Application of exogenous pancreatic acellular cell ALDH2 to preparation of medicine or health care product for preventing or treating acute pancreatitis

Abstract

The invention provides a new application of exogenous pancreatic acinus cell ALDH2 in preparation of drugs or health care products for preventing or treating acute pancreatitis. Experiments prove that on a mouse acute pancreatitis model, after pancreatic acinus cell ALDH2 expression is specifically knocked out, the pancreatic tissue damage degree is increased, and the pancreatic tissue inflammation infiltration degree is increased; the levels of AMY and LPS reflecting the severity of acute pancreatitis and the overall inflammation level in mouse serum are increased; the level of NLRP3 inflammasome closely related to occurrence and development of acute pancreatitis in pancreatic tissue is remarkably increased, and the results show that the pancreatic acellular cell ALDH2 protein has a protective effect on the severity and prognosis of acute pancreatitis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular, to the use of exogenously administering ALDH2 to pancreatic acinar cells in the preparation of a drug or health product for preventing or treating acute pancreatitis. Background Art

[0002] Acute pancreatitis (AP) is a common disease of the digestive system, with an annual incidence rate of 13-45 / 100,000 and an increasing trend. 20-30% of patients with acute pancreatitis may develop severe pancreatitis, with an overall mortality rate of 5-10%. Globally, the annual health care costs for acute pancreatitis are huge, but effective drugs for its prevention and treatment are still to be developed. Acute pancreatitis is characterized by pancreatic inflammatory reaction damage as a typical pathological feature. According to the cause of the disease, it can be divided into biliary, alcoholic, endoscopic retrograde pancreaticocholangiopancreatography-related and drug-related types. The pancreas is mainly composed of acinar cells, accounting for up to 82% of the volume. It realizes the synthesis, transportation, storage and secretion of digestive enzymes through the orderly operation of organelles such as the endoplasmic reticulum, Golgi body, lysosomes and mitochondria. Damage to the autophagy pathway in acinar cells, calcium ion transport disorders, endoplasmic reticulum stress, and mitochondrial dysfunction can lead to the activation of a series of inflammatory pathways, inducing the release of inflammatory factors and chemokines, which in turn cause inflammatory reactions in pancreatic tissue and ultimately lead to acute pancreatitis. Therefore, the regulation of the physiological process of pancreatic acinar cells is an effective means and key link in the prevention and treatment of acute pancreatitis, and is expected to become an important breakthrough in optimizing the treatment of acute pancreatitis.

[0003] The term "acute pancreatitis" refers to a common pancreatic injury disease characterized by acute inflammation. The incidence of acute pancreatitis has been increasing year by year worldwide, seriously affecting human health and quality of life. The pancreas is mainly composed of acinar cells, which realize the synthesis, transportation, storage and secretion of digestive enzymes through the orderly operation of organelles such as the endoplasmic reticulum, Golgi apparatus, lysosomes and mitochondria. Events such as damage to the autophagy pathway in acinar cells, dysregulated calcium ion transport, endoplasmic reticulum stress and mitochondrial dysfunction can lead to the activation of a series of inflammatory pathways, induce the release of inflammatory factors and chemokines, and then cause inflammatory reactions in pancreatic tissue and ultimately lead to acute pancreatitis. Therefore, the regulation of the physiological processes of pancreatic acinar cells is an effective means and key link in the prevention and treatment of acute pancreatitis. Acute pancreatitis has an acute onset, is a serious disease, has a high mortality and disability rate, and has many complications. The current clinical frontline treatment strategies for acute pancreatitis include the use of acid-suppressing drugs, somatostatin and other drugs and parenteral nutrition support. Although the current treatment drugs can alleviate the progression of acute pancreatitis to a certain extent, the clinical symptom relief effect is poor, and the long-term treatment effect is still unsatisfactory. Therefore, a lot of in-depth research is still needed for acute pancreatitis, and there is an urgent need to find new targets and develop new and effective drugs for the treatment of acute pancreatitis.

[0004] Focusing on aldehyde dehydrogenase 2 (ALDH2), an important enzyme in ethanol metabolism. Studies have shown that ALDH2 can participate in the regulation of intracellular metabolism through the autophagy pathway and play a protective role in a variety of diseases. In the animal liver ischemia-reperfusion injury model, ALDH2 activators can upregulate AMPK-related autophagy levels, thereby reducing the accumulation of reactive aldehydes and toxic protein compounds, and ultimately reducing the occurrence of liver cell apoptosis and mitochondrial dysfunction. In acute kidney injury, ALDH2 can upregulate autophagy levels through the Beclin-1 pathway to reduce tubular epithelial cell injury and renal damage. At the same time, ALDH2 also has an important regulatory effect on NLRP3 inflammasomes. In arteriosclerotic cardiovascular disease, ALDH2 can reduce oxidative stress, inflammatory response and mitochondrial damage, thereby reducing the initiation and activation of NLRP3 inflammasomes. However, it is unclear whether ALDH2 plays a relevant role in acute pancreatitis. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of exogenously administering ALDH2 to pancreatic acinar cells, which is an application in the preparation of a drug or health product for preventing or treating acute pancreatitis.

[0006] The technical solution of the present invention is: The present invention provides an application of exogenously administering ALDH2 to pancreatic acinar cells in the preparation of a drug or health product for preventing or treating acute pancreatitis, wherein the exogenously administering ALDH2 to pancreatic acinar cells comprises at least one of the following: A) exogenously administering ALDH2 recombinant protein to pancreatic acinar cells; B) exogenously administering ALDH2 encoding gene to pancreatic acinar cells; C) exogenously administering a recombinant vector containing ALDH2 encoding gene to pancreatic acinar cells; D) exogenously administering a recombinant virus containing ALDH2 encoding gene to pancreatic acinar cells.

[0007] Optionally, in the above-mentioned use of exogenous administration of ALDH2 to pancreatic acinar cells in the preparation of a drug or health product for preventing or treating acute pancreatitis, the use is an use in the preparation of a protective drug for acute pancreatitis.

[0008] Optionally, in the above-mentioned use of exogenously administering ALDH2 to pancreatic acinar cells in the preparation of a drug or health product for preventing or treating acute pancreatitis, the use is in the preparation of a health product for assisting in the prevention or treatment of acute pancreatitis.

[0009] Optionally, in the application of the above-mentioned exogenous administration of ALDH2 to pancreatic acinar cells in the preparation of drugs or health products for preventing or treating acute pancreatitis, the administration method is one of direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-coated DNA gene gun bombardment, reproduction-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA, PEG-modified protein drug injection, liposome-encapsulated protein intravenous injection or protein microsphere preparation subcutaneous injection.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the use of exogenously administered pancreatic acinar cell ALDH2 in the preparation of a drug or health product for preventing or treating acute pancreatitis. Experiments show that pancreatic acinar cell ALDH2 protein plays a protective role on the severity and prognosis of acute pancreatitis. Since pancreatic acinar cell ALDH2 is an endogenous protein of the body, it is very safe as a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0012] Figure 1 ALDH2 acinar cell-specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + Mouse) breeding model diagram; Figure 2This is a picture of the tissue morphology after HE staining taken under a microscope after specifically knocking out the expression of ALDH2 in pancreatic acinar cells in a mouse acute pancreatitis model; Figure 3 In a mouse acute pancreatitis model, after specifically knocking out the expression of ALDH2 in pancreatic acinar cells, Aldh2 + / + Mice and Aldh2 fl / fl AMY and LPS levels in Cela1-Cre mouse serum reflect the severity of acute pancreatitis and overall inflammation level; Figure 4 In a mouse acute pancreatitis model, the expression of ALDH2 in pancreatic acinar cells was specifically knocked out, and Aldh2 was detected by tissue immunofluorescence technology. + / + Mice and Aldh2 fl / fl The expression results of NLRP3 and ASC proteins associated with NLRP3 inflammasome in pancreatic tissue of Cela1-Cre mice. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0014] The experimental methods without specific conditions in the following experimental examples are usually carried out under conventional conditions or conditions recommended by the manufacturers.

[0015] The use of exogenously administering ALDH2 to pancreatic acinar cells of the present invention in the preparation of a drug or health product for preventing or treating acute pancreatitis, wherein the exogenously administering ALDH2 to pancreatic acinar cells comprises at least one of the following: A) ALDH2 recombinant protein was exogenously administered to pancreatic acinar cells; B) The gene encoding ALDH2 is exogenously administered to pancreatic acinar cells; C) A recombinant vector containing the ALDH2 encoding gene exogenously administered to pancreatic acinar cells; D) Recombinant virus containing the ALDH2 encoding gene exogenously administered to pancreatic acinar cells.

[0016] The above application is the application in the preparation of protective drugs for acute pancreatitis or the application in the preparation of health products for auxiliary prevention or treatment of acute pancreatitis. The drug administration method adopts one of the following methods: direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-encapsulated DNA gene gun bombardment, reproduction-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA, PEG-modified protein drug injection, liposome-encapsulated protein intravenous injection, or protein microsphere preparation subcutaneous injection.

[0017] Pancreatic acinar cell ALDH2 is an endogenous protein present in human pancreatic acinar cells. ALDH2 recombinant protein can be purchased from Biovision or Abcam.

[0018] The ALDH2 acinar cell-specific knockout mice used in the experiment (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + The mice were purchased from Saiye (Suzhou) Biotechnology Co., Ltd. and constructed and bred through the genetic engineering Cre-Loxp system. Based on the homologous recombination technology, loxp sites were inserted on both sides of the 3rd and 4th exons of ALDH2 to obtain Aldh2 floxp / floxp The mice were then mated with Cela1-Cre mice to obtain ALDH2 acinar cell-specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + Mice). According to previous research reports, the present invention continuously intraperitoneally injected 10 ml / kg bombesin in vivo, once every 1 hour, for a total of 7 times, to construct a mouse acute pancreatitis model, so as to study the role and mechanism of pancreatic acinar cell ALDH2 in acute pancreatitis at the whole animal level.

[0019] Experiment 1: ALDH2 acinar cell-specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + Mouse) breeding model, such as Figure 1 shown.

[0020] Experimental process: The animals in this project were constructed and bred by Saiye (Suzhou) Biotechnology Co., Ltd. The specific implementation method was: Based on the homologous recombination technology, loxp sites were inserted on both sides of the 3rd and 4th exons of ALDH2 to obtain Aldh2 floxp / floxp The mice were then mated with Cela1-Cre mice to obtain ALDH2 acinar cell-specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + Mice) (e.g. Figure 1 as shown).

[0021] Experimental results: Genotype identification confirmed that the construction was successful and can be used for subsequent experimental research.

[0022] Experiment 2: In the mouse acute pancreatitis model, after specifically knocking out the expression of ALDH2 in pancreatic acinar cells, the degree of pancreatic tissue damage and inflammatory infiltration in the pancreatic tissue increased.

[0023] Experimental process: 1. Construction and breeding of ALDH2 acinar cell-specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + Mice); 10 ml / kg bombesin was injected intraperitoneally continuously every 1 hour for a total of 7 times to establish a mouse acute pancreatitis model.

[0024] 2. After modeling, the pancreatic tissue was removed and fixed in 4% paraformaldehyde for 24 h; the degree of pancreatic tissue damage and inflammatory cell infiltration was detected by HE staining.

[0025] 3. According to the tissue morphology after HE staining taken under a microscope, the severity of acute pancreatitis pathology is scored. The scoring criteria are shown in Table 1: Table 1 Scoring criteria for pathological severity of acute pancreatitis Experimental results: When acute pancreatitis occurs, Aldh2 + / + Compared with mice, Aldh2 fl / fl The pancreatic tissue pathological change scores of Cela1-Cre mice were significantly increased, and the degree of pancreatic tissue edema, inflammatory cell infiltration, and acinar cell vacuolation were more obvious (e.g. Figure 2 as shown).

[0026] This result shows that in the mouse acute pancreatitis model, after specifically knocking out the expression of ALDH2 in pancreatic acinar cells, the degree of pancreatic tissue damage and the degree of inflammatory infiltration in the pancreatic tissue increased.

[0027] Experiment 3: In the mouse acute pancreatitis model, after specifically knocking out the expression of ALDH2 in pancreatic acinar cells, the levels of AMY and LPS in the mouse serum, which reflect the severity of acute pancreatitis and the overall level of inflammation, increased.

[0028] Experimental process: 1. Construction and breeding of ALDH2 acinar cell-specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + Mice); 10 ml / kg bombesin was injected intraperitoneally continuously every 1 hour for a total of 7 times to establish a mouse acute pancreatitis model.

[0029] 2. After the modeling was completed, the blood of the mice was taken out and centrifuged to obtain serum samples. The AMY and LPS levels that reflect the severity of acute pancreatitis and the overall level of inflammation were detected by ELISA technology.

[0030] Experimental results: When acute pancreatitis occurs, Aldh2 + / + Compared with mice, Aldh2 fl / fl The serum AMY and LPS levels of Cela1-Cre mice were significantly increased. Figure 3 shown.

[0031] This result indicates that in the mouse acute pancreatitis model, after specifically knocking out the expression of ALDH2 in pancreatic acinar cells, the levels of AMY and LPS in the mouse serum, which reflect the severity of acute pancreatitis and the overall level of inflammation, increased.

[0032] Experiment 4: In the mouse acute pancreatitis model, after specifically knocking out the expression of ALDH2 in pancreatic acinar cells, the level of NLRP3 inflammasome in pancreatic tissue, which is closely related to the occurrence and development of acute pancreatitis, increased significantly.

[0033] Experimental process: 1. Construction and breeding of ALDH2 acinar cell-specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + Mice); 10 ml / kg bombesin was injected intraperitoneally continuously every 1 hour for a total of 7 times to establish a mouse acute pancreatitis model.

[0034] 2. After modeling, the pancreatic tissue was removed and fixed in 4% paraformaldehyde for 24 h; the expression of NLRP3 and ASC proteins related to NLRP3 inflammasome in the pancreatic tissue was detected by tissue immunofluorescence technique.

[0035] Experimental results: When acute pancreatitis occurs, Aldh2 + / + Compared with mice, Aldh2 fl / fl NLRP3 inflammasome-associated NLRP3 in pancreatic tissue of Cela1-Cre mice (e.g. Figure 4 , shown in red light), ASC protein (such as Figure 4 , shown by green light) expression was significantly increased.

[0036] This result indicates that in the mouse acute pancreatitis model, after specifically knocking out the expression of ALDH2 in pancreatic acinar cells, the level of NLRP3 inflammasome in pancreatic tissue, which is closely related to the occurrence and development of acute pancreatitis, increased significantly.

[0037] In summary, the present invention experiments prove that: by genetic engineering Cre-Loxp system to construct and cultivate ALDH2 acinar cell specific knockout mice (Aldh2 fl / fl Cela1-Cre mice) and their wild-type counterparts (Aldh2 + / + The experimental group was given bombesin to establish an acute pancreatitis model in mice. After modeling, the pancreatic tissue was removed and histopathological analysis was performed by HE staining. It was found that Aldh2 + / + Compared with mice, Aldh2 fl / fl Cela1-Cre mice showed significantly increased scores of pancreatic tissue pathological changes, more obvious pancreatic tissue edema, inflammatory cell infiltration, and acinar cell vacuolation. The serum levels of AMY and LPS, which reflect the severity of acute pancreatitis and the overall level of inflammation, were significantly increased. In addition, in the mouse acute pancreatitis model, the expression of Aldh2 + / + Compared with mice, Aldh2 fl / fl The level of NLRP3 inflammasome, which is closely related to the development of acute pancreatitis, was significantly increased in the pancreatic tissue of Cela1-Cre mice. These results indicate that ALDH2 protein in pancreatic acinar cells plays a protective role in the severity and prognosis of acute pancreatitis.

[0038] The above embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify or improve the technical solutions recorded in the above embodiments within the technical scope disclosed by the present invention, or replace some of the technical features therein by equivalents; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. Use of exogenously administering ALDH2 to pancreatic acinar cells in the preparation of a drug or health product for preventing or treating acute pancreatitis, characterized in that: The exogenous administration of ALDH2 to pancreatic acinar cells includes at least one of the following: A) ALDH2 recombinant protein was exogenously administered to pancreatic acinar cells; B) The gene encoding ALDH2 is exogenously administered to pancreatic acinar cells; C) A recombinant vector containing the ALDH2 encoding gene exogenously administered to pancreatic acinar cells; D) Recombinant virus containing the ALDH2 encoding gene exogenously administered to pancreatic acinar cells.

2. The use of exogenously administered pancreatic acinar cells ALDH2 in the preparation of a drug or health product for preventing or treating acute pancreatitis according to claim 1, characterized in that: The application is application in preparing protective medicine for acute pancreatitis.

3. The use of exogenously administered pancreatic acinar cells ALDH2 in the preparation of a drug or health product for preventing or treating acute pancreatitis according to claim 1, characterized in that: The application is in the preparation of health products for assisting in the prevention or treatment of acute pancreatitis.

4. The use of exogenously administered pancreatic acinar cells ALDH2 in the preparation of a drug or health product for preventing or treating acute pancreatitis according to claim 1 or 2, characterized in that: The administration method is one of direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-encapsulated DNA gene gun bombardment, reproduction-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA, PEG-modified protein drug injection, liposome-encapsulated protein intravenous injection or protein microsphere preparation subcutaneous injection.

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