Use of a substance that specifically binds to klk8 in the preparation of a product for the prevention, treatment or diagnosis of sepsis

Abstract

The present application relates to the technical field of medicine, in particular to the use of a substance specifically binding to KLK8 in the preparation of a product for the prevention, treatment or diagnosis of sepsis, which treats sepsis by: 1) reducing endothelial cell permeability or improving capillary leakage; 2) stimulating the VE-cadherin / Akt / FOXM1 signaling pathway; preferably, selected from up-regulating the expression level of FOXM1 and its downstream target genes, up-regulating the protein level of AKT or p-AKT, or up-regulating the protein level of VE-cadherin; 3) increasing the proliferation rate of endothelial cells; 4) reducing the mortality rate of endothelial cells; 5) increasing the activity of endothelial cells. The present application provides a new treatment strategy for sepsis complicated with microvascular barrier dysfunction.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of a substance specifically binding to KLK8 in the preparation of a product for the prevention, treatment or diagnosis of sepsis. BACKGROUND

[0002] Sepsis is a common devastating complication in patients with severe infection, trauma or burn. Progressive multiple organ dysfunction syndrome (MODS) accumulates to cause the mortality rate of sepsis to rise. It is generally believed that the microvascular system, especially the endothelial cells in the microvascular lumen, is very susceptible to the harmful consequences of sepsis. Among the major organs, the lung is not only a common affected organ, but also one of the organs most susceptible to severe sepsis. Endothelial cells are the first line of defense in the pathogenesis of sepsis-induced lung injury, and the earliest morphological changes, apoptosis and intercellular junction disruption of effector cells lead to endothelial cell hyperpermeability and pulmonary edema. At the same time, a large number of toxins and inflammatory mediators in the post-sepsis circulation directly inhibit the proliferative capacity of pulmonary microvascular endothelial cells, leading to dysfunction of microvascular self-repair and further aggravating sepsis-induced lung injury. Therefore, targeting endothelial repair mechanisms is a promising therapeutic strategy to restore the integrity of the pulmonary microvascular barrier after sepsis. However, it is still unclear whether KLKs are involved in the regulation of lung endothelial regeneration and vascular repair during sepsis. SUMMARY

[0003] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide the use of a substance specifically binding to KLK8 in the preparation of a product for the prevention, treatment or diagnosis of sepsis, in order to solve the problems in the prior art.

[0004] To achieve the above-mentioned objects and other related objects, the present application provides the use of KLK8 or its encoding gene as a target in the preparation of a product for the prevention, treatment or diagnosis of sepsis.

[0005] The present application also provides the use of a substance specifically binding to KLK8 in the preparation of a product for the prevention, treatment or diagnosis of sepsis

[0006] The present application also provides the use of a substance specifically binding to KLK8 in the preparation of any one or more of the following products:

[0007] 1) a product for reducing endothelial cell permeability or a product for improving capillary leakage;

[0008] 2) a product for stimulating the VE-cadherin / Akt / FOXM1 signaling pathway; preferably, a product selected from a product for up-regulating the expression level of FOXM1 and its downstream target genes, a product for up-regulating AKT, or a product for up-regulating the protein level of p-AKT, or a product for up-regulating the protein level of VE-cadherin;

[0009] 3) products that increase the proliferation rate of endothelial cells;

[0010] 4) products that reduce the mortality rate of endothelial cells;

[0011] 5) products that increase the activity of endothelial cells.

[0012] The present application also provides the use of a VE-cadherin / Akt / FOXM1 signaling pathway upregulator in the preparation of a product for treating sepsis complicated by acute lung injury and / or complicated by endothelial injury.

[0013] As described above, the use of the substance specific to KLK8 of the present application in the preparation of a sepsis prevention, treatment or diagnosis product has the following beneficial effects: the new role of KLK8 upregulation in the development of LPS-induced lung endothelial barrier dysfunction is determined. By inhibiting KLK8 through gene knockout or using anti-KLK8 neutralizing antibodies, LPS-induced endothelial hyperpermeability, acute lung injury and mortality can be significantly alleviated. In terms of mechanism, the present application finds that KLK8-induced VE-cadherin / Akt / FOXM1 pathway inactivation plays a key role in mediating endothelial regenerative injury and subsequent lung vascular leakage in response to LPS. Therefore, inhibition of KLK8 by neutralizing antibodies can be a new therapeutic strategy for restoring microvascular barrier integrity during sepsis. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 KLK8 expression in the lungs of LPS-induced sepsis models is shown, wherein: A-B, KLKs mRNA levels in lung tissue (n=6) or MLVECs (n=4) exposed to LPS for 24 hours, respectively. C-D, KLK8 protein levels in lung tissue (n=6) or MLVECs (n=4) after LPS exposure for 24 hours. E-F, Endothelial cell marker CD31 (green) and KLK8 (red) antibodies were stained, and KLK8 + / CD31 + cells accounted for the percentage of total CD31 + cells (n=6). Data are expressed as mean ± SEM * p<0.05, ** p<0.01.

[0015] Figure 2Figure 8 shows the graphs of changes in each index after KLK8 overexpression, wherein A-C, MLVECs were treated with KLK8 adenovirus (Ad-KLK8) for 48 hours (n=4). A, Western Blot detected KLK8, CD31, ZO-1 and ICAM-1 protein levels in MLVECs. B, MTT detected MLVECs cell viability. C, FITC detected MLVECs endothelial permeability. D-G, Mice were intratracheally instilled with Ad-Vector or Ad-KLK8 for 48 hours (n=6). D, Western Blot detected KLK8, CD31, ZO-1, ICAM-1 protein levels in lung tissue. E, Evans Blue dye detected lung tissue vascular leakage. F-G, HE performed lung histopathology evaluation. Data are expressed as mean ± standard deviation. * p<0.05, ** p<0.01.

[0016] Figure 3 Figure 9 shows the graphs of results of lung tissue injury induced by LPS after inhibition of KLK8 expression, wherein A-C, MLVECs were transfected with Control-siRNA or KLK8-siRNA for 48 hours. Then LPS (1,000 ng / ml) was used to treat cells for 24 hours (n=4). A, Western Blot detected CD31, ZO-1 and ICAM-1 protein levels in MLVECs. B, MTT detected MLVECs cell viability. C, FITC detected MLVECs endothelial permeability. D-H, KLK8 knockout (KLK8 - / - ) mice and control (KLK8 + / + ) mice were intraperitoneally injected with 30 mg / kg LPS (n=6). D, Western Blot detected CD31, ZO-1, ICAM-1 protein levels in lung tissue. E, Evans Blue dye detected lung tissue vascular leakage. F-G, HE performed lung histopathology evaluation. H, Kaplan-Meier analysis of survival curve (n=30). Data are expressed as mean ± standard deviation. * p<0.05, ** p<0.01.

[0017] Figure 4Figure showing the results of LPS-induced lung tissue injury after administration of anti-KLK8 antibody, wherein A-D, mice were injected with anti-KLK8 neutralizing antibody or IgG isotype control antibody via tail vein. 30 minutes later, mice were injected with LPS of 30 mg / kg intraperitoneally, and the control group was injected with an equal amount of normal saline (n=6). A, Western Blot detection of CD31, ZO-1, ICAM-1 protein levels in lung tissue. B, Evans Blue dye lung vascular leakage. C-D, HE lung histopathology evaluation. E, Kaplan-Meier survival curve analysis (n=30). Data are expressed as mean ± standard deviation. * p<0.05, ** p<0.01.

[0018] Figure 5 Figure showing the effects on molecules in the pathway after KLK8 overexpression using Ad-Vector or Ad-KLK8 treatment of MLVECs for 48 hours. RNA-Seq analysis of abnormal genes (n=4). A, Volcano plot showing differentially expressed genes in Ad-KLK8-treated MLVECs. B, DEGs heat map. C, Molecular characteristics describing abnormal pathways, as well as the number of genes in each pathway, normalized enrichment score (NES), and normalized p-value (NOM). D, IPA predicts upstream regulators responsible for the changes in gene expression profiles in Ad-KLK8-treated MLVECs. E, Heat map of FOXM1 and its target molecules in the IPA dataset. F, mRNA and protein levels of FOXM1 in Ad-KLK8-treated MLVECs (n=4). G-L, MLVECs were infected with lentiviral vectors (Len-Vector) or FOXM1 lentiviral vectors (Len-FOXM1) for 30 min, and then treated with Ad-Vector or Ad-KLK8 for 48 h. G-H, Edu detection of cell proliferation. I, Real-time quantitative PCR detection of mRNA levels of FOXM1 target molecules Ccnd1, Cdc20, Ccnf, Bub1b

[0019] Figure 6 ​The diagram shows the results of KLK8-induced endothelial barrier dysfunction. A) Gene set enrichment analysis of genes regulated by the PI3K-AKT-MTOR signaling pathway, FOXM1 heatmap, and target genes of PI3K-AKT-MTOR signaling pathway dysregulation. B) Western blot analysis of p-Akt and Akt protein levels in MLVECs treated with Ad-KLK8 for 48 hours. C) Western blot analysis of p-Akt, Akt, and FOXM1 protein levels (10 μM) in MLVECs treated with Ad-KLK8 for 48 hours after Akt activator SC79 pretreatment. D) Western blot analysis of VE-cadherin protein levels in MLVECs (n=4) or lung tissue (n=6) after Ad-KLK8 treatment for 48 hours. E) Western blot analysis of N-terminal VE-cadherin fragments (n=4) in the culture medium after Ad-KLK8 treatment for 48 hours. F. After infecting MLVECs with lentiviral vector (Len-Vector) or VE-cadherin lentivirus (Len-VE-cadherin) for 30 min, MLVECs were treated with Ad-KLK8 for 48 h. Western blot was used to detect the protein levels of VE-cadherin, FOXM1, p-Akt, and Akt in MLVECs (n=4). G. With Len-VE-cadherin and / or the Akt inhibitor AZD5363, MLVECs were treated with Ad-KLK8 for 48 h. Western blot was used to detect the protein levels of VE-cadherin and FOXM1 in MLVECs (n=4). Data are expressed as mean ± standard deviation. * p < 0.05; ** p < 0.01.

[0020] Figure 7 The results are shown for KLK8 deletion or blockade, including A, C, and KLK8 knockout mice (KLK8). - / - ) and control group (KLK8) + / + Mice were injected intraperitoneally with LPS 30 mg / kg, and lung tissue was collected 24 hours later. (B, D) Mice were injected intravenously with anti-KLK8 neutralizing antibody or IgG isotype antibody via tail vein. 30 minutes later, mice were injected intraperitoneally with 30 mg / kg LPS, while the control group received an equal volume of physiological saline. Lung tissue was collected 24 hours later. (A, B) Western blot was used to detect the protein levels of VE-cadherin, p-Akt, Akt, and FOXM1 in lung tissue. (C, D) Real-time quantitative PCR was used to detect FOXM1 target molecules. Ccnd1, Cdc20, Ccnf, and Bub1bmRNA levels. Data are expressed as mean ± standard deviation (n=6). * p<0.05; * * p<0.01.

[0021] Figure 8 The results show immunohistochemical staining after KLK8 deletion or blockade. Mice A and C, KLK8-deficient (KLK8- / -) and control (KLK8+ / +) were intraperitoneally injected with 30 mg / kg LPS. Mice C and D were injected via tail vein with anti-KLK8 neutralizing antibody or IgG isotype antibody. Thirty minutes later, mice were intraperitoneally injected with 30 mg / kg LPS, while the control group received an equal volume of saline. Forty-eight hours later, lung tissue sections were collected for immunohistochemical staining of the anti-endothelial cell marker CD31 (red) and the cell proliferation marker Ki-67 (green). Mice B and D, KLK8... - / - and KLK8 + / + Ki67 in lung tissue sections from mice or mice treated with Anti-KLK8 / IgG + / CD31 + Cells account for a certain percentage of total CD31 + Percentage of cells. (n=6) Data are expressed as mean ± standard deviation. ** p<0.01.

[0022] Figure 9 The image shown is a map of the overexpression adenovirus vector of the present invention.

[0023] Figure 10 The image shows a vector map of the Len-Vector or FOXM1 lentivirus of this invention. Detailed Implementation

[0024] This study found that KLK8, a member of the KLK family, was significantly upregulated in LPS-exposed lung tissue or MLVECs. Combined evidence from mice treated with KLK8 knockout, pulmonary KLK8 overexpression, and KLK8 neutralizing antibodies suggests that KLK8 upregulation contributes to LPS-induced endothelial cell hyperpermeability and acute lung injury. Further transcriptomic analysis revealed that KLK8-induced inactivation of the VE-cadherin / Akt / FOXM1 pathway plays a crucial role in mediating endothelial regeneration injury and subsequent pulmonary vascular leakage, providing a target for the development of angiogenic drugs for sepsis.

[0025] This invention provides the use of KLK8 or its encoding gene as a target in the preparation of sepsis prevention, treatment or diagnostic products.

[0026] Tissue kallikrein-related peptidases (KLKs) include 15 secretory serine peptidases, numbered KLK1 to KLK15. KLK8, or tissue kallikrein-associated peptidase 8, is a trypsin-like serine protease that is highly expressed in the central nervous system.

[0027] The use of KLK8 or its encoding gene as a target in the preparation of products for the prevention, treatment or diagnosis of concentration-related diseases specifically refers to substances that can reduce KLK8 levels or inhibit the function of KLK8 by using KLK8 protein or its encoding gene as a recognition target.

[0028] In some embodiments of the invention, the use is in the preparation of products for treating sepsis complicated with acute lung injury and / or endothelial injury.

[0029] In one embodiment, the use is in the preparation of a product for treating sepsis complicated by pulmonary endothelial injury. The product for treating sepsis complicated by pulmonary endothelial injury may be a pulmonary endothelial regeneration product or a vascular repair product.

[0030] Sepsis is a life-threatening organ dysfunction caused by a dysregulation of the body's response to infection. The specific definition and diagnostic criteria are as follows: In February 2016, at the 45th Annual Meeting of the Society of Critical Care Medicine, the American Society of Critical Care Medicine (SCCM) and the European Society of Critical Care Medicine (ESICM) jointly released the 3.0 definition and diagnostic criteria for sepsis.

[0031] The acute lung injury includes early-stage acute lung injury and severe-stage acute respiratory distress syndrome.

[0032] The manifestations of acute lung injury include pulmonary vascular leakage, etc.

[0033] Endothelial damage manifests as endothelial hyperpermeability and downregulation of endothelial markers such as CD31 and ZO-1 protein levels.

[0034] The present invention also provides the use of substances that specifically bind to KLK8 in the preparation of sepsis prevention, treatment or diagnostic products.

[0035] In some embodiments of the invention, the use is in the preparation of products for treating sepsis complicated with acute lung injury and / or endothelial injury.

[0036] In one embodiment, the substance that specifically binds to KLK8 is selected from KLK8 inhibitors.

[0037] KLK8 inhibitors are molecules that inhibit KLK8. Inhibition of KLK8 includes, but is not limited to, inhibiting the level or activity of KLK8.

[0038] Inhibiting KLK8 activity means reducing KLK8 activity. Preferably, compared with before inhibition, KLK8 activity is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0039] Suppressing KLK8 levels can be achieved by inhibiting the transcription or translation of the KLK8 gene. Specifically, this can mean preventing the KLK8 gene from being transcribed, reducing its transcriptional activity, preventing its translation, or reducing its translation level.

[0040] Those skilled in the art can use conventional methods to regulate KLK8 gene expression, such as gene knockout, homologous recombination, and interfering RNA.

[0041] The gene knockout can be a complete gene knockout or a conditional gene knockout. The conditional gene knockout can be achieved using the Cre / LoxP system or the Gin / Gix system.

[0042] Preferably, compared with the wild type, KLK8 gene expression is reduced by at least 10%, more preferably by at least 30%, even better by at least 50%, better by at least 70%, even better by at least 90%, and most preferably by no expression of the KLK8 gene at all.

[0043] The KLK8 inhibitors include, but are not limited to: nucleic acid molecules, lipids, small molecule chemicals, antibody drugs, peptides, proteins, interfering lentiviruses, adeno-associated viruses, nanoparticles, liposomes, extracellular vesicles, or cells. The nucleic acid molecules include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (siRNA) or short hairpin RNA (shRNA) prepared by ribonuclease I or II.

[0044] The sepsis prevention, treatment or diagnostic products must include substances that specifically bind to KLK8, and use substances that specifically bind to KLK8 as active ingredients.

[0045] In some embodiments of the present invention, the KLK8 inhibitor is siRNA. The nucleotide sequence of the siRNA is shown in SEQ ID NO:3-4.

[0046] In some embodiments of the present invention, the KLK8 inhibitor is an anti-KLK8 neutralizing antibody.

[0047] The sepsis prevention, treatment, or diagnostic products may be single-component substances or multi-component substances.

[0048] There are no special restrictions on the form of the sepsis prevention, treatment or diagnosis product, which can be in various forms such as solid, liquid, gel, semi-liquid, aerosol, etc.

[0049] The sepsis prevention, treatment, or diagnostic products are primarily targeted at mammals. Preferred mammals include rodents, even-toed ungulates, perissodactyls, lagomorphs, and primates. Preferred primates include monkeys, apes, or humans.

[0050] The sepsis prevention, treatment, or diagnostic products are pharmaceuticals.

[0051] The sepsis prevention, treatment, or diagnostic product treats sepsis through one or more of the following methods:

[0052] Reduce endothelial cell permeability or improve capillary leakage;

[0053] Stimulate the VE-cadherin / Akt / FOXM1 signaling pathway; preferably, it is selected from upregulating the expression levels of FOXM1 and its downstream target genes, upregulating the protein levels of AKT or p-AKT, or upregulating the protein levels of VE-cadherin.

[0054] Increase the rate of endothelial cell proliferation;

[0055] Reduce endothelial cell mortality;

[0056] Enhance endothelial cell activity.

[0057] In some embodiments of the present invention, the downstream target genes of FOXM1 are, for example, Ccnd1, Cdc20, Ccnf, and Bub1b.

[0058] This invention also provides the use of substances that specifically bind to KLK8 in the preparation of one or more of the following products:

[0059] Products that reduce endothelial cell permeability or improve capillary leakage;

[0060] Products that stimulate the VE-cadherin / Akt / FOXM1 signaling pathway; preferably, products that upregulate the expression levels of FOXM1 and its downstream target genes, products that upregulate AKT, products that upregulate p-AKT protein levels, or products that upregulate the protein levels of VE-cadherin.

[0061] Products that increase the rate of endothelial cell proliferation;

[0062] Products that reduce endothelial cell mortality;

[0063] Products that enhance endothelial cell activity.

[0064] The present invention also provides the use of VE-cadherin / Akt / FOXM1 signaling pathway upregulators in the preparation of products for treating sepsis complicated with acute lung injury and / or endothelial injury.

[0065] In some embodiments of the present invention, the VE-cadherin / Akt / FOXM1 signaling pathway upregulator is selected from VE-cadherin upregulators, Akt upregulators, or FOXM1 upregulators.

[0066] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0067] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0068] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0069] Example 1: Increased KLK8 expression in the lungs in an LPS-induced sepsis model

[0070] Animal experiments:

[0071] The experimental animals were SPF-grade C57 mice, which were randomly divided into the following two groups (n=6 per group).

[0072] Control group: LPS 0 ng / ml group, fed normally.

[0073] Sepsis group: Sepsis model induced by intraperitoneal injection of different concentrations of lipopolysaccharide (LPS, 30 mg / kg), intervention for 48 hours:

[0074] LPS 10ng / ml group

[0075] LPS 100ng / ml group

[0076] LPS 1000ng / ml group

[0077] After 48 hours of intervention using an LPS-induced sepsis model, mice were anesthetized, and lung tissue was taken to detect the expression level of KLK8 protein.

[0078] In vitro experiments:

[0079] Primary lung vascular endothelial cells (MLVECs) were extracted from 3-4 week old ICR mice and treated with LPS doses (0, 10, 100, 1000 ng / ml) for 24 hours (n=4 per group).

[0080] Result: As Figure 1 As shown, LPS treatment significantly induced KLK8 expression in pulmonary vascular endothelial cells both in vitro and in vivo. Real-time quantitative PCR, Western blot, and immunohistochemical analysis of septic mouse lung tissue all indicated increased KLK8 expression. In vitro experiments also showed increased KLK8 expression in MLVECs after LPS treatment.

[0081] Example 2. Effects of KLK8 overexpression on the lungs

[0082] In vitro experiments:

[0083] MLVECs were treated for 48 hours with different MOIs (1, 3, 10), and the groups are as follows:

[0084] Ad-vector group, overexpression adenovirus vector map as follows Figure 9 As shown.

[0085] Ad-KLK8 1 MOI group, overexpression adenovirus vector map as follows Figure 9 As shown, the inserted gene coding fragment is the KLK8 coding sequence (NM_008940).

[0086] Ad-KLK8 3 MOI Group

[0087] Ad-KLK8 10 MOI Group

[0088] After MLVECs were treated with different MOIs (1, 3, 10) for 48 hours, Western Blot was used to detect the protein levels of KLK8, CD31, ZO-1, and ICAM-1, MTT assay was used to detect cell viability, and FITC assay was used to detect endothelial permeability.

[0089] Animal experiments:

[0090] SPF-grade mice were used as experimental animals. A KLK8 overexpression model in lung tissue was established by intratracheal instillation for 48 hours. The mice were randomly divided into two groups (n=6 per group):

[0091] Ad-Vector group: Ad-Vector infusion via endotracheal drip, 10 8 Droplet / each.

[0092] Ad-KLK8 group: Ad-KLK8 administered via intratracheal infusion, 10 8 Droplet / each.

[0093] Lung tissue was collected 48 hours after intratracheal instillation of adenovirus. Western blotting was used to detect the protein levels of KLK8, CD31, ZO-1, and ICAM-1. Evans Blue dye was used to detect vascular leakage in lung tissue. HE morphology was used for pathological evaluation of lung tissue.

[0094] The results are as follows Figure 2 As shown, KLK8 overexpression leads to decreased levels of CD31, ZO-1, and ICAM-1 proteins in endothelial cells, reduced cell viability, and increased cell permeability. KLK8 overexpression also leads to decreased levels of CD31, ZO-1, and ICAM-1 proteins in mouse lung tissue, increased pulmonary capillary leakage, and elevated lung tissue injury scores, indicating that KLK8 overexpression can cause acute lung injury.

[0095] Example 3. Inhibition of KLK8 expression alleviated LPS-induced lung tissue damage.

[0096] In vitro experiments:

[0097] MLVECs were pretreated with siRNA for 48 hours, followed by LPS intervention for 24 hours.

[0098] Grouping: Control siRNA group; Control siRNA sequences: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.1), 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.2)

[0099] KLK8 siRNA group, KLK8 siRNA sequence: 5'-CCUGGAUCAAGAAGACCAUTT-3' (SEQ ID NO.3), 5'-AUGGUCUUCUUGAUCCAGGTT-3' (SEQ ID NO.4)

[0100] Control siRNA+LPS group

[0101] KLK8 siRNA+LPS group

[0102] Cells were transfected with siRNA for 48 hours and then treated with LPS for 24 hours. Western blotting was used to detect the protein levels of CD31, ZO-1, and ICAM-1, MTT assay was used to detect cell viability, and FITC assay was used to detect endothelial permeability.

[0103] Animal experiments:

[0104] The experimental animals were SPF-grade KLK8 knockout mice and an LPS-induced sepsis mouse model. KLK8 knockout mice were constructed by Shanghai Southern Model Biotechnology Co., Ltd., and the construction steps were roughly as follows: KLK8-flox-tagged mice were obtained using the Cre-loxp system, and then KLK8-flox-tagged mice were mated with EIIa-Cre transgenic mice to obtain KLK8 knockout mice. KLK8flox / flox;EIIa-Cre(+) (KLK8- / -), and age-matched KLK8flox / flox;EIIa-Cre(-) littermates (KLK8+ / +) served as controls to study the effect of KLK8 deletion. Mice with the same genotype were randomly assigned in a 1:1 ratio to the KLK8 knockout group and the KLK8 knockout+LPS group. Wild-type experimental mice were randomly assigned. There were 6 mice in each group.

[0105] Wild-type control group: intraperitoneal injection of an equal dose of physiological saline.

[0106] Wild-type LPS group: Intraperitoneal injection of LPS (30 mg / kg) induced sepsis model, with a modeling time of 48 hours.

[0107] KLK8 knockout group: intraperitoneal injection of an equal dose of normal saline.

[0108] KLK8 knockout + LPS group: Intraperitoneal injection of LPS (30 mg / kg) induced sepsis model, and the modeling time was 48 hours.

[0109] Twenty-four hours after LPS-induced sepsis mouse model, Western blotting was used to detect the levels of CD31, ZO-1, and ICAM-1 proteins, and Evans Blue dye was used to detect vascular leakage in lung tissue. Forty-eight hours after LPS-induced sepsis mouse model, HE morphological examination was used for lung tissue pathological evaluation, and Kaplan-Meier analysis was used to analyze the survival status of sepsis mice.

[0110] The results are as follows Figure 3 As shown:

[0111] 1. Inhibition of KLK8 expression in MLVECs significantly improved the LPS-induced downregulation of endothelial markers CD31 and ZO-1 protein levels, reduced ICAM-1 expression, significantly increased cell viability, and markedly decreased cell permeability.

[0112] 2. In KLK8 knockout mice, LPS-induced lung endothelial markers CD31 and ZO-1 protein levels were significantly increased, while ICAM-1 expression was decreased, which improved pulmonary capillary leakage and alleviated LPS-induced lung tissue damage.

[0113] In summary, KLK8 can improve endothelial hyperpermeability and acute lung injury induced by LPS and reduce mortality caused by systemic LPS.

[0114] Example 4. Administration of anti-KLK8 antibody reduced LPS-induced lung tissue damage.

[0115] The experimental animals were SPF-grade C57 mice. Anti-KLK8 neutralizing antibody (M081-3M2, MBL International; 20 μg / kg) or IgG isotype (MBL International; 20 μg / kg) was injected via the tail vein. Thirty minutes later, LPS was injected intraperitoneally to establish a sepsis model. The mice were randomly divided into four groups (n=6 per group), and lung tissue was harvested 48 hours later.

[0116] IgG group: intraperitoneal injection of an equal dose of physiological saline.

[0117] IgG+LPS group: LPS (30 mg / kg) was injected intraperitoneally to induce a sepsis model for a total of 48 hours.

[0118] Anti-KLK8 group: intraperitoneal injection of an equal dose of physiological saline.

[0119] Anti-KLK8+LPS group: Intraperitoneal injection of LPS (30 mg / kg) induced sepsis model for 48 hours.

[0120] Twenty-four hours after LPS-induced sepsis mouse model, Western blotting was used to detect the levels of CD31, ZO-1, and ICAM-1 proteins, and Evans Blue dye was used to detect vascular leakage in lung tissue. Forty-eight hours after LPS-induced sepsis mouse model, HE morphological examination was used for lung tissue pathological evaluation, and Kaplan-Meier analysis was used to analyze the survival status of sepsis mice.

[0121] The results are as follows Figure 4As shown, tail vein injection of KLK8 neutralizing antibody significantly improved the levels of CD31 and ZO-1 protein, markers of lung endothelial tissue, in septic mice with KLK8 expression in vivo, decreased ICAM-1 expression, reduced pulmonary capillary leakage, alleviated LPS-induced lung tissue damage, and reduced mortality caused by systemic LPS.

[0122] Example 5. KLK8 overexpression induced a decrease in FOXM1 expression, and FOXM1 overexpression reversed lung injury induced by KLK8 overexpression.

[0123] MLVECs were treated with AdKLK8 (3 MOI) adenovirus for 48 hours and then analyzed by RNA-Seq.

[0124] Ad-Vector group

[0125] Ad-KLK8 group

[0126] The results are as follows Figure 5 As shown in Figure AF, RNA-Seq analysis revealed abnormal genes, molecular characteristics of abnormal pathways, and the number of genes in each pathway in MLVECs after Ad-KLK8 overexpression. Ingenuity Pathway Analysis (IPA) was used to predict the upstream regulators responsible for altered gene expression profiles in Ad-KLK8-treated MLVECs. The results showed a significant decrease in both mRNA and protein expression of FOXM1 in Ad-KLK8-treated MLVECs.

[0127] MLVECs were infected with either Len-Vector or Len-FOXM1 lentivirus at a MOI of 10 for 30 min, and then treated with Ad-Vector or Ad-KLK8 (3 MOI) for 48 h. The expression profiles of Len-Vector and Len-FOXM1 vectors are shown below. Figure 10 As shown, the coding sequence of the Len-FOXM1 insertion gene is NM_008021.

[0128] Len-Vector+Ad-Vector group

[0129] Len-Vector+Ad-KLK8 set

[0130] Len-FOXM1 + Ad-Vector group

[0131] Len-FOXM1+Ad-KLK8 set

[0132] The results are as follows Figure 5As shown in the GL, FOXM1 overexpression significantly reversed the decrease in cell proliferation and the reduction in downstream target genes (Ccnd1, Cdc20, Ccnf, Bub1b) caused by KLK8 overexpression, effectively improved endothelial marker damage, increased cell activity, and reduced endothelial permeability.

[0133] Example 6. KLK8 induces endothelial barrier dysfunction by inhibiting endothelial cell proliferation mediated by the VE-cadherin / Akt / FOXM1 signaling pathway.

[0134] MLVECs were treated with AdKLK8 adenovirus for 48 hours, and then gene set enrichment analysis (GSEA) was performed to identify the upstream signaling pathways regulating FOXM1.

[0135] MLVECs were pretreated with Akt activator SC79 (10 μM), followed by treatment with Ad-KLK8 (3 MOI) for 48 hours, and grouped as follows:

[0136] Ad-Vector group

[0137] Ad-KLK8 group

[0138] SC79+Ad-Vector group

[0139] SC79+Ad-KLK8 group

[0140] SPF-grade mice were used as experimental animals. A KLK8 overexpression model in lung tissue was established by intratracheal instillation for 48 hours. The mice were randomly divided into two groups (n=6 per group).

[0141] Ad-Vector group

[0142] Ad-KLK8 group

[0143] Using VE-cadherin lentivirus (10 μM, vector expression map as shown) Figure 10 As shown, MLVECs were pretreated with the inserted gene coding sequence NM_009868, followed by Ad-KLK8 (3 MOI) treatment for 48 hours, and the groups were as follows:

[0144] Len-Vector+Ad-Vector group

[0145] Len-Vector+Ad-KLK8 set

[0146] Len-VE-cadherin + Ad-Vector group

[0147] Len-VE-cadherin+Ad-KLK8 group

[0148] MLVECs were pretreated with the Akt inhibitor AZD5363 (10 μM), followed by treatment with Ad-KLK8 (3 MOI) for 48 hours, and grouped as follows:

[0149] Ad-Vector group

[0150] Ad-KLK8 group

[0151] AZD5363+Ad-Vector group

[0152] AZD5363+Ad-KLK8 group

[0153] The results are as follows Figure 6 As shown:

[0154] 1. GSEA screening results showed that the PI3K-AKT-mTOR signaling pathway was downregulated in MLVECs after AdKLK8 treatment, and Western blot results showed decreased p-Akt and Akt protein levels. Pretreatment of MLVECs with the Akt activator SC79, followed by Ad-KLK8 treatment, resulted in significantly improved p-Akt, Akt, and FOXM1 protein levels according to Western blot results. These results suggest that KLK8 may negatively regulate FOXM1 expression by inducing Akt signaling inactivation.

[0155] 2. The protein level of VE-cadherin in MLVECs or lung tissue was significantly reduced after Ad-KLK8 treatment. After 48 hours of Ad-KLK8 treatment, the N-terminal VE-cadherin fragment in the MLVEC culture medium significantly increased. KLK8, a secreted serine protease, has been shown to mediate the shedding of the outer membrane of VE-cadherin, thereby promoting the initiation of myocardial EndMT in the pathogenesis of diabetes-induced cardiac fibrosis. Cleavage of VE-cadherin can inactivate the Akt signaling pathway in endothelial cells. Overexpression of VE-cadherin significantly reversed the Ad-KLK8-induced decrease in the protein levels of VE-cadherin, FOXM1, p-Akt, and Akt in MLVECs. Pretreatment with the AKT inhibitor AZD5363 eliminated the protective effect of VE-cadherin against the downregulation of VE-cadherin and FOXM1 in Ad-KLK8-induced MLVECs.

[0156] The experimental results suggest that KLK8 may induce endothelial barrier dysfunction by inhibiting endothelial cell proliferation mediated by the VE-cadherin / Akt / FOXM1 signaling pathway.

[0157] Example 7. KLK8 deficiency or blockade can improve endothelial injury and cell proliferation.

[0158] Animal Experiment 1:

[0159] The experimental animals were SPF-grade KLK8 knockout mice, using an LPS-induced sepsis mouse model. The mice were randomly divided into the following 4 groups (n=6 per group):

[0160] Wild-type control group: intraperitoneal injection of an equal dose of physiological saline.

[0161] Wild-type LPS group: Intraperitoneal injection of LPS (30 mg / kg) induced sepsis model, with a modeling time of 48 hours.

[0162] KLK8 knockout group: intraperitoneal injection of an equal dose of normal saline.

[0163] KLK8 knockout + LPS group: Intraperitoneal injection of LPS (30 mg / kg) induced sepsis model, and the modeling time was 48 hours.

[0164] Animal Experiment 2:

[0165] SPF-grade C57 mice were used as experimental animals. Anti-KLK8 neutralizing antibodies were injected via the tail vein, and 30 minutes later, LPS was injected intraperitoneally to establish a sepsis model. The mice were randomly divided into four groups (n=6 per group), and lung tissue was harvested 48 hours later:

[0166] IgG group: intraperitoneal injection of an equal dose of physiological saline.

[0167] IgG+LPS group: LPS (30 mg / kg) was injected intraperitoneally to induce a sepsis model for a total of 48 hours.

[0168] Anti-KLK8 group: intraperitoneal injection of an equal dose of physiological saline.

[0169] Anti-KLK8+LPS group: Intraperitoneal injection of LPS (30 mg / kg) induced sepsis model for 48 hours.

[0170] The results are as follows Figure 7 As shown, Western blotting and real-time quantitative PCR results indicate that KLK8 deletion or blockage can improve the VE-cadherin / Akt / FOXM1 signaling pathway, upregulate the expression levels of FOXM1 downstream target genes (Ccnd1, Cdc20, Ccnf, Bub1b), and promote endothelial cell regeneration.

[0171] Immunohistochemical results as follows Figure 8 As shown, KLK8 deficiency or blockade can improve endothelial injury and cell proliferation.

[0172] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The use of a substance that specifically binds to KLK8 in the preparation of sepsis prevention and treatment products, wherein the substance that specifically binds to KLK8 is a KLK8 inhibitor, wherein the KLK8 inhibitor is an anti-KLK8 neutralizing antibody or siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO:3~4.

2. The use according to claim 1, characterized in that, The intended use is in the preparation of products for the treatment of sepsis complicated with acute lung injury and / or endothelial injury.

3. The use according to claim 1, characterized in that, The intended use is in the preparation of products for treating sepsis complicated by pulmonary endothelial injury.

4. The use according to claim 3, characterized in that, The product for treating sepsis complicated by pulmonary endothelial injury is a pulmonary endothelial regeneration product or a pulmonary microvascular repair product.

5. The use according to claim 1, characterized in that, The sepsis prevention or treatment product treats sepsis through one or more of the following methods: 1) Reduce endothelial cell permeability or improve capillary leakage; 2) Stimulate the VE-cadherin / Akt / FOXM1 signaling pathway; 3) Increase the rate of endothelial cell proliferation; 4) Reduce endothelial cell mortality; 5) Enhance endothelial cell activity.

6. The use according to claim 5, characterized in that, When the sepsis prevention or treatment product stimulates the VE-cadherin / Akt / FOXM1 signaling pathway, it upregulates the expression levels of FOXM1 and its downstream target genes, upregulates the protein levels of AKT or p-AKT, or upregulates the protein level of VE-cadherin.

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