Application of cGAS-STING signal channel inhibitor in preparation of product for preventing or treating abdominal adhesion

Abstract

The invention relates to application of a cGAS-STING signal channel inhibitor in preparation of a product for preventing or treating abdominal adhesion, and belongs to the technical field of biological medicines. The invention finds that peritoneal dialysis causes that a peritoneal mesothelial cell cGAS-STING pathway and downstream protein thereof are activated, inflammatory factors are generated, and the peritoneal mesothelial cell secretes a chemotactic factor CCL2 (MCP-1) participating in macrophage recruitment, so that migration of macrophages can be promoted, and abdominal adhesion is caused. The cGAS-STING signaling pathway inhibitor can be used for relieving the effects, so that the aim of treating abdominal adhesion is fulfilled.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a cGAS-STING signaling pathway inhibitor in the preparation of a product for preventing or treating abdominal adhesions. Background Art

[0002] Intra-abdominal adhesions are a significant cause of morbidity and mortality worldwide. The pathogenesis of intra-abdominal adhesions is multifactorial, with causes including various surgical procedures and diseases such as peritoneal dialysis (PD), tuberculous peritonitis, radiotherapy, intra-abdominal infection, and Crohn's disease. Although topically applied hydrogels are commonly used to prevent postoperative adhesions, in cases involving extensive adhesions, pharmacological interventions that can be administered intraperitoneally or systemically are needed.

[0003] Among the various causes of abdominal adhesions, encapsulating peritoneal sclerosis (EPS) is a particularly severe condition. EPS is characterized by extensive adhesions leading to intestinal obstruction, often accompanied by peritoneal thickening, sclerosis, calcification, and encapsulation of the small intestine. The most common cause of EPS is peritoneal dialysis, especially when complicated by peritonitis. There is still a lack of drugs that can effectively treat PD-induced EPS and improve patient outcomes. Therefore, in this study, we chose PD-induced EPS as our pathological model to better understand the mechanism of adhesion formation and identify potential therapeutic strategies.

[0004] The cGAS-STING signaling pathway, which consists of cyclic guanosine monophosphate-AMP synthase (cGAS) and its downstream adaptor, stimulator of interferon genes (STING), is crucial for regulating the transcription of host defense genes, including type I interferons and proinflammatory cytokines. Recent studies have shown that STING activation plays a role in the progression of fibrotic and inflammatory diseases through pathways involving NF-κB activation, cell death, and endoplasmic reticulum stress. However, its role in peritoneal adhesion formation, especially in the context of PD-induced EPS, has not been investigated. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of H151 in the preparation of a drug for treating encapsulated peritoneal sclerosis.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides the use of a cGAS-STING signaling pathway inhibitor in the preparation of a product for preventing or treating abdominal adhesions.

[0008] The present invention found that peritoneal dialysis can lead to the activation of the cGAS-STING pathway and its downstream proteins in peritoneal mesothelial cells, and secrete a chemokine CCL2 (MCP-1) that can induce macrophage recruitment, thereby promoting the aggregation of macrophages on the peritoneal surface and ultimately forming peritoneal adhesions. These effects can be alleviated by using cGAS-STING signaling pathway inhibitors, thereby achieving the purpose of treating peritoneal adhesions.

[0009] As a preferred embodiment of the first aspect, the cGAS-STING signaling pathway inhibitor is H151.

[0010] The present invention finds that inhibiting H151 by using the cGAS-STING signaling pathway can alleviate the above symptoms and achieve the effect of treating abdominal adhesions.

[0011] As a preferred embodiment of the first aspect, the abdominal adhesion is caused by encapsulating peritoneal sclerosis.

[0012] As a preferred embodiment of the first aspect, the product further comprises a medically acceptable pharmaceutical excipient.

[0013] As a preferred embodiment of the first aspect, the medically acceptable pharmaceutical excipient is selected from one or more of mannitol, trehalose, polyvinyl pyrrolidone, dodecyl-β-D-maltoside, hydroxypropyl-β-cyclodextrin, calcium phosphate, and zinc carbonate.

[0014] In a second aspect, the present invention provides the use of H151 in preparing a product for protecting peritoneal mesothelial cells.

[0015] In a third aspect, the present invention provides the use of H151 in the preparation of a product for inhibiting the activation of the cGAS-STING signaling pathway in peritoneal mesothelial cells.

[0016] As a preferred embodiment of the third aspect, the inhibition of activation of the cGAS-STING signaling pathway in peritoneal mesothelial cells is the inhibition of the expression of p-IRF3, IRF3, p-p65, p65, cGAS and STING proteins.

[0017] Compared with the existing technology, the present invention has the following beneficial effects: the existing technology for improving abdominal adhesion mainly applies anti-adhesion gel to the surgical site after surgery, mainly for physical defense. However, this method is not suitable for diffuse and extensive abdominal adhesion, because it is impossible to pad the entire abdominal cavity with anti-adhesion gel. Our technology is a soluble drug that can be dissolved in the fluid in the abdominal cavity. With the flow of the fluid in the abdominal cavity, especially the peritoneal dialysis fluid, it acts on the entire abdomen and exerts a wide range of anti-adhesion effects on the entire abdomen. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Figure 1 is a schematic diagram of the increased expression of MCP1 in mesothelial cells due to the activation of the cGAS-STING pathway (Figure A is an immunofluorescence analysis showing that the expression of MCP-1 in the peritoneum of the EPS parietal layer increased, and was mainly related to Cytokeratin7 + Schematic diagram of co-localization of mesothelial cells; Figure B shows that STING expression in the EPS parietal peritoneum is increased by immunofluorescence and is co-localized with Cytokeratin7. + Schematic diagram of co-localization of mesothelial cells; Figure C is a schematic diagram of activation of cGAS-STING and downstream proteins in EPS peritoneum confirmed by Western blotting; *** indicates p<0.001; EPS indicates model group, and Control indicates control group);

[0019] Figure 2 Schematic diagram of in vitro simulated EPS environmental stimulation inducing cGAS-STING pathway activation in mesothelial cells (Figures AB are schematic diagrams showing that STING and downstream signals in mesothelial cells are activated under EPS stimulation by WB and immunofluorescence, while the H151 treatment group can alleviate this effect; Figure C is a schematic diagram showing that qPCR shows that inflammation-related genes in mesothelial cells are upregulated under EPS stimulation, while H151 treatment can reduce the expression of inflammation-related genes; *p<0.05, **p<0.01, ***p<0.001.);

[0020] Figure 3 Schematic diagram of the regulation of macrophage chemotaxis by activation of the cGAS-STING pathway in mesothelial cells (Figure A is a schematic diagram of the transwell assay for evaluating macrophage migration; Figure B is a bright field image of macrophage migration after co-culture with EPS-stimulated mesothelial cells; Figure C is a quantitative statistical graph of migrating macrophages in different groups; **p<0.01, ***p<0.001);

[0021] Figure 4 Schematic diagram of improving EPS mouse model (Figure A is a schematic diagram of the experimental scheme for establishing EPS mouse model; Figure B is a schematic diagram of the results of macroscopic examination of the abdominal cavity; Figure C is a quantitative diagram of adhesion scores in different experimental groups; Figure D is a diagram of the dynamic changes in mouse body weight during the study; Figure E is a Kaplan-Meier survival curve);

[0022] Figure 5Schematic diagram of using H151 to inhibit cGAS-STING activation to effectively improve EPS abdominal adhesions (Figure A is a schematic diagram of H151 improving macrophage infiltration on the peritoneal surface; Figure B is a schematic diagram of gross abdominal observation and macroscopic adhesion scoring; Figure C is a schematic diagram of proving that H151 effectively relieves abdominal adhesions from a macroscopic perspective; Figure D (a) is a schematic diagram of the adhesion conditions of different groups presented by MASSON staining of the abdominal cross-section; Figure D (b) is a schematic diagram showing the area of ​​MASSON+ areas in the adhesion area; Figure E (a) is a schematic diagram of the thickness of the parietal peritoneum of the three groups shown by Masson's trichrome staining; Figure E (bd) are immunohistochemical images showing the expression of three classic ECM-related proteins COL1A1, fibronectin and α-SMA in the parietal peritoneum; Figure F is a statistical graph showing the area of ​​MASSON+ areas in the adhesion area; Figure G is a bar graph showing the corresponding peritoneal thickness statistics). DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0024] The H151 used in this example is a STING antagonist, powder, CAS No. 941987-60-6, molecular weight: 279.34.

[0025] Example 1 In vitro cell experiment

[0026] 1. Verification of H151 inhibition of cGAS-STING pathway in peritoneal mesothelial cells

[0027] 1.1 Experiment:

[0028] EPS environment was simulated in vitro by stimulating human peritoneal mesothelial cells (HPMC) with LPS (lipopolysaccharide) and SHS (surgical hygiene solution):

[0029] First, prepare the corresponding stimulants. After purchasing LPS powder, prepare the mother solution of the corresponding concentration according to the instructions; SHS is a mixed solution containing 0.1% glucose chlorhexidine + 15% ethanol. Then, culture the attached primary peritoneal mesothelial cells in vitro. When the cell density reaches about 80%, add the stimulants to simulate the in vivo environment of peritoneal dialysis. The concentrations of the stimulants are: LPS (working concentration is 1μg / mL), SHS (working concentration is 1%); observe the effects of the corresponding stimuli on the peritoneal mesothelial cells after 24 hours.

[0030] The cells were grouped into control group and EPS group to study the activation of chemokines and cGAS-STING pathway.

[0031] The cells were divided into control group, SHS+LPS group, and SHS+LPS+H151 group. The concentration of H151 used in the cell experiment was 2 μm / mL. This was used to study the effect of STING inhibitor H151 on EPS.

[0032] 1.2 Results

[0033] Given that CCL2 (MCP-1) is a known chemokine involved in macrophage recruitment, immunofluorescence showed a significant increase in MCP-1 expression in the parietal peritoneum, mainly in combination with Cytokeratin7. + Mesothelial cell colocalization ( Figure 1 A). These findings suggest that peritoneal mesothelial cells are the main source of MCP-1 secretion in EPS. We then investigated the activation of the cGAS-STING pathway, and both Western blot and immunofluorescence confirmed significant activation of the cGAS-STING pathway in the EPS peritoneum, with STING mainly co-localizing with mesothelial cells ( Figure 1 B and 1C), indicating that activation of the cGAS-STING pathway in mesothelial cells under EPS conditions could be responsible for promoting chemotaxis of macrophages.

[0034] Western blotting and immunofluorescence confirmed that STING and its downstream proteins were activated in mesothelial cells under EPS environment, and treatment with STING inhibitor H151 could partially alleviate this effect ( Figure 2 A, 2B). qPCR analysis further demonstrated that the expression of inflammation-related genes in mesothelial cells was significantly upregulated under EPS conditions, and this effect was also inhibited by H151 ( Figure 2 C).

[0035] 2. Verification of H151 inhibition of macrophage chemotaxis

[0036] 2.1 Experiment

[0037] To directly assess the effect of mesothelial cell activation on macrophage migration, we performed a transwell co-culture assay (mesothelial cells were seeded in the lower chamber, stimulated with the above stimuli, and co-cultured with macrophages in the upper chamber, Figure 3 A).

[0038] 2.2 Results

[0039] It was found that mesothelial cells in the EPS environment could significantly promote macrophage migration, while the STING inhibitor H151 reduced macrophage migration ( Figure 3 BC).

[0040] Example 2 Animal model experiment

[0041] 1. Animal model construction:

[0042] 8-week-old C57BL / 6J male mice were raised in the Experimental Animal Center of South China Agricultural University. All animal experiments complied with the ethical guidelines of the Guide for the Care and Use of Laboratory Animals of the National Research Council of the United States. The mice were randomly divided into three groups: 1) Control group: no treatment was given; 2) SHS group: mice were intraperitoneally injected with 0.3 mL surgical hygiene solution (SHS, a disinfectant composed of chlorhexidine gluconate and ethanol) daily; 3) PD+LPS+SHS group: mice were intraperitoneally injected with 100 mL / kg 4.25% peritoneal dialysis solution (PD) and 0.3 mL SHS daily, and 4 mg / kg lipopolysaccharide (LPS) was intraperitoneally injected once a week to induce occasional peritonitis ( Figure 4 A). The three groups of mice were monitored daily for signs of intraperitoneal adhesion formation and further examined at predetermined time points.

[0043] 2. Modeling results

[0044] The results are as follows Figure 4 As shown in BE, Figure 4 B shows that macroscopic examination of the abdominal cavity showed that the mesentery of mice in the control group was smooth and well-expanded, and the liver margins were sharp; the liver margins of mice in the SHS group were blunted (yellow arrows), but there were no adhesions between organs; in the PD+LPS+SHS group, there were extensive adhesions between abdominal organs (blue arrows), and the liver margins were thickened due to surface fibrosis. Figure 4 C shows the quantitative scores of adhesion scores in different groups, and the PD+LPS+SHS group has the highest score. Figure 4 D is the dynamic change diagram of mouse body weight, showing that the weight loss of mice in the PD+LPS+SHS group was the fastest. Figure 4 E Kaplan-Meier survival curve shows the survival rate of each group, indicating that the PD+LPS+SHS group had the lowest survival rate.

[0045] It can be seen that the mouse model constructed in the SHS group takes up to 8 weeks to form EPS, but in the PD+LPS+SHS group, the addition of PD and LPS successfully accelerated this process, and obvious EPS was formed in just 3 weeks. Therefore, the model mice in the PD+LPS+SHS group were used for subsequent experiments.

[0046] 3. Preparation and use of H151:

[0047] Purchase H151 powder, add each solvent in sequence: 5% DMSO → 5% Tween-80 → 90% PBS, prepare a 1mM H151 stock solution, store in a -30° refrigerator, and thaw and dissolve when used. In the animal experiment, each mouse in the drug treatment group was intraperitoneally injected with 750nmol of diluted H151 solution.

[0048] 4. Treatment results

[0049] To investigate whether inhibition of the cGAS-STING pathway could reduce the severity of EPS, we administered the STING inhibitor H151 (750 nmol / mouse) to model mice by intraperitoneal injection. The results showed that H151 treatment significantly improved the pathological characteristics of EPS.

[0050] Results from Figure 5 It can be seen that using H151 to inhibit cGAS-STING activation can effectively improve EPS abdominal adhesion. Figure 5 Figure A shows that H151 can improve macrophage infiltration on the peritoneal surface; Figure B shows that H151 is effective in relieving abdominal adhesions from a macroscopic perspective. Figure 5 C shows macroscopic adhesion scores, which show that mice treated with H151 had significantly lower adhesion scores in gross assessment, reflecting reduced intra-abdominal adhesions. Figure 5 D(a) shows the adhesion of different groups in the abdominal cross section by MASSON staining. Figure 5 D(b) shows the area of ​​MASSON+ region in the adhesion area. The histological staining results showed that the collagen fiber deposition in the adhesion area in the treatment group was significantly reduced ( Figure 5 F). Figure 5 E(a) shows Masson trichrome staining showing the thickness of the three groups of parietal peritoneum. Figure 5 G shows the corresponding bar graph of peritoneal thickness statistics using Masson's trichrome staining, showing that the thickness of the parietal peritoneum (a marker of EPS fibrosis) was significantly reduced in mice treated with H151, indicating a direct effect on the progression of fibrosis. Figure 5 E (bd) shows immunohistochemical images showing the expression of three classic ECM-related proteins (COL1A1, fibronectin (FN), and α-SMA) in the parietal peritoneum. It shows that the expression of ECM-related proteins was significantly reduced in the group treated with H151, further supporting the anti-fibrotic effect of this inhibitor.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Application of cGAS-STING signaling pathway inhibitors in the preparation of products for preventing or treating abdominal adhesions.

2. The use according to claim 1, characterized in that The cGAS-STING signaling pathway inhibitor is H151.

3. The use according to claim 1, characterized in that The abdominal adhesion is whole-abdominal adhesion caused by encapsulating peritoneal sclerosis.

4. The use according to claim 1, characterized in that The product also includes medically acceptable pharmaceutical excipients.

5. The use according to claim 4, characterized in that The medically acceptable pharmaceutical excipient is selected from one or more of mannitol, trehalose, polyvinyl pyrrolidone, dodecyl-β-D-maltoside, hydroxypropyl-β-cyclodextrin, calcium phosphate, and zinc carbonate.

6. Application of H151 in the preparation of products for protecting peritoneal mesothelial cells.

7. Application of H151 in the preparation of products that inhibit activation of cGAS-STING signaling pathway in peritoneal mesothelial cells.

8. The use according to claim 7, characterized in that The inhibition of the activation of the cGAS-STING signaling pathway in peritoneal mesothelial cells is to inhibit the expression of p-IRF3, p-p65, cGAS and STING proteins.

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