In vitro method for screening candidate drugs for treating copd emphysema phenotype and use thereof

Abstract

The application discloses an in-vitro method for screening candidate drugs for treating COPD emphysema phenotypes and application thereof, and relates to the technical field of biological medicine.The in-vitro method for screening candidate drugs for treating COPD emphysema phenotypes comprises the following steps: S1, providing an in-vitro test system for expressing LPA2 protein; S2, contacting a candidate substance with the in-vitro test system; S3, detecting the expression level of LPA2 and the activity of a downstream signal path of the in-vitro test system; and S4, if the candidate substance can up-regulate the expression level of LPA2 and activate the activity of the downstream signal path, the candidate substance is determined as a candidate drug for treating COPD emphysema phenotypes. By constructing the LPA2 expression detection system, a drug screening platform for up-regulating the expression or activity of LPA2 is provided, and the drug screening platform has important clinical application values for the disease progression and drug treatment of COPD emphysema phenotype patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an in vitro method for screening candidate drugs for treating COPD emphysema phenotypes and its application. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a heterogeneous respiratory disease that seriously endangers public health. Its main clinical phenotype, emphysema, is characterized by irreversible destruction of alveolar structure. Current treatments can only relieve symptoms and cannot halt disease progression. Current research indicates that abnormal recruitment of neutrophils in lung tissue and the release of neutrophil extracellular traps (NETs) are positively correlated with COPD severity, but the upstream regulatory mechanisms of excessive NET formation remain unclear. Lysophosphatidylcholine (LPA), as an important bioactive lipid mediator, is involved through its receptor family (LPA... 1-3 LPA2 is a unique subtype of the LPA receptor family, widely involved in inflammation regulation in lung tissue. It not only exhibits anti-apoptotic activity through interaction with Siva-1, but its activation has also been shown to significantly reduce Th2-type airway inflammation in asthma models. However, its role in the development of chronic airway inflammation, particularly the COPD emphysema phenotype, has not been reported to date. Furthermore, interleukin IL-33 and its receptor system (ST2L / sST2) are key therapeutic targets for COPD, and sST2, as a decoy receptor, can inhibit the IL-33 / ST2 signaling pathway, thereby affecting the expression of downstream chemokines (such as human IL-8 or mouse CXCL1 / CXCL2) and the formation of NETs. Therefore, current technologies lack drug screening strategies and application protocols that directly target LPA2 and inhibit NET formation and treat the COPD emphysema phenotype by regulating the IL-33 / sST2 signaling axis. Summary of the Invention

[0003] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide an in vitro method for screening candidate drugs for treating COPD emphysema phenotype and its application. By constructing an LPA2 expression detection system, it provides a drug screening platform that targets the upregulation of LPA2 expression or activity, which has important clinical application value for the disease progression and drug treatment of COPD emphysema phenotype patients.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: An in vitro method for screening candidate drugs for treating COPD emphysema phenotypes includes the following steps: S1. Provide an in vitro assay system for expressing LPA2 protein; S2. Contact the candidate substance with the in vitro test system; S3. Detect the expression level of LPA2 and the activity of its downstream signaling pathways in the in vitro test system; S4. If a candidate substance can upregulate the expression level of LPA2 and activate its downstream signaling pathway activity, then the candidate substance is determined to be a candidate drug for treating the COPD emphysema phenotype.

[0005] As a preferred embodiment: the downstream signaling pathway activity includes the expression level of soluble ST2 mediated by LPA2; if a candidate substance can upregulate the expression of sST2, then the candidate substance is determined to be a candidate drug.

[0006] As a preferred embodiment: the downstream signaling pathway activity includes the inhibitory activity of LPA2 on the IL-33 / ST2 signaling pathway, which is specifically evaluated by detecting the expression levels of IL-33 downstream chemokines CXCL1 and CXCL2; if the candidate substance can reduce the expression levels of CXCL1 and CXCL2, then the candidate substance is determined to be a candidate drug.

[0007] As a preferred embodiment: the downstream signaling pathway activity includes the inhibitory activity of LPA2 on neutrophil infiltration and neutrophil extracellular trap formation; if a candidate substance can reduce neutrophil infiltration and / or inhibit neutrophil extracellular trap formation, then the candidate substance is determined to be a candidate drug.

[0008] Application of LPA2 agonists in the preparation of drugs for treating COPD emphysema phenotype: The in vitro method described above is used to screen for LPA2 agonists that can upregulate LPA2 expression levels and activate the activity of its downstream signaling pathways.

[0009] As a preferred embodiment, the LPA2 agonist is used to upregulate the expression of soluble ST2 and inhibit the IL-33 / ST2 signaling pathway, thereby reducing the expression of neutrophil chemokines CXCL1 and CXCL2.

[0010] As a preferred embodiment, the LPA2 agonist is used to reduce neutrophil infiltration in the lungs and inhibit the formation of neutrophil traps.

[0011] The use of IL-33 inhibitors in the preparation of drugs for treating COPD emphysema phenotype, wherein the IL-33 inhibitor is an IL-33 antibody.

[0012] Application of reagents for detecting LPA2 and / or soluble ST2 expression levels in the preparation of kits for prognostic assessment of emphysema.

[0013] As a preferred embodiment: decreased expression levels of LPA2 and / or soluble sST2 indicate a poor prognosis for emphysema; the kit also includes reagents for detecting the expression levels of IL-33 downstream chemokines CXCL1 and CXCL2.

[0014] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solution: First, by constructing an LPA2 expression detection system, this invention provides a drug screening platform for targeting the upregulation of LPA2 expression or activity, filling the gap in this field where there is a lack of specific targeted screening models.

[0015] Secondly, this invention elucidates for the first time that LPA2 enhances the inhibition of the IL-33 / ST2 signaling pathway by upregulating the expression of soluble ST2 (sST2), thereby reducing the expression levels of downstream chemokines CXCL1 and CXCL2, and thus reducing neutrophil infiltration in the lungs and inhibiting the excessive formation of NETs. The revelation of this complete signal regulation chain provides a clear pharmacological basis and multiple detectable efficacy evaluation indicators for the development of novel COPD therapeutics.

[0016] Third, this invention verifies the effectiveness of LPA2 agonists and IL-33 inhibitors (such as IL-33 neutralizing antibodies) in the treatment of COPD emphysema phenotypes, providing a direct drug candidate strategy for clinical translation. In particular, LPA2 agonists can intervene from the upstream source, with better treatment precision and potential safety advantages.

[0017] Fourth, this invention also provides a prognostic assessment kit based on the expression levels of LPA2 and / or sST2, which can monitor the disease progression and treatment response of patients with COPD emphysema phenotype, and has important clinical application value.

[0018] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0019] Figure 1 This invention Lpar2 Schematic diagram of PPE-induced emphysema exacerbated by deficiency; A shows HE-stained lung tissue sections from KO and WT mice, including emphysema-inducing and non-emphysema-inducing groups, 10×, scale bar 200µm. B shows identification by PCR amplification. Lpar2 - / - genes. Bar charts C and D show the MLI and MAN levels in the four groups, respectively. MLI: mean alveolar size, MAN: mean number of alveoli. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0020] Figure 2 This invention Lpar2 A schematic diagram showing the effects of deficiency on the expression of the IL-33 / sST2 signaling axis and neutrophil chemokines; A shows the relative mRNA expression levels of IL-33, CXCL1, CXCL2, and sST2 in different groups. B shows the protein expression of CXCL1, CXCL2, and sST2 in bronchoalveolar lavage fluid; CD shows the expression and spatial localization of IL-33 protein in lung tissue detected by immunohistochemistry, magnification 20×, mean optical density (AOD). E is the Western Blot experiment, and F is the bar chart of the Western Blot experiment; (n=6). *: P<0.05, **: P<0.01, ***: P<0.001.

[0021] Figure 3 This diagram illustrates the regulation of neutrophil infiltration and NET formation in mouse emphysematous lung tissue using LPA2 according to the present invention. Immunofluorescence analysis was performed on lung tissue from WT and KO mice at week 5 after intratracheal infusion of PPE and PBS. A shows immunofluorescence staining at 40× magnification; B shows the colocalization area ratio of CitH3 / MPO; C shows immunofluorescence staining at 20× magnification, with the mean fluorescence intensity of Ly6G (n=6); D shows the quantification of the mean fluorescence intensity (MFI) of Ly6G. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0022] Figure 4 The IL-33 antibody of this invention alleviates Lpar2 A schematic diagram of lung emphysema in mice lacking lung function; Figure 4 Image A is an HE staining image of lung tissue. Figure 4 Figure B shows a statistical graph of the mean linear intercept (MLI) and the mean number of alveoli (MAN) of lung tissue, which are quantified.

[0023] Figure 5 This diagram illustrates the inhibition of chemokine expression by the IL-33 antibody of this invention. A shows the relative mRNA expression levels of IL-33, CXCL1, CXCL2, and sST2 in different groups after intraperitoneal injection of IL-33 antibody. B shows the protein expression of CXCL1, CXCL2, and sST2 in bronchoalveolar lavage fluid after intraperitoneal injection of IL-33 antibody. C and D show the expression and spatial localization of IL-33 protein in lung tissue by immunohistochemical detection after intraperitoneal injection of IL-33 antibody, magnification 20× (n=6), and average optical density (AOD). E is the Western Blot experiment, and F is the bar chart of the Western Blot experiment (n=6). *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0024] Figure 6 This diagram illustrates how the IL-33 antibody of this invention inhibits neutrophil infiltration and NET formation in mouse emphysematous lung tissue. Immunofluorescence analysis was performed on mouse lung tissue after intraperitoneal injection of the IL-33 antibody. A shows the co-localization analysis of MPO and CitH3 by immunofluorescence staining at 40× magnification; B shows the quantification of the co-localization area; C shows the average fluorescence intensity of Ly6G (n=6) by immunofluorescence staining at 20× magnification; D shows the quantification of the MFI of Ly6G by immunofluorescence staining. *: P < 0.05, **: P < 0.01, ***: P < 0.001. Detailed Implementation

[0025] The present invention is as follows Figure 1 As shown in Figure 6, an in vitro method for screening candidate drugs for treating COPD emphysema phenotypes includes the following steps: S1. Provide an in vitro assay system for expressing LPA2 protein; S2. Introduce the candidate substance into the in vitro testing system; S3. Detect the expression level of LPA2 and the activity of its downstream signaling pathways in this in vitro test system; S4. If a candidate substance can upregulate the expression level of LPA2 and activate its downstream signaling pathway activity, then the candidate substance is determined to be a candidate drug for treating the COPD emphysema phenotype.

[0026] The activity of this downstream signaling pathway includes the expression level of soluble ST2 mediated by LPA2; if a candidate substance can upregulate the expression of sST2, then the candidate substance is determined to be a candidate drug.

[0027] The downstream signaling pathway activity includes the inhibitory activity of LPA2 on the IL-33 / ST2 signaling pathway, which is specifically evaluated by detecting the expression levels of IL-33 downstream chemokines CXCL1 and CXCL2; if a candidate substance can reduce the expression levels of CXCL1 and CXCL2, then the candidate substance is determined to be a candidate drug.

[0028] The downstream signaling pathway activity includes the inhibitory activity of LPA2 on neutrophil infiltration and neutrophil extracellular trap formation; if a candidate substance can reduce neutrophil infiltration and / or inhibit neutrophil extracellular trap formation, then the candidate substance is determined to be a candidate drug.

[0029] Application of LPA2 agonists in the preparation of drugs for treating COPD emphysema phenotype: This in vitro method was used to screen for LPA2 agonists that can upregulate LPA2 expression levels and activate the activity of its downstream signaling pathways.

[0030] This LPA2 agonist upregulates the expression of soluble ST2 and inhibits the IL-33 / ST2 signaling pathway, thereby reducing the expression of neutrophil chemokines CXCL1 and CXCL2.

[0031] This LPA2 agonist is used to reduce neutrophil infiltration in the lungs and inhibit the formation of extracellular traps for neutrophils.

[0032] The application of IL-33 inhibitors in the preparation of drugs for the treatment of emphysema, wherein the IL-33 inhibitor is an IL-33 antibody.

[0033] Application of reagents for detecting LPA2 and / or soluble ST2 expression levels in the preparation of kits for prognostic assessment of COPD emphysema phenotype.

[0034] Decreased expression levels of LPA2 and / or soluble sST2 indicate a poor prognosis in the COPD emphysema phenotype; the kit also includes reagents for detecting the expression levels of IL-33 downstream chemokines CXCL1 and CXCL2.

[0035] Example: In vitro method for screening candidate drugs for treating COPD emphysema phenotype and its application Materials and Methods: animal: Lpar2 Knockout (KO) mice were bred in a BALB / c background for at least ten generations. Wild-type mice (WT) and Lpar2 KO mice (male, 8–10 weeks old) were kept in a specific pathogen-free (SPF) animal room with a 12-hour light-dark cycle and free access to water at 20–26°C.

[0036] Modeling: PPE modeling was achieved by multiple small-dose intratracheal instillations of PPE. Mice were instilled with PPE (3u / 20g) via intratracheal instillation, while control mice were instilled with an equal volume of sterile PBS via intratracheal instillation in the same manner. This procedure was performed weekly for four consecutive weeks. WT mice and... Lpar2 KO mice (n=6 per group) were sacrificed by cervical dislocation and lung tissue and bronchoalveolar lavage fluid (BALF) were collected.

[0037] Intraperitoneal injection of anti-IL-33: The intervention experiments were divided into four models: WT Model + PBS, KO Model + PBS, WT Model + anti-IL-33, and KO Model + anti-IL-33. One hour before weekly tracheal instillation of PPE, mice were intraperitoneally injected with purified IL-33 antibody diluted in sterile PBS (100 μg / mouse), while the control group received an equal volume of sterile PBS intraperitoneally. One hour after antibody administration, PPE was instilled intratracheally.

[0038] HE staining: After sampling and processing in week 5, lung tissue was separated and fixed with 4% paraformaldehyde. It was then embedded in paraffin and cut into 4µm sections. After hematoxylin and eosin (HE) staining, the HE-stained sections were used to estimate alveolar enlargement. Digital imaging of HE-stained paraffin-embedded mouse lung sections was performed at 100X magnification. A blinded researcher evaluated three random fields of view using ImagePro Plus image analysis software. Alveolar enlargement was estimated using the Mean Linear Intercept (MLI) method; the Mean Alveolar Numbers (MAN) formula was used to calculate the mean alveolar number, reflecting alveolar density.

[0039] Bronchoalveolar lavage fluid (BALF) measurement: Five weeks after mouse modeling, BALF was collected, centrifuged at 1500 g for 10 minutes, and the supernatant was used for quantitative analysis of CXCL1, CXCL2 and sST2 using the corresponding ELISA kits.

[0040] RT-qPCR: 30 mg of lung tissue was extracted using TRK Lysis Buffer from the Omega kit, followed by mechanical homogenization using a tissue homogenizer. Subsequent purification was performed according to the manufacturer's instructions. After purification, RNA purity and concentration were determined using a K5800 spectrophotometer (KAIAO). For each sample, 1 μg of total RNA was reverse transcribed into cDNA using the ReverAid First Strand cDNA Synthesis Kit. Amplification was performed using a SuperStar Universal SYBR MasterMix. Two [units of equipment were used]. −ΔΔCT Calculation results of the method.

[0041] WB experiment: The right lung of each mouse was preserved at -80°C. After homogenization of the tissue samples, protein extraction was performed using an extraction reagent supplemented with protease inhibitors. Protein concentration was determined using a BCA protein assay kit. Proteins were separated by 10% SDS-PAGE and then transferred to PVDF membranes, followed by blocking in protein-free rapid blocking buffer for 30 minutes, and then incubated overnight at 4°C with the following primary antibodies: anti-IL-33, CitH3, MPO, and β-actin. The primary antibodies were diluted with TBST, and the secondary antibodies were diluted 1:20000 with TBST. The next day, the membrane was incubated with the secondary antibody for 1 hour. Detection was performed using horseradish peroxidase-labeled secondary antibody. The membranes were then exposed to an enhanced chemiluminescent solution, and band density was quantified using ImageJ software.

[0042] Immunofluorescence: Lung tissue sections were baked at 65°C for 30 minutes, followed by deparaffining and dehydration. Samples were boiled in EDTA buffer (pH 9.0 mmol / L) for 5 minutes for antigen retrieval. The samples were then permeabilized by incubation in 0.1% Triton X-100 for 10 minutes. Next, the samples were blocked with normal bovine serum at room temperature for 1 hour. Sections were incubated overnight at 4°C with primary antibodies of Ly6G, CitH3, and MPO, followed by incubation at room temperature for 50 minutes with secondary antibodies of FITC, Alexa Fluor-488, and TRITC. Slides were counterstained with DAPI, mounted, and imaged using a fluorescence microscope (Nikon).

[0043] Immunohistochemistry: Lung tissue sections were prepared according to the previously described method. After dewaxing, the sections were retrieval with EDTA pH 9.0 antigen, treated with 3% H2O2 to inhibit endogenous peroxidase activity, blocked with 1% bovine serum, and incubated with IL-33 overnight at 4°C. The next day, the samples were re-incubated at 37°C for 30 minutes, followed by incubation at 37°C with HRP-conjugated secondary antibody for 30 minutes. DAB solution was added and incubated at room temperature, and cell nuclei were stained with hematoxylin. The stained sections were observed under a light microscope.

[0044] Statistical analysis: Statistical analysis and graphing were performed using GraphPad Prism 10.1.2 software. All data were first tested for normality using the Shapiro-Wilk test and for homogeneity of variance using the Brown-Forsythe test. For normally distributed data with homogeneous variances, one-way ANOVA was used for comparisons among multiple groups, followed by the Tukey test for subsequent multiple comparisons. If the data were normally distributed but had unequal variances, Brown-Forsythe and Welch ANOVA tests, along with Dunnett's T3 test, were used for multiple comparisons. P A value <0.05 is considered statistically significant.

[0045] Discussion of Results: Lpar2 The absence of PPE exacerbates PPE-induced emphysema To clarify the role of LPA2 in the pathogenesis of emphysema, an emphysema model was established using Lpar2 KO mice, with WT mice serving as a control. PCR detection confirmed... Lpar2 Deletion in lung tissue of KO mice ( Figure 1 (B) Compared with the WTModel group, the KO Model group showed more severe alveolar wall destruction. Figure 1 (A), this result was further quantified using MLI and MAN scores ( Figure 1 C- Figure 1 (D). This proves... Lpar2 The absence of [certain substances] significantly exacerbated the pathological damage of PPE-induced emphysema. (Note:) Lpar2 The absence of PPE exacerbates PPE-induced emphysema.

[0046] Lpar2 The absence of this signal promotes the expression of the IL-33 / sST2 signaling axis and neutrophil chemokines. To investigate the molecular mechanism by which LPA2 exacerbates emphysema, the expression of related inflammatory factors was detected. RT-qPCR results showed that, compared with the WT mouse emphysema group, the mRNA expression levels of IL-33, CXCL1, and CXCL2 in the lung tissue of the KO Model group were significantly increased, while the expression level of sST2 was significantly decreased. Figure 2 (A). ELISA testing also confirmed that the concentrations of CXCL1 and CXCL2 in the bronchoalveolar lavage fluid of the KO Model group were significantly increased, while the concentration of sST2 was decreased. Figure 2 (B). In addition, Western blot ( Figure 2 The results (C) further confirmed that the expression level of IL-33 protein in the lung tissue of the KO Model group was significantly upregulated. To further clarify the spatial expression characteristics of IL-33, immunohistochemical staining was performed on lung tissue sections (C). Figure 2 (D). Quantitative analysis of the mean optical density (AOD) of the positive regions using ImageJ showed that, compared with the WT Model group, the expression intensity of IL-33 in the lung tissue of the KO Model group was significantly increased, a result consistent with the trends observed by Western blot and RT-qPCR. In summary, Lpar2 The absence of sST2 leads to overactivation of IL-33 and its downstream neutrophil chemokine signaling by downregulating sST2 secretion; based on this embodiment, the application of LPA2 agonists in the preparation of drugs for treating COPD emphysema phenotypes is readily apparent.

[0047] LPA2 regulates neutrophil infiltration and NET formation in lung tissue of mice with emphysema. This application investigated the neutrophil infiltration in lung tissue induced by PPE endotracheal instillation model and whether it induced NETs formation. Immunofluorescence staining results showed that the degree of Ly6G infiltration was significantly higher in the KO Model group than in the WT Model group ( Figure 2 C- Figure 2 (D); Immunofluorescence staining of lung tissue was used to analyze Cit-H3 and MPO, the main markers of NETs. Their co-localization results showed that PPE induced the formation of NETs. Figure 3 China A- Figure 3 (B)

[0048] IL-33 antibody reduced Lpar2 Emphysema in mice lacking lung tissue: To verify the role of IL-33 signaling in the above process, mice were treated with IL-33 antibody. Pathological scores showed that after antibody intervention, alveolar damage in both the KO Model + anti-IL-33 group and the WT Model + anti-IL-33 mouse emphysema group was significantly reduced compared with the untreated group. Figure 4 (A), through MLI scoring, MAN ( Figure 4 The B) score was quantified. These results suggest that blocking IL-33 signaling can effectively alleviate [the condition]. Lpar2 The pathological changes in emphysema caused by the absence of these features.

[0049] IL-33 antibody inhibits chemokine expression, suggesting that LPA2 has a protective effect against emphysema in mice by promoting sST2 secretion. RT-qPCR analysis showed that the expression levels of IL-33, CXCL1, and CXCL2 mRNA in lung tissues of both the KO Model + anti-IL-33 group and the WT Model + anti-IL-33 group were significantly decreased after IL-33 antibody intervention. Figure 5(A), while sST2 mRNA expression in the WT mouse emphysema group was higher than in the other three groups; the expression of CXCL1 and CXCL2 proteins in bronchoalveolar lavage fluid was detected by ELISA, and their expression was significantly downregulated in the KO Model+anti-IL-33 group and the WT Model+anti-IL-33 group after the use of IL-33 antibody ( Figure 2 In the WT mouse emphysema group, sST2 protein expression was significantly increased in bronchoalveolar lavage fluid; after using IL-33 antibody, Western blot experiments showed that... Figure 5 China E- Figure 5 (F) and immunohistochemical experiments ( Figure 5 C- Figure 5 (D) Detection showed that IL-33 expression was significantly decreased in both the KO Model + anti-IL-33 group and the WT Model + anti-IL-33 group. In summary, the data indicate that IL-33 antibody can effectively inhibit the expression of neutrophil chemokines; furthermore, even under the pathological condition of emphysema, the sST2 level in WT mice was significantly higher than that in the KO group. This result demonstrates that LPA2 has the potential function of promoting sST2 secretion, and that sST2 binds to IL-33 to exert a protective effect in alleviating the development and progression of emphysema in vivo.

[0050] IL-33 antibody inhibited neutrophil infiltration and NET formation in emphysematous lung tissue of mice. Immunofluorescence staining was used to analyze Ly6G infiltration. After the use of IL-33 antibody, the degree of Ly6G infiltration in the KO Model + anti-IL-33 group and the WT Model + anti-IL-33 group was significantly reduced. Figure 6 C- Figure 6 (D), and the number of CitH3 and MPO co-localizations also decreased significantly ( Figure 6 China A- Figure 6 (B)

[0051] Discussion of Results: 1. In this study, NETs were formed in mouse emphysema induced by PPE. Lpar2 The absence of IL-33 increases the accumulation of neutrophil chemokines and NET formation, thus exacerbating emphysema. The use of IL-33 antibodies reduces neutrophil accumulation and NET formation, thereby alleviating emphysema. In conclusion, LPA2 plays a crucial role in PPE-induced NET formation and the development of emphysema by regulating the IL-33 / sST2 axis.

[0052] 2. LPA works by interacting with its specific receptor (LPA). 1–6LPA plays a key regulatory role in various inflammatory diseases through its binding with other molecules. Previous studies have shown that LPA primarily regulates these mechanisms through the binding of LPA molecules. 1–3 Receptors regulate airway epithelial cell function, with LPA2 exhibiting clear negative regulatory and protective effects in allergic lung inflammation and vascular endothelial barrier maintenance. Furthermore, studies have found that LPA2 can directly promote the secretion of soluble ST2 (sST2) by mouse and human bronchial epithelial cells. However, the specific regulatory role of LPA2 in the phenotypic pathological process of COPD emphysema has remained unclear. This study, through lung tissue pathological analysis, found that compared to wild-type (WT) emphysema mice, Lpar2 KO mice showed more severe alveolar wall damage, characterized by a significantly increased MLI and a significantly reduced MAN, thus confirming the important protective function of LPA2 in the phenotypic pathological evolution of COPD emphysema. Notably, the LPA receptor family members exhibit high functional heterogeneity; LPA1 typically plays a pro-inflammatory or pro-fibrotic role in the progression of pulmonary fibrosis, while the protective effect of LPA2 observed in this study contrasts sharply with this, suggesting that the role of LPA in lung pathology is highly dependent on specific receptor subtypes and the microenvironment.

[0053] 3. The data in this study indicate that, Lpar2 The absence of sST2 significantly downregulated sST2 expression in lung tissue, accompanied by overexpression of IL-33 and its downstream chemokines CXCL1 and CXCL2. Increased sST2 expression effectively reduced the expression of neutrophil chemokines. This suggests that LPA2 is a key upstream regulator for maintaining basal sST2 expression. Lpar2 The absence of LPA2 leads to reduced sST2 secretion, relieving its inhibition of the IL-33-driven inflammatory response, which in turn upregulates CXCL1 and CXCL2, recruiting a large number of neutrophils and NETs, ​​ultimately exacerbating emphysema. In summary, this study demonstrates that LPA2 plays an important role in the negative regulation of inflammation and lung protection in the COPD emphysema phenotype.

[0054] 4. This study also observed significant NET formation in the PPE-induced emphysema model, and Lpar2The absence of these genes significantly exacerbates this process. This study observed, through PPE-induced emphysema, that the greater the infiltration of neutrophils in the lungs, the greater the co-localization of CitH3 and MPO in lung tissue. This is highly consistent with previous clinical observations of elevated CitH3 and MPO expression and aggravated alveolar destruction in the lung tissue of COPD patients. In this study, we observed that the formation of neutrophils and NETs in the lungs decreased after the use of IL-33 antibody, while NETs formation was still greater in the KO Model + anti-IL-33 group than in the WT Model + anti-IL-33 mice, further suggesting that LPA2 is an upstream gene of NETs, ​​regulating NET formation.

[0055] This study reveals the key protective role of LPA2 in inhibiting excessive NET formation in a PPE-induced emphysema model by regulating the IL-33 / sST2 signaling axis. Lpar2 The deficiency leads to reduced sST2 secretion, weakening its neutralizing ability against IL-33. This, coupled with overactivation of CXCL1 / CXCL2 chemokines, exacerbates neutrophil infiltration in the lungs and NETs release, ultimately worsening alveolar structural damage. IL-33 antibodies can alleviate these pathological changes, further confirming the specificity of this signaling axis. This not only deepens our understanding of the NETs regulatory mechanism in the COPD emphysema phenotype but also provides a theoretical basis for targeting the LPA2 / IL-33 / sST2 pathway as a potential therapeutic strategy for COPD.

[0056] The key design focus of this invention is: First, by constructing an LPA2 expression detection system, this invention provides a drug screening platform for targeting the upregulation of LPA2 expression or activity, filling the gap in this field where there is a lack of specific targeted screening models.

[0057] Secondly, this invention elucidates for the first time that LPA2 enhances the inhibition of the IL-33 / ST2 signaling pathway by upregulating the expression of soluble ST2 (sST2), thereby reducing the expression levels of downstream chemokines CXCL1 and CXCL2, and thus reducing neutrophil infiltration in the lungs and inhibiting the excessive formation of NETs. The revelation of this complete signal regulation chain provides a clear pharmacological basis and multiple detectable efficacy evaluation indicators for the development of novel COPD therapeutics.

[0058] Third, this invention verifies the effectiveness of LPA2 agonists and IL-33 inhibitors (such as IL-33 neutralizing antibodies) in the treatment of COPD emphysema phenotypes, providing a direct drug candidate strategy for clinical translation. In particular, LPA2 agonists can intervene from the upstream source, with better treatment precision and potential safety advantages.

[0059] Fourth, this invention also provides a prognostic assessment kit based on the expression levels of LPA2 and / or sST2, which can monitor the disease progression and treatment response of patients with COPD emphysema phenotype, and has important clinical application value.

[0060] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. An in vitro method for screening candidate drugs for treating COPD emphysema phenotypes, characterized in that: Includes the following steps: S1. Provide an in vitro assay system for expressing LPA2 protein; S2. Contact the candidate substance with the in vitro test system; S3. Detect the expression level of LPA2 and the activity of its downstream signaling pathways in the in vitro test system; S4. If a candidate substance can upregulate the expression level of LPA2 and activate its downstream signaling pathway activity, then the candidate substance is determined to be a candidate drug for treating the COPD emphysema phenotype.

2. The in vitro method according to claim 1, characterized in that: The downstream signaling pathway activity includes the expression level of soluble ST2 mediated by LPA2; if a candidate substance can upregulate the expression of sST2, then the candidate substance is determined to be a candidate drug.

3. The in vitro method according to claim 1, characterized in that: The downstream signaling pathway activity includes the inhibitory activity of LPA2 on the IL-33 / ST2 signaling pathway, which is specifically evaluated by detecting the expression levels of IL-33 downstream chemokines CXCL1 and CXCL2; if a candidate substance can reduce the expression levels of CXCL1 and CXCL2, then the candidate substance is determined to be a candidate drug.

4. The in vitro method according to claim 1, characterized in that: The downstream signaling pathway activity includes the inhibitory activity of LPA2 on neutrophil infiltration and neutrophil extracellular trap formation; if a candidate substance can reduce neutrophil infiltration and / or inhibit neutrophil extracellular trap formation, then the candidate substance is determined to be a candidate drug.

5. The application of LPA2 agonists in the preparation of drugs for treating COPD emphysema phenotypes, characterized in that: LPA2 agonists capable of upregulating LPA2 expression levels and activating its downstream signaling pathways were screened using the in vitro method described in claim 1.

6. The application according to claim 5, characterized in that: The LPA2 agonist is used to upregulate the expression of soluble ST2 and inhibit the IL-33 / ST2 signaling pathway, thereby reducing the expression of neutrophil chemokines CXCL1 and CXCL2.

7. The application according to claim 5, characterized in that: The LPA2 agonist is used to reduce neutrophil infiltration in the lungs and inhibit the formation of neutrophil traps.

8. The application of IL-33 inhibitors in the preparation of drugs for treating COPD emphysema phenotypes, characterized in that: The IL-33 inhibitor is an IL-33 antibody.

9. Application of reagents for detecting LPA2 and / or soluble ST2 expression levels in the preparation of kits for prognostic assessment of COPD emphysema phenotype.

10. The application according to claim 9, characterized in that: Decreased expression levels of LPA2 and / or soluble sST2 indicate a poor prognosis in the COPD emphysema phenotype; the kit also includes reagents for detecting the expression levels of IL-33 downstream chemokines CXCL1 and CXCL2.

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