Application of Amuc1409 protein in prevention and treatment of acute pancreatitis
Amuc1409 protein increases intestinal stem cell proliferation, alleviates acinar cell damage and inflammatory infiltration, reduces inflammatory cytokines, and increases regulatory T cells, thus solving the problem of prevention and treatment of acute pancreatitis and showing superior effects compared to Amuc_1100.
Patent Information
- Application Number
- CN202510722639.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-01
AI Technical Summary
Currently, there are no effective drugs for the prevention and treatment of acute pancreatitis, especially severe acute pancreatitis, and the existing Akkermansia membrane protein Amuc_1100 has limited effectiveness in this regard.
Amuc1409 protein was used to increase the proliferation and regeneration of intestinal stem cells in a cadherin-dependent manner, reduce serum amylase concentration, alleviate acinar cell damage and inflammatory cell infiltration, reduce the transcription of inflammatory cytokines in the pancreas, and increase the proportion of regulatory T cells in peripheral blood lymphocytes.
Amuc1409 protein significantly reduced serum amylase concentration in mice, decreased pancreatic acinar cell damage and inflammatory infiltration, reduced inflammatory cytokines in the pancreas, and increased the proportion of regulatory T cells, demonstrating better preventive and therapeutic effects on acute pancreatitis than Amuc_1100.
Smart Images
Figure CN120392965A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and in particular to the application of Amuc1409 protein in the prevention and treatment of acute pancreatitis. Background Art
[0002] Acute pancreatitis (AP) is one of the most common diseases in the digestive system currently, and its incidence shows an increasing trend year by year. AP can be divided into mild acute pancreatitis (MAP) and severe acute pancreatitis (SAP) according to the degree of inflammation of the disease; among them, MAP is mainly characterized by pancreatic interstitial edema and rarely results in death, and 15-25% of MAP patients will further develop into SAP; while SAP is characterized by persistent organ failure and has a mortality rate as high as 36-50%, and there is currently no effective clinical treatment drug.
[0003] Amuc_1100 is one of the most representative outer membrane proteins of Akkermansia currently, and it can maintain intestinal homeostasis and regulate the host immune response. Akkermansia and its membrane protein Amuc_1100 can activate Toll-like receptor 2 (TLR2), TLR4 to produce a large amount of IL-10, improve intestinal barrier function, maintain host intestinal homeostasis, improve intestinal inflammatory stress, and accelerate the injury repair of intestinal epithelial cells. Recent studies have found that the Akkermansia membrane protein Amuc_1100 can relieve acute pancreatitis in mice by reducing macrophage and neutrophil infiltration and regulating the intestinal flora.
[0004] Amuc_1409 is the most abundantly reported secreted protein of Akkermansia currently. Different from the Amuc_1100 protein, the Akkermansia secreted protein Amuc_1409 increases the proliferation and regeneration of intestinal stem cells in a cadherin (E-cadherin)-dependent manner and plays a key role in intestinal homeostasis. However, currently, there is no report on the effect of this Akkermansia secreted protein Amuc_1409 on acute pancreatitis, nor is there any research on exploring its immunological effect and its effect on the intestinal barrier using this protein. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide the application of Amuc1409 protein in the prevention and treatment of acute pancreatitis.
[0006] The present invention adopts the following technical solutions to solve the above technical problems:
[0007] The application of an Amuc1409 protein in the prevention and treatment of acute pancreatitis.
[0008] As one of the preferred embodiments of the present invention, the Amuc1409 protein is a commercially available protein, purchased from Nanjing Ainuodi Biotechnology Co., Ltd., which increases the proliferation and regeneration of intestinal stem cells in a cadherin-dependent manner and maintains intestinal homeostasis.
[0009] As one of the preferred embodiments of the present invention, when used for the prevention or treatment of acute pancreatitis, the Amuc1409 protein exerts its effects through at least one of the following pathways:
[0010] (1) Reducing the concentration of amylase in the serum;
[0011] (2) Alleviating acinar cell damage and inflammatory cell infiltration;
[0012] (3) Reducing the transcription of inflammatory cytokines in the pancreas;
[0013] (4) Increasing the proportion of regulatory T cells in peripheral blood lymphocytes.
[0014] As one of the preferred embodiments of the present invention, in the above-mentioned pathway (3), the inflammatory cytokines refer to interleukin-1β and interleukin-6.
[0015] As one of the preferred embodiments of the present invention, in the above-mentioned pathway (3), it also includes increasing the transcription of anti-inflammatory factors in the pancreas.
[0016] As one of the preferred embodiments of the present invention, the anti-inflammatory factor refers to interleukin-10.
[0017] The advantages of the present invention compared with the prior art are as follows:
[0018] The present invention for the first time discovers and confirms the role of the secreted protein Amuc_1409 of Akkermansia in the prevention and treatment of acute pancreatitis, and its prevention and treatment effects are better than those of the Akkermansia membrane protein Amuc_1100.
[0019] After intragastric administration of 5 μg / day of Amuc_1409 to mice in the present invention, cerulein was intraperitoneally injected into the mice to establish a pancreatitis model, and the effect of Amuc_1409 in preventing acute pancreatitis was studied. The experimental results showed that: compared with the control group, the serum amylase concentration in the Amuc_1409 intragastric administration group of mice was significantly reduced, the damage of pancreatic acinar cells was reduced, the inflammatory infiltration was reduced, the inflammatory cytokines in the pancreas were reduced, and the proportion of regulatory T cells in peripheral blood lymphocytes was increased, and it showed a better preventive effect than Amuc_1100; it was shown that Amuc_1409 could prevent the occurrence and development of acute pancreatitis.
[0020] After establishing a pancreatitis model by intraperitoneal injection of L-arginine in mice, Amuc_1409 at a dose of 5 μg / day was administered by gavage on the 1st, 2nd, and 3rd days after modeling to study the effect of Amuc_1409 on the treatment of acute pancreatitis. The experimental results showed that compared with the control group, the serum amylase concentration in the mice in the Amuc_1409 gavage group was significantly decreased, the damage of pancreatic acinar cells was reduced, the inflammatory infiltration was decreased, the inflammatory cytokines in the pancreas were decreased, the anti-inflammatory factors were increased, and the proportion of regulatory T cells in peripheral blood lymphocytes was increased, and it showed a better therapeutic effect than Amuc_1100; it was shown that Amuc_1409 could treat the occurrence and development of acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 It is the results of the serum amylase concentration, the nucleic acid transcription level of inflammatory factors in pancreatic tissue, and the expression of serum interleukin-6 in each group of mice in Example 1 (in the figure, Figure A is the concentration of mouse serum amylase Amylase, Figure B is the nucleic acid transcription level of inflammatory factors "interleukin-6, interleukin 1β" in pancreatic tissue, and Figure C is the expression of serum interleukin-6);
[0022] Figure 2 It is the results of pancreatic pathological sections of each group of mice in Example 1 (in the figure, Figure A is the result of pathological sections, and Figure B is the corresponding statistical result); <x
[0023] Figure 3 It is the results of flow cytometry staining to detect the proportion of regulatory T cells in peripheral blood lymphocytes in each group of mice in Example 1 (in the figure, Figure A is the result of flow cytometry staining, and Figure B is the corresponding statistical result);
[0024] Figure 4 It is the results of the serum amylase concentration and the nucleic acid transcription levels of inflammatory factors and anti-inflammatory factors in pancreatic tissue in each group of mice in Example 2 (in the figure, Figure A is the concentration of mouse serum amylase Amylase, and Figure B is the nucleic acid transcription levels of inflammatory factors "interleukin-6, interleukin 1β" and anti-inflammatory factor "interleukin-10" in pancreatic tissue);
[0025] Figure 5 It is the results of pancreatic pathological sections of each group of mice in Example 2;
[0026] Figure 6 It is the results of flow cytometry staining to detect the proportion of regulatory T cells in peripheral blood lymphocytes in each group of mice in Example 2 (in the figure, Figure A is the result of flow cytometry staining, and Figure B is the corresponding statistical result). DETAILED DESCRIPTION OF THE INVENTION
[0027] The embodiments of the present invention will be described in detail below. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, unless otherwise specified, the reagents, methods, and equipment used in the present invention are all conventional reagents, methods, and equipment in the technical field, and will not be elaborated herein.
[0028] Example 1: Acute pancreatitis prevention experiment of Amuc1409 protein:
[0029] In this example, after intragastric administration of 5 μg / day of Amuc_1409 to mice, caerulein was intraperitoneally injected into the mice to establish a pancreatitis model, and the effect of Amuc_1409 in preventing acute pancreatitis was studied as follows:
[0030] (1) Grouping
[0031] A total of 20 healthy C57BL / 6J mice were selected and evenly divided into four groups: control group (NC), model group (Vehical+SAP), Amuc_1100 experimental group (Amuc_1100+SAP), and Amuc_1409 experimental group (Amuc_1409+SAP).
[0032] (2) Treatment
[0033] Control group (NC): Healthy mice, without any treatment;
[0034] Amuc_1100 experimental group (Amuc_1100+SAP): First, the mice in this group were intragastrically administered 5 μg / day of Amuc_1100 (purchased from Nanjing Ainuodi Biotechnology Co., Ltd.) for one week continuously. At the same time, an antibiotic mixture composed of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg), and vancomycin (16.7 mg) was intragastrically administered every day for 5 days to exclude the interference of intestinal flora; subsequently, caerulein was intraperitoneally injected into the mice to establish a pancreatitis model.
[0035] Amuc_1409 experimental group (Amuc_1409+SAP): First, the mice in this group were intragastrically administered 5 μg / day of Amuc_1409 (purchased from Nanjing Ainuodi Biotechnology Co., Ltd.) for one week continuously. At the same time, an antibiotic mixture composed of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg), and vancomycin (16.7 mg) was intragastrically administered every day for 5 days to exclude the interference of intestinal flora; subsequently, caerulein was intraperitoneally injected into the mice to establish a pancreatitis model.
[0036] Model group (Vehical+SAP): First, the mice in this group were gavaged with PBS (equivalent to the same amount of Amuc_1100 / Amuc_1409 in the previous groups) for one week continuously. At the same time, they were gavaged with an antibiotic mixture composed of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg), and vancomycin (16.7 mg) every day for 5 days to exclude the interference of intestinal flora. Subsequently, the mice were intraperitoneally injected with cerulein to establish a pancreatitis model.
[0037] The process of establishing the pancreatitis model was as follows: Cerulein was dissolved in sterile physiological saline, and one injection of cerulein (at a dose of 50 μg / kg of mouse body weight) was intraperitoneally injected every hour for 7 consecutive injections, and one injection of LPS (10 mg / kg of body weight) was intraperitoneally injected.
[0038] Starting from the first injection of cerulein, the mice were sacrificed 12 h later.
[0039] (3) Index determination
[0040] Samples such as pancreatic tissues and peripheral blood were taken from the mice in each group.
[0041] The blood samples were placed at room temperature for 30 min and centrifuged at 3000 rpm for 10 min. The serum samples were aspirated into new EP tubes, and the content of amylase in the serum was measured using an amylase assay kit (EPS substrate method, Zhong Sheng Beikong) according to the standard procedures in the instruction manual.
[0042] The pancreatic tissues were placed in 4% paraformaldehyde solution overnight, trimmed, dehydrated step by step with ethanol solutions of different concentration gradients, cleared with xylene, and then placed in paraffin and embedded using an embedding machine. The embedded tissue blocks were sliced using a slicer, the paraffin sections were dewaxed to water, stained with hematoxylin and eosin, and finally dehydrated and sealed. The tissue lesions were observed under a microscope.
[0043] An interleukin-6 (IL-6) ELISA kit (Linker Biotech) was used to measure the content of interleukin-6 (IL-6) in the serum according to the standard procedures in the instruction manual.
[0044] Total RNA of pancreatic tissues was extracted using RNAiso Plus (Takara) reagent, and the nucleic acid transcription levels of inflammatory factors (interleukin-1β, interleukin-6) were measured using the HiScript II One Step qRT-PCR SYBR Green kit (Vazyme).
[0045] Mouse peripheral anticoagulant blood was used to isolate peripheral blood lymphocytes according to the standard procedure described in the peripheral blood lymphocyte separation kit (TDB). Flow cytometry staining was performed using CD4, CD25, and Foxp3 antibodies (BioLegend) according to the recommended method in the instruction manual, and then detected and recorded using a flow cytometer. Statistical analysis was performed using FlowJo software.
[0046] (4) Measurement results
[0047] The results are as Figures 1 to 3 shown.
[0048] It can be seen from Figure 1 that compared with Vehical+SAP mice, the amylase concentration in the serum of mice in the Amuc_1100+SAP and Amuc_1409+SAP groups was significantly decreased ( Figure 1 A), and the expression of pancreatic local interleukin 1β (IL-1β) and interleukin-6 (IL-6) was significantly decreased ( Figure 1 B), as well as the expression of IL-6 in the systemic circulation ( Figure 1 C); and compared with the treatment of Amuc_1100 (Amuc_1100+SAP), the treatment with Amuc_1409 protein (Amuc_1409+SAP group) showed a better effect in inhibiting inflammation.
[0049] It can be seen from Figure 2 that compared with Vehical+SAP mice, the treatment of the Amuc_1100+SAP or Amuc_1409+SAP group significantly alleviated pancreatic edema and inflammatory cell infiltration.
[0050] It can be seen from Figure 3 that compared with Vehical+SAP mice, the treatment of the Amuc_1100+SAP or Amuc_1409+SAP group significantly increased the proportion of regulatory T cells in peripheral blood lymphocytes; and compared with the treatment of Amuc_1100 (Amuc_1100+SAP), the treatment with Amuc_1409 protein (Amuc_1409+SAP group) more significantly increased the proportion of regulatory T cells in peripheral blood lymphocytes.
[0051] Example 2. Experimental study on the treatment of acute pancreatitis with Amuc1409 protein:
[0052] In this example, after establishing a model of pancreatitis by intraperitoneal injection of L-arginine into mice, 5 μg / day of Amuc_1409 was administered by gavage within the first 1, 2, and 3 days after modeling to study the effect of Amuc_1409 in the treatment of acute pancreatitis, as follows:
[0053] (1) Grouping
[0054] Twenty healthy C57BL / 6J mice were selected and divided into four groups on average: control group (NC), model group (Vehical+SAP), Amuc_1100 experimental group (Amuc_1100+SAP), and Amuc_1409 experimental group (Amuc_1409+SAP).
[0055] (2) Treatment
[0056] Control group (NC): Healthy mice without any treatment.
[0057] Amuc_1100 experimental group (Amuc_1100+SAP): First, the mice in this group were gavaged with an antibiotic mixture composed of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg), and vancomycin (16.7 mg) every day for 5 consecutive days to exclude the interference of intestinal flora; subsequently, the mice were modeled with arginine; on the 1st, 2nd, and 3rd days after the start of arginine modeling, Amuc_1100 (purchased from Nanjing Ainuodi Biotechnology Co., Ltd.) was gavaged at a dose of 5 μg / day.
[0058] Amuc_1409 experimental group (Amuc_1409+SAP): First, the mice in this group were gavaged with an antibiotic mixture composed of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg), and vancomycin (16.7 mg) every day for 5 consecutive days to exclude the interference of intestinal flora; subsequently, the mice were modeled with arginine; on the 1st, 2nd, and 3rd days after the start of arginine modeling, Amuc_1409 (purchased from Nanjing Ainuodi Biotechnology Co., Ltd.) was gavaged at a dose of 5 μg / day.
[0059] Model group (Vehical+SAP): First, the mice in this group were gavaged with an antibiotic mixture composed of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg), and vancomycin (16.7 mg) every day for 5 consecutive days to exclude the interference of intestinal flora; subsequently, the mice were modeled with arginine; on the 1st, 2nd, and 3rd days after the start of arginine modeling, PBS (equivalent to the same amount of the aforementioned Amuc_1100 / Amuc_1409) was gavaged.
[0060] The process of arginine modeling was as follows: Arginine was dissolved in sterile physiological saline, and one injection of arginine (at a dose of 4 g / kg body weight of the mouse) was intraperitoneally injected every hour for 2 consecutive injections, and at the 68th hour after the start of arginine modeling, one injection of LPS (10 mg / kg body weight) was intraperitoneally injected.
[0061] The mice were sacrificed 72 hours after the first injection of arginine was started.
[0062] (3) Index determination
[0063] Samples such as pancreatic tissues and peripheral blood were taken from each group of mice.
[0064] The blood samples were placed at room temperature for 30 min and centrifuged at 3000 rpm for 10 min. The serum samples were aspirated into new EP tubes, and the amylase content in the serum was measured using an amylase assay kit (EPS substrate method, Zhong Sheng Beikong) according to the standard procedure in the instruction manual.
[0065] The pancreatic tissues were placed in 4% paraformaldehyde solution overnight, trimmed, dehydrated step by step with ethanol solutions of different concentration gradients, cleared with xylene, then placed in paraffin and embedded using an embedding machine. The embedded tissue blocks were sectioned using a microtome. The paraffin sections were dewaxed to water, stained with hematoxylin and eosin, and finally dehydrated and sealed. Tissue lesions were observed under a microscope.
[0066] Total RNA of pancreatic tissues was extracted using RNAiso Plus (Takara) reagent, and the nucleic acid transcription levels of inflammatory factors (interleukin-1β, interleukin-6) were measured using the HiScript II One Step qRT-PCR SYBR Green kit (Vazyme). Meanwhile, the transcription level of the anti-inflammatory factor (interleukin-10) was measured.
[0067] Peripheral anticoagulated blood of mice was separated to obtain peripheral blood lymphocytes according to the standard procedure described in the peripheral blood lymphocyte separation kit (TDB). Flow cytometry staining was performed using CD4, CD25, and Foxp3 antibodies (BioLegend) according to the recommended method in the instruction manual, detected and recorded on a flow cytometer, and statistical analysis was performed using FlowJo software.
[0068] (4) Measurement results
[0069] The results were as Figures 4 to 6 shown.
[0070] It was Figure 4 found that compared with the Vehical+SAP mice, the amylase concentrations in the sera of the Amuc_1100+SAP and Amuc_1409+SAP group mice were significantly decreased ( Figure 4 A), and the expressions of pancreatic local interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly decreased, while the expression of the anti-inflammatory factor (interleukin-10) was increased ( Figure 4 B); and compared with the treatment with Amuc_1100 (Amuc_1100+SAP), the treatment with Amuc_1409 protein (Amuc_1409+SAP group) showed a better effect in inhibiting inflammation.
[0071] It wasFigure 5 It can be seen that compared with the Vehical+SAP mice, the treatments in the Amuc_1100+SAP or Amuc_1409+SAP groups significantly alleviated pancreatic edema and inflammatory cell infiltration.
[0072] It can be seen from Figure 6 It can be seen that compared with the Vehical+SAP mice, the treatments in the Amuc_1100+SAP or Amuc_1409+SAP groups significantly increased the proportion of regulatory T cells in peripheral blood lymphocytes; and compared with the treatment with Amuc_1100 (Amuc_1100+SAP), the treatment with Amuc_1409 protein (Amuc_1409+SAP group) more significantly increased the proportion of regulatory T cells in peripheral blood lymphocytes.
[0073] In summary, the Akkermansia muciniphila secreted protein Amuc_1409 of the present invention plays an important role in the prevention and treatment of acute pancreatitis, and its prevention and treatment effect is better than that of the Akkermansia muciniphila membrane protein Amuc_1100.
[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. Use of Amuc1409 protein in preventing and treating acute pancreatitis.
2. Use of the Amuc1409 protein according to claim 1 in the prevention and treatment of acute pancreatitis, characterized in that, The Amuc1409 protein increases the proliferation and regeneration of intestinal stem cells in a cadherin-dependent manner and maintains intestinal homeostasis.
3. Use of the Amuc1409 protein according to claim 1 in the prevention and treatment of acute pancreatitis, characterized in that, When used for the prevention or treatment of acute pancreatitis, the Amuc1409 protein exerts its function through at least one of the following pathways: (1) Reducing the concentration of amylase in the serum; (2) Alleviating acinar cell injury and inflammatory cell infiltration; (3) Reducing the transcription of inflammatory cytokines in the pancreas; (4) Increasing the proportion of regulatory T cells in peripheral blood lymphocytes.
4. Use of the Amuc1409 protein according to claim 3 in the prevention and treatment of acute pancreatitis, characterized in that, In the said pathway (3), the inflammatory cytokines refer to interleukin-1β and interleukin-6.
5. Use of the Amuc1409 protein according to claim 3 in the prevention and treatment of acute pancreatitis, characterized in that, In the said pathway (3), it also includes increasing the transcription of anti-inflammatory factors in the pancreas.
6. Use of the Amuc1409 protein according to claim 5 in the prevention and treatment of acute pancreatitis, characterized in that, The said anti-inflammatory factor refers to interleukin-10.