Application of TMPRSS11E gene in the preparation of drugs for acute lung injury

Abstract

This invention belongs to the field of drug preparation, specifically relating to the application of the TMPRSS11E gene in the preparation of drugs for acute lung injury. This invention establishes an acute lung injury model by constructing gene knockout mice and combining molecular biology and pathological analysis methods to study changes in related gene expression and inflammatory responses. The results show that TMPRSS11E expression is upregulated under inflammatory conditions. Further analysis using the TMPRSS11E gene knockout mouse model revealed that TMPRSS11E gene deletion significantly reduces lung tissue inflammation and damage, and to some extent improves mouse survival.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation, specifically involving the application of the TMPRSS11E gene in the preparation of drugs for acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is a severe lung disease caused by infection, trauma, and various inflammatory stimuli. Its typical characteristics include damage to the alveolar epithelial and vascular endothelial barriers, extensive infiltration of inflammatory cells, and increased alveolar permeability, resulting in high morbidity and mortality. While progress has been made in intensive care and supportive treatment, the molecular pathogenesis of ALI remains incompletely understood, making the search for new molecular targets crucial. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides the application of the TMPRSS11E gene in the preparation of drugs for acute lung injury. Deletion of the TMPRSS11E gene can significantly reduce lung tissue inflammation and damage, and improve the survival of mice to some extent.

[0004] The technical solution provided by this invention is as follows:

[0005] Application of the TMPRSS11E Gene in the Preparation of Drugs for Acute Lung Injury: Both the TMPRSS11E and SERPINE3 genes are known genes, and their nucleotide sequence information is available in publicly available databases. The TMPRSS11E gene sequence can be found in the NCBI or Ensembl databases (NCBI Gene: 28983 Ensembl: ENSG00000087128). The SERPINE3 gene sequence can be found in the NCBI Gene or Ensembl databases (NCBI Gene: 647174 Ensembl: ENSG00000253309).

[0006] Furthermore, the acute lung injury drug is used to inhibit the expression of the TMPRSS11E gene.

[0007] Furthermore, acute lung injury can be a trigger for sepsis.

[0008] Furthermore, the acute lung injury drug is used to alleviate lung tissue damage induced by sepsis.

[0009] Furthermore, the lung tissue damage includes alveolar structure destruction, significant thickening of alveolar septa, extensive infiltration of inflammatory cells, or alveolar hemorrhage.

[0010] Furthermore, the acute lung injury drug is used to reduce the expression level of pro-inflammatory factor mRNA induced by sepsis.

[0011] Furthermore, the pro-inflammatory factors include TNF-α, IL-1β, or IL-6.

[0012] Furthermore, the acute lung injury drug is used to reduce sepsis mortality.

[0013] Furthermore, the acute lung injury drug is used to reduce the inflammatory response and tissue damage induced by sepsis.

[0014] Furthermore, the acute lung injury drug is used to reduce the degree of pulmonary edema induced by sepsis.

[0015] Beneficial effects

[0016] In a CLP-induced acute lung injury mouse model, TMPRSS11E expression was significantly upregulated under inflammatory conditions, suggesting its potential involvement in the regulation of the inflammatory response. CLP models are widely used to simulate systemic inflammatory responses induced by severe infections and can stably induce lung inflammatory damage. This invention establishes an acute lung injury model by constructing gene knockout mice and combining molecular biology and pathological analysis methods to study changes in related gene expression and the inflammatory response. The results show that TMPRSS11E gene deletion significantly reduces lung tissue inflammation and improves mouse survival to some extent. Attached Figure Description

[0017] Figure 1 The expression of TMPRSS11E in lung tissue of CLP-induced septic mice; (A) Behavioral comparison between the Sham group and the CLP group 24 h after CLP modeling; (B) RT-qPCR detection of mRNA levels of TNF-α, IL-1β and IL-6 in lung tissue; (C) HE staining of lung tissue showing alveolar structure and inflammatory infiltration in the Sham group and the CLP modeling group 24 h after modeling; (D) qPCR detection of TMPRSS11E mRNA level; (E) Western blot and its quantitative analysis statistical graph; (F) Detection of TMPRSS11E protein expression in lung tissue from 0 h to 24 h after CLP modeling;

[0018] Figure 2 Establishment and identification of TMPRSS11E gene knockout mice (A) Genotype identification PCR electrophoresis image; (B) Western Blot detection of TMPRSS11E protein expression in lung tissues of WT and KO mice;

[0019] Figure 3 HE staining results of lung tissues from adult WT mice (A) and TMPRSS11E- / - mice (B);

[0020] Figure 4 The basic phenotypic analysis of TMPRSS11E gene deletion mice includes: (A) Genotypic distribution of mice at weaning; (B) Genotypic distribution of mice at birth; (C) Average number of mice per litter; (D) Survival curves of mice; (E) Age-related weight changes in male mice; and (F) Age-related weight changes in female mice.

[0021] Figure 5 Analysis of neonatal body weight changes in Tmprss11e gene-deleted mice; (A) Schematic diagram of neonatal WT mice; (B) Schematic diagram of neonatal mice; (C) Body weight change curves of WT and TMPRSS11E- / - neonatal mice within 0-5 hours. Body weight is expressed as a percentage of initial body weight. Blue dots represent WT mice (n=8), and red squares represent TMPRSS11E- / - mice (n=8); data are expressed as mean ± SEM. Statistical analysis showed significant differences between the two groups (p<0.005).

[0022] Figure 6 To alleviate acute lung injury induced by CLP modeling by knocking out the TMPRSS11E gene; (AB) HE staining of lung tissues from the WT+CLP group and the TMPRSS11E- / -+CLP group; (C) Statistical analysis of lung injury histological scores; (D) RT-qPCR detection of mRNA levels of TNF-α, IL-1β and IL-6 inflammatory factors in lung tissue; (E) Wet / dry weight ratio (W / D ratio) of lung tissue in each group of mice; (F) Survival curves of mice in each group after CLP modeling (n=16); Data are expressed as Mean±pm SD;

[0023] Figure 7 To verify the interaction between TMPRSS11E and SERPINE3 in a co-immunoprecipitation (CO-IP) assay;

[0024] Figure 8 To verify the formation of a complex between TMPRSS11E and SERPINE3 using Western blotting experiments; (A) SERPINE3 protein expression was detected using anti-MYC antibody; (B) TMPRSS11E protein expression was detected using anti-FLGA antibody; (C) SERPINE3 expression was increased (1:1 and 1:2) under the condition of TMPRSS11E expression level.

[0025] Figure 9 To analyze the expression of SERPINE3 in different tissues of mice using RT-PCR; (A) Expression of SERPINE3 in multiple tissues of Sham group mice; (B) Changes in SERPINE3 expression in multiple tissues after CLP-induced inflammation;

[0026] Figure 10SERPINE3 expression was upregulated in lung and liver tissues under inflammatory conditions; (A) Immunohistochemical staining of liver tissue; (B) Immunohistochemical staining of lung tissue;

[0027] Figure 11 The protein expression level of SERPINE3 was elevated in BALF samples from clinical patients; (A) Immunohistochemical detection of SERPINE3 protein expression; (BD) ELISA detection of TNF-α, IL-1β and IL-6 protein levels in BALF samples;

[0028] Figure 12 To investigate the upregulation of TMPRSS11E expression in an LPS-induced THP-1 cell inflammation model; (A) RT-PCR detection of SERPINE3 mRNA level; (B) Western blot detection of SERPINE3 protein level; (C) Immunohistochemistry detection of SERPINE3 expression;

[0029] Figure 13 To investigate the upregulation of TMPRSS11E expression in an LPS-induced THP-1 cell inflammation model; (A) RT-PCR detection of SERPINE3 mRNA level; (B) Western blot detection of SERPINE3 protein level; (C) Immunohistochemistry detection of SERPINE3 expression.

[0030] Figure 14 The expression level of SERPINE3 was increased in primary macrophages of LPS-stimulated lung tissue; (A) qPCR detection of SERPINE3 mRNA level; (B) cellular immunochemistry detection of SERPINE3 expression; (C) Western blot detection of TMPRSS11E protein level.

[0031] Figure 15 Establishment and identification of SERPINE3 gene knockout mice; (A) Genotype identification PCR electrophoresis image; (B) Western Blot detection of SERPINE3 protein expression in lung tissues of WT and KO mice;

[0032] Figure 16To exacerbate CLP-induced lung injury and reduce survival rate in CLP-induced septic mice by knocking out the SERPINE3 gene; (AB) HE staining analysis of pathological changes in lung tissue of WT mice and SERPINE3- / - mice 24 h after CLP modeling; (C) Statistical analysis of lung injury histological scores; (D) RT-qPCR detection of mRNA transcription levels of key pro-inflammatory factors in lung tissue of each group of mice; (E) Evaluation of pulmonary edema by wet / dry weight ratio (W / D ratio) of lung tissue (n=6); (F) Log-rank (Mantel-Cox) test analysis to statistically analyze the 168-hour survival rate of mice;

[0033] Figure 17 To replenish SERPINE3 and alleviate CLP-induced lung injury; (A) HE staining showing pathological changes in lung tissue; (B) Lung injury score; (C) Lung wet / dry ratio (Wet / D) mRNA expression levels of inflammatory factors TNF-α, IL-1β and IL-6 in lung tissue;

[0034] Figure 18 To investigate the regulatory role of SERPINE3 in TMPRSS11E-mediated macrophage M1 polarization. (A) Western blot analysis of TMPRSS11E protein expression in lung tissues of WT and SERPINE3⁻ / ⁻ mice under basal and LPS stimulation conditions. (B) Western blot analysis of SERPINE3 expression in cell lysates and culture supernatant. (C) Western blot analysis of iNOS and CD86 expression.

[0035] Figure 19 To investigate the roles of the TMPRSS11E and SERPINE3 genes in acute lung injury. Detailed Implementation

[0036] Example 1

[0037] The experimental mice used in this invention were all of the C57BL / 6 background strain. TMPRSS11E knockout mice (KO), SERPINE3 knockout mice (KO), and wild-type mice (WT) were all purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All experimental mice were housed in the SPF-grade barrier animal facility of Southeast University School of Medicine. The housing environment was kept at a constant temperature and humidity, following a 12-hour light-dark cycle. Sufficient high-pressure sterilized standard rodent experimental feed and drinking water were provided. Bedding was changed weekly. All procedures complied with the "3R" principles of laboratory animal welfare and use. All animal experiments were conducted according to protocols approved by the Southeast University Laboratory Animal Ethics Committee.

[0038] This invention utilizes CRISPR / Cas9 technology to construct a TMPRSS11E gene knockout (KO) mouse model. Analysis of the Ensembl database (ENSMUST00000161306.2) revealed that the TMPRSS11E gene contains only one transcript. The second and third exons (exon2-exon3) were designed as the target knockout region. This region contains a 247 bp protein-coding sequence (CDS), which is the core functional region of the TMPRSS11E protein. Knocking out this region leads to the complete loss of the protein's functional domain, thus achieving gene knockout.

[0039] The construction process followed standard technical procedures: Cas9 mRNA was mixed with sgRNA designed for a specific site and then introduced into fertilized eggs of C57BL / 6JGpt mice via microinjection. After the fertilized eggs developed to the two-cell stage, they were transplanted into pseudopregnant surrogate mice to obtain F0 generation mice. PCR amplification and sequencing were performed on the F0 generation mice to screen for positive individuals with successfully edited target genes. Subsequently, the positive F0 generation mice were backcrossed with wild-type C57BL / 6JGpt mice, and a genotype-stable experimental population was established in the F1 generation.

[0040] This invention utilizes CRISPR / Cas9 technology to construct a systemic knockout (KO) mouse model of the SERPINE3 gene. Transcript structure analysis from the Ensembl database (ENSMUST00000171692.2) revealed only one major transcript for the SERPINE3 gene. Based on the coding region distribution, exons 3 to 5 (exon3-exon5) were identified as the target knockout region. This region contains a 628 bp protein-coding sequence (CDS), which is the core functional region of the SERPINE3 protein. Knocking out this region will result in a frameshift mutation and complete inactivation of the SERPINE3 protein.

[0041] Cecal ligation and puncture-induced sepsis model (CLP model):

[0042] (1) Mice were fasted for 24 hours before surgery. Adult C57BL / 6 mice aged 8-10 weeks were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Surgical instruments were sterilized by high-pressure steam.

[0043] (2) The skin and peritoneum of the mouse were longitudinally cut along the midline of the abdomen (the incision length was about 2 cm). The abdominal cavity was opened to locate the cecum. The distal end of the cecum was ligated about 50% below the ileocecal valve, and a 21G sterile needle was used to perform a penetrating puncture to expel a small amount of fecal material to induce abdominal infection. After the operation, 1.0 mL of preheated saline was given for fluid resuscitation. The sham surgery group (Sham) only underwent abdominal exploration without ligation and puncture.

[0044] LPS-induced acute lung injury model in mice:

[0045] Six- to eight-week-old C57BL / 6 mice (weighing 20-25g) were randomly divided into a control group and an LPS experimental group. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The anesthetized mice were placed on a 60° inclined plate, and a specialized mouse endotracheal cannula was used to insert into the trachea. LPS (2 mg / kg, 40 µl) was slowly instilled into the trachea of ​​the experimental group mice, while the control group mice received an equal volume of autoclaved PBS instilled into the trachea. Immediately after instillation, the mice were rotated vertically, and then returned to their cages to await recovery.

[0046] Collection of primary macrophages from mouse lung tissue stimulated by LPS:

[0047] Twelve hours after LPS stimulation, mice were euthanized by cervical dislocation. The neck skin was disinfected with alcohol, and the glands and muscles were bluntly dissected to expose the trachea. The endotracheal tube was carefully inserted and secured with fine sutures to prevent leakage. 1 mL of pre-cooled sterile PBS was drawn into the lungs using a medical syringe and slowly injected. Once the lungs were full, the mouse's chest was gently compressed, followed by slow aspiration. This irrigation was repeated 3-5 times, with a recovery rate typically exceeding 80%. The above steps were repeated 3-5 times. All recovered fluid was combined into a single centrifuge tube and placed on ice. The collected BALF was centrifuged at 600 rpm for 10 minutes, the supernatant was discarded, and the medium was resuspended in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin. The medium was then seeded into six-well plates and incubated at 37°C for 4 hours. Non-adherent cells were washed away with PBS, leaving macrophages as the remaining adherent cells.

[0048] Extraction of mouse peritoneal macrophages:

[0049] For four days prior to the experiment, mice were intraperitoneally injected with 2 ml of sterile 3% mercaptoacetate medium daily. Mice were euthanized by cervical dislocation and immersed in 75% alcohol for 5 minutes. In a laminar flow hood, mice were immobilized in a supine position, and 5 mL of DMEM medium was injected intraperitoneally. The abdomen was gently massaged for 5 minutes. The irrigation fluid was slowly aspirated using a syringe, transferred to a sterile centrifuge tube, and placed on ice. The recovered irrigation fluid was centrifuged at 1000 rpm for 10 minutes, the pellet was resuspended in DMEM medium, and seeded into culture dishes. After incubation at 37 °C for 4 hours, non-adherent cells were washed away with PBS, and the adherent cells were identified as macrophages.

[0050] TMPRSS11E expression is upregulated in CLP-induced acute lung injury.

[0051] Acute lung injury (ALI) is a severe inflammatory disease of the lungs induced by various inflammatory stimuli. Its core pathological features are inflammatory cell infiltration, disruption of the alveolar-capillary barrier, and pulmonary edema. In the course of sepsis induced by the CLP model, lung tissue, due to its rich capillary network, often becomes one of the earliest target organs attacked by inflammatory mediators. Circulating inflammatory factors can rapidly act on lung tissue, thereby triggering a local inflammatory response and leading to damage to lung tissue structure.

[0052] To further clarify the changes of TMPRSS11E under inflammatory conditions, this invention first detected changes in TMPRSS11E expression in a CLP-induced mouse model of acute lung injury. A CLP model was established, and mouse lung tissue samples were collected at different time points for protein expression detection. The results showed that, compared with the sham group, mice 24 hours after CLP modeling exhibited typical clinical symptoms of sepsis, including general weakness, significantly reduced spontaneous activity, insensitivity to external physical stimuli, sluggishness, and increased respiratory rate. Figure 1 A). Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) are important biomarkers of inflammatory responses, and their expression levels can reflect the degree of inflammatory activation in the body and are closely related to disease progression. To assess the degree of inflammatory response induced by CLP, this invention detected the mRNA expression levels of TNF-α, IL-1β, and IL-6 in mouse lung tissue using RT-qPCR. The results showed that compared with the Sham group, the mRNA expression levels of TNF-α, IL-1β, and IL-6 were significantly increased after CLP treatment, and gradually increased with the extension of modeling time. Figure 1 B), indicating that the inflammatory response was effectively activated. Further histological changes were observed through HE staining of lung tissue. The results showed that the lung tissue of the Sham group mice was structurally intact, with clear alveolar morphology and no obvious inflammatory cell infiltration. In contrast, the lung tissue of the CLP group mice showed obvious pathological changes, including alveolar wall thickening, extensive neutrophil infiltration, and alveolar hemorrhage—typical features of acute lung injury. Figure 1 C). The above results histologically confirm that the CLP model successfully induced acute lung injury in mice. After confirming the successful establishment of the model, we further examined the expression changes of TMPRSS11E under inflammatory conditions. RT-qPCR results showed that, compared with the Sham group, the mRNA expression level of TMPRSS11E in lung tissue was significantly increased after CLP treatment ( Figure 1 D). Western blot analysis further confirmed that TMPRSS11E expression was also significantly increased at the protein level, reaching a peak approximately 24 hours after CLP surgery. Figure 1(E and F). In summary, this invention successfully established a CLP-induced mouse model of sepsis and acute lung injury, and verified that TMPRSS11E is induced to express under inflammatory conditions.

[0053] Example 2

[0054] TMPRSS11E gene deletion reduces acute lung injury

[0055] 1. Construction and identification of TMPRSS11E gene knockout mice

[0056] After confirming the upregulation of TMPRSS11E expression under inflammatory conditions, this invention constructed a systemic TMPRSS11E gene knockout mouse model and systematically identified it to further explore its biological function in the development of acute lung injury. The TMPRSS11E gene was knocked out using CRISPR / Cas9 gene editing technology, and a stable TMPRSS11E- / - mouse strain was established by deleting the key coding regions of Exon2–Exon3. Changes in inflammatory response and lung tissue damage were observed by observing the deletion of this gene, thus evaluating the functional role of TMPRSS11E in the disease process at a holistic level.

[0057] First, PCR was used to identify the mouse genotype. The results showed that PCR electrophoresis clearly distinguished the wild-type and knockout gene bands, indicating that the TMPRSS11E gene editing was successful and could be stably inherited. Figure 2 A). Subsequently, we further examined the protein expression of TMPRSS11E in lung tissue. An acute lung injury (ALI) model was established in mice via intratracheal instillation of LPS, and Western blot analysis was used to further detect the protein expression of TMPRSS11E in lung tissue. The results showed that TMPRSS11E protein was highly expressed in the lung tissue of LPS-induced wild-type mice; in stark contrast, the expression level of the target protein was significantly downregulated or even completely absent in the lung tissue of the knockout group mice. Figure 2 B). Successful gene knockout was confirmed at the protein level. As shown in the figure, qPCR and western blot results indicate successful TMPRSS11E knockdown, allowing for subsequent experiments. To further evaluate the impact of gene knockout on basic lung morphology, we compared adult WT and TMPRSS11E... - / - Histological examination of the lung tissue of mice was performed. HE staining results showed that, under unstimulated basal physiological conditions, the lung tissue structure of both groups of mice remained intact, alveoli were well-developed, and no obvious inflammatory cell infiltration was observed. Figure 3 This confirms that the absence of TMPRSS11E does not affect normal lung development and basic morphological structure.

[0058] Next, we will work on TMPRSS11E - / - The basic phenotype of the mice was further evaluated. We found that TMPRSS11E knockout mice could grow and reproduce normally, and the newborn mice had good survival rates. Specifically, TMPRSS11E knockout mice at birth ( Figure 4 A) and during weaning ( Figure 4 The genotype distribution of B) was not significantly different from that of WT mice, indicating that gene deletion had no significant effect on the birth and growth of mice. The number of mice per litter also did not differ significantly between the TMPRSS11E knockout group and the WT group. Figure 4 (C) indicates that the gene deletion did not significantly affect reproductive capacity. Furthermore, the survival curves of the TMPRSS11E knockout mice were similar to those of the WT mice ( Figure 4 (D) indicates that there was no significant difference in survival probability between the two groups of mice during their lifespan. Weight observation results showed that the weight gain trends of TMPRSS11E knockout mice and WT mice were similar across different sexes and age groups. Figure 4 E and Figure 4 (F) No significant difference in body weight was observed. Therefore, the deletion of the TMPRSS11E gene did not have a significant impact on the normal growth, reproduction, or survival of mice.

[0059] The aforementioned experimental results indicate that TMPRSS11E gene deletion mice did not show significant abnormalities in basic phenotypes such as birth rate, survival rate, and growth and development. Members of the TMPRSS11E family are expressed in skin epithelial tissue and may be involved in the regulation of skin barrier function and water homeostasis. Therefore, to further assess whether TMPRSS11E gene deletion affects surface water loss in newborn mice, we examined the weight changes of newborn mice over a certain period after birth. In the experiment, wild-type and TMPRSS11E mice were compared... - / - Mice were placed under constant environmental conditions, and their body weight changes were recorded over 0-5 hours. Normalized analysis was performed using the initial body weight as a percentage of total body weight. Figure 5 The results showed that both groups of mice were initially set to 100% body weight. Over time, the WT mice showed only a slight decrease in body weight, while the TMPRSS11E mice... - / - The weight loss in mice was more pronounced. At 5 hours, the body weight of WT mice remained at approximately 98.5%, while that of TMPRSS11E mice was significantly lower. - / - The weight loss in mice decreased to approximately 95.5%. Statistical analysis showed a significant difference between the two groups (p<0.005). This result suggests that the deletion of the TMPRSS11E gene may lead to increased water loss from the skin surface of newborn mice, resulting in accelerated weight loss. This phenomenon indicates that TMPRSS11E may play a role in the skin barrier function or water retention of newborn mice.

[0060] After successfully establishing the TMPRSS11E gene knockout mouse model, to further investigate the role of TMPRSS11E in sepsis-induced acute lung injury (ALI), we compared WT mice with TMPRSS11E. - / - Pathological changes in lung tissue of mice after CLP modeling.

[0061] First, histological changes were observed by HE staining of lung tissue. The results showed that CLP-treated WT mice exhibited significant pathological damage in their lung tissue, including alveolar structure destruction, marked thickening of alveolar septa, extensive inflammatory cell infiltration, and alveolar hemorrhage—typical features of acute lung injury. In contrast, TMPRSS11E- / - mice showed significantly reduced pathological changes, relatively intact alveolar structure, and a significantly decreased degree of inflammatory cell infiltration. Figure 6 A and Figure 6 B). Quantitative analysis of the lung injury score further confirmed that, compared with the WT group, TMPRSS11E - / - The lung injury scores of the mice in the group were significantly reduced. Figure 6 C). Subsequently, we examined changes in the expression of inflammatory factors. RT-qPCR results showed that CLP-induced pro-inflammatory factors TNF-α, IL-1β, and IL-6 were expressed in TMPRSS11E. - / - mRNA expression levels in mouse lung tissue were significantly downregulated compared to WT mice. Figure 6 D). The lung wet / dry weight ratio (W / D ratio) results indicate that TMPRSS11E - / - The degree of pulmonary edema in mice after modeling was significantly less than that in WT mice. Figure 6 E). Finally, survival curve analysis showed that during the 168-hour observation period, mice in the WT group began to die 12 hours post-surgery, with the mortality rate rising rapidly thereafter, ultimately resulting in a survival rate of only about 45%. In contrast, TMPRSS11E... - / - Mice in the first group experienced later mortality, and the rate of decline in the survival curve was significantly slower, with the final survival rate stabilizing at around 60%. The Log-rank (Mantel-Cox) test showed a statistically significant difference in survival between the two groups (P=0.002). This result confirms at the overall level that deletion of the TMPRSS11E gene significantly reduces the risk of death in septic mice. These results collectively indicate that deletion of TMPRSS11E exerts a significant protective effect against CLP-induced acute lung injury by inhibiting the inflammatory response and alleviating tissue damage.

[0062] Example 3

[0063] Interaction analysis between TMPRSS11E and SERPINE3

[0064] 1. TMPRSS11E interacts with SERPINE3.

[0065] During the body's inflammatory response, the dynamic balance between proteases and their inhibitors plays a crucial role in maintaining immune homeostasis. Numerous studies have shown that the activity of serine proteases is typically tightly regulated by members of the SERPIN (serine protease inhibitor) family. This regulation limits excessive protease activation by forming stable enzyme-inhibitor complexes, thereby preventing tissue damage caused by uncontrolled inflammatory responses. Therefore, we hypothesize that endogenous protease inhibitors may exert negative feedback regulation on TMPRSS11E during its role in inflammatory regulation.

[0066] Based on previous protein interaction screening results, this invention discovered that SERPINE3, a member of the SERPIN family, may potentially bind to TMPRSS11E. To verify whether a direct protein-protein interaction exists between the two, this invention constructed TMPRSS11E-FLAG and SERPINE3-MYC expression vectors in 293T cells and co-transfected them. Forty-eight hours after transfection, total cell protein was extracted, and co-immunoprecipitation (Co-IP) experiments were performed using MYC antibody. Western blot results showed that stable expression of TMPRSS11E-FLAG and SERPINE3-MYC was detected in the Input group, and the internal control protein GAPDH band was clear and consistent, indicating that the experimental system was stable and reliable. The TMPRSS11E-FLAG signal was detected in the complex precipitated with MYC antibody, while no corresponding band was detected in the IgG control group. Figure 7 This result indicates that there is a specific protein-protein interaction between TMPRSS11E and SERPINE3 under cellular conditions.

[0067] SERPIN family proteins typically inhibit the activity of target proteases by forming stable enzyme-inhibitor complexes. Therefore, the above results suggest that SERPINE3 may regulate its function by directly binding to TMPRSS11E. Notably, under co-expression conditions, we observed a complex band with a molecular weight higher than the monomeric protein, suggesting that SERPINE3 may form a stable complex with TMPRSS11E through a typical SERPIN family inhibitory mechanism. This invention further verifies whether a direct molecular link exists between the two. To verify whether this binding is a direct interaction, we performed in vitro protein incubation experiments. Similarly, we transfected 293T cells with both TMPRSS11E-FLAG and SERPINE3-MYC expression vectors. Forty-eight hours after transfection, total cell protein was extracted and Western blot experiments were performed. Figure 8 As shown, the results revealed a distinct new band when the two proteins were co-incubated, the location of which roughly corresponded to the sum of the molecular weights of the two proteins. Further concentration gradient experiments showed that the formation of this complex was dose-dependent. This finding suggests that SERPINE3 may act as an endogenous regulator of TMPRSS11E, and can directly bind to and form a stable complex, exerting a negative regulatory role in the inflammatory response by inhibiting its protease activity.

[0068] Example 4

[0069] SERPINE3 expression levels are significantly upregulated under inflammatory conditions.

[0070] 1. SERPINE3 expression in lung and liver tissues under inflammatory conditions

[0071] Having established the pro-damage effect of TMPRSS11E, this invention further seeks its potential regulatory factors. In the aforementioned study, we verified the interaction between SERPINE3 and TMPRSS11E through protein-protein interaction prediction and Western blot experiments. Therefore, we further focus on the expression changes of SERPINE3 under inflammatory conditions to explore its potential role in the inflammatory response. Because CLP surgery can induce severe systemic inflammatory responses and lead to multi-organ damage, this invention first examines the expression of SERPINE3 in different tissues in a mouse CLP sepsis model.

[0072] First, we used RT-PCR to detect SERPINE3 expression in various tissues. We extracted total RNA from several important organs in the Sham and CLP groups for RT-PCR. Figure 9As shown, electrophoresis results revealed that in the Sham group, SERPINE3 was largely absent in major organs such as the heart, liver, spleen, lung, kidney, salivary glands, esophagus, and reproductive organs, except for basal expression in the eyes. However, under CLP-induced sepsis, the expression pattern of SERPINE3 changed significantly. While expression was maintained in the eyes, it was significantly activated and highly expressed in the lungs and liver, while other tissues (salivary glands, esophagus, testes, penis, heart, spleen, and kidneys) maintained extremely low or no expression. The internal control showed stable expression, ensuring the comparability of the results. These results indicate that SERPINE3 expression is significantly activated in specific organs such as the lungs and liver during CLP-induced systemic inflammatory responses. The lungs and liver are the most frequently affected key organs in sepsis-associated multiple organ dysfunction syndrome (MIDS), and the significant upregulation of SERPINE3 in these two important organs suggests its potential involvement in the regulation of inflammatory responses and related tissue damage.

[0073] After detecting a significant upregulation of SERPINE3 mRNA expression in lung and liver tissues under CLP-induced inflammatory conditions, this invention employed immunohistochemistry (IHC) to detect changes in SERPINE3 protein expression at the tissue level and observe its localization within tissues in order to further verify these changes. Figure 10 As shown, the results indicated that in the normal control group, SERPINE3 protein expression levels were low, with only a small number of cells exhibiting weak positive staining. However, under inflammatory conditions, the positive signal of SERPINE3 in tissues was significantly enhanced, mainly manifested as an increase in brownish granular deposits in the cytoplasm. In liver tissue, the positive signal was mainly located in the hepatocytes and surrounding sinusoidal regions, appearing as punctate or granular staining. The spatial distribution and cellular morphology suggest that these positive cells may be Kupffer cells. In lung tissue, the positive signal was mainly distributed along the alveolar septa, and scattered positive mononuclear-like cells were also observed in some alveolar cavities. These results indicate that under inflammatory conditions, the protein expression level of SERPINE3 in lung and liver tissues is significantly increased, consistent with the aforementioned RT-PCR results, further confirming that SERPINE3 may be involved in inflammation-related tissue responses.

[0074] 2. SERPINE3 expression levels are elevated in BALF from patients with clinical pulmonary inflammation.

[0075] In the aforementioned animal experiments, we observed a significant upregulation of SERPINE3 expression in lung tissue in a CLP-induced acute lung injury model. To further verify whether this result also exists in clinical inflammatory states, this invention first analyzed clinical samples. Bronchoalveolar lavage fluid (BALF) is an important sample source reflecting the local inflammatory microenvironment of the lungs, containing various inflammation-related cells and secreted proteins, and is therefore often used to assess the inflammatory response status of the lungs. As shown in Table 1, this invention included bronchoalveolar lavage fluid (BALF) samples from 10 patients with clinical lung inflammation as the experimental group, and selected bronchoalveolar lavage fluid (BALF) samples from 10 patients with pulmonary nodules as clinical controls. This invention used ELISA to measure the levels of pro-inflammatory cytokines in the BALF of 20 subjects (…). Figure 11 B). Data showed that the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the BALF of patients with pulmonary inflammation were significantly higher than those in the control group. The cellular components in the BALF were then centrifuged to form cell slides, which were subsequently subjected to immunohistochemical (IHC) staining using a specific antibody against SERPINE3. Figure 11 A). The brownish areas represent the expression sites of the SERPINE3 protein. Morphologically, positive staining is mainly concentrated in immune cells (primarily alveolar macrophages) within the BALF. Clinical sample analysis revealed significantly elevated SERPINE3 levels in the BALF of patients with pulmonary nodules or tumors. Considering that BALF directly reflects the local inflammatory environment of the lungs, these results further suggest that SERPINE3 may be involved in the regulation of pulmonary inflammatory responses.

[0076] 3. SERPINE3 expression levels were elevated in an LPS-induced THP-1 cell inflammation model.

[0077] In the aforementioned study, we observed significantly elevated SERPINE3 expression levels in both a mouse CLP sepsis model and BALF samples from patients with clinical pulmonary inflammation. To further validate the effect of inflammatory stimulation on SERPINE3 expression at the in vitro cellular level, this invention uses the human mononuclear cell line THP-1 as an experimental model. An in vitro inflammation model was established using lipopolysaccharide (LPS) stimulation, and cell samples were collected at different time points to detect changes in SERPINE3 expression. THP-1 cells are a human mononuclear cell line commonly used to study inflammatory responses and macrophage-related immune regulatory mechanisms. Lipopolysaccharide (LPS) is an important component of the cell wall of Gram-negative bacteria and can induce inflammatory responses by activating the TLR4 signaling pathway; therefore, it is widely used to construct in vitro inflammation models.

[0078] Following LPS stimulation, we harvested THP-1 cells at different time points and extracted RNA and protein. RT-PCR results showed that in the unstimulated control group, SERPINE3 mRNA was almost not expressed. After 12 and 24 hours of LPS stimulation, the SERPINE3 mRNA band was significantly enhanced, while the internal control GAPDH expression remained stable. Figure 12 A). Western blot results were consistent with the trend of transcriptional level changes. SERPINE3 protein expression was almost undetectable in MH-S cells in the unstimulated and early 6 hours after stimulation; however, the SERPINE3 protein band was significantly enhanced after 12 and 24 hours of LPS treatment, indicating that its protein expression level gradually accumulated with prolonged inflammatory stimulation. Figure 12 B). Furthermore, immunocytochemical staining was used to further observe the expression and distribution of SERPINE3 in cells. The results showed that almost no obvious positive staining signal was observed in the control group THP-1 cells. However, after 24 hours of LPS treatment, the cell morphology changed significantly, the cell volume increased, and a large number of brownish-red positive granules were visible in the cytoplasm. Figure 12 (C) This suggests that SERPINE3 accumulates significantly intracellularly under inflammatory stimulation. These results indicate that inflammatory stimulation can also induce upregulation of SERPINE3 expression in human mononuclear cell lines. This finding is consistent with observations in animal models and clinical samples, further supporting the potential role of SERPINE3 in inflammatory responses.

[0079] 4. SERPINE3 expression levels were elevated in an LPS-induced MHS cell inflammation model.

[0080] In the aforementioned experiments, we observed a significant upregulation of SERPINE3 expression in an LPS-induced THP-1 cell inflammation model. To investigate the biological function of SERPINE3 in lung macrophage-mediated inflammatory responses, we selected the mouse alveolar macrophage line MH-S as our experimental subject and established an in vitro cellular inflammation model using lipopolysaccharide (LPS). MH-S cells, derived from mouse alveolar macrophages, are a commonly used cell model for studying lung inflammatory responses and immune regulation mechanisms. Since macrophages play a crucial role in the development and progression of acute lung injury, detecting changes in SERPINE3 expression in this cell model helps to further understand its potential function in lung inflammatory responses.

[0081] Following LPS stimulation, we collected MH-S cells at different time points and extracted total RNA and protein. RT-PCR results showed that, compared with the control group, the mRNA level of SERPINE3 was significantly increased after 12 and 24 hours of LPS treatment. Figure 13A). This suggests that inflammatory stimulation can induce upregulation of SERPINE3 expression at the transcriptional level. Western blot results were consistent with the trend of changes at the transcriptional level. Compared with the control group, the SERPINE3 protein band was significantly enhanced after 12 and 24 hours of LPS treatment, indicating that its protein expression level gradually accumulated with prolonged inflammatory stimulation. Figure 13 B). Furthermore, immunocytochemical staining was used to further observe the expression and distribution of SERPINE3 in cells. No obvious positive staining signal was observed in the control group, while a large number of brownish-red positive granules were observed 24 hours after LPS treatment. Figure 13 (C) This suggests that SERPINE3 is expressed under inflammatory stimulation. In summary, LPS stimulation can significantly induce increased expression of SERPINE3 at both the mRNA and protein levels in MH-S cells, suggesting that SERPINE3 may play an important role in alveolar macrophage-mediated inflammatory responses.

[0082] 5. SERPINE3 expression levels were elevated in LPS-stimulated primary macrophages of lung tissue.

[0083] In the aforementioned experiments, we observed that LPS stimulation significantly induced the upregulation of SERPINE3 expression in both THP-1 cell and MH-S cell inflammation models. To further verify whether this result also exists in immune cells that are closer to the physiological state in vivo, this invention first established a mouse acute lung injury model by intratracheal instillation of lipopolysaccharide (LPS), and then isolated primary lung macrophages from the lung tissue of the model mice to detect changes in SERPINE3 expression.

[0084] After successfully isolating primary lung macrophages, we extracted total RNA and protein from the cells and detected the expression level of SERPINE3. RT-PCR results showed that, compared with the control group, the mRNA expression level of SERPINE3 in mouse primary lung macrophages was significantly increased after 24 hours of LPS treatment. Figure 14 A). Further analysis of protein expression using Western blot revealed that SERPINE3 protein expression was low in the control group, but significantly increased after LPS stimulation. Figure 14(C) This suggests that SERPINE3 is significantly induced by inflammatory stimulation at both the transcriptional and protein levels. Furthermore, to further observe the expression and intracellular localization of SERPINE3 in primary lung macrophages, we performed immunocytochemical staining on the isolated macrophages. The results showed that almost no obvious positive staining signal was observed in the control group of primary lung macrophages; however, after 24 hours of LPS treatment, obvious brownish-red positive granules were observed in the cytoplasm, indicating significant intracellular accumulation of SERPINE3 under inflammatory conditions. Simultaneously, the LPS-treated macrophages increased in size and became more robust, exhibiting a typical state of inflammatory activation. In summary, in the primary lung macrophages isolated from the LPS-induced acute lung injury model, SERPINE3 was significantly upregulated at both the mRNA and protein levels, accompanied by a significant increase in intracellular protein signaling. This result is consistent with observations in THP-1 cell and MH-S cell inflammation models, further suggesting that SERPINE3 may play an important role in macrophage-mediated inflammatory responses.

[0085] Example 5

[0086] Construction and identification of SERPINE3 gene knockout mice

[0087] In the aforementioned experiments, we observed that SERPINE3 was significantly upregulated under inflammatory conditions, exhibiting a consistent trend of increased expression in mouse acute lung injury models, clinical inflammatory samples, and various inflammatory cell models. To further investigate the biological function of SERPINE3 in the development and progression of acute lung injury, this invention constructed a systemic SERPINE3 gene knockout mouse model and systematically identified it. To further clarify the function of SERPINE3 in inflammation, this invention knocked out the SERPINE3 gene using CRISPR / Cas9 gene editing technology, establishing a stable SERPINE3- / - mouse strain by deleting the key coding regions of Exon3–Exon5. Changes in inflammatory responses and lung tissue damage were observed by deleting this gene, evaluating the functional role of SERPINE3 in the disease process at a holistic level.

[0088] First, PCR was used to identify the mouse genotype. Two sets of specific primers were designed for the knockout site to perform genotyping. Figure 15 As shown, the results indicated that PCR electrophoresis bands could clearly distinguish between wild-type (WT) and knockout (KO) mice, and Western blot results further confirmed the absence of SERPINE3 protein expression in the knockout mice. These results demonstrate the successful establishment of the SERPINE3 gene knockout model.

[0089] Basic histological observations revealed that SERPINE3 deficiency has some impact on the development of certain tissues, such as changes in eye tissue structure, but the mice as a whole can survive normally and be used for disease model research.

[0090] Example 6

[0091] SERPINE3 gene knockout exacerbates acute lung injury induced by CLP modeling.

[0092] To clarify the biological function of SERPINE3 in acute lung injury (ALI), we compared wild-type (WT) mice with SERPINE3 using a CLP-induced sepsis model. - / - Lung tissue damage in mice after inflammatory stimulation.

[0093] First, pathological changes in lung tissue were observed using HE staining. The results showed that after CLP treatment, WT mice exhibited significant inflammatory changes in their lung tissue, including inflammatory cell infiltration and thickening of the alveolar septa. Meanwhile, in SERPINE3... - / - In mice, lung tissue damage was further aggravated, manifested as extensive alveolar structural destruction, massive infiltration of inflammatory cells, and diffuse hemorrhage, among other pathological changes. Figure 16 (A) Further semi-quantitative scoring analysis was performed to assess the degree of lung tissue damage. (SERPINE3) - / - The lung injury score of the group was significantly higher than that of the WT group, suggesting that SERPINE3 deficiency exacerbates the severity of CLP-induced acute lung injury. Figure 16 C). Furthermore, we examined changes in the inflammatory response in lung tissue. qPCR results showed that in SERPINE3… - / - In mouse lung tissue, the mRNA levels of pro-inflammatory factors such as IL-6, TNF-α, and IL-1β were significantly higher than those in the WT group. Figure 16 D). Meanwhile, the significantly increased lung wet / dry weight ratio further confirms that SERPINE3 deficiency exacerbates inflammation-induced pulmonary edema. Figure 16 E). Finally, survival curve analysis was used to assess the impact of SERPINE3 deficiency on overall survival in mice. The results showed that in the CLP-induced sepsis model, SERPINE3... - / - The survival rate of mice was significantly lower than that of WT mice. Figure 16 F). In summary, in the CLP-induced sepsis model, SERPINE3 deficiency significantly aggravated lung tissue inflammation, promoted the expression of inflammatory factors, and exacerbated pulmonary edema, while also reducing mouse survival, suggesting that SERPINE3 may play an important protective role in acute lung injury.

[0094] Example 7

[0095] Replenishment of the SERPINE3 plasmid alleviated CLP-induced acute lung injury and improved survival in mice.

[0096] Previous studies have demonstrated that knocking out the SERPINE3 gene exacerbates CLP-induced acute lung injury and reduces the survival rate of mice. To further verify the protective effect of SERPINE3 in acute lung injury, we reintroduced exogenous SERPINE3 gene (pCMV-SERPINE3) into SERPINE3 knockout mice and observed its alleviating effect on CLP-induced sepsis and lung injury.

[0097] First, the pathological changes in the lung tissues of mice in each group were observed by HE staining. The results showed that both CLP-treated WT mice and SERPINE3 knockout mice exhibited significant alveolar wall thickening and inflammatory cell infiltration. Specifically, SERPINE3... - / - The pathological damage was particularly severe in mice. However, in SERPINE3... - / - After recombining exogenous SERPINE3 in mice, lung tissue structure was significantly improved, and alveolar collapse and inflammatory exudation were significantly reduced compared with the knockout group. Figure 17 A). Further semi-quantitative analysis of the degree of histological damage using lung injury scoring showed that the score in the replenishment group was significantly lower than that in the knockout group ( Figure 17 (B) This confirmed that SERPINE3 has the function of reducing histopathological damage. Furthermore, the degree of pulmonary edema was assessed by detecting the wet / dry weight ratio of lung tissue. The results showed that after CLP treatment, the wet / dry weight ratio of the lungs in SERPINE3- / - mice was significantly higher than that in WT mice, while after SERPINE3 reinjection, the wet / dry weight ratio of the lung tissue in mice decreased significantly, showing a significant difference compared to the SERPINE3- / - group. Figure 17 (C) indicates that the restoration of SERPINE3 expression can effectively alleviate CLP-induced pulmonary edema. Subsequently, we examined the expression changes of inflammatory factors in lung tissue. qPCR results showed that after CLP stimulation, the mRNA levels of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 in the lung tissue of SERPINE3- / - mice were significantly higher than those in the WT group. However, after SERPINE3 gene restoration, the expression levels of the above pro-inflammatory factors were significantly lower than those in the SERPINE3- / - group (C). Figure 17 (D) indicates that restoring SERPINE3 expression can effectively inhibit the enhancement of the inflammatory response. In conclusion, SERPINE3 restoration effectively reduced pulmonary inflammation levels and alleviated acute lung injury.

[0098] Example 8

[0099] SERPINE3 inhibits TMPRSS11E-induced macrophage M0-M1 polarization.

[0100] To further explore the mechanism of action of SERPINE3 in the inflammatory response, this invention first examines whether SERPINE3 exerts its effect by regulating the expression level of TMPRSS11E. To this end, we compared wild-type and SERPINE3... - / - Changes in the expression of TMPRSS11E in mouse-derived macrophages. For example... Figure 18 As shown in the Western blot results, under basal conditions and LPS stimulation, SERPINE3 deficiency did not significantly alter the protein expression level of TMPRSS11E. This result suggests that SERPINE3 regulates TMPRSS11E not by affecting its expression level, but by modulating its functional activity. Considering that some SERPIN family proteins have secretory properties and can exert protease inhibitory effects extracellularly, we further investigated whether SERPINE3 can be secreted by cells. SERPINE3 expression plasmids or GFP control plasmids were transfected into 293T cells, and cell lysates and culture supernatants were collected for Western blot analysis. The results showed that a clear SERPINE3 protein band was detected in the culture supernatant of the SERPINE3 transfected group, while no corresponding signal was detected in the supernatant of the GFP group and the untreated group. This result indicates that SERPINE3 can be secreted into the extracellular environment by cells. To further investigate the role of SERPINE3 in macrophage inflammatory responses, LPS and IFN-γ were added to TMPRSS11E-overexpressing THP-1 cells to induce M1 polarization, followed by SERPINE3 intervention. Western blot results showed that the expression of M1 marker proteins iNOS and CD86 was significantly increased after LPS and IFN-γ stimulation, while the expression levels of iNOS and CD86 were significantly decreased after SERPINE3 treatment. These results suggest that SERPINE3 can inhibit the TMPRSS11E-enhanced macrophage M1 polarization response.

[0101] This invention systematically studies the role of TMPRSS11E and its endogenous inhibitor SERPINE3 in acute lung injury. By constructing a gene knockout mouse model and combining it with CLP and LPS-induced acute lung injury models, the functions of related molecules were comprehensively evaluated at both the whole animal and cellular levels. The following conclusions were drawn: TMPRSS11E expression is upregulated under inflammatory conditions. TMPRSS11E knockout mice showed significantly reduced inflammatory responses and lung tissue damage. Inflammatory stimulation significantly induced upregulation of SERPINE3 expression. Increased SERPINE3 expression was observed in BALF and THP-1 cells, MH-S cells, and mouse primary macrophages from patients with clinical lung inflammation. SERPINE3 gene knockout mice exhibited more severe inflammatory responses and tissue damage in the acute lung injury model. SERPINE3 can interact with TMPRSS11E. By inhibiting TMPRSS11E-mediated macrophage M1 polarization, it weakens the inflammatory response and protects lung tissue.

[0102] This invention systematically evaluated the pro-inflammatory role of TMPRSS11E in acute lung injury. It also discovered the protective function of SERPINE3 in lung inflammation. Furthermore, it proposes that TMPRSS11E–SERPINE3 constitute a novel protease-inhibitor regulatory axis and participate in the regulation of inflammatory responses.

[0103] The development of acute lung injury (ALI) is often accompanied by complex imbalances in the immune microenvironment and excessive activation of the inflammatory response. In inflammatory states such as infection or sepsis, the release of large amounts of inflammatory mediators can rapidly damage alveolar structure and lead to impaired gas exchange. Macrophage-mediated inflammatory signaling also plays a crucial role in disease progression. Therefore, elucidating the regulatory mechanisms of key molecules in the inflammatory process is of great significance for understanding the development of ALI. This invention focuses on the role of the transmembrane serine protease TMPRSS11E and its potential regulator SERPINE3 in the pulmonary inflammatory response, and provides a comprehensive analysis of their functions at the molecular, cellular, and animal levels.

[0104] First, this invention observed a significant upregulation of TMPRSS11E expression in a CLP-induced acute lung injury mouse model under inflammatory conditions, suggesting its potential involvement in the regulation of the inflammatory response. The CLP model is widely used to simulate systemic inflammatory responses induced by severe infections and can stably induce lung inflammatory damage. Further analysis using a TMPRSS11E gene knockout mouse model revealed that TMPRSS11E gene deletion significantly reduced lung tissue inflammatory damage and improved mouse survival to some extent. These results suggest that TMPRSS11E may play a promoting role in the amplification of the inflammatory response.

[0105] Regarding the exploration of mechanisms, considering that SERPIN family proteins typically inhibit their activity by forming complexes with serine proteases, this invention, based on previous screening results, further focused on SERPINE3 (reference). Through immunoprecipitation and Western blotting experiments, this invention confirmed that SERPINE3 can form a stable complex with TMPRSS11E, suggesting a direct functional interaction between the two.

[0106] As one of the key findings of this invention, we identified and validated the potential protective role of SERPINE3 in pulmonary inflammation. We examined changes in SERPINE3 expression in the same model and found that SERPINE3 is expressed at extremely low levels in normal lung tissue, but its expression is significantly upregulated under inflammatory stimulation. As another key finding of this invention, we systematically evaluated the role of SERPINE3 in pulmonary inflammation. The results showed that under normal physiological conditions, SERPINE3 expression is low in lung tissue, while its expression is significantly upregulated under inflammatory stimulation. This inflammatory damage-induced expression pattern suggests that SERPINE3 may participate in the negative feedback regulation of the body's inflammatory response, thereby playing a protective role in the progression of inflammation. Similar inflammatory damage-induced expression characteristics have been reported in other members of the SERPIN family.

[0107] To further clarify the expression distribution of SERPINE3 in vivo, this invention examined its expression in multiple tissues. RT-PCR results showed that under normal physiological conditions, SERPINE3 expression levels were low in most tissues, with basal expression only observed in the eye. However, under CLP-induced inflammation, SERPINE3 expression was significantly induced in lung and liver tissues. Immunohistochemical results further confirmed that the positive signal of SERPINE3 protein was significantly enhanced in lung and liver tissues after CLP treatment. These results indicate that SERPINE3 expression has a significant inflammatory induction characteristic and suggests its potential involvement in inflammation-related organ damage. The lung and liver are the most vulnerable organs to inflammatory responses, and the simultaneous upregulation of SERPINE3 in these two key organs suggests its potential role in regulating inflammatory responses. To verify the effect of inflammatory stimulation on SERPINE3 expression, this invention established an LPS-induced inflammatory cell model in vitro. The results showed that in THP-1 cells, both SERPINE3 mRNA and protein levels were significantly upregulated after LPS stimulation, exhibiting a time-dependent increasing trend. To further verify the prevalence of this phenomenon in lung immune cells, the same experiment was performed in the mouse alveolar macrophage line MH-S. The results showed that LPS stimulation significantly increased both SERPINE3 mRNA and protein expression. Since macrophages are important effector cells in lung inflammation, this result suggests that SERPINE3 may be involved in macrophage-mediated inflammatory regulation. Furthermore, to more closely approximate the physiological state in vivo, this invention further validated the phenomenon in primary mouse lung macrophages. The results showed that LPS stimulation also significantly induced an increase in SERPINE3 expression. These results indicate that SERPINE3 exhibits consistent inflammation-induced expression characteristics in macrophages from different sources, suggesting that it may play an important role in the regulation of inflammatory responses.

[0108] Having established that SERPINE3 expression is upregulated under inflammatory conditions, this invention further validated its function using a SERPINE3 gene knockout mouse model. We found that in an acute lung injury model, SERPINE3 deficiency significantly aggravated lung tissue inflammation, accompanied by increased inflammatory cell infiltration and elevated levels of pro-inflammatory cytokines, thus reducing mouse survival. However, exogenous SERPINE3 replenishment alleviated the degree of inflammation to some extent. These results suggest that SERPINE3 may play a protective role in the inflammatory response.

[0109] Furthermore, this invention demonstrated in 293T cells that SERPINE3 can be secreted into the extracellular environment, suggesting that it may participate in the regulation of the inflammatory microenvironment as a secreted protein. Further research revealed that in macrophages overexpressing TMPRSS11E, LPS stimulation significantly promoted the expression of M1 polarization markers iNOS and CD86, while SERPINE3 treatment significantly inhibited this process. These results suggest that SERPINE3 may suppress macrophage M1 polarization and attenuate the inflammatory response by inhibiting TMPRSS11E-related signaling pathways. Figure 19 As shown. In summary, this invention systematically reveals the important protective role of SERPINE3 in inflammation-related acute lung injury and proposes that SERPINE3 may participate in the disease process by regulating TMPRSS11E-mediated macrophage inflammatory responses.

[0110] A significant aspect of this invention lies in its expansion of the inflammatory regulatory mechanisms in acute lung injury from the perspective of protease-inhibitor balance. Previous studies have largely focused on the role of single inflammatory molecules or signaling pathways, with relatively little attention paid to the synergistic effects of membrane-bound proteases and their endogenous inhibitory systems in inflammatory regulation. The results of this invention suggest that TMPRSS11E and SERPINE3 may constitute a dynamic regulatory system, jointly participating in the regulation of the intensity of the inflammatory response under inflammatory stimulation. Building upon our previous research, TMPRSS11E, as a type II transmembrane serine protease, is primarily expressed in lung epithelial cells and macrophages under inflammatory conditions. It can cleave and activate the substrate protein PAR1 through its proteolytic activity, thereby triggering the activation of the downstream STAT3 signaling pathway and participating in the regulation of the inflammatory response. The results of this invention are consistent with the aforementioned mechanism, further supporting the crucial regulatory role of TMPRSS11E in the inflammatory response. Based on this, this invention systematically evaluated the functional roles of TMPRSS11E and SERPINE3 in acute lung injury at the overall level by constructing a gene deletion model of TMPRSS11E and SERPINE3. The results showed that TMPRSS11E and SERPINE3 have an antagonistic regulatory relationship in the inflammatory response, suggesting that SERPINE3 may play a negative regulatory role in the above-mentioned inflammatory-related processes by inhibiting the protease activity of TMPRSS11E.

[0111] It should be noted that this invention mainly explores the functions of related molecules based on gene deletion models and inflammatory phenotype analysis, and further in-depth research is needed on their specific molecular mechanisms. Furthermore, the differences in the roles of TMPRSS11E and SERPINE3 in different cell types and their translational significance in clinical diseases also warrant further investigation. Therefore, this invention not only provides a new perspective for understanding the regulatory mechanisms of excessive inflammatory responses in acute lung injury, but also provides a theoretical basis for conducting intervention research on related diseases from the perspective of protease-inhibitor balance. Although this invention still requires further exploration at the mechanistic level, through multi-model and multi-level experimental verification, it has systematically elucidated the roles of TMPRSS11E and SERPINE3 in acute lung injury from the perspective of functional and regulatory relationships, and has certain theoretical significance and research value.

Claims

1. Application of the TMPRSS11E gene in the preparation of drugs for acute lung injury.

2. The application according to claim 1, characterized in that, The acute lung injury drug is used to inhibit the expression of the TMPRSS11E gene.

3. The application according to claim 1, characterized in that, Acute lung injury is induced by sepsis.

4. The application according to claim 1, characterized in that, The acute lung injury drug is used to reduce lung tissue damage induced by sepsis.

5. The application according to claim 4, characterized in that, The lung tissue damage includes alveolar structure destruction, significant thickening of alveolar septa, extensive infiltration of inflammatory cells, or alveolar hemorrhage.

6. The application according to claim 1, characterized in that, The acute lung injury drug is used to reduce the expression of pro-inflammatory factor mRNA induced by sepsis.

7. The application according to claim 6, characterized in that, The pro-inflammatory factors include TNF-α, IL-1β, or IL-6.

8. The application according to claim 1, characterized in that, The acute lung injury drug is used to reduce the mortality rate of sepsis.

9. The application according to claim 1, characterized in that, The acute lung injury drug is used to reduce the inflammatory response and tissue damage induced by sepsis.

10. The application according to claim 1, characterized in that, The acute lung injury drug is used to reduce the degree of pulmonary edema induced by sepsis.

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