Vagus nerve electrical stimulation regulation method capable of relieving ARDS lung injury

Abstract

The invention discloses a vagus nerve electrical stimulation regulation method capable of relieving ARDS lung injury, and relates to the technical field of ARDS lung injury. In order to improve the therapeutic effect and prognosis of ARDS patients; comprising the following steps that 1, an ARDS animal model is established, and the animal model is constructed by instilling lipopolysaccharide (LPS) into the trachea; 2, after the ARDS model is established, separating and exposing the left vagus nerve of the animal; and 3, performing electrical stimulation on the left vagus nerve by using an electrical stimulation device, wherein the stimulation parameters comprise the voltage of 5V, the frequency of 5Hz and the pulse width of 2ms, and the stimulation lasts for 10 minutes. By implementing a vagus nerve electrical stimulation (VNS) regulation method, lung tissue pathological changes of an ARDS rat model can be remarkably improved, such as alveolar wall thickening, alveolar cavity stenosis and inflammatory cell infiltration, the lung injury score and the lung moisture-to-dry ratio are remarkably reduced, and the pulmonary edema condition is effectively relieved.

Description

Technical Field

[0001] The present invention relates to the technical field of ARDS lung injury, and in particular to a vagus nerve electrical stimulation regulation method capable of alleviating ARDS lung injury. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is an fulminant disease with a high mortality rate, characterized by acute, diffuse and inflammatory lung damage, which may lead to a mortality rate of more than 40%. Although the medical community has conducted in-depth research on the pathogenesis of ARDS, it has not yet been fully clarified. Existing studies have pointed out that the occurrence and outcome of ARDS involve the activation of multiple inflammatory cells and the release of inflammatory mediators. However, the excessive recruitment and activation of these inflammatory cells often lead to an imbalance of inflammatory response in the lungs and even the whole body, becoming a key factor in the deterioration of ARDS.

[0003] Currently, the main treatment for ARDS is still based on mechanical ventilation. Mechanical ventilation is done by inserting a plastic tube into the trachea through the nose or mouth, or directly inserting it into the trachea through a tracheotomy, and connecting it to a ventilator to ensure that the patient's lungs get enough oxygen. However, mechanical ventilation is not a perfect solution. It may cause complications such as pneumothorax and infection, and cannot effectively control infection and extrapulmonary organ failure, both of which are the main causes of death in ARDS. In addition, mechanical ventilation also requires feeding through a gastric tube to maintain the patient's nutrition, which increases the complexity of treatment and the pain of the patient.

[0004] In addition to mechanical ventilation, drug therapy is also an important means of treating ARDS. Hormone therapy has received much attention, but research results are divergent. Although hormone therapy can reduce the infection rate of complications and shorten the duration of mechanical ventilation, its applicability is not widespread and not all early patients are suitable for it. Therefore, hormone therapy for ARDS requires a personalized plan.

[0005] In addition, there are other drug treatment options, such as vitamins C and D, aspirin, heparin, inhaled nitric oxide, inhaled prostaglandins, β2 receptor agonists, etc. However, the effects of these drug treatments have not been clearly verified, and there is no clear ARDS drug that can significantly reduce short-term or long-term mortality;

[0006] Given the limitations of existing treatments, it is particularly important to find new and more effective treatments for ARDS. Vagus nerve stimulation (VNS), as an innovative treatment, was initially used to treat refractory epilepsy and long-term and recurrent depression in adults. VNS may have the ability to sense inflammation and inhibit inflammatory responses by affecting the hypothalamic pituitary-adrenal axis and regulating the autonomic nervous system that controls major immune organs. Therefore, applying VNS to the treatment of ARDS is expected to bring new treatment hope to ARDS patients by regulating the balance of inflammatory responses.

[0007] In summary, the present invention aims to provide a method for treating ARDS based on vagus nerve stimulation (VNS) to overcome the shortcomings of existing treatment methods and improve the treatment effect and prognosis of ARDS patients. Summary of the invention

[0008] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a vagus nerve electrical stimulation regulation method that can alleviate ARDS lung damage.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A vagus nerve electrical stimulation regulation method for reducing ARDS lung injury comprises the following steps:

[0011] 1: Establishing an ARDS animal model, wherein the animal model is established by tracheal instillation of lipopolysaccharide (LPS);

[0012] 2: After establishing the ARDS model, the left vagus nerve of the animal was isolated and exposed;

[0013] 3: Use an electrical stimulation device to electrically stimulate the left vagus nerve, with stimulation parameters including voltage 5V, frequency 5Hz, pulse width 2ms, and last for 10 minutes;

[0014] 4: Collect bronchoalveolar lavage fluid (BALF) and lung tissue samples from animals for subsequent assessment of inflammation and lung injury;

[0015] 5: Detect the levels of total protein and inflammatory cytokines in BALF, and perform histological evaluation and lung injury scoring.

[0016] Preferred: In step 2, the salivary glands are isolated through a midline incision in the neck and retracted laterally, the left carotid artery is exposed and the vagus nerve is carefully isolated.

[0017] Further: In step 3, the collected lung tissue samples are embedded in paraffin, sliced, and stained with H&E, and the lung wet-to-dry ratio (W / D) is measured to evaluate the degree of pulmonary edema.

[0018] Further: In step 5, the total protein level in BALF was determined by BCA kit, the TNF-α and IL-1β levels were determined by enzyme-linked immunosorbent assay (ELISA) kit, and the lung inflammation was scored using the Szapiel criteria.

[0019] As a preferred scheme of the present invention: it also includes measuring the levels of Th17 and Treg cells and their related transcription factors RORγt, FOXP3 and cytokines IL-17, IL-10 by flow cytometry, protein immunoblotting (WB) and ELISA to evaluate the regulatory effect of VNS on immune cells.

[0020] As a further scheme of the present invention: it also includes conducting afferent and efferent VNS experiments, in which the left cervical vagus nerve is physically cut off, and electrical stimulation is performed at the afferent end (central end) and the efferent end (peripheral end) respectively, to evaluate the effects of different vagus nerve pathways on ARDS lung injury and inflammatory response.

[0021] As a further scheme of the present invention: it also includes establishing an ARDS model and performing VNS after splenectomy (SPX) to evaluate the role of the spleen in VNS in alleviating ARDS lung injury and inflammatory response.

[0022] On the basis of the above scheme: it also includes adrenalectomy (ADX), then establishing an ARDS model and performing VNS to evaluate the role of the adrenal gland in VNS alleviating ARDS lung injury and inflammatory response.

[0023] Based on the above protocol: the animals are Sprague-Dawley rats, weighing between 200-250 g.

[0024] Based on the above scheme: the method also includes performing statistical analysis on the collected data to verify the effect of VNS in alleviating ARDS lung injury and inflammatory response, and whether the regulatory effect of VNS on Th17 and Treg cells is statistically significant.

[0025] The beneficial effects of the present invention are:

[0026] 1. A vagus nerve stimulation regulation method that can reduce ARDS lung injury. By implementing the vagus nerve stimulation (VNS) regulation method, the lung tissue pathological changes of the ARDS rat model, such as alveolar wall thickening, alveolar cavity stenosis and inflammatory cell infiltration, were significantly improved, the lung injury score and lung wet-to-dry ratio were significantly reduced, and the pulmonary edema was effectively alleviated.

[0027] 2. A vagus nerve electrical stimulation regulation method that can alleviate ARDS lung damage. VNS can significantly reduce the total protein level and the levels of inflammatory cytokines TNF-α and IL-1β in the bronchoalveolar lavage fluid (BALF) in the ARDS rat model, indicating that VNS has a strong anti-inflammatory effect.

[0028] 3. A vagus nerve electrical stimulation regulation method that can reduce ARDS lung damage. VNS achieves the balance of the immune system by regulating the expression levels of Th17 and Treg cells and their related transcription factors and cytokines. In the ARDS rat model, VNS reduced the percentage of Th17 cells and the expression level of IL-17, and increased the percentage of Treg cells and the expression level of IL-10, which helps to reduce inflammation in the lungs and promote tissue repair.

[0029] 4. A vagus nerve electrical stimulation regulation method that can reduce ARDS lung damage. Through afferent and efferent VNS experiments, the mechanism of action of VNS in the treatment of ARDS was revealed. Both afferent and efferent electrical stimulation can improve lung pathological tissue, reduce lung injury scores and inflammatory cytokine levels, indicating that the effect of VNS is not limited to a specific part of the vagus nerve, but has a wide range of regulatory effects.

[0030] 5. A vagus nerve electrical stimulation regulation method that can reduce ARDS lung damage. Through splenectomy and adrenalectomy experiments, the physiological basis required for VNS to reduce ARDS lung damage and inflammatory response was explored. The results showed that the effect of VNS requires the participation of the spleen but is not affected by the adrenal glands. This provides a new idea for the clinical treatment of ARDS, that is, while preserving spleen function, using neuroregulation methods such as VNS to improve patient prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the improvement of lung tissue pathological changes in a vagus nerve electrical stimulation regulation method proposed by the present invention that can alleviate ARDS lung injury;

[0032] Figure 2 It is a schematic diagram of regulating the ratio of Th17 and Treg cells in a vagus nerve electrical stimulation regulation method for alleviating ARDS lung injury proposed by the present invention;

[0033] Figure 3 It is a schematic diagram comparing the afferent and efferent VNS effects of a vagus nerve electrical stimulation regulation method proposed by the present invention that can alleviate ARDS lung injury;

[0034] Figure 4 It is a schematic diagram of the regulation mechanism of Th17 and Treg cells and related factors of a vagus nerve electrical stimulation regulation method for alleviating ARDS lung injury proposed by the present invention;

[0035] Figure 5 This is a schematic diagram of the effect change of a vagus nerve electrical stimulation regulation method proposed by the present invention that can reduce lung damage in ARDS after splenectomy;

[0036] Figure 6 It is a schematic diagram showing that a vagus nerve electrical stimulation regulation method proposed by the present invention that can reduce lung injury in ARDS fails to regulate Th17 and Treg cells after splenectomy;

[0037] Figure 7 It is a schematic diagram of the effect of the vagus nerve electrical stimulation regulation method proposed by the present invention for alleviating lung injury in ARDS after adrenalectomy;

[0038] Figure 8 It is a schematic diagram of a vagus nerve electrical stimulation regulation method proposed in the present invention that can alleviate ARDS lung injury and regulate Th17 and Treg cells stably after adrenalectomy. DETAILED DESCRIPTION

[0039] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0040] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0041] Embodiment 1:

[0042] A vagus nerve electrical stimulation method that can reduce lung damage in ARDS, such as Figures 1 to 8 As shown, the following steps are included:

[0043] S1: Establishment of ARDS rat model and vagus nerve stimulation (VNS)

[0044] S11: Establishment of ARDS rat model

[0045] Male Sprague-Dawley rats (weight 200-250 g) were anesthetized with 3% sodium pentobarbital injected intraperitoneally;

[0046] The rats were fixed, the trachea was exposed, and LPS (E. coli LPS serotype 0111:B4 dissolved in phosphate buffered saline (PBS)) at a dose of 2 mg / kg was administered by tracheal instillation;

[0047] S12: Vagus nerve stimulation (VNS)

[0048] The rats were anesthetized again, and a midline incision was made in the cervix and abdomen. The salivary glands were separated and retracted laterally to expose the left carotid artery and carefully separate the vagus nerve.

[0049] Place the vagus nerve on a bipolar hook electrode connected to an electrical stimulation device (such as the Huatuo brand electroacupuncture therapy device);

[0050] Six hours after the establishment of the ARDS rat model, the vagus nerve was stimulated at 5 V, 5 Hz, and 2 ms for 10 min;

[0051] The neck incision was sutured, and the rat's body temperature was maintained with a warming pad. The rat was closely observed until it woke up;

[0052] The left vagus nerve was exposed but not stimulated;

[0053] S2: Collection of bronchoalveolar lavage fluid (BALF) and lung tissue samples

[0054] After anesthetizing the rats, endotracheal intubation was performed;

[0055] Use 2 ml of PBS to flush the alveoli back and forth three times and collect bronchoalveolar lavage fluid;

[0056] Centrifuge at 500 × g for 20 min at 4 °C, collect the supernatant and store in a −80 °C refrigerator;

[0057] The right upper lobe was collected, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E; at the same time, the right middle lobe was taken out and weighed, and the dry weight was measured after drying in a 55°C oven for 72 hours, and the wet / dry (W / D) ratio was calculated;

[0058] S3: Detection of total protein and inflammatory cytokines in BALF

[0059] Total protein levels in BALF were determined using a BCA kit;

[0060] TNF-α and IL-1β levels were measured by enzyme-linked immunosorbent assay (ELISA) kits;

[0061] S4: Histological evaluation and lung injury scoring

[0062] H&E-stained sections were observed under an optical microscope, and histological evaluation was performed based on 10 randomly selected high-power fields (×200) in each section. The Szapiel criteria were used to score lung inflammation, including four levels: 0-normal lung morphology, 1-inflammatory damage <20%, 2-inflammatory damage 20%-50%, and 3-inflammatory damage >50%. The histopathological score of lung injury was calculated by a pathologist who was unaware of the grouping.

[0063] S5: Determination of Th17 and Treg cells and their related transcription factors and cytokines

[0064] S51: Preparation of lung single cell suspension

[0065] Cut the lung tissue into small pieces and grind them with a grinder;

[0066] Filter using a 70 μm nylon cell mesh to remove tissue debris;

[0067] Add red blood cell lysis buffer to the treated cell suspension to lyse red blood cells;

[0068] Resuspend the cells in PBS for later use;

[0069] S52: Flow cytometry

[0070] Cells were immunolabeled for surface proteins using FITC-labeled anti-CD4 and PE-labeled anti-CD25;

[0071] Cells were fixed and permeabilized using the Cytofix / Cytoperm kit;

[0072] Intracellular proteins were immunolabeled with PE-labeled anti-IL-17A and APC-labeled anti-FOXP3;

[0073] Immunostained cell data were collected and analyzed on a flow cytometer;

[0074] S53: Western blot (WB)

[0075] Proteins from spleen and lung tissues were extracted using a whole protein extraction kit;

[0076] Protein concentration was determined using BCA protein assay reagent;

[0077] Equal amounts of protein were separated by SDS-PAGE electrophoresis;

[0078] The proteins were transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 1 h;

[0079] Incubate with primary antibodies (RORγt, FOXP3, GAPDH) overnight at 4°C;

[0080] After washing with TBST, the cells were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG antibodies for 1 h at room temperature, then washed with TBST and visualized with an enhanced chemiluminescence reagent;

[0081] S54: ELISA to detect cytokine levels

[0082] Cytokine IL-17 and IL-10 levels were measured using ELISA kits;

[0083] S6: Incoming and outgoing VNS experiments

[0084] S61: Modeling and stimulation

[0085] The ARDS rat model was established according to the same steps as in the first part. After waiting for 6 hours, a midline incision was made in the neck, the salivary glands were separated, and the left cervical vagus nerve was found. The left cervical vagus nerve was physically cut, and then electrical stimulation was performed at the afferent end (central end) and the efferent end (peripheral end) respectively, with the same stimulation parameters as above.

[0086] S62: Sample collection and testing

[0087] Rats were euthanized 2 hours after stimulation, and lung tissue samples and bronchoalveolar lavage fluid were collected; the specific experimental methods were the same as in the first part, including H&E staining, pulmonary edema analysis, and detection of inflammatory cytokines in BALF;

[0088] S7: Splenectomy (SPX) and VNS Experiment

[0089] S71: Splenectomy

[0090] After anesthetizing the rats, an incision was made on the left abdomen, the splenic vessels were ligated and the spleen was removed; the wound was sutured and the rats recovered for 7 days.

[0091] S72: ARDS model construction and VNS

[0092] The ARDS model was established 7 days after splenectomy, and full VNS was performed 6 hours later. The rats were euthanized 2 hours later and lung tissues and BALF were collected. The specific steps were the same as in the first part.

[0093] S8: Adrenalectomy (ADX) and VNS Experiment

[0094] S81: Adrenalectomy

[0095] After anesthetizing the rats, an incision was made on the back and the bilateral adrenal glands were removed. The back wounds were sutured and the rats recovered for 7 days. The rats were given free access to 0.9% NaCl drinking water.

[0096] S82: ARDS model construction and VNS

[0097] The ARDS model was established 7 days after adrenalectomy; the rest of the steps were the same as in the first part.

[0098] Example 2

[0099] A vagus nerve electrical stimulation regulation method that can reduce ARDS lung damage. This embodiment is combined with the method in Example 1 to conduct experiments. The experimental items, methods, experimental results and conclusions are as follows:

[0100] Experimental Project 1: Experiment on the effect of VNS on the lung damage of ARDS

[0101] Experimental methods:

[0102] Establishment of ARDS rat model: Male Sprague-Dawley rats were anesthetized and LPS was instilled into the trachea;

[0103] Vagus nerve stimulation (VNS): The left vagus nerve is isolated and stimulated using an electrical stimulation device;

[0104] Lung tissue samples and bronchoalveolar lavage fluid (BALF) were collected;

[0105] Experimental results:

[0106] Figure 1 A and 1B: H&E staining showed pathological changes in lung tissue. Compared with the control group, the LPS group showed thickening of alveolar walls, narrowing of alveolar cavities, inflammatory cell infiltration, and increased lung injury scores. Compared with the LPS group, the LPS+VNS group significantly improved these conditions.

[0107] Figure 1 C: The lung wet-to-dry ratio (W / D) reflects the condition of pulmonary edema. Compared with the control group, the pulmonary edema in the LPS group was significantly more severe, while the lung wet-to-dry ratio in the LPS+VNS group was significantly lower than that in the LPS group.

[0108] Figure 1 D: BALF total protein level; compared with the control group, the BALF total protein level in the LPS group was significantly increased, while that in the LPS+VNS group was significantly decreased compared with that in the LPS group;

[0109] Figure 1 E and 1F: Levels of inflammatory cytokines TNF-α and IL-1β; compared with the control group, they were significantly increased in the LPS group, indicating that the inflammation in the lungs was aggravated, while after VNS, the levels of inflammatory cytokines decreased significantly;

[0110] in conclusion:

[0111] VNS can reduce lung injury and pulmonary inflammatory response in ARDS model rats;

[0112] Experimental Project 2: VNS regulates Th17 and Treg cells to alleviate ARDS experiment

[0113] Experimental methods:

[0114] Th17 and Treg cells and their related transcription factors and cytokines were determined using flow cytometry, western blotting (WB), and ELISA;

[0115] Experimental results:

[0116] Figure 2AD: Flow cytometry was used to detect the percentages of Th17 and Treg cells. Compared with the control group, the percentage of Th17 cells increased and the percentage of Treg cells decreased in the LPS-treated group; in the LPS+VNS-treated group, the percentage of Th17 cells decreased and the percentage of Treg cells increased.

[0117] Figure 2 E and 2F: ELISA detected the levels of cytokines IL-17 and IL-10. Compared with the control group, the expression level of IL-17 in the LPS group was increased, while the expression level of IL-10 was decreased. Compared with the LPS group, the expression level of IL-17 in the LPS+VNS group was decreased, while the expression level of IL-10 was increased.

[0118] Figure 2 GJ: WB detected the expression levels of RORγt and FOXP3. Compared with the control group, the expression level of RORγt in the LPS group was increased, while the expression level of FOXP3 was decreased. Compared with the LPS group, the expression level of RORγt in the LPS+VNS group was decreased, while the expression level of FOXP3 was increased.

[0119] in conclusion:

[0120] VNS can reduce Th17 cells and increase Treg cells, thereby alleviating inflammation in the lungs of ARDS;

[0121] Experimental Project 3: Experiment on the reduction of lung injury and inflammatory response in ARDS by inflow and outflow VNS

[0122] Experimental methods:

[0123] The left cervical vagus nerve was physically cut and electrically stimulated at both the afferent and efferent ends;

[0124] Lung tissue samples and BALF were collected for H&E staining and ELISA testing;

[0125] Experimental results:

[0126] Figure 3 A and 3B: H&E staining showed changes in lung pathological tissue; compared with the LPS group, complete, afferent, and efferent VNS could improve lung pathological tissue and reduce lung injury scores;

[0127] Figure 3 C: Lung wet-to-dry ratio (W / D) reflects the status of pulmonary edema; compared with the LPS group, complete, afferent, and efferent VNS can reduce W / D;

[0128] Figure 3 D and 3E: ELISA was used to detect the levels of inflammatory cytokines TNF-α and IL-1β in BALF; compared with the LPS group, intact, afferent, and efferent VNS could reduce the levels of these inflammatory cytokines;

[0129] in conclusion:

[0130] Both afferent and efferent VNS can reduce lung injury and inflammatory response in ARDS;

[0131] Experimental Project 4: Whether incoming and outgoing VNS can regulate Th17 and Treg cells to alleviate ARDS

[0132] Experimental methods:

[0133] Th17 and Treg cells and their related factors were determined using flow cytometry, ELISA, and WB;

[0134] Experimental results:

[0135] Figure 4 AJ: Flow cytometry, ELISA and WB results; compared with the LPS group, intact, incoming and outgoing VNS could reduce the percentage of Th17 cells, the expression levels of IL-17 and RORγt, and increase the percentage of Treg cells, the expression levels of IL-10 and FOXP3;

[0136] in conclusion:

[0137] Both incoming and outgoing VNS can regulate Th17 and Treg cells;

[0138] Experimental Project 5: Does VNS need the spleen to participate in the experiment to reduce lung injury and inflammatory response in ARDS?

[0139] Experimental methods:

[0140] Rats underwent splenectomy (SPX), and seven days later, the ARDS model was established and VNS was performed;

[0141] Lung tissues and BALF were collected for H&E staining and ELISA testing;

[0142] Experimental results:

[0143] Figure 5 A and 5B: H&E staining showed changes in lung pathological tissues. Compared with the LPS+VNS group, the SPX+LPS+VNS group showed thickening of alveolar walls, narrowing of alveolar cavities, obvious infiltration of inflammatory cells, and increased lung injury scores.

[0144] Figure 5 C: Lung wet-to-dry ratio (W / D) reflects the status of pulmonary edema; compared with the LPS+VNS group, lung W / D increased in the SPX+LPS+VNS group;

[0145] Figure 5D and 5E: ELISA was used to detect the levels of inflammatory cytokines TNF-α and IL-1β in BALF; the levels of these inflammatory cytokines were increased in SPX+LPS+VNS compared with the LPS+VNS group;

[0146] in conclusion:

[0147] VNS reduces lung injury and inflammatory response in ARDS, which requires the participation of the spleen;

[0148] Experimental Project 6: Does VNS regulate Th17 and Treg cells require the participation of the spleen? Experimental methods:

[0149] Th17 and Treg cells and their related factors were determined by flow cytometry, ELISA and WB (the method is the same as experimental project 2);

[0150] Experimental results:

[0151] Figure 6 AJ: Flow cytometry, ELISA, and WB results; compared with the LPS+VNS group, the percentage of Th17 cells, the expression levels of IL-17, and RORγt increased, while the percentage of Treg cells, the expression levels of IL-10, and FOXP3 decreased in the SPX+LPS+VNS group;

[0152] in conclusion:

[0153] Splenectomy abolishes the regulatory capacity of VNS on Th17 and Treg cells;

[0154] Experimental Project 7: Does VNS need adrenal involvement to reduce lung injury and inflammation in ARDS?

[0155] Experimental methods:

[0156] Rats underwent adrenalectomy (ADX), and seven days later, the ARDS model was established and VNS was performed;

[0157] Lung tissues and BALF were collected for H&E staining and ELISA testing;

[0158] Experimental results:

[0159] Figure 7 A and 7B: H&E staining showed no obvious changes in lung pathological tissue and no significant changes in lung injury scores;

[0160] Figure 7 C: There was no significant change in lung wet-to-dry ratio (W / D);

[0161] Figure 7 D and 7E: There was no significant change in the levels of inflammatory cytokines TNF-α and IL-1β in BALF detected by ELISA;

[0162] in conclusion:

[0163] VNS reduces lung injury and inflammatory response in ARDS without the involvement of the adrenal glands;

[0164] Experimental Project 8: Does VNS regulate Th17 and Treg cells require the participation of the adrenal glands?

[0165] Experimental methods:

[0166] Th17 and Treg cells and their related factors were determined by flow cytometry, ELISA and WB (the method is the same as experimental project 2);

[0167] Experimental results:

[0168] Figure 8 AJ: Flow cytometry, ELISA, and WB results; compared with the LPS+VNS group, there were no significant changes in the expression levels of Th17 and Treg cells and their related cytokines and transcription factors in the ADX+LPS+VNS group;

[0169] in conclusion:

[0170] Adrenalectomy does not abolish the regulatory capacity of VNS on Th17 and Treg cells;

[0171] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. A vagus nerve electrical stimulation regulation method for alleviating ARDS lung damage, characterized in that: The following steps are involved: 1: Establishing an ARDS animal model, wherein the animal model is established by tracheal instillation of lipopolysaccharide (LPS); 2: After establishing the ARDS model, the left vagus nerve of the animal was isolated and exposed; 3: Use an electrical stimulation device to electrically stimulate the left vagus nerve, with stimulation parameters including voltage 5V, frequency 5Hz, pulse width 2ms, and last for 10 minutes; 4: Collect bronchoalveolar lavage fluid (BALF) and lung tissue samples from animals for subsequent assessment of inflammation and lung injury; 5: Detect the levels of total protein and inflammatory cytokines in BALF, and perform histological evaluation and lung injury scoring.

2. A vagus nerve electrical stimulation regulation method for alleviating ARDS lung damage according to claim 1, characterized in that: In step 2, the salivary glands are isolated through a midline incision in the neck and retracted laterally, exposing the left carotid artery and carefully isolating the vagus nerve.

3. The method for vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 2, characterized in that: In step 3, the collected lung tissue samples are embedded in paraffin, sliced, and stained with H&E, and the lung wet-to-dry ratio (W / D) is measured to evaluate the degree of pulmonary edema.

4. The method for vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 3, characterized in that: In step 5, the total protein level in BALF was measured by BCA kit, TNF-α and IL-1β levels were measured by enzyme-linked immunosorbent assay (ELISA) kits, and lung inflammation was scored using the Szapiel criteria.

5. The method for regulating vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 4, characterized in that: It also includes measuring the levels of Th17 and Treg cells and their related transcription factors RORγt, FOXP3 and cytokines IL-17 and IL-10 by flow cytometry, western blotting (WB) and ELISA to evaluate the regulatory effect of VNS on immune cells.

6. The method for regulating vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 5, characterized in that: It also includes afferent and efferent VNS experiments, in which the left cervical vagus nerve is physically cut and electrically stimulated at the afferent end (central end) and efferent end (peripheral end) respectively to evaluate the effects of different vagus nerve pathways on lung injury and inflammatory response in ARDS.

7. The method for regulating vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 6, characterized in that: It also includes splenectomy (SPX), establishment of ARDS model and VNS to evaluate the role of spleen in VNS alleviating ARDS lung injury and inflammatory response.

8. The method for regulating vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 7, characterized in that: It also includes adrenalectomy (ADX), establishment of the ARDS model and VNS to evaluate the role of the adrenal gland in VNS alleviating ARDS lung injury and inflammatory response.

9. The method for vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 8, characterized in that: The animals were Sprague-Dawley rats, weighing between 200-250 g.

10. The method for regulating vagus nerve electrical stimulation for alleviating ARDS lung injury according to claim 9, characterized in that: The method also includes performing statistical analysis on the collected data to verify the effect of VNS in alleviating ARDS lung injury and inflammatory response, and whether the regulatory effect of VNS on Th17 and Treg cells is statistically significant.

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