Application of IL-4 secreted by type 2 innate lymphocytes in preparation of medicine for treating sepsis heart injury

Abstract

The invention finds that type 2 innate lymphocytes and cytokines thereof can recover the cardiac function of sepsis. The invention discusses a related mechanism of regulating autophagosome-lysosome fusion of sepsis heart endothelial cells by the type 2 innate lymphocyte in sepsis heart injury; the influence of the type 2 innate lymphocyte and the generated cell factor IL-4 on the sepsis heart function and endothelial cells is clarified, and a new immune regulation mechanism is expected to be provided for sepsis heart function impairment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of IL-4 secreted by type 2 innate lymphocytes in the preparation of a medicine for treating sepsis-induced cardiac injury. Background Art

[0002] Sepsis is an uncontrolled immune response to infection that causes organ dysfunction. Septic shock is a severe form of sepsis with severe circulatory, cellular, and metabolic dysfunction and is a common disease in intensive care units. Sepsis can cause acute organ dysfunction, and patients with impaired cardiac function have a mortality risk of up to 70%. Current treatments for sepsis-induced cardiomyopathy focus on inflammation suppression and hemodynamic stabilization, but often prove to be insufficient because cytokine storms not only exacerbate cardiac damage but also reduce the efficacy of traditional therapies to stabilize cardiac function. Therefore, effectively targeting cardiac inflammation and thereby reducing myocardial damage is a challenge for current treatment strategies. Summary of the invention

[0003] The present invention aims to overcome the above defects. Studies have found that type 2 innate lymphocytes and their cytokines can restore cardiac function in sepsis.

[0004] Application of type 2 innate lymphocytes in the preparation of drugs for treating, alleviating or preventing sepsis-induced cardiac injury.

[0005] Application of type 2 innate lymphocytes in the preparation of drugs for restoring or protecting cardiac function.

[0006] Application of type 2 innate lymphocytes in the preparation of drugs for sepsis-induced cardiac dysfunction and cardiac cell apoptosis.

[0007] Application of type 2 innate lymphocytes in the preparation of agents regulating IL-4.

[0008] Application of IL-4 in the preparation of drugs for treating, alleviating or preventing sepsis-induced cardiac injury.

[0009] Application of IL-4 in the preparation of drugs for restoring or protecting cardiac function.

[0010] Application of IL-4 in the preparation of drugs for treating and alleviating cardiac dysfunction and cardiac cell apoptosis caused by sepsis.

[0011] Application of IL-4 secreted by type 2 innate lymphocytes in the preparation of drugs for treating, alleviating or preventing sepsis-induced cardiac injury.

[0012] Application of IL-4 secreted by type 2 innate lymphocytes in the preparation of drugs for restoring or protecting cardiac function.

[0013] Application of IL-4 secreted by type 2 innate lymphocytes in the preparation of drugs for sepsis-induced cardiac dysfunction and cardiac cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :Septic cardiac injury is closely related to the expression of type 2 innate lymphocytes and their downstream cytokine IL-4

[0015] (A) Changes in the proportion of type 1 innate lymphocytes in the hearts of septic mice; (B) Statistics of the proportion of type 1 innate lymphocytes in the hearts of septic mice; (C) Statistics of the proportion of type 3 innate lymphocytes in the hearts of septic mice; (D) Changes in the proportion of type 3 innate lymphocytes in the hearts of septic mice; (E) Changes and statistics of the proportion of type 2 innate lymphocytes in the hearts of mice in the sepsis group and the sham operation group over time; (F) Changes and statistics of the proportion of IL-4 secreted by type 2 innate lymphocytes in the hearts of mice in the sepsis group and the sham operation group over time; (G) Changes and statistics of the proportion of IL-4 secreted by type 2 innate lymphocytes in the hearts of mice in the sham operation group over time; (H) Changes in mouse plasma lactate dehydrogenase LDH and CK-MB; (I) HE staining to detect changes in the tissue structure of mouse heart sections, scale bar = 50μm; data are expressed as mean ± standard deviation. (n=3-5; **P<0.01 and ***P<0.0001).

[0016] Figure 2 IL-4, a cytokine secreted by type 2 innate lymphocytes, protects cardiac function and alleviates cell apoptosis in sepsis

[0017] (A) Schematic diagram of septic mice and injection of Anti-CD90.2 neutralizing antibody; (B) Flow cytometric analysis of changes in the expression of type 2 innate lymphocytes in the mouse heart after using the in vivo antibody of Anti-CD90.2; (C) Flow cytometric analysis of changes in the expression of type 2 innate lymphocytes and secreted IL-4 in the mouse heart after using the in vivo antibody of Anti-CD90.2; (D) M-mode echocardiography of mice; (E) Quantitative echocardiographic analysis of cardiac output (CO), ejection fraction (EF) ) and shortening fraction (FS); (F) Changes of plasma lactate dehydrogenase LDH and CK-MB in mice; (G) TUNEL staining to detect apoptosis in mouse heart sections, scale bar = 50 μm; (H) Immunohistochemistry to detect changes in C-caspased-3 positive cells in mouse heart sections, scale bar = 50 μm; Data are expressed as mean ± SD (n = 4-5; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

[0018] Figure 3:IL-4 derived from ILC2 alleviates impaired autophagic flux in cardiac endothelial cells

[0019] (A) Immunoblotting to detect the expression of LAMP2, P62, and LC3 proteins; (B) Quantification of (A); (C) Immunofluorescence to detect the colocalization of LC3 and P62, scale bar = 50 μm; (D) Analysis of the colocalization of LC3 and P62; (E) Immunofluorescence to detect the colocalization of LC3 and LAMP2, scale bar = 50 μm; (F) Analysis of the fluorescence intensity of LAMP2; (G) Immunoblotting to detect the expression of P62 and LC3 proteins; (H) Quantification of (G); Data are expressed as mean ± SD (n = 4-8; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001). DETAILED DESCRIPTION

[0020] Specific experimental methods:

[0021] Experimental subjects: Male C57BL / 6 mice, 8-12 weeks old.

[0022] The mice were randomly divided into 4 groups: sham operation group and experimental group (12, 24, and 48 hours).

[0023] Experimental Example 1. A mouse model of sepsis was established by cecal ligation and puncture, and flow cytometry analysis was used to detect changes in the expression of cardiac type 2 innate lymphocytes (ILC2) and cytokine IL-4 within 48 hours after sepsis;

[0024] Intrinsic lymphocytes, including ILC1, ILC2, and ILC3, play key roles in regulating immunity, inflammation, and tissue homeostasis. However, the specific phenotypes and functions of intrinsic lymphocytes in septic cardiac tissue remain unclear.

[0025] like Figure 1 To identify subsets of innate lymphocytes in septic heart tissue, we first examined the expression of ILCs in a mixture of cardiac lymphocytes isolated from the hearts of CLP mice. We identified a population of cardiac resident lymphocytes expressing the surface marker Lin-CD45+CD90.2+. The percentage of ILC2s in cardiac tissue after CLP was significantly increased compared with sham-operated mice, while there was no significant change in the percentage of ILC1s or ILC3s.

[0026] In addition, flow cytometry was used to further detect ILC2s in the heart after sham surgery or at specific time points of CLP. There was no significant difference in ILC2s after sham surgery, and the percentage in septic hearts was upregulated in a time-dependent manner, reaching a peak at 24h after sepsis and gradually decreasing at 48h. Compared with the sham surgery group, ILC2 secreted more cytokine IL-4 in the hearts of septic mice, showing the same trend of change as ILC2.

[0027] LDH and CK-MB are markers of myocardial function. Compared with the sham operation group, the levels of LDH and CK-MB in the serum of mice treated with CLP for 12-24h were significantly increased, which is consistent with the changes of ILC2s in sepsis heart tissue.

[0028] H&E staining histopathological examination showed abnormal myocardial tissue structure and morphology after CLP, including disordered cell arrangement, extensive inflammatory infiltration, and enlarged intercellular spaces.

[0029] These results suggest that ILC2 and the downstream cytokine IL-4 in septic hearts may play a role in cardiac dysfunction in sepsis.

[0030] Experimental Example 2. After depletion of type 2 innate lymphocytes using an in vivo antibody against CD90.2 and exogenous supplementation of IL-4, a small animal ultrasound machine was used to detect cardiac function in mice, and TUNEL staining and immunohistochemistry for CIeaved Caspased-3 were used to detect cell apoptosis.

[0031] like Figure 2 As shown in the results, IL-4 is the main cytokine secreted by ILC2. To further confirm the role of IL-4 in septic heart, mice were intraperitoneally injected with anti-CD90.2 neutralizing antibodies every 2 days after CLP surgery for 14 days to deplete ILC2s, and IL-4 treatment was given. After the in vivo neutralizing antibody of Anti-CD90.2 was used to eliminate ILC2, the expression of IL-4 in the mouse heart decreased significantly.

[0032] As shown by echocardiography, cardiac function deteriorated with the progression of sepsis, and the deterioration worsened after the elimination of ILC2. Subsequently, cardiac function was improved to a certain extent after exogenous supplementation of IL-4c. At the same time, similar dynamic changes were observed in cardiac injury biomarkers (LDH, CK-MB).

[0033] In addition, the hearts were stained with TUNEL and cleaved-Caspase-3 (a key enzyme for apoptosis activity), which is a marker of apoptotic cell death. The number of apoptotic cells in the septic heart increased significantly, while almost no apoptotic cells were seen in the sham group. After IL-4 treatment, the number of apoptotic cells in the hearts of CLP mice decreased. These results indicate that ILC2-derived IL-4 alleviates cardiac dysfunction during the progression of sepsis.

[0034] Therefore, after using CD90.2 antibodies in vivo to eliminate type 2 innate lymphocytes, the expression of IL-4 in the mouse heart decreased significantly. The echocardiographic results showed that the damage of cardiac function in sepsis was aggravated. Intraperitoneal injection of exogenous IL-4 effectively alleviated the cardiac function in sepsis and reduced cell apoptosis, indicating that IL-4 derived from type 2 innate lymphocytes plays a protective role in cardiac function in sepsis.

[0035] Experimental Example 3. LPS / TNF-α was used to stimulate primary cardiac endothelial cells of neonatal mice as an in vitro injury model of sepsis, and the expressions of autophagy-related proteins P62, LC3, and Lamp2 were detected by Western blot and immunofluorescence staining.

[0036] like Figure 3 As shown in the figure, in order to further study the specific mechanism of IL-4 in regulating cardiac function in sepsis, LPS / TNF-a co-stimulated primary cardiac endothelial cells in vitro to simulate sepsis injury. Western Blot and fluorescence co-localization results showed that after LPS / TNF-α stimulation, the expression of autophagy-related proteins LC3-Ⅱ and P62 increased, and the expression of lysosome-related membrane protein Lamp2 decreased. The LPS / TNF-α+IL-4 treatment group could upregulate Lamp2 expression, reduce P62 expression, and increase LC3-Ⅱ expression, indicating that after LPS / TNF-a stimulation of primary cardiac endothelial cells in vitro, autophagic flow was enhanced, but autophagic degradation was blocked. IL-4 can improve the expression of lysosomal proteins and alleviate the impaired autophagic flow of endothelial cells. After exogenous supplementation of IL-4, IL-4 can improve its effect and alleviate the impaired autophagic flow and enhance the expression of lysosomal proteins. These results show that ILC2-derived IL-4 alleviates the impaired autophagic flow of cardiac endothelial cells.

[0037] Using the autophagy inhibitor chloroquine (which acts on the fusion stage of autophagosomes and lysosomes), it was found that the autophagy expression in the LPS / TNF-a+chloroquine group remained unchanged compared with the LPS / TNF-a group. The blocked autophagy flow may be related to the obstruction of autophagosome formation, that is, reversing the autophagy protective effect of IL-4, suggesting that IL-4 exerts its autophagy alleviating effect by regulating the autophagosome-lysosome fusion stage of septic cardiac endothelial cells.

[0038] In summary, this example demonstrates that type 2 innate lymphocytes and their cytokines can restore cardiac function in sepsis. In addition, impaired clearance of autophagosomes in sepsis hearts is closely related to sepsis heart damage. This experiment explores the mechanism of type 2 innate lymphocytes regulating autophagosome-lysosome fusion in septic heart endothelial cells in sepsis heart damage; clarifies the effects of type 2 innate lymphocytes and the production of cytokine IL-4 on sepsis heart function and endothelial cells, in order to provide a new immune regulation mechanism for sepsis heart function damage.

Claims

Application of type 1.2 innate lymphocytes in the preparation of drugs for treating, alleviating or preventing sepsis-induced cardiac injury. Application of type 2.2 innate lymphocytes in the preparation of drugs for restoring or protecting cardiac function. Application of type 3.2 innate lymphocytes in the preparation of drugs for sepsis-induced cardiac dysfunction and cardiac cell apoptosis. Application of type 4.2 innate lymphocytes in the preparation of preparations for regulating IL-4.

5. Application of IL-4 in the preparation of drugs for treating, alleviating or preventing sepsis-induced cardiac damage.

6. Application of IL-4 in the preparation of drugs for restoring or protecting cardiac function.

7. Application of IL-4 in the preparation of drugs for treating and alleviating cardiac dysfunction and cardiac cell apoptosis caused by sepsis. Application of IL-4 secreted by type 8.2 innate lymphocytes in the preparation of drugs for treating, alleviating or preventing septic cardiac damage. Application of IL-4 secreted by type 9.2 innate lymphocytes in the preparation of drugs for restoring or protecting cardiac function.

10. Application of IL-4 secreted by type 2 innate lymphocytes in the preparation of drugs for sepsis-induced cardiac dysfunction and cardiac cell apoptosis.

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