Method for studying the mechanism of action of cxcr3a on acute respiratory distress syndrome

Abstract

The application discloses a research method for the mechanism of CXCR3A in acute respiratory distress syndrome, and relates to the technical field of biomedicine; and comprises the following steps: establishing an animal ARDS model and setting an intervention group; experiment animals are randomly divided into at least four groups, namely a normal control group, an ARDS model group, a CXCR3 broad-spectrum inhibitor intervention group and a CXCR3A selective inhibitor intervention group; a damaging agent is applied to the model group and the two inhibitor intervention groups of animals to establish an ARDS model; the CXCR3 broad-spectrum inhibitor intervention group and the CXCR3A selective inhibitor intervention group are respectively pretreated with corresponding inhibitors before modeling. The method of the application constructs a complete research framework from the whole animal level to the molecular level, covers multiple key aspects of pathological morphology, inflammatory response, oxidative stress and target gene expression, and can reveal the comprehensive mechanism of CXCR3A in the occurrence and development of ARDS.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to research methods for the mechanism of action of CXCR3A on acute respiratory distress syndrome. Background Technology

[0002] Acute respiratory distress syndrome (ARDS) is an acute, progressive respiratory failure syndrome caused by multiple factors both inside and outside the lungs. Its incidence is rising globally, with a mortality rate as high as 30%-40%. Current treatments for ARDS include mechanical ventilation, fluid management, and pharmacological therapy, but these methods all have limitations. From a pathogenic mechanism perspective, ARDS involves multiple stages, including a complex inflammatory cascade, oxidative stress, and apoptosis. Persistent inflammation and tissue damage can also trigger pulmonary fibrosis. Given the severity of ARDS and the limitations of current treatments, many aspects of its pathogenic mechanisms remain under-explored and require further in-depth investigation. This is crucial for improving patient prognosis and reducing mortality. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by proposing a research method for the mechanism of action of CXCR3A on acute respiratory distress syndrome.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: The research methodology for studying the mechanism of action of CXCR3A in acute respiratory distress syndrome includes the following steps: S1: Establishing an animal ARDS model and setting up intervention groups: Experimental animals were randomly divided into at least four groups, namely, a normal control group, an ARDS model group, a CXCR3 broad-spectrum inhibitor intervention group, and a CXCR3A selective inhibitor intervention group; the model group and the two inhibitor intervention groups were given a causative agent to establish an ARDS model; the CXCR3 broad-spectrum inhibitor intervention group and the CXCR3A selective inhibitor intervention group were pretreated with the corresponding inhibitors before modeling; S2: Dynamic collection of biological samples: Lung tissue, peripheral blood and bronchoalveolar lavage fluid samples were collected from each group of animals at multiple different time points after injury. S3: Perform multi-dimensional indicator detection: For the samples collected in S2, detect indicators including the degree of pathological damage to lung tissue, the degree of fibrosis in lung tissue, the level of oxidative stress, the level of inflammatory factors, the level of fibrosis-related factors, and the gene expression levels of CXCR3, CXCR3A and CXCR3B in lung tissue. S4: Data Analysis and Mechanism Determination: Compare the differences in each indicator in S3 among the normal control group, ARDS model group, CXCR3 broad-spectrum inhibitor intervention group, and CXCR3A selective inhibitor intervention group. Based on the specific or superior regulatory effect of CXCR3A selective inhibitor on the indicators compared with CXCR3 broad-spectrum inhibitor, determine the core mechanism of action of CXCR3A in the occurrence and development of ARDS.

[0005] Preferably, in S1, the experimental animal is a C57BL / 6J mouse; the injury agent is lipopolysaccharide, administered via intranasal drip at a dose of 5-15 mg / kg body weight.

[0006] Preferably, in step S1, the broad-spectrum CXCR3 inhibitor is AMG487; and the selective CXCR3A inhibitor is SCH546738.

[0007] Preferably, in step S1, AMG487 is pretreated by intraperitoneal injection at a dose of 3-7 mg / kg / day for 7-14 consecutive days; SCH546738 is pretreated by intraperitoneal injection at a dose of 8-12 mg / kg / day for 7-14 consecutive days.

[0008] Preferably, the pretreatment dose of AMG487 is 5 mg / kg / day, and the pretreatment is carried out continuously for 10 days; the pretreatment dose of SCH546738 is 10 mg / kg / day, and the pretreatment is carried out continuously for 10 days.

[0009] Preferably, in S2, the multiple different time points include day 1, day 3, day 5, day 7, and day 10 after the injury.

[0010] Preferably, in step S3, the detection of oxidative stress level is performed by enzyme-linked immunosorbent assay (ELISA) to detect the concentration of reactive oxygen species and the activity of superoxide dismutase in peripheral blood and bronchoalveolar lavage fluid.

[0011] Preferably, in step S3, the levels of inflammatory factors are detected by using ELISA to detect the concentrations of interleukin-1β, interleukin-6, and gamma-interferon in peripheral blood and bronchoalveolar lavage fluid.

[0012] A method for screening or evaluating CXCR3A inhibitors for the treatment of acute respiratory distress syndrome (ARDS) involves comparing the results of a candidate compound intervention group with those of the ARDS model group and a CXCR3 broad-spectrum inhibitor intervention group. If the candidate compound is as effective as or better than the CXCR3 broad-spectrum inhibitor in reducing lung tissue pathological damage, decreasing oxidative stress marker ROS, increasing antioxidant enzyme SOD activity, decreasing the levels of inflammatory factors IL-1β, IL-6, and IFN-γ, decreasing the levels of fibrosis markers KL-6 and TGF-β1, and / or specifically downregulating CXCR3A mRNA expression in lung tissue, then the candidate compound is identified as a potential CXCR3A inhibitor.

[0013] The method is described in its application in the preparation of tools or kits for studying the pathogenesis of acute respiratory distress syndrome or screening related therapeutic drugs.

[0014] The beneficial effects of this invention are as follows: 1. The method of this invention constructs a complete research framework from the whole animal level to the molecular level, covering multiple key aspects such as pathological morphology (damage and fibrosis), inflammatory response, oxidative stress and target gene expression, which can comprehensively and three-dimensionally reveal the integrated mechanism of CXCR3A in the occurrence and development of ARDS.

[0015] 2. By setting multiple consecutive time points (such as days 1, 3, 5, 7, and 10) for sample collection and testing, this invention can dynamically track the complete pathological process of ARDS from the acute inflammatory phase to the late potential fibrotic stage, thereby clarifying the spatiotemporal variation characteristics of CXCR3A and revealing its core regulatory role in different stages of the disease.

[0016] 3. This invention uses parallel intervention groups of broad-spectrum CXCR3 inhibitor (AMG487) and CXCR3A selective inhibitor (SCH546738) for comparison. This method can effectively distinguish and identify the functional differences between the total CXCR3 receptor pathway and specific CXCR3A subtypes, directly and strongly demonstrating that CXCR3A is the core target mediating ARDS pathological damage, rather than CXCR3B or other factors.

[0017] 4. The multi-dimensional detection index system established by the method of this invention (such as lung tissue pathological score, level of specific inflammatory / oxidative / fibrotic factors, CXCR3A / B gene expression ratio, etc.) can not only be used for basic mechanism exploration, but is also a mature evaluation standard in itself. This system can be directly applied to screen and evaluate the effectiveness of candidate drugs or treatment strategies targeting CXCR3A, providing a reliable and directly translatable experimental tool for new drug development and the development of clinical precision treatment plans. Attached Figure Description

[0018] Figure 1 This figure shows the time-dependent effect of the inhibition of CXCR3 and CXCR3A on lipopolysaccharide-induced pathological damage in lung tissue of mice with acute respiratory distress syndrome. Figure 2 This is a graph showing the time-dependent effects of the inhibition of CXCR3 and CXCR3A on lipopolysaccharide-induced pulmonary fibrosis in mice with acute respiratory distress syndrome. Figure 3 This figure shows the time-dependent effect of the inhibition of CXCR3 and CXCR3A on the levels of reactive oxygen species (ROS) in peripheral blood and bronchoalveolar lavage fluid of mice with lipopolysaccharide-induced acute respiratory distress syndrome. Figure 4 This figure shows the time-dependent effect of the inhibition of CXCR3 and CXCR3A on the activity of superoxide dismutase (SOD) in peripheral blood and bronchoalveolar lavage fluid (BALF) of mice with lipopolysaccharide-induced acute respiratory distress syndrome (ARDS). Figure 5 This is a first schematic diagram illustrating the time-dependent effects of the present invention on the inhibition of CXCR3 and CXCR3A on the levels of pro-inflammatory factors (IL-1β, IL-6, IFN-γ) in peripheral blood and bronchoalveolar lavage fluid (BALF) of mice with lipopolysaccharide-induced acute respiratory distress syndrome (ARDS). Figure 6 This is a second schematic diagram illustrating the time-dependent effects of CXCR3 and CXCR3A on the levels of pro-inflammatory factors (IL-1β, IL-6, IFN-γ) in peripheral blood and bronchoalveolar lavage fluid (BALF) of mice with lipopolysaccharide-induced acute respiratory distress syndrome (ARDS). Figure 7 This figure shows the time-dependent effect of the inhibition of CXCR3 and CXCR3A on the levels of fibrosis-related markers (KL-6 and TGF-β1) in peripheral blood and bronchoalveolar lavage fluid (BALF) of mice with lipopolysaccharide-induced acute respiratory distress syndrome (ARDS). Figure 8 This diagram illustrates the time-dependent expression of CXCR3 and its subtypes (CXCR3A, CXCR3B) in the lung tissue of mice with acute respiratory distress syndrome induced by lipopolysaccharide (LPS) of this invention, and the regulatory effects of its inhibitors. Figure 9 This is a technical roadmap of the present invention; Figure 10 This is a flowchart of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0020] Example 1: Research Methods: Sixty-four 8-week-old male C57BL / 6J mice were randomly divided into three groups: normal group, model group, model group + CXCR3 inhibitor (AMG487 group), and model group + CXCR3A inhibitor (SCH546738) group (n=16). The model group was induced into an ARDS model by intranasal instillation of LPS (10 mg / kg). The model group + CXCR3 inhibitor (AMG487) group underwent pretreatment with intraperitoneal injection of AMG487 (5 mg / kg) for 10 days before ARDS modeling. The model group + CXCR3A inhibitor (SCH546738) group underwent pretreatment with intraperitoneal injection of SCH546738 (10 mg / kg) for 10 days before ARDS modeling. The normal group received an equal volume of sterile phosphate-buffered saline (PBS) intraperitoneally. Mice were sacrificed 24 hours after the last administration on day 1 (d1), day 3 (d3), day 5 (d5), day 7 (d7), and day 10 (d10). Peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected. Lung tissue damage was assessed by HE staining; the degree of lung fibrosis was assessed by Masson staining; oxidative stress markers (ROS levels, SOD activity), inflammatory cytokine levels (IL-1β, IL-6, INF-γ), and fibrosis markers (KL-6, TGF-β1) in BALF and peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA); and the mRNA expression levels of CXCR3, CXCR3A, and CXCR3B in lung tissue were detected by RT-qPCR (quantitative real-time PCR).

[0021] Data Analysis Methods: All data in this study were derived from at least three independent experiments. The Brown-Forsythe test was used to test for homogeneity of variance among the four groups. If the variances were homogeneous, one-way ANOVA was used, followed by Tukey's post-hoc test for pairwise comparisons between groups. If the variances were unequal, Welch ANOVA was used, followed by Games-Howell's post-hoc test for pairwise comparisons between groups. p < 0.05 was considered statistically significant. For all experiments with error bars, the standard deviation (SD) of the mean was calculated to represent the variation within each experiment.

[0022] Example 2: Experimental Method 1. Animal source and feeding SPF-grade male C57BL / 6J mice, weighing 18–25 g (64 mice, 8 weeks old), were purchased from Chongqing Enbi Biotechnology Co., Ltd. and housed in the animal facility of the Central Laboratory of the Second Affiliated Hospital of Chongqing Medical University. The mice had free access to food and water, and their bedding was changed regularly. All procedures were conducted in accordance with animal ethics requirements.

[0023] 2. Establish an acute respiratory distress syndrome model. Mouse preparation: After one week of standardized rearing in the animal room, the purchased C57 mice were observed to be in good mental and physical condition and weight. Then, they underwent intraperitoneal drug pretreatment. Drug pretreatment preparation: AMG487 and SCH546738 were injected intraperitoneally before mouse modeling. Modeling: C57 mice were correctly held with the left hand and anesthetized with an intraperitoneal injection of 100 mg / kg sodium pentobarbital. A single intranasal infusion of 10 mg / kg LPS was then administered to establish the ARDS model, and the mice were continued to be fed under standard conditions.

[0024] 3. Animal handling (1) Mice were randomly divided into four groups, with 16 mice in each group. According to the literature review, the dosage of AMG487 was 5 mg / kg, and the dosage of SCH546738 was 10 mg / kg. The specific grouping and treatment protocols were as follows: Control group: intraperitoneal injection of an equal volume of sterile phosphate-buffered saline (PBS); Model group (LPS): intranasal instillation of LPS (10 mg / kg) for modeling; Model group + CXCR3 inhibitor (AMG487) group: pretreatment with intraperitoneal injection of AMG487 (5 mg / kg) for 10 days before LPS modeling; Model group + CXCR3A allosteric inhibitor (SCH546738) group: pretreatment with intraperitoneal injection of SCH546738 (10 mg / kg) for 10 days before LPS modeling. ARDS modeling was performed after treatment.

[0025] (2) Mice were treated according to the above protocol, and on day 1 (d1), day 3 (d3), day 5 (d5), day 7 (d7), and day 10 (d10) 24 hours after the last administration, peripheral blood samples were collected by enucleation. The trachea was exposed, lavaged, and bronchoalveolar lavage fluid was collected. Peripheral serum and bronchoalveolar lavage fluid were collected from 3 mice in each group for the detection of oxidative stress indicators, inflammatory factor levels, and fibrosis indicators.

[0026] (3) 24 hours after the last administration, mice were euthanized on day 1 (d1), day 3 (d3), day 5 (d5), day 7 (d7), and day 10 (d10). The lung tissue was carefully dissected to obtain the complete lungs, which were then stored in 4% paraformaldehyde for further H&E and Masson staining analysis. The lungs were also stored in liquid nitrogen for further RT-qPCR (quantitative real-time PCR) analysis.

[0027] Experimental results and conclusions: I. Method: (1) Tissue section preparation: After routine paraffin embedding of tissue, adjust the section thickness to 5mm and slice it, spreading it evenly on the glass slide. Finally, bake it at a constant temperature of 60℃ for 1 hour.

[0028] (2) HE staining: Dewaxing paraffin sections to water: Dewaxing in environmentally friendly dewaxing agent (1), environmentally friendly dewaxing agent (2), and environmentally friendly dewaxing agent (3) for 10 minutes each, then in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each. Rinse with tap water for 1 minute.

[0029] Stain with Harris hematoxylin solution for 4 minutes, then wash with tap water for 2 minutes until no excess staining solution is removed from the slide.

[0030] Differentiate with 0.8% hydrochloric acid alcohol for 2 seconds, then rinse with tap water. Alternatively, use lithium carbonate aqueous solution to turn blue, then rinse with water for 2 minutes.

[0031] Stain with eosin dye solution (alcohol-soluble) for 20 seconds without rinsing with water. Then, directly add 95% ethanol for 5 seconds to adjust the color. Finally, dehydrate with anhydrous ethanol (1) and anhydrous ethanol (2) for 2 minutes.

[0032] Environmentally friendly transparent agent for transparency, sealing, and microscopic examination.

[0033] (3) Observe and photograph the above HE-stained sections under a microscope. Image results analysis (HE staining) Figure 1 The lung tissue pathological changes in four groups of mice (Control, ARDS, ARDS+AMG487, ARDS+SCH546738) on days 1, 3, 5, 7, and 10 after LPS-induced ARDS are shown: Control group At each time point, the alveolar structure was intact, the alveolar septa were thin and not significantly thickened, there was no inflammatory cell infiltration, no hemorrhage or edema, and the lung tissue structure was clear.

[0034] 2. ARDS Model Group (ARDS) Day 1: The alveolar structure was severely damaged, with extensive infiltration of inflammatory cells, significant thickening of the alveolar septa, and exudate and hemorrhage visible in the alveolar cavities. Lung injury was the most severe. Model construction was successful.

[0035] Day 3: Inflammatory infiltration and alveolar septal thickening remained significant, and lung injury persisted.

[0036] Day 5: The damage has slightly improved, but there is still significant inflammatory infiltration and structural disorder.

[0037] Days 7 and 10: Lung tissue gradually repairs, but irregular alveolar structure and interstitial fibrosis are still visible, indicating incomplete recovery.

[0038] 3. ARDS + AMG487 group (broad-spectrum CXCR3 inhibitor) The degree of lung injury at each time point was significantly less than that in the model group.

[0039] Inflammatory infiltration, alveolar septal thickening, and alveolar structural damage were significantly reduced, and the recovery rate was faster than that of the model group. By Day 10, the lung tissue was close to normal.

[0040] 4. ARDS + SCH546738 group (CXCR3A allosteric inhibitor) The improvement in lung injury was comparable to that of the AMG487 group, and even showed a more significant protective effect at some time points (such as Day 1 and Day 3).

[0041] Inflammatory infiltration and alveolar structural damage were significantly reduced, and lung tissue was basically restored to normal by Day 10, suggesting that CXCR3A is a key regulatory target for lung injury in ARDS.

[0042] in conclusion 1. LPS induction can successfully construct a mouse ARDS model, which shows severe pathological damage to lung tissue, and the damage shows a trend of first aggravation and then gradual repair over time.

[0043] 2. Inhibition of CXCR3 (especially CXCR3A subtype) can significantly reduce LPS-induced ARDS pathological damage to lung tissue, reduce inflammatory cell infiltration, improve alveolar structural integrity, and promote lung tissue repair.

[0044] 3. The protective effect of the CXCR3A allosteric inhibitor (SCH546738) is comparable to that of the broad-spectrum CXCR3 inhibitor (AMG487), suggesting that CXCR3A is a potential important target for the treatment of ARDS.

[0045] II. Method: (1) Tissue section preparation: After routine paraffin embedding of tissue, adjust the section thickness to 5mm and slice it, spreading it evenly on the glass slide. Finally, bake it at a constant temperature of 60℃ for 1 hour.

[0046] (2) Masson staining: ① Routinely dewax tissue sections to water, immerse the dehydrated sections in Bouin's solution or Zenker's solution overnight, and rinse thoroughly with running water.

[0047] ② Stain with Harris hematoxylin or iron hematoxylin for 5-10 minutes, then rinse briefly with running water.

[0048] ③ Differentiate with 0.8%-1% hydrochloric acid alcohol, rinse with running water for several minutes; return to blue with lithium carbonate for a few seconds, then rinse with running water. ④ Stain with Ponceau S acid fuchsin solution for 5-10 minutes, then rinse briefly with running water.

[0049] ⑤ Treat with phosphomolybdic acid solution for about 5 minutes, do not rinse with water, and counterstain directly with aniline blue solution for 5 minutes. ⑥ Treat with 1% glacial acetic acid for 1 minute, and dehydrate repeatedly with 95% alcohol.

[0050] ⑦ Dehydrate with anhydrous alcohol, clear with xylene, and seal with neutral resin.

[0051] (3) Observe and photograph the Masson stained sections under a microscope.

[0052] Image results analysis (Masson staining) Figure 2 The changes in lung fibrosis (collagen deposition) in four groups of mice (Control, ARDS, ARDS+AMG487, ARDS+SCH546738) on days 1, 3, 5, 7, and 10 after LPS-induced ARDS are shown: 1. Control Group Only a very small amount of blue collagen fibers were observed at each time point, the alveolar structure remained intact, and there were no obvious signs of fibrosis.

[0053] 2. ARDS Model Group (ARDS) Day 1: Collagen deposition was not obvious; the main symptom was acute inflammatory response.

[0054] Days 3 and 5: Collagen fibers (blue staining) begin to increase significantly, mainly distributed around the alveolar septa and bronchi, indicating the initiation of fibrosis.

[0055] Days 7 and 10: Collagen deposition reaches its peak, with extensive and dense blue-stained areas. The alveolar structure is replaced by a large number of collagen fibers, resulting in obvious pulmonary fibrosis.

[0056] 3. ARDS + AMG487 group (broad-spectrum CXCR3 inhibitor) Collagen deposition at each time point was significantly less than that in the model group.

[0057] The blue-stained areas were significantly reduced and their distribution was limited, the alveolar structure remained relatively intact, and the degree of fibrosis was significantly reduced by Day 10, approaching that of the normal group.

[0058] 4. ARDS + SCH546738 group (CXCR3A allosteric inhibitor) The fibrosis inhibition effect was significantly greater than that of the AMG487 group, with outstanding performance on Day 7 and 10.

[0059] Collagen deposition was significantly reduced and alveolar structure damage was mild, suggesting that CXCR3A is a key subtype that regulates pulmonary fibrosis in the later stages of ARDS.

[0060] in conclusion 1. LPS-induced ARDS mice showed significant pulmonary fibrosis in the later stages (Day 7-10), characterized by extensive deposition of collagen fibers.

[0061] 2. Inhibition of CXCR3 (especially the CXCR3A subtype) can significantly reduce the degree of pulmonary fibrosis in ARDS mice, reduce collagen deposition, and protect the integrity of alveolar structure.

[0062] 3. The CXCR3A allosteric inhibitor (SCH546738) showed significantly better anti-fibrotic effects than the broad-spectrum CXCR3 inhibitor (AMG487), further supporting the potential of CXCR3A as a therapeutic target for ARDS fibrosis.

[0063] III. Sample Collection: Preparation of peripheral blood serum from mice: After anesthetizing mice, peripheral blood was collected via the orbital venous plexus and placed in sterile centrifuge tubes without anticoagulant. The blood was allowed to clot naturally at room temperature for 10-20 minutes, then centrifuged at 2-8°C for approximately 20 minutes (2000-3000 rpm). The supernatant was carefully collected. If precipitation occurred during storage, centrifugation was repeated. After aliquoting, the serum was stored at -80°C until analysis, avoiding repeated freeze-thaw cycles.

[0064] Bronchoalveolar lavage fluid (BALF) collection: Anesthetize mice and fix them on a sterile surgical board. Expose the thoracic cavity and trachea, insert a blunt needle or indwelling needle into the trachea and ligate it. Inject 1 mL of pre-cooled 4°C sterile PBS solution into the trachea, aspirate repeatedly 3 times, and collect all the lavage fluid. Centrifuge at 2-8°C for about 20 minutes (2000-3000 rpm), carefully collect the supernatant. If precipitation occurs during storage, centrifuge again. Take the supernatant, aliquot it, and store at -80°C for later analysis.

[0065] Methods: Following the kit instructions, enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of reactive oxygen species (ROS) in mouse serum and bronchoalveolar lavage fluid. The steps are as follows: (1) Remove serum and bronchoalveolar lavage fluid samples from the -80℃ freezer and place them on an ice box to thaw.

[0066] (2) Remove the kit from the refrigerator, allow it to return to room temperature, and dilute the standard according to the instructions.

[0067] (3) Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent, all other steps are the same), standard wells, and sample wells. Accurately add 50 μl of standard to the enzyme-labeled plate. Add 40 μL of sample diluent to the sample wells, and then add 10 μl of sample to the sample wells (the final sample dilution is 5 times). Add the sample to the bottom of the wells, avoiding contact with the well walls as much as possible, and gently shake to mix.

[0068] (4) Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0069] (5) Solution preparation: Dilute the 30-fold concentrated washing solution with distilled water 30 times and set aside.

[0070] (6) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing liquid, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0071] (7) Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.

[0072] (8) Incubation: Same procedure as 4.

[0073] (9) Washing: Same as 6.

[0074] (10) Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0075] (11) Termination: Add 50 μl of stop solution to each well to terminate the reaction (at this time, the blue color will immediately turn yellow).

[0076] (12) Measurement: Zero the instrument with the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0077] In subsequent experiments, the steps for measuring superoxide dismutase (SOD), inflammatory factors interleukin-6 (IL-6), IL-1β, IFN-γ, KL-6, and TGF-β by ELISA were the same as above, and the collection methods for peripheral blood and bronchoalveolar lavage fluid were the same as before.

[0078] Image Results Analysis (ROS Oxidative Stress Factor) Figure 3The changes in reactive oxygen species (ROS) levels in peripheral blood (A) and bronchoalveolar lavage fluid (B) in four groups of mice (Control, ARDS, ARDS+AMG487, ARDS+SCH546738) on days 1, 3, 5, 7, and 10 after LPS-induced ARDS are shown. 1. Control Group ROS levels remained at a low level at all time points without significant fluctuations, indicating that the baseline oxidative stress level was stable.

[0079] 2. ARDS Model Group (ARDS) Peripheral blood (A): ROS levels were significantly elevated on Day 1, peaked on Day 3, and then gradually decreased (Day 5-10), but remained significantly higher than those in the normal group.

[0080] BALF(B): The trend of ROS level changes is consistent with that of peripheral blood, reaching its peak on Day 3, and the overall level is higher than that of peripheral blood, suggesting that local oxidative stress damage in the lungs is more severe.

[0081] 3. ARDS + AMG487 group (broad-spectrum CXCR3 inhibitor) ROS levels in peripheral blood and BALF were significantly lower than those in the model group at all time points.

[0082] The peak value (Day 3) decreased significantly and the rate of decline was faster, reaching the level of the normal group by Day 10, suggesting that broad-spectrum inhibition of CXCR3 can effectively alleviate systemic and local pulmonary oxidative stress.

[0083] 4. ARDS + SCH546738 group (CXCR3A allosteric inhibitor) The ROS level reduction effect was comparable to that of the AMG487 group, and even showed a stronger inhibitory effect at some time points (such as Day 3 and Day 5).

[0084] It effectively reduced the levels of oxidative stress in the lungs (BALF) and the whole body (peripheral blood), suggesting that CXCR3A is a key subtype that regulates oxidative stress in ARDS.

[0085] in conclusion 1. LPS-induced ARDS can lead to a significant increase in systemic and local pulmonary oxidative stress in mice, manifested by a sharp increase in ROS levels, which peaked on Day 3 and then gradually decreased.

[0086] 2. Inhibition of CXCR3 (especially the CXCR3A subtype) can significantly reduce ROS levels in peripheral blood and BALF of ARDS mice, effectively alleviating oxidative stress damage.

[0087] 3. The CXCR3A allosteric inhibitor (SCH546738) showed significantly better anti-oxidative stress effects than the broad-spectrum CXCR3 inhibitor (AMG487), further supporting the potential of CXCR3A as a therapeutic target for ARDS.

[0088] IV. Analysis of Image Results (SOD Antioxidant Enzyme) Figure 4 The changes in superoxide dismutase (SOD) activity in peripheral blood (C) and bronchoalveolar lavage fluid (D) of four groups of mice (Control, ARDS, ARDS+AMG487, ARDS+SCH546738) on days 1, 3, 5, 7, and 10 after LPS-induced ARDS are shown: 1. Control Group SOD activity remained at a high and stable level at all time points, indicating that the body's antioxidant capacity was normal.

[0089] 2. ARDS Model Group (ARDS) Peripheral blood (C): SOD activity decreased significantly on Day 1, reached its lowest point on Day 3, and then gradually recovered (Day 5-10), but remained significantly lower than that of the normal group.

[0090] BALF(D): The trend of SOD activity changes is consistent with that of peripheral blood, reaching the lowest value on Day 3, and the overall level is lower than that of peripheral blood, suggesting that the local antioxidant capacity of the lungs is more severely impaired.

[0091] 3. ARDS + AMG487 group (broad-spectrum CXCR3 inhibitor) SOD activity in peripheral blood and BALF was significantly higher than that in the model group at all time points.

[0092] The lowest value (Day 3) increased significantly and recovered faster, approaching the level of the normal group by Day 10, suggesting that broad-spectrum inhibition of CXCR3 can effectively restore the body's and lung's local antioxidant capacity.

[0093] 4. ARDS + SCH546738 group (CXCR3A allosteric inhibitor) The SOD activity recovery effect was significantly better than that of the AMG487 group, and even showed a stronger recovery effect at some time points (such as Day 7 and Day 10).

[0094] It effectively enhanced the activity of antioxidant enzymes in the lungs (BALF) and systemic (peripheral blood), suggesting that CXCR3A is a key subtype that regulates the antioxidant capacity of ARDS.

[0095] in conclusion 1. LPS-induced ARDS can lead to a significant decrease in the antioxidant capacity of mice in the whole body and lungs, manifested by a sharp decrease in SOD activity, which reaches its lowest value on Day 3 and then gradually recovers.

[0096] 2. Inhibition of CXCR3 (especially the CXCR3A subtype) can significantly increase the activity of SOD in peripheral blood and BALF of ARDS mice, effectively restoring the body's antioxidant defense system.

[0097] 3. The CXCR3A allosteric inhibitor (SCH546738) showed significantly better results than the broad-spectrum CXCR3 inhibitor (AMG487) in restoring antioxidant capacity, further supporting the potential of CXCR3A as a therapeutic target for ARDS.

[0098] V. Analysis of Image Results (Inflammatory Factors IL-1β, IL-6, IFN-γ) Figure 5 , Figure 6 The changes in the levels of IL-1β, IL-6, and IFN-γ in peripheral blood (A, C, E) and bronchoalveolar lavage fluid (B, D, F) in four groups of mice (Control, ARDS, ARDS+AMG487, ARDS+SCH546738) on days 1, 3, 5, 7, and 10 after LPS-induced ARDS are shown. 1. Control Group The levels of the three inflammatory factors remained at extremely low levels at each time point without significant fluctuations, indicating that the underlying inflammatory state was stable.

[0099] 2. ARDS Model Group (ARDS) IL-1β (A, B): Levels were significantly elevated on Day 1, peaked on Day 3, and then gradually decreased (Day 5-10), but remained significantly higher than those in the normal group; levels in BALF were higher than those in peripheral blood, suggesting a more severe local inflammatory response in the lungs.

[0100] IL-6 (C, D): The trend is consistent with IL-1β, with a significant increase on Day 1-3, peaking on Day 3, and then slowly decreasing, with higher levels in BALF.

[0101] IFN-γ (E, F): Significantly increased on Day 1-5, peaking on Day 5, and then decreased, suggesting that the Th1 immune response is activated in ARDS.

[0102] 3. ARDS + AMG487 group (broad-spectrum CXCR3 inhibitor) The levels of the three inflammatory factors in peripheral blood and BALF at each time point were significantly lower than those in the model group.

[0103] The peak value was significantly reduced and the rate of decline was faster, reaching levels close to the normal group by Day 10, suggesting that broad-spectrum inhibition of CXCR3 can effectively suppress systemic and local pulmonary inflammatory responses.

[0104] 4. ARDS + SCH546738 group (CXCR3A allosteric inhibitor) The inhibitory effect on inflammatory factors was significantly greater than that of the AMG487 group, and even showed stronger inhibitory effects at some time points (such as Day 1-3 of IL-1β and IL-6).

[0105] It effectively reduced the levels of inflammatory factors in the lungs (BALF) and systemic (peripheral blood), suggesting that CXCR3A is a key subtype that regulates the inflammatory response in ARDS.

[0106] in conclusion 1. LPS-induced ARDS can lead to a significant enhancement of systemic and local pulmonary inflammatory responses in mice, manifested by a substantial increase in the levels of IL-1β, IL-6, and IFN-γ, which peaked on Day 1-5 and then gradually decreased.

[0107] 2. Inhibition of CXCR3 (especially the CXCR3A subtype) can significantly reduce the levels of inflammatory factors in peripheral blood and BALF of ARDS mice, effectively inhibiting the inflammatory cascade response.

[0108] 3. The CXCR3A allosteric inhibitor (SCH546738) showed significantly better anti-inflammatory effects than the broad-spectrum CXCR3 inhibitor (AMG487), further supporting the potential of CXCR3A as a therapeutic target for ARDS.

[0109] VI. Analysis of Image Results (Fibrosis Indicators KL-6, TGF-β1) Figure 7 The changes in KL-6 and TGF-β1 levels in peripheral blood (A, C) and bronchoalveolar lavage fluid (B, D) in four groups of mice (Control, ARDS, ARDS+AMG487, ARDS+SCH546738) on days 1, 3, 5, 7, and 10 after LPS-induced ARDS are shown. 1. Control Group At each time point, the levels of KL-6 and TGF-β1 remained at extremely low levels without significant fluctuations, suggesting no significant progression of pulmonary fibrosis.

[0110] 2. ARDS Model Group (ARDS) KL-6 (A, B): Levels were significantly elevated on Day 1, peaked on Day 3, and then gradually decreased (Days 5-10), but remained significantly higher than in the normal group; levels in BALF were higher than in peripheral blood, suggesting lung epithelial cell damage and the initiation of fibrosis.

[0111] TGF-β1 (C, D): The levels continued to rise from Day 1 to Day 7, reaching a peak from Day 7 to Day 10, indicating that fibrosis-related signaling pathways were continuously activated in the later stages of ARDS. The levels were even higher in BALF, suggesting that the local fibrotic microenvironment in the lungs was more significant.

[0112] 3. ARDS + AMG487 group (broad-spectrum CXCR3 inhibitor) At each time point, the levels of KL-6 and TGF-β1 in peripheral blood and BALF were significantly lower than those in the model group.

[0113] The peak value decreased significantly and the rate of decline was faster, reaching levels close to the normal group by Day 10, suggesting that broad-spectrum inhibition of CXCR3 can effectively suppress the progression of pulmonary fibrosis.

[0114] 4. ARDS + SCH546738 group (CXCR3A allosteric inhibitor) The inhibitory effect on fibrosis indicators was significantly greater than that of the AMG487 group, and even showed a stronger inhibitory effect at some time points (such as Day 7-10 of TGF-β1).

[0115] It effectively reduced the levels of fibrosis-related factors in the lungs (BALF) and systemic blood (peripheral blood), suggesting that CXCR3A is a key subtype that regulates fibrosis in the later stages of ARDS.

[0116] in conclusion 1. LPS-induced ARDS can lead to a significant increase in the levels of pulmonary fibrosis-related factors (KL-6, TGF-β1) in mice. KL-6 increases in the early stage (Day 1-3), indicating lung epithelial damage; TGF-β1 peaks in the later stage (Day 7-10), indicating the continued progression of fibrosis.

[0117] 2. Inhibition of CXCR3 (especially the CXCR3A subtype) can significantly reduce the levels of KL-6 and TGF-β1 in peripheral blood and BALF of ARDS mice, effectively inhibiting the progression of pulmonary fibrosis.

[0118] 3. The CXCR3A allosteric inhibitor (SCH546738) showed significantly better anti-fibrotic effects than the broad-spectrum CXCR3 inhibitor (AMG487), further supporting the potential of CXCR3A as a therapeutic target for ARDS fibrosis.

[0119] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome, characterized in that, Includes the following steps: S1: Establishing an animal ARDS model and setting up intervention groups: Experimental animals were randomly divided into at least four groups, namely, a normal control group, an ARDS model group, a CXCR3 broad-spectrum inhibitor intervention group, and a CXCR3A selective inhibitor intervention group; the model group and the two inhibitor intervention groups were given a causative agent to establish an ARDS model; the CXCR3 broad-spectrum inhibitor intervention group and the CXCR3A selective inhibitor intervention group were pretreated with the corresponding inhibitors before modeling; S2: Dynamic collection of biological samples: Lung tissue, peripheral blood and bronchoalveolar lavage fluid samples were collected from each group of animals at multiple different time points after injury. S3: Perform multi-dimensional indicator detection: For the samples collected in S2, detect indicators including the degree of pathological damage to lung tissue, the degree of fibrosis in lung tissue, the level of oxidative stress, the level of inflammatory factors, the level of fibrosis-related factors, and the gene expression levels of CXCR3, CXCR3A and CXCR3B in lung tissue. S4: Data Analysis and Mechanism Determination: Compare the differences in each indicator in S3 among the normal control group, ARDS model group, CXCR3 broad-spectrum inhibitor intervention group, and CXCR3A selective inhibitor intervention group. Based on the specific or superior regulatory effect of CXCR3A selective inhibitor on the indicators compared with CXCR3 broad-spectrum inhibitor, determine the core mechanism of action of CXCR3A in the occurrence and development of ARDS.

2. The method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome according to claim 1, characterized in that, In S1, the experimental animal is a C57BL / 6J mouse; the injury agent is lipopolysaccharide, which is administered via intranasal drip at a dose of 5-15 mg / kg body weight.

3. The method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome according to claim 1, characterized in that, In S1, the broad-spectrum CXCR3 inhibitor is AMG487; the selective CXCR3A inhibitor is SCH546738.

4. The method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome according to claim 3, characterized in that, In S1, AMG487 is pretreated by intraperitoneal injection at a dose of 3-7 mg / kg / day for 7-14 consecutive days; SCH546738 is pretreated by intraperitoneal injection at a dose of 8-12 mg / kg / day for 7-14 consecutive days.

5. The method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome according to claim 4, characterized in that, The pretreatment dose of AMG487 was 5 mg / kg / day for 10 consecutive days; the pretreatment dose of SCH546738 was 10 mg / kg / day for 10 consecutive days.

6. The method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome according to claim 1, characterized in that, In S2, the multiple different time points include day 1, day 3, day 5, day 7, and day 10 after the injury.

7. The method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome according to claim 1, characterized in that, In step S3, the detection of oxidative stress level is performed by enzyme-linked immunosorbent assay (ELISA) to detect the concentration of reactive oxygen species and the activity of superoxide dismutase in peripheral blood and bronchoalveolar lavage fluid.

8. The method for studying the mechanism of action of CXCR3A on acute respiratory distress syndrome according to claim 1, characterized in that, In S3, the levels of inflammatory factors are detected by using ELISA to detect the concentrations of interleukin-1β, interleukin-6, and gamma interferon in peripheral blood and bronchoalveolar lavage fluid.

9. A method for screening or evaluating CXCR3A inhibitors for the treatment of acute respiratory distress syndrome, characterized in that, Using the research method described in any one of claims 1-8, by comparing the detection results of the candidate compound intervention group with the ARDS model group and the CXCR3 broad-spectrum inhibitor intervention group described in claim 1, if the candidate compound has the same or better effect as the CXCR3 broad-spectrum inhibitor in alleviating lung tissue pathological damage, reducing oxidative stress index ROS, increasing antioxidant enzyme SOD activity, reducing the levels of inflammatory factors IL-1β, IL-6, and IFN-γ, reducing the levels of fibrosis indicators KL-6 and TGF-β1, and / or specifically downregulating CXCR3A mRNA expression in lung tissue, then the candidate compound is determined to be a potential CXCR3A inhibitor.

10. The use of the method according to any one of claims 1-8 in the preparation of tools or kits for studying the pathogenesis of acute respiratory distress syndrome or screening related therapeutic drugs.

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