Application of RIPK1 inhibitor in preparation of medicine for treating acute respiratory distress syndrome

Abstract

The invention discloses application of an RIPK1 inhibitor in preparation of a medicine for treating acute respiratory distress syndrome. Under the condition of same bronchial LPS administration stimulation, the lung state and function of a mouse injected with the RIPK1 inhibitor are obviously superior to those of a mouse without injection of the inhibitor, the lung tissue edema of the mouse is obviously weakened, the number of leukocytes in alveolar lavage fluid and the proportion of neutrophil polymorphic leukocytes are obviously reduced, the lung inflammation is weakened, and the RIPK1 inhibitor can be used for treating lung diseases. The pathological staining result of the lung tissue section also shows that immune cell infiltration is reduced, and the lung form is recovered. The RIPK1 kinase targeting the lung tissue is expected to become a target for screening drugs for treating ARDS, and an inhibitor of the RIPK1 kinase is suitable for preparing the drugs for treating ARDS.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the use of a RIPK1 inhibitor in the preparation of a drug for treating acute respiratory distress syndrome. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is a disease caused by lung damage and subsequent inflammatory response, characterized by pulmonary edema, decreased pulmonary permeability, and hypoxemia. After infection, trauma, inhalation and other stimuli, inflammatory factors accumulate in the lungs, while pulmonary capillaries are damaged, permeability changes, and tissue fluid enters the alveoli, leading to the formation of dead zones in the lungs. The accumulation of hyaline membranes (pulmonary edema) disrupts gas exchange between alveoli and capillaries, leading to low oxygen supply (hypoxemia), which can cause severe shortness of breath, weakness, cough, fever, and alveolar collapse.

[0003] The pathogenesis of ARDS involves a complex process of lung injury and inflammation, which can be divided into multiple stages. First, after the lungs are damaged under stimulation, alveolar blood flow decreases, platelets begin to aggregate, and inflammatory mediators such as histamine are released. Inflammatory mediators damage the alveolar capillary membrane, increase the permeability of the alveolar capillary membrane, and lead to edema. At the same time, the release of inflammatory mediators and the activation of immune cells recruit neutrophils and macrophages, further leading to lung injury and aggravated inflammatory response. As lung tissue damage worsens, capillary permeability increases further, leading to protein and fluid exudation, a sudden increase in interstitial fluid osmotic pressure, and the formation of pulmonary edema. This impairs pulmonary capillary function, reduces pulmonary blood flow, destroys surfactant in alveolar fluid, causes alveolar collapse and atelectasis, restricts gas exchange, and decreases lung compliance. Finally, excessive inflammatory response leads to pulmonary fibrosis, disease exacerbation, hardening of lung tissue, and long-term respiratory disorders.

[0004] The pathogenesis of ARDS mainly involves two categories: one is direct lung injury, such as pneumonia, mechanical injury or aspiration of gastric contents, which can cause damage to the alveolar capillary membrane, leading to inflammation, edema and impaired gas exchange; the other is indirect lung injury, which is mainly caused by non-pulmonary factors, such as sepsis, trauma or pancreatitis, leading to systemic inflammation, and then causing damage to the alveolar capillary membrane, leading to ARDS.

[0005] Receptor-interacting protein 1 (RIP1 or RIPK1) is an important molecule that regulates cell fate. It is involved in regulating many important signaling pathways and cellular effects, including the NF-κB signaling pathway, apoptosis and necrosis, inflammasome activation, and pyroptosis. The structure of RIPK1 includes an N-terminal death domain (DD), an intermediate binding enzyme domain (kinase-like domain, KD), and a C-terminal serine / threonine kinase domain. The DD domain can interact with other proteins containing DD domains to form a signaling complex; the KD domain has kinase activity, which can cause autophosphorylation and cross-phosphorylation of other target proteins, and is also the final catalytic part of RIPK1.

[0006] RIPK1 is an important component of the TNF signaling pathway. In the TNF-α-activated signaling pathway, RIPK1 recruits and binds to downstream signaling molecules as a scaffold protein through ubiquitination, thereby mediating the activation of NF-κB and promoting cell survival. When RIPK1 is deubiquitinated or ubiquitination is inhibited, RIPK1 binds to caspase-8 and FADD proteins to induce cell apoptosis; when caspase-8 is inhibited or missing by inhibitors, RIPK1 will undergo autophosphorylation, bind to and phosphorylate RIPK3, and promote programmed cell necrosis. Therefore, factors such as the expression level, phosphorylation and ubiquitination modification of RIPK1, and its interaction with other protein molecules determine the fate of cells. Summary of the invention

[0007] The purpose of the present invention is to provide the use of a RIPK1 inhibitor in the preparation of a drug for treating acute respiratory distress syndrome.

[0008] Furthermore, the RIPK1 inhibitor described in the present invention is a RIPK1 inhibitor targeting lung tissue.

[0009] The RIPK1 inhibitors of the present invention include but are not limited to small molecule compounds that can inactivate RIPK1 kinase, or siRNA that can inhibit RIPK1 gene expression and / or RIPK1 kinase inactivation.

[0010] Specifically, the siRNA is a double-stranded siRNA that targets RIPK1 and interferes with RIPK1 expression and kinase inactivation. It is injected into the human body to inactivate RIPK1 kinase through RNA interference to treat acute respiratory distress syndrome.

[0011] Specifically, the small molecule compounds described in the present invention target RIPK1 and can specifically inhibit the kinase activity of RIPK1, including but not limited to RIPK1 kinase activity inhibitors such as Nec-1, Nec-1s, and RIPA56.

[0012] The present invention found that under the same bronchial LPS administration stimulation, the lung status and function of mice injected with RIPK1 inhibitors were significantly better than those of mice not injected with inhibitors, and their lung tissue edema was significantly reduced, the number of white blood cells and the proportion of neutrophils and polymorphonuclear leukocytes in the alveolar lavage fluid were significantly reduced, and lung inflammation was reduced. The results of pathological staining of lung tissue sections also showed that immune cell infiltration was reduced and lung morphology was restored. RIPK1 kinase targeting lung tissue is expected to become a drug target for screening and treating ARDS, and its inhibitors can be used to prepare drugs for treating ARDS. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a graph showing the ratio of wet weight to dry weight of mouse lung tissue.

[0014] Figure 2 Figure 2 shows the number of white blood cells (WBC) and the ratio of neutrophils and polymorphonuclear leukocytes (PMN) in the bronchoalveolar lavage fluid of mice (A).

[0015] Figure 3 Figure 2 shows TUNEL staining of mouse lung tissue sections (A) and positive cell counts (B).

[0016] Figure 4 This is a hematoxylin and eosin (H&E) stained image of mouse lung tissue sections. DETAILED DESCRIPTION

[0017] The present invention will be further described in detail below by way of examples and accompanying drawings.

[0018] Example 1

[0019] Therapeutic effect of RIPK1 inhibitor on LPS-induced ARDS

[0020] 1. Grouping and building ARDS model

[0021] The mice were divided into three groups, namely the control group (PBS + DMSO), the modeling group (LPS + DMSO) and the treatment group (LPS + inhibitor). First, the mice in the treatment group were intraperitoneally injected with RIPK1 inhibitors at a dose of 10 mg / kg mouse body weight, and the other two groups were injected with the same volume of PBS. One hour later, LPS was injected into the mice in the experimental group and the treatment group by bronchial choking at a dose of 10 mg / kg mouse body weight, and the control group was injected with the same volume of DMSO. The specific method of administration is as follows: straighten the mouse vertically, pull out the mouse's tongue with tweezers so that it cannot swallow, and then use a pipette to inject the liquid deep into the mouse's mouth, block the mouse's nose until the mouse starts to cough, and inhale the liquid into the trachea. After 24 hours of administration, the mice were killed.

[0022] 2. Sample acquisition

[0023] After the mice were killed by eye blood sampling and spinal dislocation, the skin and flesh of the mouse neck were cut open to expose the trachea, and then the syringe needle was inserted into the trachea, pre-cooled PBS was injected, and the liquid was drawn out after blowing and aspirating twice, which was the alveolar lavage fluid. The mice were then dissected and the lung tissue was removed.

[0024] 3. Detection Methods

[0025] (3.1) Take some fresh lung tissue from mice, weigh it, and then dry it in a 60°C oven for 24 hours. Weigh it again and calculate the wet weight / dry weight ratio.

[0026] (3.2) After obtaining the bronchoalveolar lavage fluid, it should be sent to the hospital in time to test the number of white blood cells (WBC) and the ratio of neutrophils and polymorphonuclear leukocytes (PMN).

[0027] (3.3) Hematoxylin and eosin staining (HE staining)

[0028] (1) Lung tissue sections

[0029] Part of the fresh lung tissue of the mouse was fixed in 4% paraformaldehyde solution for 24 hours, then embedded in paraffin, and single slices were cut using an automatic slicer and placed on a glass slide. The slices were slightly baked with an alcohol lamp to flatten the slices, and the attached slices were placed in a 60°C oven to dry for 2 hours and stored.

[0030] (2) Dewaxing of paraffin sections

[0031] Put the slices into xylene I for 10 min - xylene II for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 95% alcohol for 5 min - 90% alcohol for 5 min - 80% alcohol for 5 min - 70% alcohol for 5 min - and wash with distilled water.

[0032] (3) Dyeing

[0033] The sections were stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water. The sections were then stained with eosin solution for 1-3 minutes.

[0034] (4) Dehydration and sealing

[0035] The slices were placed in 95% alcohol I for 5 min-95% alcohol II for 5 min-absolute ethanol I for 5 min-absolute ethanol II for 5 min-xylene I for 5 min-xylene II for 5 min to dehydrate and make them transparent, and the slices were taken out of xylene to dry slightly, and then sealed with neutral gum. Microscope examination and image acquisition and analysis were performed.

[0036] (3.4) TUNEL staining

[0037] (1) After dewaxing, the lung tissue sections were washed with PBS solution.

[0038] (2) Transparent treatment

[0039] Use filter paper to absorb the liquid near the sample on the slice, draw a circle around the sample with a histochemical pen (hydrophobic pen), put it in a wet box, and then add 100μL 20μg / mL Proteinase K to each sample to cover the sample, and leave it at room temperature for 20 minutes. Then soak and wash it with PBS for 3 times, 5 minutes each time.

[0040] (3) Use paper to absorb the liquid near the sample, place it in a wet box, add 100 μL of 1× Equilibration Buffer to cover each sample, and equilibrate at room temperature for 20 minutes. At this time, prepare the labeling solution according to the instructions of the TUNEL staining kit.

[0041] (4) Use paper to absorb the 5× Equilibration Buffer, then add 50 μL of labeling solution to the sample and incubate at room temperature in a wet box for 60 min.

[0042] (5) DAPI nuclear staining

[0043] The sections were washed by soaking in PBS for 3 times, 5 minutes each time, and then DAPI staining solution was added to cover the samples and placed in a wet box at room temperature for 5 minutes.

[0044] (6) Sealing

[0045] Wash the slices by soaking in PBS for 3 times, 5 minutes each time, then remove the liquid around the sample with paper, add anti-fluorescence quenching mounting medium, and cover the slices with coverslips, taking care not to leave bubbles above the sample.

[0046] (7) Microscopic examination

[0047] The sections were photographed with a fluorescence microscope. DAPI emitted blue fluorescence under 460nm excitation light, and BrightRed emitted red fluorescence under 620±20nm excitation light. The two fluorescence images were then fused using the Adobe Photoshop CC 2019 program to count the number of valid red positive points.

[0048] (3.5) Data processing

[0049] GraphPad Prism was used to analyze and plot the data, and the Ordinary one-way ANOVA method was used for statistical analysis of the data. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates no significant difference. All data are expressed in the form of "mean ± standard deviation".

[0050] 4. Results

[0051] The present invention constructs an ARDS model by administering LPS intrabronchially to wild-type C57 mice and constructs a treatment model thereof by intraperitoneal injection of RIPK1 inhibitors before administration. The results show that the dry-wet ratio of lung tissue in the treatment group is significantly reduced, indicating that RIPK1 inactivation can significantly alleviate LPS-induced pulmonary edema and restore the lung function of mice (such as Figure 1 shown).

[0052] By detecting the white blood cells in the alveolar lavage fluid of mice, it was found that the number of white blood cells in the lavage fluid of the treatment group was significantly reduced, and the proportion of neutrophils and polymorphonuclear leukocytes was also significantly reduced, indicating that the lung damage and infection in the RIPK1 inhibitor treatment group were alleviated (such as Figure 2 shown).

[0053] Lung tissue sections of mice were taken for TUNEL apoptosis staining. The results showed that RIPK1 inhibitor treatment significantly reduced LPS-induced lung cell death and alleviated the degree of lung tissue damage (such as Figure 3 shown).

[0054] HE staining of mouse lung tissue sections showed that the infiltration of immune cells in the lung tissue of the RIPK1 inhibitor-treated group was reduced, and the alveolar structure was restored, indicating that inhibiting RIPK1 kinase activity can weaken the inflammatory response in the ARDS model and restore the lung structure and function (such as Figure 4 shown).

Claims

1. Application of RIPK1 inhibitors in the preparation of drugs for the treatment of acute respiratory distress syndrome.

2. The use according to claim 1, characterized in that: The RIPK1 inhibitor is a RIPK1 inhibitor targeting lung tissue.

3. The use according to claim 1, characterized in that: The RIPK1 inhibitor is a small molecule compound capable of inactivating RIPK1 kinase, or a siRNA capable of inhibiting RIPK1 gene expression and / or RIPK1 kinase inactivation.

4. The use according to claim 1, characterized in that: The small molecule compound is Nec-1, Nec-1s or RIPA56.

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