Application of ENOBlock in preparation of medicine for treating sepsis

Abstract

The invention relates to the field of biological medicine, and discloses application of ENOBlock in preparation of a medicine for treating sepsis. The drug provided by the invention can effectively relieve the immunosuppression state in the course of sepsis by targeting and blocking the interaction of NETs-MPO and ENO1, and provides a new intervention target for the immunoregulation treatment of sepsis.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to the use of ENOBlock in preparing a drug for treating sepsis. Background Art

[0002] During sepsis, inflammatory responses and immunosuppression may occur sequentially or simultaneously. Standardized antibiotic therapy, adequate fluid resuscitation, and multi-organ support can effectively suppress the excessive inflammatory response in the early stages of sepsis and reduce mortality. However, sepsis often develops into a persistent inflammation-immunosuppression-catabolism syndrome in the later stages. The immunosuppression caused by sepsis significantly increases the patient's susceptibility to secondary infections, leading to life-threatening multi-organ dysfunction and circulatory disturbances, and is the main cause of poor prognosis and even death.

[0003] Reversing immunosuppression can improve the survival rate of animals in sepsis models, but the therapeutic effects of current related immunotherapies remain mixed. Studies have shown that regulatory T cells (Tregs) are an important cause of immunosuppression in sepsis. At the same time, over-activated neutrophils and excessive release of neutrophil extracellular traps (NETs) play an important role in the progression of sepsis. However, the role of NETs in sepsis-induced immunosuppression has not been elucidated, especially whether and how NETs regulate Treg differentiation.

[0004] Although the NETs degrader DNase I currently used in research can decompose the chromatin DNA of NETs, ​​it cannot specifically regulate its downstream immune regulatory function. This limitation seriously restricts the clinical application value of NETs targeted therapy. Summary of the Invention

[0005] The purpose of the invention of this application is to provide a precise treatment strategy for targeted intervention of the NETs-MPO-ENO1 signaling pathway, inhibit the differentiation of Treg regulated by NETs, ​​effectively correct the immune imbalance of sepsis patients, and improve clinical prognosis.

[0006] This application reveals that NETs-MPO promotes the CD4 +The new mechanism of T cell differentiation into Treg cells clarifies the key role of NETs in sepsis immunosuppression. Based on this discovery, this application developed a targeted intervention plan: the enolase inhibitor ENOBlock (AP-III-a4) was used to directly block the ENO1 active site, effectively destroying the molecular interaction between NETs-MPO and ENO1. Experimental data showed that this strategy can significantly inhibit NETs-induced Treg differentiation (inhibition rate 30%, p<0.001), and in the second-hit sepsis model, the survival rate of mice was increased from 30% in the control group to 70% (p<0.05), and significantly improved pathological damage to multiple organs such as the lungs, liver, and kidneys.

[0007] Based on this, in a first aspect, the present application provides the use of ENOBlock in the preparation of a drug for treating sepsis.

[0008] Furthermore, the drug is a drug that alleviates the progression of immunosuppression in sepsis.

[0009] Furthermore, the drug effectively destroys the molecular interaction between NETs-MPO and ENO1 by directly blocking the active site of ENO1, thereby inhibiting NETs-induced Treg differentiation.

[0010] In summary, this application has the following beneficial effects:

[0011] MPO in NETs can recognize and bind to the C-terminal domain (amino acids 139-434) of ENO1 on the cell surface, thereby promoting the differentiation and function of Treg cells. Targeted blocking of the interaction between NETs-MPO and ENO1 can effectively alleviate the immunosuppressive state during sepsis, providing a new intervention target for the immunomodulatory treatment of sepsis.

[0012] Precisely targeting the newly discovered immune regulatory axis NETs-MPO-ENO1; effectively correcting the immune imbalance in sepsis patients by regulating the differentiation and function of Treg cells; suitable for sepsis patients with excessive NETs formation and abnormally increased Treg cell proportions, providing new ideas for precise immunotherapy of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :NETs-induced upregulation of ENO1 expression in Treg cells differentiated in vitro;

[0014] A. The proportion of regulatory T cells induced to differentiate in vitro after 12-48 hours of stimulation with NETs (0-100 ng / mL), n=3; B. The expression of ENO1 in regulatory T cells induced to differentiate in vitro after 24-48 hours of stimulation with solvent or NETs (100 ng / mL), n=3; C. The expression of ENO1 in regulatory T cells induced to differentiate in vitro after 24-48 hours of stimulation with solvent or NETs (100 ng / mL), n=3. ENO1 expression in CD4+ T cells; D. Regulatory T cell differentiation conditions after 48h stimulation with solvent or NETs (100ng / mL) Expression of ENO1 in the cell membrane of CD4+ T cells; E. Expression of ENO1 in regulatory T cells induced and differentiated in vitro in the control group treated with solvent or DNase I (2 μg / mL) and the group treated with NETs (100 ng / ml) (24-48 h), n=3; F. Proportion of regulatory T cells induced and differentiated in vitro in the control group infected with ShNC or shENO1 lentivirus and the group treated with NETs (100 ng / mL) (24-48 h), n=3.

[0015] Figure 2 : Inhibition of ENO1 alleviates the progression of immunosuppression in sepsis;

[0016] A. The proportion of regulatory T cells in the spleen of the Sham group and the CLP group during the immunosuppressive period, which were intraperitoneally injected with the solvent or ENOBlock (10 mg / kg) every other day, n=6; B. The expression of CD152 in the spleen of the Sham group and the CLP group during the immunosuppressive period, which were intraperitoneally injected with the solvent or ENOBlock (10 mg / kg) every other day, n=6; C. Kaplan-Meier survival curves of the Sham group and the CLP group 72 hours after the second challenge with intranasal inoculation of Pseudomonas aeruginosa, which were intraperitoneally injected with the solvent or ENOBlock (10 mg / kg) every other day; Sham+Vehicle group, n=5, CLP+Vehicle group, n=10, Sham+ENOBlock group, n=5, CLP+ENOBlock group, n=10; D. The expression of CD152 in the spleen of the Sham group and the CLP group, which were intraperitoneally injected with the solvent or ENOBlock (10 mg / kg) every other day, which were intranasally inoculated with Pseudomonas aeruginosa E. HE staining of the lungs 72 hours after the second attack of Pseudomonas aeruginosa (scale bar: 50 μm); E. HE staining of the livers 72 hours after the second attack of Pseudomonas aeruginosa by intranasal injection of solvent or ENOBlock (10 mg / kg) every other day and CLP group mice (scale bar: 50 μm); F. HE staining of the kidneys 72 hours after the second attack of Pseudomonas aeruginosa by intranasal injection of solvent or ENOBlock (10 mg / kg) every other day and CLP group mice (scale bar: 50 μm). DETAILED DESCRIPTION

[0017] The technical solutions and effects of the present application are further described in detail below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0018] Example 1: NETs-MPO recognizes and binds to the C-terminal domain of ENO1 on the cell surface to promote CD4 + T cells differentiate into Treg cells

[0019] In this example, bone marrow cells were collected from the femur and tibia of 6-8 week old normal wild-type mice. After erythrocytes were lysed with erythrocyte lysis buffer, the cells were resuspended in Hanks' balanced salt solution. Neutrophils were then isolated and purified using Histopaque 1119 and Histopaque 1077 density gradient centrifugation. The purified neutrophils (1×10 6 / mL) and resuspended in RPMI-1640 complete medium. Phorbol ester (20 nM) was then added to the medium for stimulation for 3.5 hours to induce NET formation. After stimulation, the cells were removed by centrifugation at 300 g for 5 minutes, followed by high-speed centrifugation at 18,000 g for 15 minutes and the supernatant discarded. The resulting precipitate was purified NETs.

[0020] In addition, in this example, the spleen of 8-10 week old normal wild-type mice was separated and prepared into single cell suspension, and then the mouse CD4 + Isolation by negative sorting with the T Cell Isolation Kit CD4 + T cells, purified CD4 + T cells (2×10 5 / mL) were resuspended in RPMI 1640 complete medium and inoculated into a 48-well plate coated with anti-CD3 antibody (10μg / mL). Anti-CD3 antibody (2μg / mL), anti-CD28 antibody (2μg / mL), anti-IFN-γ antibody (10μg / mL), anti-IL-4 antibody (10μg / mL), TGF-β1 (1.5ng / mL), and IL-2 (200U / mL) were added to stimulate the cells. The cells were cultured in a humidified incubator at 37°C and 5% CO2 ventilation conditions. CD4 + After 48-84 hours, NETs (25-500 ng / mL) and / or DNase I (2 μg / mL) for NET degradation were added to the culture medium according to the experimental purpose. After 12-48 hours, the cells were collected for the next step of detection and analysis. CD4 + After 24 hours of T cell infection, the amount of virus required to be added to each well was calculated using a multiplicity of infection of 40. pSLenti-U6-shENO1-CMV-EGFP-F2A-Puro-WPRE (Heyuan Biotechnology) and pSLenti-U6-shNC-CMV-EGFP-F2A-Puro-WPRE (Heyuan Biotechnology) were added to the culture system, respectively. Two replica wells were set up in each group for cell infection. After 16 hours of viral infection, each group was centrifuged at 500 g for 5 minutes to discard the original culture medium, and fresh RPMI-1640 complete culture medium was added. The cells were inoculated into a new anti-CD3 antibody-coated 24-well plate to induce in vitro differentiation of Treg cells.

[0021] The results showed that compared with the solvent control group, NETs significantly promoted the differentiation of Treg cells in vitro, and showed obvious time-dependent and dose-dependent effects ( Figure 1 A). In addition, we observed that the expression level of ENO1 in Treg cells induced in vitro by NETs was significantly increased, and this upregulation was very obvious in the early stage of NETs treatment ( Figure 1 B) Western blotting analysis further confirmed that under the conditions of Treg cell differentiation, NETs stimulation led to The expression level of ENO1 protein in CD4+T cells increased ( Figure 1 C), especially the upregulation of ENO1 protein level in the cell membrane ( Figure 1 D) When treated with DNase I to degrade NET-DNA, the expression level of ENO1 in Treg cells induced in vitro was reduced ( Figure 1 E) We used lentivirus to knock down solvent or NETs. CD4 + The Eno1 gene of T cells was detected by flow cytometry and it was found that infection with shENO1 lentivirus significantly weakened the role of NETs in promoting CD4 + The role of T cells in differentiating into Treg cells in vitro Figure 1 F) These results indicate that NETs promote the differentiation and function of Treg cells through ENO1 on the cell surface, and knockdown CD4 + The Eno1 gene of T cells can effectively weaken the regulatory effect of NETs.

[0022] Example 2: ENOBlock inhibits NETs' recognition and binding to ENO1

[0023] In this example, a sepsis model was established by cecal ligation and puncture. Male C57 / BL6 mice aged 8-10 weeks and 20-25 g were selected and anesthetized with 50 mg / kg 1% sodium pentobarbital. After skin preparation, a midline abdominal incision was made to expose the cecum. The cecum was ligated at the center of the distal ileocecal valve. A 22G needle was used to penetrate once from the mesentery to the anti-mesentery direction, and a small amount of feces was squeezed out. After the cecum was returned, the muscle layer and skin layer were sutured in sequence. The sham group (Sham) mice received the same operation plan without the cecal ligation and puncture step. All mice received 0.9% saline and analgesia after the operation, and had free access to water and food. The success of the CLP model was judged by observing the survival of the mice, detecting changes in the ratio of neutrophils and lymphocytes in the peripheral blood of the mice, and evaluating the damage of multiple organs in the mice. In this example, one week after the CLP model was used as the main observation time node for sepsis immunosuppression.

[0024] To evaluate the effect of ENO1 inhibition on the progression of immunosuppression in sepsis, the experimental group was given ENOBlock (10 mg / kg) by intraperitoneal injection every 24 hours starting from the third day after surgery, while the control group was injected with an equal volume of PBS buffer. On the seventh day after surgery, mice were anesthetized with halothane and fixed vertically with their heads facing upwards. Pseudomonas aeruginosa (Pseudomonas aeruginosa, bacterial concentration 4×10 7 A secondary challenge model was established using 1000 CFU / mouse. Survival rates of mice were then assessed 72 hours after the secondary challenge. Quant-iT PicoGreen dsDNA was used to quantify cfDNA levels in peripheral blood plasma. Flow cytometry was used to determine the proportion of splenic Treg cells. HE staining was used to assess the extent of histopathological damage in vital organs, including the lungs, liver, and kidneys.

[0025] The results showed that compared with the solvent control group, the proportion of Treg cells in the spleen of mice in the immunosuppressive phase of sepsis in the ENOBlock-treated group was reduced ( Figure 1 A), and the expression of CD152 functional molecules in Treg cells was also reduced ( Figure 1 B). The results of survival analysis of CLP model mice showed that ENOBlock intervention significantly improved the survival rate of septic mice ( Figure 1 C), and effectively improved multiple organ damage in the lungs, liver, and kidneys ( Figure 1 DF). These findings collectively suggest that inhibiting NETs' recognition and binding to ENO1 using ENOBlock may help alleviate the progression of immunosuppression in sepsis.

[0026] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. Application of ENOBlock in the preparation of drugs for the treatment of sepsis.

2. The use according to claim 1, characterized in that The drug is a drug for alleviating the progression of immunosuppression in sepsis.

3. The use according to claim 1, characterized in that The drug effectively destroys the molecular interaction between NETs-MPO and ENO1 by directly blocking the active site of ENO1, thereby inhibiting NETs-induced Treg differentiation.

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