Application of glycolysis regulation gene PFKFB3 in bacterial lung injury

Abstract

The invention provides an application of a glycolysis regulation gene PFKFB3 in bacterial lung injury, and belongs to the field of biomedicine. The application finds that PFKFB3-mediated glycolysis flux in pulmonary vascular endothelial cells is increased due to pseudomonas aeruginosa infection, and PFKFB3 promotes damage of endothelial barriers through the way; the glycolysis regulatory gene PFKFB3 is an important target for maintaining the integrity of an endothelial barrier after infectious lung injury occurs; besides, the baicalin can be used as an effective adjuvant therapy for treating pulmonary vascular endothelial barrier destruction caused by pseudomonas aeruginosa infection, and when the PFKFB3 inhibitor and the baicalin are combined for use, the pulmonary vascular endothelial barrier destruction caused by pseudomonas aeruginosa infection can be synergistically protected.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to application of glycolysis regulatory gene PFKFB3 in bacterial lung injury. BACKGROUND

[0002] The lung is the core organ of the respiratory system, which continuously supplies O2 and removes CO2 for the body by its external respiration function, maintaining blood gas balance and internal environment stability. Many pathological factors can cause changes in lung function, leading to lung diseases, and even endangering life. At present, severe lung infection leads to lung injury, even sepsis or ARDS, with high mortality and poor treatment drug effect, only improving the quality of life of patients, and some severe patients ultimately need to rely on lung or heart transplantation surgery.

[0003] In-depth exploration of the pathogenesis of lung injury diseases and development of effective treatment strategies and drugs have become the mission to be completed in the medical field. Vascular endothelial cells play a crucial role in lung infection, not only as a physical barrier to maintain the integrity of lung structure, but also involved in regulating immune response, inflammatory process and tissue repair. The vascular endothelium acts as a semipermeable barrier between vascular smooth muscle cells and vascular lumen, and provides a non-thrombotic lining for the vascular system. In sepsis lung injury, the integrity of this barrier is destroyed, leading to fluid and cell extravasation, causing pulmonary edema and inflammatory cell infiltration, and the clinical manifestations are pulmonary edema (characteristic of acute lung injury) and high altitude pulmonary edema. So far, no drug has been approved for the treatment of this life-threatening endothelial injury disease. SUMMARY

[0004] The present application provides an application of glycolysis regulatory gene PFKFB3 in bacterial lung injury, to solve the problem in the prior art that the key regulatory target of lung vascular endothelial barrier damage in bacterial lung injury caused by Pseudomonas aeruginosa infection and effective treatment strategy have not been clear, and the endothelial barrier damage and related inflammatory process cannot be effectively reversed.

[0005] In a first aspect, the embodiments of the present application provide any one of the following applications of glycolysis regulatory gene PFKFB3 in bacterial lung injury: (1) application in the preparation of a drug for preventing or treating lung vascular endothelial barrier damage caused by Pseudomonas aeruginosa infection; (2) application as a therapeutic target in the preparation of a drug for repairing the integrity of lung vascular endothelial barrier; (3) application in the preparation of a drug for reducing the content of lung tissue inflammatory factors TNF-α, IL-6 and IL-1β.

[0006] Optionally, the application is achieved by inhibiting the glycolysis pathway mediated by PFKFB3.

[0007] Optionally, the PFKFB3-mediated glycolysis pathway is inhibited in any of the following manners: (1) reducing the glycolysis flux; (2) up-regulating the expression of vascular endothelial cadherin; (3) increasing the trans-endothelial electrical resistance by 50% or more; (4) reducing the vascular permeability.

[0008] In a second aspect, the embodiments of the present application provide any of the following uses of a PFKFB3 inhibitor: (1) for preparing a drug for preventing or treating lung vascular endothelial barrier damage caused by P. aeruginosa infection; (2) for preparing a drug for repairing the continuity of VE-cadherin protein; (3) for preparing a drug for reducing the content of inflammatory factors TNF-α, IL-6 and IL-1β in the lung lavage fluid.

[0009] Optionally, the PFKFB3 inhibitor includes one or more of 3-(3-pyridyl)-1-(4-pyridyl)-2-propen-1-one, N-((2-chloro-6-fluorophenyl)sulfonyl)-6-(4-morpholinyl)-1H-indazole-3-carboxamide and siRNA targeting PFKFB3.

[0010] Optionally, the PFKFB3 inhibitor is administered by intraperitoneal injection.

[0011] In a third aspect, the embodiments of the present application provide any of the following uses of a combination of a PFKFB3 inhibitor and baicalin: (1) for preparing a drug for treating lung vascular endothelial barrier damage caused by P. aeruginosa infection; (2) for preparing a drug for reducing the content of TNF-α, IL-6 and IL-1β in the lung lavage fluid; (3) for preparing a drug for repairing the continuity of VE-cadherin protein in lung tissue.

[0012] Optionally, the uses are achieved through the following synergistic mechanisms: (1) the PFKFB3 inhibitor inhibits the glycolysis flux, and the extracellular acidification rate is reduced by 30% or more; (2) baicalin enhances the stability of intercellular junction proteins in endothelial cells, and the expression of VE-cadherin is increased by 40% or more; (3) the combination of the two drugs reduces the lung vascular permeability by 60% or more.

[0013] Optionally, the mass ratio of the PFKFB3 inhibitor to the baicalin is 1:(1-3).

[0014] Optionally, the dosage of the baicalin is 50-150 mg / kg.

[0015] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art: (1) The key regulatory role of PFKFB3 in lung vascular endothelial barrier damage caused by Pseudomonas aeruginosa infection is first determined: The present application first discloses the specific mechanism of glycolysis regulatory gene PFKFB3 in promoting the destruction of lung vascular endothelial barrier after Pseudomonas aeruginosa infection by mediating the increase of glycolytic flux, and determines it as an important target for maintaining the integrity of endothelial barrier after infectious lung injury, which provides a new direction for the mechanism research and treatment of bacterial lung injury.

[0016] (2) Comprehensive and accurate verification system improves result reliability: In view of the defects of existing research in cell metabolism and barrier function verification, the present application uses pH-XtraGlycolysis test to prove the change of glycolytic flux, and combines in-vivo and in-vitro permeation test and transmembrane electrical resistance detection to reflect endothelial barrier function, instead of relying only on lactic acid detection and single VE-cadherin expression analysis, so that the conclusion is more scientific and convincing.

[0017] (3) PFKFB3 inhibitor and baicalin are used in combination to achieve synergistic effect: The present application finds that when PFKFB3 inhibitor and baicalin are used in combination, they can improve metabolic disorder and enhance the stability of junction proteins respectively, synergistically reduce lung vascular permeability by more than 60%, significantly improve the anti-inflammatory and endothelial barrier protection effect, and determine the optimal mass ratio and dosage, which provides a more efficient treatment strategy for bacterial lung injury and expands the application value of existing drugs.

[0018] (4) Targeted therapy improves the safety and pertinence of clinical application: The present application determines that PFKFB3 inhibitor can directly inhibit the glycolysis pathway mediated by PFKFB3, and precisely intervene in the endothelial barrier damage caused by Pseudomonas aeruginosa infection, and ensures that the drug is efficiently targeted to the lung by intraperitoneal injection, avoiding the side effects caused by non-specific action, which provides a potential scheme with strong pertinence and high safety for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings, which are incorporated into and form part of the specification, illustrate embodiments consistent with the present application and, together with the specification, serve to explain the principles of the application.

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows, and obviously, other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 The related experimental results figure of the lung infection of mice provided in Embodiment 1 of the present application is provided; Figure 2 The related experimental results figure of the lung infection of mice provided in Embodiment 1 of the present application is provided; Figure 3 The related experimental results figure of the lung infection of mice provided in Embodiment 1 of the present application is provided; Figure 4 The related experimental results figure of the lung infection of mice provided in Embodiment 1 of the present application is provided; Figure 5 The related experimental results figure of the lung infection of mice provided in Embodiment 1 of the present application is provided; Figure 6 The related experimental results figure of the lung infection of mice provided in Embodiment 1 of the present application is provided; Figure 7 The related experimental results figure of the lung infection of mice provided in Embodiment 1 of the present application is provided. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0023] The ranges described herein, such as ranges of values, ranges of ratios, and the like, encompass all possible subranges and individual numerical values within that range, e.g. a range from 1 to 6 or 1-6 should be read to include all possible subranges, e.g. 1 to 3, 2 to 5, etc. and individual numbers within that range, e.g. 1, 2, 3, 4, 5, and 6. Unless otherwise stated, the terms "include", "comprise", and the like as used herein are used in their open, conventional sense, and should be construed to mean "including but not limited to"; the terms "first", "second", and the like, are used merely as labels, and are not intended to impose numerical preferences or order of importance; the term "and / or" as used herein refers to individual or combined action of the referenced items; the terms "at least one", "one or more", and the like, refer to any combination of one or more items, including individual or combined action of one or more items. The proportional relationships, such as mass ratio, molar ratio, and the like, should be understood as the corresponding relationship between the former and the latter in the order of description. The raw materials, reagents, instruments and equipment used herein can be purchased or prepared by existing methods.

[0024] The technical idea of the present application is as follows: Endothelial cells themselves have metabolic activity and have an important influence on the supply of nutrients and oxygen to all tissues of the body. The main energy source of endothelial cells is glycolysis. The mechanism of action of glycolysis in pulmonary inflammatory diseases is complex and diverse, involving cell proliferation, apoptosis, inflammatory response, and many other aspects. The glycolysis level of endothelial cells is comparable to that of tumor cells, much higher than that of other healthy cells. Among them, lung microvascular endothelial cells play a key role in the integrity of the lung gas exchange interface, and they have a strong glycolytic capacity. PFKFB3 (6-phosphofructo-2-kinase / fructose-2, 6-bisphosphatase 3) is an important regulatory enzyme in the glycolysis pathway, which plays an important role in various physiological and pathological processes.

[0025] PFKFB3 gene is located on human chromosome 10p15.1, encoding a bifunctional enzyme containing 6-phosphofructo-2-kinase (PFK-2) and fructose-2, 6-bisphosphatase (FBPase-2) two active domains. PFKFB3 regulates glycolysis by catalyzing the synthesis and degradation of fructose-2, 6-bisphosphate (F2, 6BP). In the process of glycolysis, 85% of ATP is produced by the conversion of glucose to lactic acid with the help of PFKFB3. In the oxygen-induced retinopathy model, the growth of neovascularization in the retina is inhibited after the endothelial PFKFB3 gene is deleted or treated with PFKFB3 inhibitor in neonatal mice. In addition, tumors implanted in mice lacking endothelial cell PFKFB3 grow more slowly and have less blood flow. Therefore, the authors believe that blocking or deleting endothelial cell PFKFB3 will reduce angiogenesis in vitro and in vivo. Some research teams use chronic hypoxia to induce endothelial cell and smooth muscle cell-specific Pfkfb3 knockout mice to replicate the pulmonary arterial hypertension (PAH) model. The results show that after endothelial cell-specific Pfkfb3 is knocked out, the glycolysis level of endothelial cells is significantly reduced, the production of glycolysis metabolites-pyruvate is inhibited, and ultimately the expression of growth factors, pro-inflammatory cytokines and cell adhesion factors is reduced, thereby inhibiting the abnormal proliferation of pulmonary vascular smooth muscle cells and the infiltration of inflammatory cells around the pulmonary vessels, and inhibiting the development of hypoxia-induced pulmonary arterial hypertension. Some studies have found that endothelial cell-specific Pfkfb3 knockout can protect mice from lipopolysaccharide (LPS)-induced sepsis and acute lung injury, and endothelial cell PFKFB3 can regulate the expression of adhesion factors ICAM-1 and VCAM-1 and the release of inflammatory factors IL-1β and TNF-α through the NF-κB signaling pathway, thereby promoting tissue damage due to excessive inflammatory response. In diabetic nephropathy, enhancing PFKFB3-mediated endothelial glycolysis promotes the progression of diabetic nephropathy. Wang Litao's research team found that endothelial PFKFB3-mediated glycolysis promotes thoracic aortic vascular remodeling by regulating endothelial mesenchymal transition (EndoMT). In summary, PFKFB3 is a multifunctional metabolic regulator that plays an important role in glycolysis, cell proliferation in various cells in various diseases. The development and application of PFKFB3 are expected to provide new strategies and potential targets for the mechanism research and treatment of pulmonary infectious diseases.

[0026] Endothelial cells are attached to the basement membrane composed of collagen, fibronectin, laminin and glycosaminoglycans by cell-to-cell lateral connections and basal-lateral parts of the cells. Several different proteins are assembled on endothelial cells to form cohesive structures in the membrane, thus achieving the barrier properties of the vascular wall. Among the adhesive structures forming the barrier, the most important are adhesive junctions (AJ), gap junctions (GJ) and tight junctions (TJ). AJ is mainly composed of VE-cadherin and mediates cell-to-cell contact between all vascular endothelial cells. Maintaining the integrity of the endothelial barrier is essential for maintaining vascular homeostasis. Recent studies have reported some emerging therapeutic targets based on inhibiting excessive endothelial permeability, such as dynein light chain 1 and CXC chemokine ligand (CXCL) 13. Overexpression of dynein light chain 1 prevents LPS-induced endothelial hyperpermeability in rats; inhibition of dynein light chain 1 can significantly increase LPS-induced permeability. CXCL13 in human umbilical vein endothelial cells can significantly promote LPS-induced endothelial permeability by regulating the p38 MAPK signaling pathway.

[0027] The present application finds that after Pseudomonas aeruginosa (PA) infection in the lung, lung vascular endothelial cells are damaged, which is manifested as decreased expression of VE-cadherin on vascular endothelial cells, disrupted endothelial tight junctions, increased cell permeability, and impaired cell barrier function. At the same time, the expression of PFKFB3 in endothelial cells is increased, the production of lactic acid by cells is increased, the acidification in cells is obvious, and the glycolytic flux in cells is increased. In in vivo and in vitro experiments, after inhibiting the expression of PFKFB3 by using various PFKFB3 inhibitors, the expression of VE-cadherin in endothelial cells is restored, and the tight junctions of the cells are repaired. Therefore, the present application considers that the glycolytic regulatory gene PFKFB3 is an important target for maintaining the integrity of the endothelial barrier after lung infection.

[0028] Scutellaria baicalensis Georgi is a perennial herb of the Lamiaceae family. It has been used for at least 2000 years for the treatment of influenza, pneumonia, dysentery, and cancer. Baicalin, the most abundant flavonoid in the roots of Scutellaria baicalensis Georgi, has a variety of special biological activities. Baicalin shows a variety of pharmacological effects in vitro and in vivo, including antioxidant, anti-inflammatory effects, anti-apoptosis, anti-cancer, anti-diabetic, antibacterial, and antiviral effects. Studies have found that baicalin can protect mice from staphylococcal pneumonia caused by Staphylococcus aureus, reducing mortality from 80% to 28%, and protecting the lungs from the accumulation of cell infiltration. After PA infection, baicalin can regulate the formation of Pseudomonas aeruginosa biofilm by down-regulating the quorum sensing system and type VI secretion system to regulate the components related to Pseudomonas aeruginosa biofilm. Studies have also found that baicalin can alleviate the inflammatory damage of acute pneumonia rats induced by MDR Pseudomonas aeruginosa through arginine biosynthesis associated with intestinal microbiota. However, the application of baicalin in reducing lung injury caused by PA and playing a role in endothelial barrier protection has not been reported. The present application found that baicalin alone had no obvious effect on maintaining vascular endothelial injury caused by PA, but the combination of PFKFB3 small molecule inhibitor and traditional Chinese medicine small molecule baicalin could significantly improve the inflammatory infiltration of lung injury mice after PA infection, and play a significant anti-inflammatory role to protect the endothelial barrier function.

[0029] The existing research results show that PFKFB3 is a multifunctional metabolic regulatory enzyme, which plays an important role in glycolysis, cell proliferation in various cells of various diseases. Although the present application has reported that after PAO1 infects endothelial cells, autophagy key gene ATG5 regulates PFKFB3-mediated metabolic reprogramming, regulates cell adhesion junction, and maintains the barrier function of cells. The present application finds that after PAO1 infects the lung, the lung vascular endothelial cells are damaged, which is manifested as the expression of VE-cadherin on the vascular endothelial cells is reduced, the tight junction of the endothelial cells is destroyed, and the inflammation of the tissue is aggravated. At the same time, PFKFB3 expression increases in PAO1 infected HULEC-5a cells, and the cell produces increased lactic acid. After PFKFB3 is knocked down, the expression of VE-cadherin in the cell is partially restored. The present application believes that PAO1 infection leads to the increase of PFKFB3-mediated glycolysis metabolism, which destroys the tight junction of endothelial cells, and may cause lung tissue edema and aggravate inflammation in mice. Therefore, the present application proposes that PFKFB3 may become a potential target for treating lower respiratory tract infection. However, previous research has serious defects in cell metabolism and barrier function verification, only analyzes the increase of intracellular lactic acid after PA infection, and cannot completely prove the increase of cell glycolytic flux; only detects the expression of VE-cadherin, and cannot fully reflect the damage of endothelial barrier function. At the same time, the previous research lacks key verification data of PFKFB3 as a therapeutic target, and there is no application research of related PFKFB3 inhibitors. Therefore, in order to make up for the above shortcomings, the present application uses pH-Xtra Glycolysis test to detect the degree of extracellular acidification after PA infection of cells, further proves that PA infection promotes the increase of cell glycolytic flux. The change of endothelial cell permeability after PA infection is analyzed by in vivo and in vitro permeability test, and the transmembrane electrical resistance of the cell membrane is detected, which together proves the change of endothelial cell barrier function after PA infection. Importantly, the present application uses a variety of PFKFB3 inhibitors in in vivo and in vitro experiments to verify the hypothesis that PFKFB3 is a target for reducing endothelial cell glycolysis and maintaining the integrity of endothelial barrier after lung infection. As an important active molecule of Huangqi, Huangqin has anti-inflammatory effect on Pseudomonas aeruginosa infection when used alone, but has no obvious effect on maintaining the integrity of endothelial barrier. However, PFKFB3 inhibitors combined with Huangqin show a significant synergistic effect in the treatment of bacterial lung injury, and play an obvious protective role in the integrity of vascular endothelium. Therefore, the present application finds a new use of the two drugs in the treatment of bacterial lung injury.

[0030] Based on the above technical concept, the technical content of the present application is as follows: In a first aspect, the embodiments of the present application provide any one of the following applications of glycolysis regulatory gene PFKFB3 in bacterial lung injury: (1) the use in the preparation of a drug for preventing or treating lung vascular endothelial barrier damage caused by Pseudomonas aeruginosa infection; (2) the use as a therapeutic target in the preparation of a drug for repairing the integrity of the lung vascular endothelial barrier; (3) the use in the preparation of a drug for reducing the content of inflammatory factors TNF-α, IL-6 and IL-1β in lung tissue.

[0031] Based on the key role of glycolysis regulatory gene PFKFB3 in bacterial lung injury caused by Pseudomonas aeruginosa infection, its specific applications revolve around the core goal of improving lung vascular endothelial function and reducing lung inflammation, mainly in the following aspects: First, PFKFB3 can be used to prepare a drug for preventing or treating lung vascular endothelial barrier damage caused by Pseudomonas aeruginosa (PA) infection. After PA infection, the lung vascular endothelial cell barrier will exhibit core pathological features such as reduced VE-cadherin expression and damaged tight junctions. By regulating PFKFB3, the damage process can be directly intervened, thereby preventing or reversing the abnormality of endothelial barrier function.

[0032] Second, PFKFB3 can be used as a therapeutic target to prepare a drug for repairing the integrity of the lung vascular endothelial barrier. As a key molecule that mediates the abnormality of endothelial cell glycolysis and barrier destruction after PA infection, targeting PFKFB3 can precisely target the core mechanism of endothelial barrier damage, providing a clear direction for the development of related drugs.

[0033] In addition, PFKFB3 can also be used to prepare a drug for reducing the content of inflammatory factors in lung tissue. PA infection can activate PFKFB3, which in turn promotes the release of inflammatory factors (such as TNF-α, IL-6 and IL-1β). By regulating PFKFB3, the production of these inflammatory factors can be reduced, thereby alleviating lung inflammation and reducing lung tissue damage.

[0034] In some embodiments, the application is achieved by inhibiting the glycolysis pathway mediated by PFKFB3.

[0035] In some embodiments, the inhibition of the glycolysis pathway mediated by PFKFB3 has any of the following manifestations: (1) reducing the glycolysis flux; (2) up-regulating the expression of vascular endothelial cadherin; (3) increasing the transendothelial electrical resistance value by ≥50%; (4) reducing vascular permeability.

[0036] It should be noted that the implementation of the above application lies in the effective inhibition of the glycolysis pathway mediated by PFKFB3. PA infection can lead to increased expression of PFKFB3, which in turn drives the hyperglycolysis, ultimately damaging the endothelial barrier, so inhibiting this pathway is the core mechanism to reverse the above pathological process. This inhibitory effect can be reflected by a number of specific indicators, which collectively reflect the improvement of endothelial barrier function and the relief of metabolic disorders: first, reducing glycolytic flux directly reduces the excessive glycolysis metabolism of endothelial cells after PA infection (such as increased lactic acid production and extracellular acidification), thereby alleviating the toxic effects of metabolic disorders on endothelial cells; second, up-regulating the expression of vascular endothelial cadherin (VE-cadherin), which is a key protein that maintains the adhesion connection between endothelial cells, and its up-regulation can repair tight junctions and restore the physical integrity of the endothelial barrier; third, increasing the transendothelial electrical resistance value ≥ 50%, the transendothelial electrical resistance (TEER) is a quantitative indicator of endothelial barrier function, and the improvement of this parameter indicates that the integrity of the endothelial cell layer is significantly restored, and the ability of the barrier to block material penetration is enhanced; fourth, reducing vascular permeability directly improves the "leakage" phenomenon of the vascular endothelium after PA infection, reduces the extravasation of fluid and inflammatory cells, and relieves pulmonary edema and inflammatory infiltration.

[0037] In a second aspect, the embodiments of the present application provide the following applications of the PFKFB3 inhibitor: (1) in the preparation of a drug for preventing or treating lung vascular endothelial barrier damage caused by P. aeruginosa infection; (2) in the preparation of a drug for repairing the continuity of VE-cadherin protein; (3) in the preparation of a drug for reducing the content of inflammatory factors TNF-α, IL-6 and IL-1β in lung lavage fluid.

[0038] The application of the PFKFB3 inhibitor in the present application mainly reflects the following aspects: First, it can be used for preventing or treating lung vascular endothelial barrier damage caused by P. aeruginosa infection. By directly inhibiting the activity or expression of PFKFB3, it blocks the hyperglycolysis mediated by this gene, thereby interfering with the pathological mechanism of endothelial barrier damage from the upstream.

[0039] Second, it can repair the continuity of VE-cadherin protein. The destruction of VE-cadherin continuity is a hallmark of endothelial barrier damage, and the PFKFB3 inhibitor can restore this continuity and thereby rebuild the intercellular connection structure.

[0040] In addition, the content of inflammatory factors in the alveolar lavage fluid can also be reduced. By inhibiting PFKFB3, the activation of the inflammatory signaling pathway can be reduced, thereby reducing the levels of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β in the alveolar lavage fluid, and alleviating the local inflammatory response in the lungs.

[0041] In some embodiments, the PFKFB3 inhibitor comprises one or more of 3-(3-pyridyl)-1-(4-pyridyl)-2-propen-1-one, N-((2-chloro-6-fluorophenyl)sulfonyl)-6-(4-morpholinyl)-1H-indazole-3-carboxamide, and siRNA targeting PFKFB3.

[0042] The present application selects PFKFB3 inhibitors of the type 3-(3-pyridyl)-1-(4-pyridyl)-2-propen-1-one, N-((2-chloro-6-fluorophenyl)sulfonyl)-6-(4-morpholinyl)-1H-indazole-3-carboxamide, and siRNA, covering both small molecule chemical inhibitors and gene silencing tools, which work by inhibiting enzyme activity or reducing gene expression, respectively, to adapt to different drug development needs.

[0043] In some embodiments, the PFKFB3 inhibitor is administered by intraperitoneal injection.

[0044] The present application selects intraperitoneal injection administration, which can ensure efficient absorption of the inhibitor and target action on the lungs, rapidly exerting the effects of inhibiting PFKFB3 and repairing the endothelial barrier.

[0045] In a third aspect, the present application provides any one of the following uses of a combination of a PFKFB3 inhibitor and baicalin: (1) in the preparation of a drug for treating lung vascular endothelial barrier damage caused by P. aeruginosa infection; (2) in the preparation of a drug for reducing the content of TNF-α, IL-6, and IL-1β in alveolar lavage fluid; (3) in the preparation of a drug for repairing the continuity of lung tissue VE-cadherin protein.

[0046] The combination of PFKFB3 inhibitor and baicalin is mainly reflected in the following aspects: Firstly, it can be used for treating lung vascular endothelial barrier damage caused by P. aeruginosa infection. The two can synergistically act on different links of endothelial barrier damage, and can more efficiently repair the barrier function compared with single drug use.

[0047] Secondly, it can reduce the content of inflammatory factors in bronchoalveolar lavage fluid. The combination therapy inhibits the inflammatory response through a dual mechanism: the PFKFB3 inhibitor reduces the production of inflammatory factors, and baicalin enhances the anti-inflammatory effect, thereby significantly reducing the levels of TNF-α, IL-6, and IL-1β.

[0048] In addition, it can restore the continuity of VE-cadherin protein in lung tissue. Among them, PFKFB3 inhibitors alleviate the damage of VE-cadherin caused by metabolic abnormalities, while baicalin enhances the stability of the protein, and the two work together to promote the restoration of protein continuity.

[0049] In some implementations, the application is achieved through the following collaborative mechanism: (1) PFKFB3 inhibitors suppress glycolysis flux and reduce extracellular acidification rate by ≥30%; (2) Baicalin enhances the stability of intercellular junction proteins in endothelial cells, and increases the expression level of VE-cadherin by ≥40%; (3) Combined medication reduces pulmonary vascular permeability by ≥60%.

[0050] The synergistic mechanism of the combined use of PFKFB3 inhibitors and baicalin can be demonstrated by the following quantitative indicators: The reduction in glycolysis flux and extracellular acidification rate by ≥30% was mainly driven by PFKFB3 inhibitors, which provided the necessary metabolic basis for endothelial barrier repair by improving metabolic disorders. The increase in VE-cadherin expression by ≥40% was due to baicalin enhancing the stability of connective proteins and working synergistically with PFKFB3 inhibitors to strengthen the connections between endothelial cells. The reduction in pulmonary vascular permeability by ≥60% was the result of comprehensive metabolic regulation and connection repair, which significantly improved the vascular "leakage" phenomenon and was also the core quantitative manifestation of the synergistic effect of the two.

[0051] In some embodiments, the mass ratio of the PFKFB3 inhibitor to the baicalin is 1:(1~3).

[0052] The mass ratio of PFKFB3 inhibitor to baicalin is limited to 1:(1~3). At this ratio, the two components can exert the best synergistic effect, which can not only ensure the metabolic regulation effect of PFKFB3 inhibitor, but also enhance anti-inflammatory and joint repair capabilities through baicalin.

[0053] In some embodiments, the dosage of baicalin is 50–150 mg / kg.

[0054] Therefore, the core findings of this application include the following: Pseudomonas aeruginosa infection leads to an increase in glycolytic flux mediated by PFKFB3 in pulmonary vascular endothelial cells, and PFKFB3 promotes endothelial barrier disruption through this pathway; the glycolysis regulatory gene PFKFB3 is an important target for maintaining endothelial barrier integrity after infectious lung injury; in addition, baicalin can be used as an effective adjuvant therapy for treating pulmonary vascular endothelial barrier disruption caused by Pseudomonas aeruginosa infection, and when PFKFB3 inhibitors are used in combination with baicalin, they can synergistically protect against pulmonary vascular endothelial barrier disruption caused by Pseudomonas aeruginosa infection.

[0055] Therefore, the advantages of this application are mainly reflected in the following aspects: (1) Clear core target and clear mechanism: For the first time, it was clearly identified that the glycolysis regulatory gene PFKFB3 is a key regulatory target for pulmonary vascular endothelial barrier damage caused by Pseudomonas aeruginosa (PA) infection. The specific mechanism by which it destroys the endothelial barrier (such as reduced VE-cadherin expression and impaired tight junctions) by mediating increased glycolysis flux was revealed. This provides a precise target for the treatment of bacterial lung injury and breaks through the limitation of the existing research on the unclear mechanism of endothelial damage.

[0056] (2) Comprehensive verification methods and reliable results: In response to the deficiencies of existing studies in the verification of cell metabolism and barrier function, several key experiments were added: the pH-XtraGlycolysis test was used to directly prove that the glycolysis flux increased after PA infection, rather than relying solely on lactate detection; the combination of in vitro and in vivo permeation test and transmembrane impedance detection comprehensively reflected the changes in endothelial barrier function, rather than simply detecting VE-cadherin expression, making the conclusions more scientific and convincing.

[0057] (3) Innovative drug application with strong targeting: It is clear that PFKFB3 inhibitors (such as 3-(3-pyridyl)-1-(4-pyridyl)-2-propen-1-one, etc.) can directly intervene in endothelial barrier damage caused by PA infection by inhibiting glycolysis pathway, repairing VE-cadherin continuity, reducing inflammatory factor levels, and providing a highly targeted potential therapeutic drug for clinical use, filling the gap in the existing lack of specific therapeutic drugs for endothelial damage.

[0058] (4) Synergistic effect of combined drug use and significant therapeutic effect: It was found that the combination of PFKFB3 inhibitor and baicalin has a significant synergistic effect: PFKFB3 inhibitor mainly improves metabolic disorders, while baicalin enhances the stability of connexins. Together, they reduce pulmonary vascular permeability by ≥60%, and the anti-inflammatory and barrier protection effects are far superior to those of single drugs. The optimal mass ratio of 1:(1~3) and the dosage of baicalin of 50~150mg / kg were also determined, providing a specific reference for clinical combination drug regimens and expanding the application scenarios of existing drugs.

[0059] (5) Clear application scenarios and high clinical value: It covers multiple links such as prevention, treatment and repair. It can be used to prepare drugs to prevent / treat endothelial barrier damage caused by PA infection, reduce inflammatory factors and repair VE-cadherin continuity. It directly targets the core pathological process of bacterial lung injury (endothelial barrier destruction and hyperinflammatory), and is expected to improve the treatment effect of critically ill patients and reduce mortality. It has important clinical translational value.

[0060] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0061] Example 1: Verifying the integrity of vascular endothelium and changes in PFKFB3 expression in a mouse model of lung infectious disease. Experimental Materials: *Pseudomonas aeruginosa* strain PA14 was provided by Dr. Min. Wu of the Wenzhou Institute of the Chinese Academy of Sciences and is currently held at the Chengdu Institute of Respiratory Health. VE-cadherin mouse monoclonal antibody (14-1449-82, Invitrogen, USA), PFKFB3 antibody (ab181861, ABCAM, USA), PECAM antibody (Invitrogen, USA), goat anti-mouse Alexa Fluor™ 594 antibody (Invitrogen, USA), goat anti-rabbit Alexa Fluor™ 555 antibody (Invitrogen, USA), Alexa Fluor 488-labeled goat anti-rabbit antibody (A-11001, Thermo Fisher Scientific, USA), DAPI (4′6-diamidinyl-2-phenylindole, Thermo Fisher Scientific, USA), dextran fluorescein, 10000MW (D1821, Invitrogen, USA), and an FV3000 laser confocal microscope (OLYMPUS, Japan). The following equipment was used: an Agilent Cary 60 UV spectrophotometer (Agilent Technologies, USA), an Innova® 40 / 40R benchtop rocker (New Brunswick, USA), and a Leica CM1860 cryostat (Leica Biosystems, Germany). Six- to eight-week-old male C57BL / 6J mice were purchased from Chongqing Tengxin Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the guidelines of the Animal Protection and Utilization Committee of Southwest Jiaotong University.

[0062] Experimental methods: Animal model construction: C57BJ / 6J female mice were randomly divided into two groups of six each. In this embodiment, *Pseudomonas aeruginosa* (PA), a common clinical pathogen causing lung infections, was selected to construct a mouse lung infection model. PA14 is a common laboratory reference strain that can mimic the infection phenotype of highly virulent strains. Mice infected with PA14 were labeled as the PA group, while the control group was treated with PBS and labeled as the Ctrl group. After one week of acclimatization, mice in the PA group were anesthetized with ketamine (80 mg / kg) and toluenethiazide (10 mg / kg), and each mouse was administered 30 μL of PA14 (1 × 10⁻⁶ mg / kg) intranasally. 7 CFU was used to induce lung infection. The Ctrl group was given an equal volume of PBS. After 24 hours, both groups of mice were anesthetized with CO2 and then euthanized by cervical dislocation. Lung tissue was collected for subsequent experiments.

[0063] Hematoxylin-eosin (HE) staining of lung tissue: Freshly extracted left mouse lungs were taken, rinsed with PBS to remove blood stains, soaked in 4% paraformaldehyde for 48 hours, dehydrated with graded ethanol, embedded in paraffin, and cut into 5 μm thick sections. The cytoplasm and nuclei were stained sequentially with hematoxylin and eosin. After destaining and mounting, the inflammatory cell infiltration around blood vessels and airways in the lung tissue was observed under a microscope, photographed, and scored.

[0064] Immunofluorescence staining of lung tissue: Mouse lung tissue was fixed by soaking in 4% paraformaldehyde for 48 hours, then dehydrated by soaking in 30% sucrose solution for 24 hours. After embedding in OCT, the lung tissue was frozen for 24 hours and sectioned to a thickness of 5 µm. The sections were then permeabilized with 0.05% Tween 20 for 10 minutes, incubated with 10% goat serum at room temperature for 1 hour to block non-specific antibodies, and then incubated overnight at 4°C with antibodies (VE-cadherin mouse monoclonal antibody, 1:400 dilution; PFKFB3 rabbit monoclonal antibody, 1:200 dilution; PECAM mouse monoclonal antibody, 1:400 dilution). Finally, the sections were incubated at room temperature with the corresponding species' secondary antibodies (1:1000 dilution) for 2 hours. Cell nuclei were stained with DAPI, and the sections were mounted with an anti-fluorescence quencher. The tissue sections were then observed and photographed using a laser confocal microscope.

[0065] In vivo vascular permeability assay: The dosage per mouse was calculated at 10 mg / kg body weight of FITC-glucan (10000 MW). After anesthesia, mice were held upright with nostrils facing upwards, and 20 μL of FITC-glucan was slowly inhaled through each nostril. The mice were kept in this upright position for 1-2 minutes until respiration stabilized. One hour later, mice were deeply anesthetized with isoflurane, and blood was collected from the heart. The collected blood was injected into EP tubes pre-moistened with EDTA (60 mg / mL) and centrifuged at 550g for 10 minutes. The supernatant was collected. Subsequently, the fluorescence OD values ​​of plasma FITC-glucan were detected using a multi-well microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0066] Experimental results: 1) Lung infection in mice led to disruption of vascular endothelial integrity. Figure 1 Figure 1 shows the experimental results related to the disruption of vascular endothelial integrity after lung infection in mice, as provided in Example 1. Captions: A. HE staining of lung tissue sections from two groups of mice under a microscope, scale bar: 20 μm. B. Bar chart showing the inflammation score of the left lobe lung tissue of mice, n=4, ****: P<0.0001 C. Confocal microscopy observation of paraffin sections of lung tissue from mice in the Ctrl and PA groups, immunofluorescence staining: PECAM-labeled endothelial cells were green, VE-cadherin-labeled cells were red, and DAPI-stained cell nuclei were blue. Left icon scale: 20 μm; right icon scale: 5 μm. D. Bar chart showing the results of FITC-glucan permeability analysis of vascular endothelium in mice under different treatments (n=4).

[0067] Depend on Figure 1 It can be seen that the results of HE staining of lung tissue ( Figure 1 A) showed that the lung tissue of PA group mice exhibited extensive inflammatory cell infiltration, alveolar wall structure destruction, diffuse red blood cells in the alveolar walls and alveolar cavities, and widened alveolar septa, while no such abnormalities were observed in Ctrl group. The inflammation score of PA group was significantly higher than that of Ctrl group, and the difference was statistically significant. P<0.0001 ) ( Figure 1 B). Detecting the expression of adhesion proteins on vascular endothelium can reflect endothelial integrity. Endothelial cells were labeled with PECAM antibody, and the immunofluorescence results of lung tissue are as follows: Figure 1 C showed that, compared with the Ctrl group, the PA group had reduced VE-cadherin expression on vascular endothelial cells and disrupted endothelial continuity. The levels of FITC-glucan-labeled FITC in plasma were measured to reflect changes in vascular endothelial permeability. Results ( Figure 1 D) showed that, compared with the Ctrl group, the FITC fluorescence in the plasma of mice in the PA group was increased ( P=0.001This indicates increased vascular endothelial permeability. These results suggest that the integrity of the vascular endothelial barrier is disrupted after lung infection.

[0068] 2) Lung infection in mice leads to increased PFKFB3 expression in vascular endothelial cells. The expression of PFKFB3 on vascular endothelium was detected by immunofluorescence. Endothelial cells were labeled with PECAM antibody.

[0069] Figure 2 Figure 1 shows the experimental results of increased PFKFB3 expression in vascular endothelial cells caused by lung infection in mice, as provided in Example 1. Caption: Immunofluorescence staining of paraffin sections of lung tissue from mice in the Ctrl and PA groups observed under a confocal microscope. PECAM-labeled endothelial cells are green, PFKFB3 is red, and DAPI-stained cell nuclei are blue. Left icon ruler: 20 μm; Right icon ruler: 5 μm.

[0070] like Figure 2 As shown, compared with the Ctrl group, the expression of PFKFB3 on vascular endothelial cells in the PA group was significantly increased.

[0071] Example 2: PA infection promotes increased PFKFB3 expression in vascular endothelial cells and increases glycolysis flux.

[0072] Experimental materials: Mouse vascular endothelial cells C166 and human lung microvascular endothelial cells (HULEC-5a) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). DMEM medium (SH30081.FS, HyClone, USA), MCDB131 medium (10372019, Gibco, USA), epidermal growth factor (EGF, AF-100-15, PeproTech, USA), L-glutamine (A2916801, Gibco, USA), and hydrocortisone (HY-N0583R, MCE, USA) were used. Trizol reagent (Invitrogen, USA), reverse transcription kit (Takara Bio, Japan), real-time quantitative polymerase chain reaction kit (RT-qPCR, Applied Biosystems, USA), and Lipofectamine RNAiMAX transfection reagent (13778150, Invitrogen, USA) were also used. PFKFB3 antibody (ab181861, ABCAM, USA), goat anti-rabbit Alexa Fluor™ 594 antibody (Invitrogen, USA). DAPI (4′,6-diamidinyl-2-phenylindole, Thermo Fisher Scientific, USA). Laser confocal microscope (OLYMPUS, Japan), CFX Connect™ real-time PCR detection system (Bio-Rad, USA). L-LactateAssay kit (ab65330, Abcam, USA), pH-Xtra glycolysis analysis kit (PH-200-4, Agilent Technologies, USA).

[0073] Experimental methods: For cell culture, mouse endothelial cells C166 and human lung vascular endothelial cells HULEC-5a were selected. C166 cells adhered and grew in high-glucose DMEM containing 10% fetal bovine serum (FBS) and 1% streptomycin / penicillin antibiotics, and were stably passaged in a 5% CO2, 37°C, constant temperature and humidity cell culture incubator. HULEC-5a cells adhered and grew in MCDB131 containing 10% FBS, 10 ng / mL EGF, 1 μg / mL hydrocortisone, 10 mM L-glutamine, and 1% streptomycin / penicillin antibiotics, and were stably passaged in a 5% CO2, 37°C, constant temperature and humidity cell culture incubator. 2.5 × 10⁵ cells were evenly seeded in each well of a 6-well plate and cultured stably overnight. Cells were then treated with PA14 at MOI=10 for 2 hours, and the cells were collected for subsequent experiments.

[0074] siRNA transfection of cells: HULEC-5a cells were seeded evenly in 6-well plates at a density of 2.5 × 10⁵ cells per well. After 12 h, following the RNAiMAX transfection reagent instructions, 30 pmol of siRNA was diluted with 150 μL of opti-MEM low-serum medium and incubated at room temperature for 5 minutes; the RNAiMAX transfection reagent was then diluted with 150 μL of opti-MEM low-serum medium and incubated at room temperature for 3 minutes. Subsequently, two solutions of opti-MEM containing PFKFB3 siRNA and the transfection reagent were gently mixed and incubated at room temperature for 15 minutes. Then, the mixture was carefully added to the cell culture medium, and after culturing the cells for 6-8 hours, the medium was replaced with normal medium. Subsequent experiments were performed 24 h after transfection.

[0075] RT-qPCR experiment: Cells were collected, total RNA was extracted using Trizol, and complementary DNA (cDNA) was synthesized according to the TAKARA reverse transcription kit procedure. The cDNA was then used as a template for RT-qPCR. Reaction conditions: 95 ℃ for 30 s, followed by denaturation at 95 ℃ for 5 s, and annealing at 60 ℃ for 34 s, for a total of 40 cycles. Primer sequences were as follows: 5'-GATCTGGGTGCCCGTCGATCACCG-3' upstream of mouse Pfkfb3, 5'-CAGTTGAGGTAGCGAGTCAGCTTC-3' downstream of mouse Pfkfb3; 5'-GGCTGTATTCCCCTCCATCG-3' upstream of mouse β-actin, 5'-CCAGTTGGTAACAATGCCATGT-3' downstream of mouse β-actin. Human β-Actin upstream 5'-AAATCTGGCACCACACCTTC-3', human β-Actin downstream 5'-GGGGTGTTGAAGGTCTCAAA-3'. Human PFKFB3 upstream 5'-CCATGAAAGTCCGGAAGCAATG-3', human PFKFB3 downstream 5'-GCTTTTGACATCTCTCAAGGCAG-3'.

[0076] Immunofluorescence staining of cells: at 10×10 4HULEC-5a / C166 cells were evenly seeded in 24-well plates for inoculation. After infection, the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.02% Tween 20 for 10 minutes, blocked with 10% goat serum at room temperature for 1 hour, and then incubated overnight at 4˚C with PFKFB3 rabbit monoclonal antibody (1:500 dilution). Following this, the cells were incubated with goat anti-rabbit Alexa Fluor™ 594 antibody (1:1000 dilution) at room temperature for 2 hours. The cell nuclei were stained with DAPI, mounted with an anti-fluorescence quencher, and the sections were observed and photographed using a laser confocal microscope.

[0077] L-Lactate Assay: Prepare the necessary reagents according to the L-lactate assay kit. Wash 5 × 10⁶ HULEC-5a cells infected with PA with cold PBS, then rapidly lyse the cells with lactate assay buffer and collect the cell lysate supernatant. Deproteinize the cell lysate with perchloric acid and potassium hydroxide, adjust the pH, and then load the sample according to the L-lactate assay kit procedure. Measure the OD value using a fluorescence microplate reader. Apply the corrected OD value to the lactate standard curve to obtain the amount of lactate in the sample wells.

[0078] pH-Xtra Glycolysis Assay: 80,000 HULEC-5a cells / well were evenly seeded into 96-well plates. Two hours before the glycolysis assay, the plates were placed in a CO2-free incubator to remove CO2 from the cell culture medium. The plate arrangement and control wells were strictly followed according to the pH-Xtra glycolysis assay kit instructions. After sample addition, the plates were immediately placed in a multi-plate reader for analysis.

[0079] Experimental results: PA infection promotes PFKFB3-mediated glycolysis in vascular endothelial cells. This study investigated the effect of PA infection on PFKFB3 expression in mouse and human vascular endothelial cells. RT-qPCR was used to detect the expression of Pfkfb3 mRNA in mouse C166 cells.

[0080] Figure 3Figure 1 shows the experimental results related to the increase in PFKFB3-mediated glycolysis flux in endothelial cells after infection, as provided in Example 2. Figure captions: A. Bar chart showing the expression of Pfkfb3 mRNA after PA treatment of C166 cells (n=3) detected by RT-qPCR. B. Fluorescence confocal microscopy observation of PFKFB3 protein expression after PA treatment of C166 cells. Pfkfb3 is labeled with red fluorescence, and the cell nucleus is stained blue with DAPI. Scale bar: 5 μm. C. Bar chart showing the expression of PFKFB3 mRNA after PA treatment of HULEC-5a cells (n=3) detected by RT-qPCR. D. Fluorescence confocal microscopy observation of PFKFB3 protein expression in PA-treated HULEC-5a cells. PFKFB3 is labeled with red fluorescence, and the cell nucleus is stained blue with DAPI. Scale bar: 5 μm. E. Bar chart showing the L-lactic acid levels in HULEC-5a cells treated with different methods (n=4). L-Lactate: L-lactic acid. F. Line graph showing extracellular pH-Xtra fluorescence signals of HULEC-5a cells under different treatments. TRF signal (AU): Time-resolved fluorescence measurement.

[0081] like Figure 3 As shown, the results ( Figure 3 A) showed that, compared with the Ctrl group, the expression of Pfkfb3 mRNA in the PA group was significantly increased ( P =0.0005 Immunofluorescence assay results showed that, compared with the Ctrl group, PFKFB3 fluorescence was significantly enhanced after PA treatment of C166. Figure 3 B). HULEC-5a cells were treated with PA for 2 hours, and the expression of PFKFB3 mRNA was detected by RT-qPCR. The results were as follows: Figure 3 C) showed that, compared with the Ctrl group, the PA group had significantly increased PFKFB3 mRNA expression ( P=0.006 Immunofluorescence results showed that, compared with the Ctrl group, the PFKFB3 fluorescence in the PA group was significantly enhanced. Figure 3 D). The above results indicate that PA infection of endothelial cells led to a significant increase in PFKFB3 expression. Cells underwent glycolysis followed by anaerobic respiration to produce lactic acid. This example measured the lactic acid content produced by HULEC-5a cells after PA treatment and assessed extracellular acidification. L-lactic acid analysis results ( Figure 3 E) showed that, compared with the Ctrl group, the PA group had increased cellular lactate ( P=0.001 After knocking down PFKFB3, compared with the PA group, the lactate levels in the siPFKFB3 + PA group were significantly reduced. P=0.007The intensity of the fluorescence signal in the pH-Xtra glycolysis assay reflects the degree of extracellular acidification. The pH-Xtra glycolysis analysis results in this example (…). Figure 3 F) shows that, compared with the Ctrl group, the fluorescence signal of HULEC-5a treated with PA increased with time, indicating increased extracellular acidification. After PFKFB3 knockdown, the fluorescence signal of cells in the siPFKFB3 + PA group gradually decreased with time compared with the PA group. These results indicate that PFKFB3-mediated glycolysis flux increases after endothelial cell infection.

[0082] Example 3: PA infection leads to decreased expression of VE-cadherin in vascular endothelial cells.

[0083] Experimental materials: Mouse vascular endothelial cells C166 and human lung microvascular endothelial cells (HULEC-5a) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). DMEM medium (SH30081.FS, HyClone, USA), MCDB131 medium (10372019, Gibco, USA), epidermal growth factor (EGF, AF-100-15, PeproTech, USA), L-glutamine (A2916801, Gibco, USA), and hydrocortisone (HY-N0583R, MCE, USA) were used. Trizol reagent (Invitrogen, USA), reverse transcription kit (Takara Bio Inc., Japan), real-time quantitative polymerase chain reaction kit (RT-qPCR, Applied Biosystems, USA), vascular endothelial cadherin (VE-cadherin, Cell Signaling, USA), goat anti-rabbit Alexa Fluor™ 594 antibody (Invitrogen, USA) and goat anti-rabbit Alexa Fluor™ 488 antibody (Invitrogen, USA). DAPI (4′,6-diamidinyl-2-phenylindole, Thermo Fisher Scientific, USA). Laser confocal microscope (OLYMPUS, Japan), CFX Connect™ real-time PCR detection system (Bio-Rad, USA). Dextran fluorescein, 3000MW (D3305, Invitrogen, USA).

[0084] Experimental methods: The methods for cell culture and cell immunofluorescence staining are the same as above.

[0085] The RT-qPCR procedure is the same as above, with β-actin as an internal control, and the relative expression level of CDH5 calculated using the formula 2–ΔΔCt. Some primer sequences are as follows: human β-actin upstream 5'-AAATCTGGCACCACACCTTC-3', human β-actin downstream 5'-GGGGTGTTGAAGGTCTCAAA-3'; human CDH5 upstream 5'-TTGGAACCAGATGCACATTGAT-3', human CDH5 downstream 5'-TCTTGCGACTCACGCTTGAC-3'.

[0086] Experimental results: PA infection led to decreased VE-cadherin expression in vascular endothelial cells. Figure 4 Figure 3 shows the experimental results related to the decrease in VE-cadherin expression in vascular endothelial cells caused by PA infection, as provided in Example 3. Captions: A. Bar chart showing Cdh5 mRNA expression detected by RT-qPCR after PA treatment of C166. B. Laser confocal microscopy observation of VE-cadherin protein expression after PA treatment of C166; VE-cadherin is labeled with red fluorescent dye, and DAPI stains the cell nucleus blue. Scale bar: 5 μm. C. Bar chart showing CDH5 mRNA expression detected by RT-qPCR after PA treatment of HULEC-5a. D. Laser confocal microscopy observation of VE-cadherin protein expression after PA treatment of HULEC-5a; VE-cadherin is labeled with green fluorescent dye, and DAPI stains the cell nucleus blue. Scale bar: 5 μm.

[0087] like Figure 4 As shown, C166 cells were infected with PA14 (MO1=10) for 2 hours, and the expression of Cdh5 mRNA was detected by RT-qPCR. The results were as follows: Figure 4 A) showed that, compared with the Ctrl group, the Cdh5 mRNA expression in the PA group was significantly reduced ( P=0.023 Immunofluorescence was used to detect the expression of VE-cadherin (a protein encoded by the Cdh5 gene), and the results were as follows ( Figure 4 B) showed that, compared with the Ctrl group, the VE-cadherin fluorescence in the PA group was significantly weakened, and its continuity was disrupted. HULEC-5a cells were infected with PA14 (MOI=10) for 2 hours, and the expression of CDH5 mRNA was detected by RT-qPCR. The results were... Figure 4 C) showed that, compared with the Ctrl group, the CDH5 mRNA expression in the PA group was significantly reduced ( P=0.0115 CDH5 mRNA expression was significantly reduced in the 30 ng / mL hOSM group. P=0.03) Immunofluorescence was used to detect VE-cadherin protein expression, and the results were ( Figure 4 D) shows that, compared with the Ctrl group, VE-cadherin fluorescence was significantly weakened after PA treatment of HULEC-5a, and cell membrane continuity was disrupted.

[0088] Example 4: Inhibition of PFKFB3 in endothelial cells reversed endothelial barrier disruption caused by PA infection.

[0089] Experimental materials: PFK-158 (HY-12203, MCE Corporation, USA), the rest are the same as above.

[0090] Experimental methods: In this embodiment, human pulmonary microvascular endothelial cells - HULEC-5a were selected, and the cell culture method was the same as above.

[0091] siRNA cell transfection: HULEC-5a cells were seeded evenly in 6-well plates at a density of 2.5 × 10⁵ cells per well. After 12 hours, PFKFB3 siRNA and RNAiMAX transfection reagent were diluted separately in opti-MEM medium according to the RNAiMAX transfection reagent manufacturer's instructions, mixed well, and incubated at room temperature for 5 minutes. Then, the two doses of opti-MEM containing siRNA and transfection reagent were gently mixed and incubated at room temperature for 15 minutes. The mixture was then carefully added to the cell culture medium, and the cells were cultured for 6-8 hours before being replaced with normal medium. The control group (NC group) received the same treatment with the negative control siRNA. PFK-158 is a specific small molecule inhibitor of PFKFB3. Transformed HULEC-5a cells were treated with PFK-158 (10 μM) and / or PA14 for 24 hours before subsequent experiments.

[0092] Immunofluorescence staining: HULEC-5a / C166 cells were evenly seeded in 24-well plates at a density of 10 × 10⁴ cells / well. After infection, the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.05% Tween 20 for 10 minutes, and blocked with 10% goat serum at room temperature for 1 hour. Then, VE-cadherin mouse monoclonal and PFKFB3 rabbit monoclonal antibodies (1:500) were diluted and incubated overnight at 4˚C. Goat anti-mouse Alexa Fluor™ 594 antibody and goat anti-rabbit Alexa Fluor™ 488 antibody (1:1000) were diluted and incubated at room temperature for 1 hour. Cell nuclei were stained with DAPI, and after mounting with an anti-fluorescence quencher, the sections were observed and photographed using a laser confocal microscope.

[0093] In vitro cell permeability assay: 2 × 10^5 HULEC-5a cells were evenly seeded in the upper transwell compartment of a 24-well plate. The 12 wells were randomly divided into four groups: NC, PA, siPFKFB3, and siPFKFB3+PA, with three wells in each group. Cells in the NC and PA groups were transfected with negative control siRNA; cells in the siPFKFB3 and siPFKFB3+PA groups were transfected with PFKFB3 siRNA and cultured at 37°C and humidified until cell confluence formed a monolayer. The monolayers of cells in the PA and siPFKFB3+PA groups were treated with PA for 2 hours. Subsequently, 100 μL of FITC-glucan (1 μg / mL) was added to each upper compartment, and 500 μL of FITC-glucan-free PBS was added to the lower compartment. After incubation at 37°C for 5 minutes, the fluorescence intensity of the FITC-glucan transferred to the lower compartment was measured using a multi-well microplate reader.

[0094] Transmembrane electrical resistance assay: 12 × 10^4 HULEC-5a cells were evenly seeded in the upper transwell chambers (3.0 μm pore size) of a 24-well plate. The 15 wells were randomly divided into three groups: NC, PA, and siPFKFB3+PA, with three wells in each group. Cells in the NC and PA groups were transfected with negative control siRNA; cells in the siPFKFB3+PA group were transfected with PFKFB3 siRNA. After transfection, the upper chambers were transferred to a transmembrane electrical resistance analyzer and cultured at 37°C under constant temperature and humidity until the cells formed a monolayer. The monolayers in the PA and siPFKFB3+PA groups were treated with PA for 2 hours, and the transepithelial electrical resistance (TEER) value of each well was measured in real time.

[0095] Experimental results: Figure 5 Figure 4 shows the experimental results related to PA promoting endothelial barrier damage via PFKFB3 as provided in Example 4. Figure captions: A, B. Laser confocal microscopy observation of VE-cadherin protein expression in HULEC-5a cells under different treatments. VE-cadherin is labeled red, PFKFB3 is labeled green, and the cell nucleus is labeled blue with DAPI. Scale bar: 5 μm. C. Bar chart showing the results of FITC-glucan relative permeability analysis of HULEC-5a cells under different treatments (n=3). D. Line graph showing the trend of transmembrane electrical impedance values ​​of different endothelial cells over time (*: P<0.05 ,**: P<0.01 (n=3).

[0096] likeFigure 5 As shown, HULEC-5a cells were treated with PA for 2 hours, and VE-cadherin protein expression was detected by immunofluorescence. The results showed that compared with the Ctrl group, VE-cadherin fluorescence was significantly weakened and its integrity was disrupted after PA treatment of HULEC-5a cells. To investigate whether endothelial cell PA affects VE-cadherin protein expression through PFKFB3, this example used siRNA to knock down PFKFB3 in HULEC-5a cells. Immunofluorescence results showed that compared with the NC group, VE-cadherin fluorescence in HULEC-5a cells in the PA group was weakened and its continuity was disrupted; compared with the PA group, VE-cadherin fluorescence in the siPFKFB3+PA group was enhanced and its continuity was somewhat restored. Figure 5 A). In this example, after inhibiting PFKFB3 with PFK-158, the expression of VE-cadherin was detected. Immunofluorescence results ( Figure 5 B) showed that, compared with the PA group, the PFK-158+PA group showed enhanced VE-cadherin fluorescence and restored membrane continuity. The in vitro FITC-glucan permeability assay reflects endothelial permeability by measuring the permeability of FITC-glucan in monolayer endothelial cells. The results of the cell permeability assay ( Figure 5 C) showed that, compared with the NC group, the relative permeability of cells in the PA group was increased ( P=0.0002 Compared with the PA group, the permeability of cells in the siPFKFB3+PA group was decreased (P=0.002). Endothelial cell transmembrane electrical impedance assay results ( Figure 5 D) showed that, compared with the NC group, the TEER value of the PA group was significantly lower ( P<0.05 Compared with the PA group, the TEER value of cells in the siPFKFB3+PA group increased ( P<0.01 This indicates that the integrity of the cell barrier has been somewhat restored. Therefore, the above results suggest that PA promotes a decrease in the expression of intercellular adhesion proteins in endothelial cells and an increase in endothelial permeability through PFKFB3, leading to endothelial barrier disruption.

[0097] Example 5: PFKFB3 inhibitor combined with baicalin reduced pulmonary vascular endothelial barrier damage in mice caused by PA infection.

[0098] Experimental materials: Baicalin (BAI, HY-N0197, MCE Pharmaceuticals, USA), 3PO (HY-19824, MedChemExpress, USA), dexamethasone (DEX, HY-14648, MCE Pharmaceuticals, USA). Mouse IL-1β ELISA kit (EK201B, Lianke Biotechnology, China), Mouse IL-6 ELISA kit (EK206, Lianke Biotechnology, China), Mouse TNF-α ELISA kit (EK282, Lianke Biotechnology, China). Dextran fluorescein, 10000MW (D1821, Invitrogen, USA), the rest are the same as above.

[0099] Experimental methods: Animal model establishment: Thirty-six 6-week-old male C57BL / 6J mice, weighing 22±2g, were randomly divided into six groups: CTRL group, PA group, 3PO+PA group, BAI+PA group, 3PO+BAI+PA group, and DEX+PA group. 3PO is a specific PFKFB3 small molecule inhibitor. BAI is baicalin, the main active ingredient of the traditional Chinese medicine Scutellaria baicalensis. DEX is dexamethasone. One day before infection, mice in the BAI+PA group and the 3PO+BAI+PA group were administered BAI solution (100mg / kg) by gavage. Two hours before infection, mice in the DEX+PA group were administered DEX solution (5mg / kg) intraperitoneally. Thirty minutes before infection, mice in the 3PO+PA group were administered 50mg / kg of 3PO solution intraperitoneally, and mice in the PA group were administered an equal volume of diluted DMSO within the safe range. Mice were anesthetized and administered 30 μL PPA14 (1 × 10^7 CFU) intranasally to induce lung infection. The CTRL group received the same treatment with an equal volume of PBS. After 24 hours, mice in all groups were anesthetized with CO2 and euthanized by cervical dislocation, and lung tissue was collected for subsequent experiments.

[0100] The method for hematoxylin-eosin (HE) staining of lung tissue is the same as above.

[0101] Enzyme-linked immunosorbent assay (ELISA): After anesthetizing mice, the trachea was exposed by making a cervical incision, and the upper end of the trachea was ligated. 0.8 mL of 0.9% physiological saline was drawn up using a 1 mL syringe and injected into the lungs from below the ligation point. The lungs were slowly and repeatedly irrigated three times, with a 30-second pause after each infusion to ensure adequate fluid collection. The collected fluid was centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant was collected. The concentrations of TNF-α, IL-6, and IL-1β in the bronchoalveolar lavage fluid (BALF) were detected using the supplier's ELISA kit, following the manufacturer's instructions.

[0102] Immunofluorescence staining of lung tissue: The left lung of mice was fixed by soaking in 4% paraformaldehyde for 48 hours, dehydrated by soaking in 30% sucrose solution for 24 hours, embedded in OCT, frozen for 24 hours, and then sectioned to a thickness of 5 µm. The sections were then permeabilized with 0.05% Tween 20 for 10 minutes, incubated with 10% goat serum at room temperature for 1 hour to block non-specific antibodies, and then incubated overnight at 4˚C with antibody (VE-cadherin mouse monoclonal antibody, 1:400 dilution). Finally, they were incubated with goat anti-mouse Alexa Fluor™ 594 antibody (1:1000 dilution) at room temperature for 2 hours to specifically bind the primary antibody. Cell nuclei were stained with DAPI, and the sections were mounted with an anti-fluorescence quencher. The tissue sections were then observed and photographed using a laser confocal microscope.

[0103] In vivo vascular permeability assay: The dosage per mouse was calculated at 10 mg / kg body weight of FITC-glucan (10000 MW). After anesthesia, mice were held upright with nostrils facing upwards, and 20 μL of FITC-glucan was slowly inhaled through each nostril. The mice were kept in this upright position for 1-2 minutes until respiration stabilized. One hour later, mice were deeply anesthetized with isoflurane, and blood was collected from the heart. The collected blood was injected into EP tubes pre-moistened with EDTA (60 mg / mL), centrifuged, and the supernatant plasma was collected. Subsequently, the fluorescence OD values ​​of plasma FITC-glucan were detected using a multi-well microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0104] Experimental results: 1. Baicalin combined with PFKFB3 inhibitors can reduce lung inflammation in mice caused by PA infection.

[0105] Figure 6 Figure 1 shows the experimental results related to the significant anti-inflammatory effect of the PFKFB3 inhibitor combined with baicalin provided in Example 5. Figure captions: A. HE staining of mouse lung tissue pathological sections under a microscope, scale bar: 20 μm. B. Bar chart showing lung injury score of the left lobe of mouse lung tissue (n=4). C. Bar chart showing the content of cytokines such as TNF-α, IL6, and IL1β in mouse bronchoalveolar lavage fluid (n=4).

[0106] like Figure 6 As shown, the results of HE staining of mouse lung tissue ( Figure 6A) shows that in the PA group, the lung tissue of mice exhibited extensive inflammatory cell infiltration, alveolar wall structure disruption, diffuse red blood cells in the alveolar walls and alveolar cavities, and widened alveolar septa. These changes were not observed in the CTRL group. Compared to the PA group, the 3PO+PA and BAI+PA groups showed inflammatory cell infiltration in the alveolar cavities and blood vessels, and widened alveolar septa. The 3PO+BAI+PA and DEX+PA groups showed significantly reduced inflammatory cell infiltration in the lung tissue compared to the PA group. Lung injury score results ( Figure 6 B) shows that, compared with the CTRL group, the PA group had a significantly increased lung injury score, and the difference was statistically significant. P< 0.0001 Compared with the PA group, there was no significant change in lung injury scores in the 3PO+PA group and the BAI+PA group; however, the lung injury score in the 3PO+BAI+PA group was significantly lower, consistent with the DEX+PA group, and the difference was statistically significant. P=0.0006 Compared with the 3PO+PA group, the lung injury score of the 3PO+BAI+PA group was significantly lower, and the difference was statistically significant. P=0.004 The levels of TNF-α, IL-6, and IL-1β in bronchoalveolar lavage fluid (BALF) were detected by ELISA. The results were as follows: Figure 6 C) shows that, compared with the CTRL group, TNF-α, IL-6, and IL-1β were significantly increased in the BALF of mice in the PA group. Compared with the PA group, TNF-α and IL-1β were significantly decreased in the BAI+PA group, while IL-6 was not significantly decreased; IL-1β was significantly decreased in the 3PO+PA group, but TNF-α and IL-6 did not change significantly; TNF-α, IL-6, and IL-1β were significantly decreased in the BALF of the 3PO+BAI+PA group and the DEX+PA group. Furthermore, this example found that, compared with the 3PO+PA and BAI+PA groups alone, the combined 3PO+BAI+PA group showed a significant decrease in TNF-α, IL-6, and IL-1β in the BALF, with statistically significant differences.

[0107] Therefore, the above results indicate that 3PO and baicalin alone cannot reduce inflammatory infiltration of lung tissue after lung infection, while the combined use of 3PO and baicalin can reduce inflammatory infiltration and tissue edema of lung tissue, reduce the secretion of inflammatory factors, and has a significant anti-inflammatory effect.

[0108] 2. Baicalin combined with PFKFB3 inhibitors reduced pulmonary vascular endothelial barrier damage in mice induced by PA infection.

[0109] To investigate the effects of baicalin and PFKFB3 inhibitors on pulmonary vascular endothelial injury induced by PA infection in mice, this study used tissue immunofluorescence to detect the expression of VE-cadherin in the vascular endothelium and the continuity of the endothelium, and used FITC-labeled dextran to detect the permeability of the endothelium.

[0110] Figure 7 Figure 5 shows the experimental results of how baicalin combined with 3PO protected against PA-induced pulmonary vascular endothelial barrier damage in mice. Figure captions: A. Confocal microscopy observation of VE-cadherin immunofluorescence staining in lung tissue sections from mice under different treatments. VE-cadherin is marked in red, and DAPI staining of cell nuclei is marked in blue. Left icon bar: 20 μm; right icon bar: 5 μm. B. Bar chart showing the results of FITC-glucan permeability analysis of vascular endothelium in mice under different treatments (n=4).

[0111] Depend on Figure 7 It can be seen that the immunofluorescence results ( Figure 7 A) showed that the red fluorescence of VE-cadherin-labeled endothelial cells in the lung tissue of mice in the PA group was significantly reduced compared to the CTRL group. Compared with the PA group, the endothelial continuity of the 3PO+PA group and the BAI+PA group was not fully restored. After treating mice with 3PO combined with BAI, the red fluorescence of VE-cadherin-labeled endothelial cells in the 3PO+BAI+PA group was increased compared with the PA group, and the vascular continuity was restored. The permeability of vascular endothelium was reflected by detecting FITC-glucan-labeled FITC in plasma. The results ( Figure 7 B) showed that, compared with the PA group, there was no significant change in FITC fluorescence in the plasma of mice in the 3PO+PA group and the BAI+PA group. P> 0.05 However, FITC fluorescence in the plasma of mice in the 3PO+BAI+PA group was significantly reduced. P=0.003 The difference was statistically significant.

[0112] The above results indicate that the PFKFB3 inhibitors 3PO and BAI alone have no significant protective effect against pulmonary vascular endothelial damage caused by PA infection. However, the combination of 3PO and baicalin can reduce vascular endothelial permeability and maintain endothelial integrity. Based on the comprehensive analysis of pathological and lung injury scores, this embodiment suggests that the combination of PFKFB3 inhibitors and baicalin provides better protection against infection-induced vascular endothelial barrier damage than 3PO and BAI alone.

[0113] In summary, glycolytic metabolic imbalance is a key characteristic of endothelial cell injury. Previous studies have shown that glycolytic metabolites, such as lactate and pyruvate, exacerbate endothelial cell damage by activating inflammatory signaling pathways (such as NF-κB) and promoting the release of inflammatory factors. PFKFB3-mediated glycolysis plays a crucial role in the development of PAH. Knockout of PFKFB3 in endothelial and smooth muscle cells can significantly inhibit the development of hypoxia-induced pulmonary hypertension. Although patents have reported significantly elevated expression of the key glycolytic enzyme PFKFB3 in bone marrow vascular endothelial cells in patients with hematopoietic suppression after chemotherapy and poor engraftment function after transplantation, the inventors believe that PFKFB3 can serve as a biomarker for vascular endothelial cell injury. Another patent proposes a method to repair endothelial cell damage by inhibiting the key glycolytic enzyme PFKFB3. *Pseudomonas aeruginosa*, a common pathogen of lower respiratory tract infections in hospitals, is an important pathogen in patients with severe pneumonia, ventilator-associated pneumonia, and structural lung disease. Traditional Chinese medicine (TCM) is a treasure of my country's traditional culture, occupying a unique and indispensable position in the prevention and treatment of lung infections. Scutellaria baicalensis, used alone, has been reported to be beneficial in treating colds and bacterial pneumonia. Baicalin, a class of flavonoids, is the main bioactive chemical component of Scutellaria baicalensis, possessing broad biological activities, including anti-inflammatory, antioxidant, antiviral, and significant in vitro and in vivo antibacterial effects. Baicalin has been developed into a new drug (Huang Qing'an tablets) for the treatment of acute and chronic hepatitis. In LPS-induced lung injury, baicalin can inhibit the levels and expression of TNF-α, IL-1β, and IL-6 in vitro and in vivo. Studies have also found that baicalin can alleviate inflammatory damage in rats with MDR-induced acute pneumonia through arginine biosynthesis associated with the gut microbiota. However, the evidence for the protective effect of baicalin on vascular endothelium remains insufficient. The regulatory role of the key glycolysis gene PFKFB3 and the application of baicalin have not been reported after pulmonary endothelial barrier damage caused by Pseudomonas aeruginosa infection.

[0114] This embodiment preliminarily demonstrates that PA infection of pulmonary vascular endothelial cells regulates PFKFB3 expression via ATG5, promoting anaerobic glycolysis, leading to cellular metabolic disorders, disruption of intercellular junctions, and resulting in pulmonary edema and exacerbated inflammation in mice. However, based on the preliminary research, this embodiment selected the more virulent PA14 as the pathogen, increasing the infection pressure. Simultaneously, two species (mouse and human) of vascular endothelial cells were selected as research subjects, expanding species diversity. The degree of extracellular acidification after PA14 infection was detected using a pH-Xtra Glycolysis assay, further demonstrating that PA14 infection promotes an increase in endothelial cell glycolytic flux. In vivo and in vitro permeability assays were used to analyze the changes in endothelial cell permeability after PA14 infection, and transmembrane electrical impedance was measured, comprehensively demonstrating the changes in endothelial cell barrier function after PA14 infection. Importantly, this embodiment utilizes two PFKFB3 inhibitors in in vivo and in vitro experiments to demonstrate that PFKFB3 is a target for maintaining endothelial barrier integrity after Pseudomonas aeruginosa infection, and to explore the potential application of PFKFB3 inhibitors in combination with baicalin. This embodiment enriches the application scope of PFKFB3 in regulating endothelial cell glycolytic metabolism, and for the first time proposes that baicalin is an effective adjuvant therapy for treating pulmonary vascular endothelial damage caused by Pseudomonas aeruginosa infection, specifically demonstrating the application of PFKFB3 inhibitors in combination with baicalin in repairing pulmonary endothelial cell damage after Pseudomonas aeruginosa infection.

[0115] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Any of the following applications of the glycolysis regulatory gene PFKFB3 in bacterial lung injury: (1) Application in the preparation of drugs for the prevention or treatment of pulmonary vascular endothelial barrier damage caused by Pseudomonas aeruginosa infection; (2) Application as a therapeutic target in the preparation of drugs that repair the integrity of the pulmonary vascular endothelial barrier; (3) Application in the preparation of drugs that reduce the content of inflammatory factors TNF-α, IL-6 and IL-1β in lung tissue.

2. The application according to claim 1, characterized in that, The application is achieved by inhibiting the PFKFB3-mediated glycolysis pathway.

3. The application according to claim 2, characterized in that, The inhibition of the PFKFB3-mediated glycolysis pathway exhibits any of the following characteristics: (1) Reduce glycolysis flux; (2) Upregulates the expression of vascular endothelial cadherin; (3) Increase the transendothelial electrical impedance value by ≥50%; (4) Reduce vascular permeability.

4. Any of the following applications of PFKFB3 inhibitors: (1) Application in the preparation of drugs for the prevention or treatment of pulmonary vascular endothelial barrier damage caused by Pseudomonas aeruginosa infection; (2) Application in the preparation of drugs that repair the continuity of VE-cadherin protein; (3) Application in the preparation of drugs that reduce the content of inflammatory factors TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid.

5. The application according to claim 4, characterized in that, The PFKFB3 inhibitors include one or more of the following: 3-(3-pyridyl)-1-(4-pyridyl)-2-propen-1-one, N-((2-chloro-6-fluorophenyl)sulfonyl)-6-(4-morpholinyl)-1H-indazole-3-carboxamide, and siRNAs targeting PFKFB3.

6. The application according to claim 5, characterized in that, The PFKFB3 inhibitor is administered via intraperitoneal injection.

7. Any of the following applications of the combination of PFKFB3 inhibitor and baicalin: (1) Application in the preparation of drugs for treating pulmonary vascular endothelial barrier damage caused by Pseudomonas aeruginosa infection; (2) Application in the preparation of drugs that reduce the content of TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid; (3) Application in the preparation of drugs that repair the continuity of VE-cadherin protein in lung tissue.

8. The application according to claim 7, characterized in that, The application is implemented through the following collaborative mechanism: (1) PFKFB3 inhibitors suppress glycolysis flux and reduce extracellular acidification rate by ≥30%; (2) Baicalin enhances the stability of intercellular junction proteins in endothelial cells, and increases the expression level of VE-cadherin by ≥40%; (3) Combined medication reduces pulmonary vascular permeability by ≥60%.

9. The application according to claim 8, characterized in that, The mass ratio of the PFKFB3 inhibitor to the baicalin is 1:(1~3).

10. The application according to claim 9, characterized in that, The dosage of baicalin is 50–150 mg / kg.

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