodiment, the nebulization process is as follows: add sterile saline or 6% OVA activation solution or other solvents, connect to the nebulization box, and use a Yuwell nebulizer (model 403C, source: Suzhou Yuwell Medical Equipment Co., Ltd.) for nebulization.

[0035] The following examples use tribromoethanol as an anesthetic for experimental animals. Its advantages include: (1) high safety: wide dosing window, making it extremely difficult for experimental animals to die due to dosage error; (2) long-lasting effect: a single dose can maintain the anesthetic state for about 30 minutes, meeting the operational requirements.

[0036] Preparation of anesthetic: Mix 25g of tribromoethanol powder with 15.5mL of 2-methyl-2-butanol, vortex until completely dissolved, to prepare a concentrated stock solution of 1.6g / mL; allow to stand at room temperature for ≥12h under light protection to ensure complete dissolution before aliquoting and storage. Preparation of tribromoethanol working solution: Dilute 0.5mL of the stock solution and add it to 39.5mL of phosphate buffered saline (PBS, pH 7.4), mix well in a shaker at 4℃ for 30min; the working solution should be stored in the dark (4℃).

[0037] Anesthesia procedure: Tribromoethanol working solution is injected intraperitoneally at a standard rate of 20 μL / g body weight; Onset time: 3-5 minutes after injection to enter the anesthetic state; Duration: 20-30 minutes.

[0038] The ultrasound stimulation method included: 1) After hair removal on the left side of the mouse's abdomen, the spleen was positioned at the lower edge of the left costal arch, close to the abdominal wall. The equivalent localization area for the mouse was set at the midpoint of the line connecting the shoulder and hip joints; 2) Sterile ultrasound gel was used to fill the gap between the probe and the skin to avoid sound energy attenuation. The probe was gently pressed perpendicularly to the skin to ensure that the sound beam axis passed through the center of the spleen's long axis; 3) A pulse mode was selected, with a stimulation duration of 1 second, an interval of 5 seconds, a frequency of 650 kHz, a specific electrical power, and a total action time. The device used was a low-power focused ultrasound experimental device (UPG series), sourced from Shenzhen Huanying Medical Technology Co., Ltd.

[0039] Example 1 A non-invasive treatment system for asthma includes a coupling agent filling module and an abdominal ultrasound treatment module; the coupling agent filling module is used to fill the gap between the probe and the skin with coupling agent, and the abdominal ultrasound treatment module is used to perform ultrasound stimulation on the spleen of the subject; the coupling agent is a sterile ultrasound gel; The abdominal ultrasound therapy module includes an abdominal ultrasound generator module, an abdominal ultrasound probe module, and an abdominal ultrasound control module. The abdominal ultrasound generator module is used to generate mechanical vibration waves, i.e., ultrasound waves. The abdominal ultrasound probe module is used to transmit ultrasound waves into the subject's body to provide ultrasound stimulation to the subject's abdomen. The abdominal ultrasound control module is used to control the ultrasound parameters output by the abdominal ultrasound generator module. The abdominal ultrasound probe module is pressed perpendicularly to the subject's skin to ensure that the sound beam axis passes through the center of the spleen's long axis. The ultrasound stimulation is performed at 1W power for 10 minutes or at 2W power for 10 minutes; the ultrasound stimulation is performed in pulse mode, with a stimulation duration of 1 second, an interval of 5 seconds, and a frequency of 650KHz.

[0040] Example 2 1. Effects of LIFUS stimulation of the spleen on airway resistance in asthmatic mice (1) Preparation of sensitizing solution Sensitization solution: For each mouse, 20 μg of ovalbumin (OVA) and 2 mg of aluminum hydroxide powder were dissolved in 300 μL of 0.9% sodium chloride solution to prepare a 300 μL OVA sensitization mixture. The total amount of sensitization solution was prepared according to the number of sensitized mice. After preparation, the solution was incubated at 4°C on a shaker for 1 hour. The control group was treated with an equal volume of 0.9% sodium chloride solution instead of the OVA sensitization mixture.

[0041] (2) Preparation of activating solution 6% OVA activation solution: Dissolve OVA (600mg) in 0.9% sodium chloride solution (10mL), mix thoroughly and place on an ice box. The activation solution should be prepared fresh for use.

[0042] (3) Establishment of asthma model and experimental grouping Eight-week-old female BALB / c mice were randomly divided into six groups: normal control (NC), model group (OVA), low-intensity focused ultrasound (HIFU) stimulation group (1 week 10 min), HIFU stimulation group (1 week 20 min), HIFU stimulation group (2 weeks 10 min), HIFU stimulation group (2 weeks 20 min), and dexamethasone (DEX) group. The modeling process and ultrasound stimulation were as follows: Figure 1 As shown. The experimental grouping and processing are as follows: Normal control group: Mice were intraperitoneally injected (ip) with 300 μl of sterile saline on days 1, 8 and 15; mice were nebulized with sterile saline for 30 min each time from day 22 to 26 for 5 consecutive days, once a day.

[0043] Model group (ovalbumin group): Mice were sensitized by intraperitoneal injection (ip) of 300 μl of OVA sensitization mixture on days 1, 8, and 15. Mice were then challenged by nebulization with 6% OVA challenge solution for 30 min each time from day 22 to 26, once a day.

[0044] Low-intensity focused ultrasound stimulation - 1W10min group (LIFUS 1W10min): Based on the model group, after 30min of stimulation with 6% OVA stimulation solution every day, the spleen was stimulated with low-intensity focused ultrasound at 1W power for 10min at 1h interval. The ultrasound stimulation method is as described above.

[0045] Low-intensity focused ultrasound stimulation - 1W20min group (LIFUS 1W20min): Based on the model group, after 30min of stimulation with 6% OVA stimulation solution every day, the spleen was stimulated with low-intensity focused ultrasound at 1W power for 20min at 1h interval. The ultrasound stimulation method is as described above.

[0046] Low-intensity focused ultrasound stimulation - 2W10min group (LIFUS 2W10min): Based on the model group, after 30min of stimulation with 6% OVA stimulation solution every day, the spleen was stimulated with low-intensity focused ultrasound at 2W power for 10min at 1h interval. The ultrasound stimulation method is as described above.

[0047] Low-intensity focused ultrasound stimulation - 2W20min group (LIFUS 2W20min): Based on the model group, after 30min of stimulation with 6% OVA stimulation solution every day, the spleen was stimulated with low-intensity focused ultrasound at 1W power for 20min at 1h interval. The ultrasound stimulation method is as described above.

[0048] Dexamethasone group: Based on the model group, the dexamethasone group was nebulized with 6% OVA stimulation solution for 30 minutes every day, followed by intraperitoneal injection of DEX 3 mg / kg every 1 hour.

[0049] 2. Airway resistance measurement After the final treatment of each group in step 1 above, airway resistance was measured 24 hours later.

[0050] (1) Animal anesthesia and pulmonary function testing Experimental mice were induced to anesthetize via intraperitoneal injection of tribromoethanol working solution. After entering a stable anesthesia state, the tests were performed according to the standard operating procedures of the Buxco pulmonary function testing system. Airway responsiveness was stimulated by nebulized inhalation of gradient concentrations of methacholine (0, 3.125, 6.25, 12.5, 25, and 50 mg / mL, respectively), and airway resistance parameters of each group of mice were collected and analyzed in real time.

[0051] (2) Endotracheal intubation procedure 24 hours after the last nebulization challenge, endotracheal intubation was performed according to the following steps: Preoperative preparation: After anesthetizing the mice, the surgical area of ​​the neck was disinfected with 75% ethanol; Tracheal exposure: The skin was incised along the midline of the neck, and the subcutaneous tissue and muscle layer were bluntly dissected to fully expose the trachea; A 2mm transverse incision was made in the middle of the trachea, and an 18G cannula (approximately 5mm deep) was inserted and fixed with silk sutures; Spontaneous breathing and vital signs were observed to ensure that the intubation position was correct.

[0052] (3) Measurement of pulmonary function parameters The ventilator parameters were set as follows: frequency 140 breaths / min, tidal volume 0.25 mL, and equilibration time 5 min.

[0053] Detection procedure: baseline value determination: record resting airway resistance for 1 min; challenge test: 15 μL of PBS and 15 μL of different concentrations of methacholine (3.125, 6.25, 12.5, 25, 50 mg / mL) were nebulized sequentially, and the resistance value (Rn) was recorded for 3 min after each challenge.

[0054] Airway resistance is a core indicator of asthma pathophysiology, reflecting the degree of obstruction encountered by airflow as it passes through the airways. Figure 2The experimental results showed that, compared with the NC group, the airway resistance of mice in the OVA group was significantly increased. After stimulation of the spleen with LIFUS-2W10 or LIFUS-2W20 parameters, the airway resistance value of mice was significantly reduced, indicating that stimulation of the spleen with LIFUS-2W10 or LIFUS-2W20 parameters can reduce airway resistance in asthmatic mice and improve airway hyperresponsiveness and airflow limitation.

[0055] 3. Blood biochemical analysis of the effects of different ultrasound stimulation parameters on liver, kidney, and cardiac function in mice. Serum Collection: Blood samples were collected by enucleation while the pulmonary function tests were completed and the experimental animals were still under anesthesia. After collecting blood using EDTA anticoagulant blood collection tubes, the tubes were immediately and gently inverted 8-10 times to ensure adequate anticoagulation. The collected blood samples were then placed vertically in a 4°C refrigerator for 1 hour to allow for complete coagulation. Subsequently, the serum components were separated by centrifugation at 3000×g for 10 minutes at 4°C. The pale yellow supernatant formed after centrifugation was carefully aspirated and transferred to pre-chilled sterile centrifuge tubes. The samples were immediately labeled with the sample number and processing date and stored at -80°C.

[0056] When assessing the safety of ultrasound stimulation of the spleen, liver, kidney, and cardiac function are key indicators. By detecting these indicators in mice, the potential toxic effects of ultrasound stimulation can be effectively evaluated. Indicators for evaluating liver function include alanine aminotransferase (ALT) and aspartate aminotransferase (AST); indicators for evaluating kidney function include urea (UREA); and indicators for evaluating cardiac function include creatine kinase (CK) and lactate dehydrogenase (LDH). The kits used were: Alanine aminotransferase (ALT) assay kit (S03030, 20240918, Redu), Aspartate aminotransferase (AST) assay kit (S03040, 20241008, Redu), Urea assay kit (S03036, 20240813, Redu), Creatine kinase assay kit (S03024, 20241225, Redu), and Lactate dehydrogenase assay kit (S03034, 20250108, Redu).

[0057] Figure 3 The results showed that, compared with the NC group, the levels of ALT, AST, UREA, CK, and LDH in mice treated with low-intensity focused ultrasound (HIFU) did not change significantly, and the results were not statistically significant (P>0.05). These results indicate that HIFU stimulation of the spleen does not cause significant hepatotoxicity, nephrotoxicity, or cardiotoxicity.

[0058] 3. Effects of LIFU stimulation of the spleen on pathological changes in lung tissue of asthmatic mice HE staining and PAS staining were used to observe pathological changes in lung tissue in order to determine whether low-intensity focused ultrasound stimulation of the spleen could improve pathological changes in the lungs of asthmatic mice.

[0059] (1) HE staining of lung tissue Tissue fixation: Mouse lung tissue samples were immediately immersed in 4% paraformaldehyde fixative, ensuring complete tissue immersion, and fixed at 4°C for 48 hours. After fixation, the samples were rinsed with deionized water to remove residual fixative. Gradient dehydration procedure: The washed tissue samples were placed in a dedicated dehydration box and dehydrated sequentially through a gradient of 30%, 50%, 70%, 80%, 95%, and 100% ethanol, with each concentration dehydrating for 1 hour. Paraffin embedding process: After dehydration, the tissue samples were cleared with xylene for 30 minutes and then immersed in molten paraffin at 60°C for 3 hours. Oriented embedding was performed using embedding molds, and the sample number and embedding orientation were marked on the surface of the paraffin block. Section preparation process: The embedded paraffin block was pre-cooled at -20℃ for 30 min. The block was trimmed using a microtome to expose the complete tissue section. The section thickness was adjusted to 5 μm for continuous sections. The slide was preheated to 37℃, and deionized water was added to form a liquid film. The paraffin strip was gently spread, avoiding wrinkles, and finally dried in a 42℃ oven for 2 h. Section pretreatment: The prepared lung tissue paraffin sections were placed in a 60℃ constant temperature oven for 1 h of paraffin melting to ensure complete melting. Subsequently, two dewaxing treatments were performed using xylene solution, each soaking for 10 min to thoroughly remove the paraffin components from the tissue. Gradient hydration treatment: The dewaxed tissue sections were sequentially hydrated with a gradient of 100% ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 5 min at each concentration. Finally, the sections were rinsed with running tap water for 5 min to complete the hydration process. Staining procedure: Hematoxylin staining for 5 min to stain the nuclei; differentiation with 5% acetic acid solution for 10 s to remove non-specific staining; rinsing with running distilled water for 3 min to restore the blue color of the nuclei; staining with eosin solution for 3 min to make the cytoplasm red; finally rinsing with running tap water for 4 min to remove excess staining. Dehydration and mounting: Stained sections were sequentially dehydrated using a gradient of 70%, 80%, 95%, and 100% ethanol, each concentration treated for 10 s. After air drying, mounting was performed using neutral resin to ensure long-term preservation of the tissue sections.

[0060] The results are as follows Figure 4 A and Figure 4As shown in Figure C, HE staining pathological analysis of lung tissue revealed that OVA-sensitized mice exhibited typical airway inflammation characteristics: ① significant inflammatory cell aggregation around the trachea and blood vessels; ② marked narrowing of the bronchial lumen. After LIFUS intervention, these pathological changes were significantly improved, manifested as: ① reduced degree of inflammatory infiltration around the airways / blood vessels; ② effective relief of airway narrowing. The above HE staining results suggest that low-intensity focused ultrasound stimulation of the spleen can reduce the infiltration of inflammatory cells around the airways in asthmatic mice and slow the spread of airway inflammation.

[0061] (2) PAS staining of lung tissue sections To investigate the effect of LIFUS on goblet cell proliferation in airway remodeling in asthmatic mice, PAS staining was performed on mouse lung tissue. The steps are as follows: Dewaxing: Place tissue sections in a 60℃ incubator for 1 hour to dewax. Dewaxing and Hydration: Dewax paraffin sections in xylene for 10 minutes each time, twice; then hydrate sequentially with a gradient of ethanol (100%, 90%, 80%, 70%) for 5 minutes each time, and finally rinse with distilled water. Periodic Acid Oxidation: Immerse sections in a 1% periodic acid solution and oxidize at room temperature for 10 minutes. The recommended oxidation temperature is 18-22℃. Then rinse sections thoroughly with distilled water to remove excess periodic acid. Schiff Reagent Staining: Immerse sections in Schiff reagent and stain at room temperature in the dark for 10 minutes. The staining time can be adjusted according to room temperature; it can be extended to 20 minutes when the room temperature is low. Then rinse sections with distilled water until the running water changes from red to colorless. Hematoxylin Counterstaining: Immerse sections in hematoxylin staining solution to stain cell nuclei for 1-3 minutes, then rinse with running water for 5 minutes to allow the cell nuclei to turn blue again. Dehydration and mounting: The slides were sequentially dehydrated with a gradient of ethanol (70%, 80%, 95%, 100%), each time soaking for 3 minutes; cleared with xylene, each time soaking for 5 minutes, for a total of 2 times; mounted with neutral resin, and then air-dried before microscopic observation.

[0062] The results showed that, compared with the normal control group, goblet cells were significantly increased in the lung tissue of OVA-sensitized mice, and these cells showed a PAS-positive reaction. However, LIFUS was able to significantly inhibit the excessive proliferation of goblet cells. Figure 4 B). Further PAS staining analysis showed that the percentage of PAS-positive cells decreased significantly after LIFUS treatment ( Figure 4 D). These results suggest that LIFUS can significantly reduce airway inflammation and abnormal goblet cell proliferation in asthmatic mice.

[0063] The results suggest that LIFUS-2W20 parameter stimulation of the spleen can significantly reduce airway inflammation and inhibit goblet cell proliferation in asthmatic mice.

[0064] 4. HE staining to assess the effects of different ultrasound stimulation parameters on mouse spleen HE staining of the spleen is an important pathological assessment method when evaluating the safety of ultrasound stimulation of the spleen. HE staining of the spleen allows observation of microstructural changes in the spleen tissue, including cell morphology, tissue integrity, and the presence of inflammatory cell infiltration. Splenic samples from mice stimulated with different ultrasound parameters were collected for HE staining to observe spleen damage. The HE staining procedure was the same as in step 3(1).

[0065] Figure 5 HE staining results showed that the spleen tissue structure in the NC, LIFUS-1W10, and LIFUS-2W10 groups was normal, with neatly arranged cells and no obvious inflammatory cell infiltration, hemorrhage, or necrosis. In the LIFUS-1W20 group, a few isolated lymphocytes showed punctate necrosis with pyknosis (black arrows) in the white pulp. In the LIFUS-2W20 group, a small number of lymphocytes showed punctate necrosis with pyknosis (black arrows) and a small amount of brownish-yellow pigment deposition (blue arrows) in the white pulp. This indicates that ultrasound stimulation of the spleen with LIFUS-1W20 and LIFUS-2W20 parameters caused splenic cell damage, while LIFUS-1W10 and LIFUS-2W10 did not cause splenic cell damage and are safe ultrasound stimulation parameters. Based on safety, LIFUS-2W10 showed better efficacy and is the optimal indicator.

[0066] In summary, the experimental results show that no histological damage to the spleen or functional abnormalities in adjacent organs (such as the pancreas and kidneys) were observed in ovalbumin-induced asthmatic mice after LIFUS intervention, confirming the effectiveness and safety of this technology. These findings not only fill the research gap in the field of peripheral neuroimmunomodulation using LIFUS, but also provide a new tool for personalized treatment of asthma.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of low intensity focused ultrasound in the preparation of a product for non-invasive treatment of asthma.

2. Use according to claim 1, wherein The low intensity focused ultrasound targets and regulates the activity of the splenic nerve to treat asthma.

3. The use according to claim 1, wherein The low intensity focused ultrasound has a power of 0.5-2.5 W and a total time of 8-12 min.

4. The use according to claim 1, wherein The low intensity focused ultrasound selects a pulse mode in which the stimulation duration is 0.5-1.5 s, the interval is 4-6 s, and the frequency is 640-660 KHz.

5. A non-invasive treatment system for asthma, characterized by, The system comprises a coupling agent filling module and an abdominal ultrasound treatment module; the coupling agent filling module is used to fill the gap between the probe and the skin, and the abdominal ultrasound treatment module is used to stimulate the spleen of the subject.

6. The non-invasive treatment system for asthma of claim 5, wherein, The coupling agent comprises one or more of sterile ultrasound gel, electrolyte hydrogel, bioadhesive gel, ionic liquid gel, and biodegradable gel.

7. The non-invasive treatment system for asthma as claimed in claim 5 wherein, The abdominal ultrasound treatment module comprises an abdominal ultrasound generator module, an abdominal ultrasound probe module, and an abdominal ultrasound control module. The abdominal ultrasound generator module is used to generate mechanical vibration waves; the abdominal ultrasound probe module is used to transmit ultrasound waves into the body of the subject to stimulate the abdomen of the subject; and the abdominal ultrasound control module is used to control the parameters of the ultrasound waves output by the abdominal ultrasound generator module.

8. The non-invasive treatment system for asthma of claim 7, wherein, The abdominal ultrasound probe module is in contact with the skin of the subject to ensure that the sound beam axis passes through the center of the long diameter of the spleen.

9. The non-invasive treatment system for asthma as claimed in claim 5 wherein, The ultrasound stimulation comprises stimulation at a power of 0.5-2.5 W for 8-12 min; and the ultrasound stimulation selects a pulse mode in which the stimulation duration is 0.5-1.5 s, the interval is 4-6 s, and the frequency is 640-660 KHz.

10. Use of the system for non-invasive treatment of asthma according to any one of claims 5-9 in the preparation of a product for treatment of asthma.

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