Use of xanthine nucleosides for the preparation of a medicament for the prevention and treatment of acute pancreatitis

Abstract

The application relates to the field of biological medicine, and particularly relates to application of xanthine nucleoside in preparation of a drug for preventing and treating acute pancreatitis. In the application, it is found that the xanthine nucleoside can significantly improve pancreas inflammation, necrosis and systemic inflammatory response caused by acute pancreatitis, the xanthine nucleoside is used in preparation of the drug for preventing and treating acute pancreatitis, a new strategy is provided for preventing and treating acute pancreatitis, and the application has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of xanthine nucleoside in the preparation of drugs for the prevention and treatment of acute pancreatitis. Background Technology

[0002] Acute pancreatitis (AP) is a common acute abdominal condition in clinical practice, with severe acute pancreatitis (SAP) still having a mortality rate as high as 30%. Studies have shown that obesity is not only an independent risk factor for AP, but also a key driver of its progression to severe illness, the occurrence of complications, and poor prognosis. Obese patients have a significantly higher incidence of organ failure and mortality, longer hospital stays, and a heavier medical burden; however, there are currently no specific treatments for AP, especially obesity-related AP.

[0003] Currently, the pathological mechanisms of obesity-related acute pancreatitis (SAP) are believed to mainly include lipid metabolism disorders, adipokines imbalance, and organelle dysfunction. Specifically, excessive lipolysis of visceral and peripancreatic adipose tissue mediated by pancreatic lipase and triglyceride lipase releases large amounts of free fatty acids, inducing systemic lipotoxic damage; elevated levels of pro-inflammatory adipokines (such as resistin and leptin) and decreased levels of anti-inflammatory factors (such as adiponectin) trigger an inflammatory cascade; endoplasmic reticulum stress and mitochondrial dysfunction cause pancreatic acinar cells to shift from protective apoptosis to pro-inflammatory necrosis. Therefore, although obesity-related AP and AP of other etiologies both lead to pancreatic injury and inflammation, they have significantly different pathophysiological mechanisms, suggesting that different treatment strategies should be adopted for the two.

[0004] Current traditional AP treatments fail to effectively address the underlying causes of obesity-related AP. Most medications used in clinical practice focus on improving clinical symptoms and maintaining organ function, lacking specific drugs. Summary of the Invention

[0005] Based on this, the present invention provides the application of xanthine nucleoside in the preparation of drugs for the prevention and treatment of acute pancreatitis, thereby solving at least one problem in the prior art.

[0006] In a first aspect, the present invention provides the use of xanthine nucleoside in the preparation of drugs for the prevention and treatment of acute pancreatitis.

[0007] Understandably, the drugs mentioned for the prevention and treatment of acute pancreatitis refer to drugs used for the prevention, treatment, and / or adjunctive treatment of acute pancreatitis. Xanthine nucleoside can downregulate serum amylase expression levels, improve the early cytokine storm in acute pancreatitis, reduce pancreatic tissue edema, reduce pancreatic inflammatory cell infiltration, reduce pancreatic acinar cell necrosis, inhibit pancreatic acinar cell panapoptosis, and reduce the level of inflammatory factors in pancreatic tissue.

[0008] Secondly, the present invention provides a drug for the prevention and treatment of acute pancreatitis, which includes xanthine nucleoside.

[0009] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: it has been discovered that xanthine nucleosides can significantly improve pancreatic inflammation, necrosis and systemic inflammatory response caused by acute pancreatitis; using xanthine nucleosides to prepare drugs for the prevention and treatment of acute pancreatitis provides a new strategy for the prevention and treatment of acute pancreatitis and has good clinical application prospects. Attached Figure Description

[0010] Figure 1 The images show the H&E staining results of mouse pancreatic tissues in each group in Example 1 of this invention.

[0011] Figure 2 This is a statistical chart of the pathological scores of pancreatic tissues in each group of mice in Example 1 of the present invention.

[0012] Figure 3 This is a statistical chart of serum amylase levels in mice in each group in Example 1 of the present invention.

[0013] Figure 4 The image shows the MPO staining results of the pancreatic tissues of mice in each group in Example 1 of this invention.

[0014] Figure 5 This is a statistical chart of the MPO immunohistochemical scores of the pancreatic tissues of mice in each group in Example 1 of the present invention.

[0015] Figure 6 The images show the F4 / 80 staining results of mouse pancreatic tissues in each group in Example 1 of this invention.

[0016] Figure 7 This is a statistical chart of the F4 / 80 immunohistochemical scores of pancreatic tissues from each group of mice in Example 1 of the present invention.

[0017] Figure 8 This is a statistical graph showing the expression levels of IL-6, IL-1β, and TNF-α in the pancreatic tissues of mice in each group in Example 1 of the present invention, as detected by RT-PCR.

[0018] Figure 9 This is a statistical graph showing the expression levels of IL-6 and TNF-α in the pancreatic tissues of mice in each group of the present invention, as detected by ELISA.

[0019] Figure 10 The image shows the TUNEL staining results of the pancreatic tissues of mice in each group in Example 1 of this invention.

[0020] Figure 11 The image shows the Western blot results of the pancreatic tissues of mice in each group in Example 1 of this invention.

[0021] Figure 12 The images show the H&E staining, Masson staining, and Sirius red staining results of pancreatic tissue from mice in each group 48 hours after intraperitoneal injection of taurine in Example 2 of this invention.

[0022] Figure 13 The images show the H&E staining, Masson staining, and Sirius red staining results of pancreatic tissue from mice in each group 96 hours after intraperitoneal injection of taurine in Example 2 of this invention.

[0023] Figure 14 This is a diagram showing the results of an in vitro TCA stimulation experiment on primary mouse pancreatic acinar cells to induce acute pancreatitis in Example 3 of this invention.

[0024] In the statistical chart, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0025] The following will provide a clear and complete description of the concept and technical effects of the present invention, so as to fully explain the purpose, solution and effects of the present invention.

[0026] Xanthosine (Xa), an important intermediate molecule in the purine nucleoside metabolic pathway, is widely involved in nucleic acid synthesis, energy metabolism, and cell signal transduction. The inventors discovered that xanthosine can reduce pancreatic tissue edema, decrease pancreatic inflammatory infiltration, reduce pancreatic acinar cell apoptosis, promote acinar cell regeneration and repair, and inhibit fibrosis in obesity-related acute pancreatitis. Based on these findings, this invention utilizes xanthosine in the preparation of drugs for the prevention and treatment of acute pancreatitis.

[0027] In a first aspect, the present invention provides the use of xanthine nucleoside in the preparation of drugs for the prevention and treatment of acute pancreatitis.

[0028] In some implementations, the acute pancreatitis is severe acute pancreatitis or obesity-related acute pancreatitis.

[0029] Secondly, the present invention provides a drug for the prevention and treatment of acute pancreatitis, which includes xanthine nucleoside.

[0030] The drug for preventing and treating acute pancreatitis uses xanthine nucleoside as its active ingredient and can be formulated into any pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

[0031] In some implementations, the drug for preventing and treating acute pancreatitis also includes a pharmaceutically acceptable carrier.

[0032] In some embodiments, the pharmaceutically acceptable carrier includes one or more of the following commonly used medical fillers, binders, humectants, disintegrants, lubricants, and flavoring agents.

[0033] In some implementations, the pharmaceutically acceptable carrier is DMSO, water, or physiological saline.

[0034] In some embodiments, the dosage form of the drug for preventing and treating acute pancreatitis includes, but is not limited to, tablets, capsules, solutions, granules, injections, powder for injection, or aerosols. Preferably, the dosage form of the drug for preventing and treating acute pancreatitis is dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, soft capsules, or pills.

[0035] In some embodiments, when using the drug for the prevention and treatment of acute pancreatitis, the pharmaceutically effective amount of xanthine nucleoside is 40-60 mg / kg. Preferably, when using the drug for the prevention and treatment of acute pancreatitis, the pharmaceutically effective amount of xanthine nucleoside is 50 mg / kg.

[0036] The effectiveness of this invention was verified through the following examples. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used are all commercially available. Specifically, SPF-grade 5-6 week old male C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; xanthosine (Xa) was purchased from MCE; Caerulein (Cer) was purchased from Tocris and used to induce severe acute pancreatitis in mice; sodium taurocholate (TCA) was purchased from Sigma; and a 60% high-fat diet was purchased from Jiangsu Nantong Trofi Feed Co., Ltd.

[0037] Example 1: Validation experiment on the effect of preventive supplementation of xanthine nucleoside on alleviating acute pancreatitis in obese mice SPF-grade 5-6 week old male C57BL / 6 mice were randomly divided into a high-fat diet group (Vehicle_cer group) and a high-fat diet combined with xanthine nucleoside group (Xa_cer group), with 5 mice in each group. The mice in the Vehicle_cer group and Xa_cer group were fed a 60% high-fat diet. The Xa_cer group mice were administered xanthine nucleoside (dissolved in DMSO and diluted with physiological saline, 50 mg / kg body weight per mouse) by gavage daily, while the Vehicle_cer group mice were administered an equal volume of physiological saline by gavage daily for 4 weeks. After 4 weeks, obesity-related acute pancreatitis was induced in the mice by intraperitoneal injection of vitexin (vitexin diluted with physiological saline to 0.01 mg / mL, 100 μg / kg body weight per mouse), thus establishing a mouse model of obesity-related acute pancreatitis.

[0038] Pancreatic tissue was taken from model mice and stained with H&E. The results are as follows: Figure 1 and Figure 2As shown, compared with the Vehicle_cer group mice, the Xa_cer group mice showed significantly reduced pancreatic damage in multiple dimensions, including pancreatic tissue edema, inflammatory infiltration, and acinar necrosis, and a significantly lower pathological score (total decrease of 34.15%). The results were statistically significant, indicating that xanthine nucleoside has a protective effect on pancreatic tissue in a high-fat environment.

[0039] Blood was taken from the model mice, and serum amylase levels were measured. The results are as follows: Figure 3 As shown, the serum amylase level in the Xa_cer group mice was significantly reduced, decreasing by 17.21% compared to the Vehicle_cer group (p<0.05), suggesting that xanthine nucleoside intervention can significantly reduce pancreatic tissue damage in mice with obesity-related acute pancreatitis.

[0040] Pancreatic tissue was taken from the model mice and subjected to immunohistochemical analysis. The results are as follows: Figures 4-7 As shown, the expression of the neutrophil marker MPO in the pancreatic tissue of Xa_cer group mice ( Figure 4 and Figure 5 ) and macrophage marker F4 / 80 expression ( Figure 6 and Figure 7 The levels of [specific markers] were significantly lower in the model mice than in the Vehicle_cer group, decreasing by 50.00% and 37.50%, respectively. Real-time quantitative PCR was performed on pancreatic tissue from the model mice, and the results are as follows: Figure 8 As shown, the expression levels of inflammatory cytokines TNF-α, IL-6, and IL-1β in the pancreatic tissue of mice in the Xa_cer group were significantly lower than those in the Vehicle_cer group, decreasing by 32.62%, 62.58%, and 62.54%, respectively. These results indicate that xanthine nucleoside intervention can significantly reduce the level of inflammation in pancreatic tissue.

[0041] Blood was collected from model mice and subjected to an ELISA test. The results were as follows: Figure 9 As shown, the serum expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in mice of the Xa_cer group were significantly reduced, decreasing by 40.50%, 27.76%, and 55.24% respectively compared to the Vehicle_cer group (p<0.05), indicating that xanthine nucleoside intervention can significantly reduce the systemic inflammatory response in mice with obesity-related acute pancreatitis.

[0042] Pancreatic tissue was taken from model mice and stained with TUNEL. The results are as follows: Figure 10 As shown, the proportion of pancreatic tissue cells in the Xa_cer group mice was significantly reduced, decreasing by 55.89% compared to the Vehicle_cer group mice (p<0.05).

[0043] Pancreatic tissue was taken from model mice and subjected to Western blot experiments. The results are as follows: Figure 11 As shown, the expression levels of panapoptotic marker proteins ZBP1, Cleaved-GSDMD, p-MLKL, Cleaved-Caspase3, and p-RIPK3 in the pancreatic tissue of Xa_cer group mice were significantly reduced, by 66.93%, 31.49%, 43.35%, 36.21%, and 27.85% respectively compared with Vehicle_cer group (p<0.05).

[0044] Example 2: Validation experiment on the therapeutic supplementation of xanthine nucleoside to alleviate acute pancreatitis in obese mice. SPF-grade 5-6 week old male C57BL / 6 mice were randomly divided into a control group (Vehicle group), a high-fat diet group (Vehicle_cer group), and a high-fat diet combined with xanthine nucleotides group (Xa_cer group), with 5 mice in each group. The Vehicle group mice were fed a normal diet, while the Vehicle_cer and Xa_cer groups were fed a 60% high-fat diet for 4 weeks. After 4 weeks, Vehi was induced by intraperitoneal injection of vitexin (vitexin diluted with physiological saline to 0.01 mg / mL, 100 μg / kg body weight per mouse). Mice in the cle_cer and Xa_cer groups developed obesity-related acute pancreatitis, thus establishing a mouse model of obesity-related acute pancreatitis. One hour after injection of crotonin, mice in the Xa_cer group were administered xanthine nucleoside (dissolved in DMSO and diluted with physiological saline, with a gavage dose of 50 mg / kg body weight per mouse). Mice in the Vehicle_cer group were administered an equal volume of physiological saline daily. Subsequently, mice in the Xa_cer group were administered xanthine nucleoside and mice in the Vehicle and Vehicle_cer groups were administered an equal volume of physiological saline once daily.

[0045] Pancreatic tissues were collected from mice in each group 48 h and 96 h after intraperitoneal injection of bacitracin, and subjected to H&E staining, Masson staining, and Sirius red staining. The results are as follows: Figure 12 and Figure 13 As shown. Among them, H&E staining results of mouse pancreatic tissue after 48 hours showed that, compared with the Vehicle_cer group, the Xa_cer group mice had reduced edema, necrotic cell and inflammatory cell infiltration, and an increased proportion of regenerated normal acinar cells (as shown). Figure 12 ). H&E staining of mouse pancreatic tissue after 96 hours showed that, compared with the Vehicle_cer group, the Xa_cer group mice had a further increased proportion of regenerated acinar cells that fused together, with regular cell arrangement, largely restored lobular structure, and smaller residual necrotic foci. Figure 13Masson staining and Sirius red staining of mouse pancreatic tissue showed that, compared with the Vehicle_cer group, the Xa_cer group mice had less pancreatic tissue fibrosis and significantly reduced collagen fiber deposition. These results indicate that xanthine nucleoside has a therapeutic effect on obesity-related acute pancreatitis mice, significantly promoting acinar cell regeneration and repair and alleviating fibrosis.

[0046] Example 3: In vitro TCA stimulation of primary mouse pancreatic acinar cells to induce acute pancreatitis Pancreas cells were harvested from normal C57BL / 6 mice. Primary pancreatic acinar cells were isolated using collagenase digestion. The isolated primary pancreatic acinar cells were seeded into culture plates. After cell stabilization, cells were treated with PBS (Control group), 5 mM TCA (TCA group), and 5 mM TCA combined with 10 μM Xa (TCA+Xa group). After 1 h of treatment, cell viability and death were assessed using Calcein AM / PI kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). Results are as follows: Figure 14 As shown, the proportion of pancreatic acinar cell death was significantly reduced in the TCA+Xa group, decreasing by 32.10% compared to the TCA group (p<0.05), indicating that xanthine nucleoside intervention can significantly reduce pancreatic acinar cell death.

[0047] In summary, xanthine nucleoside can alleviate pancreatic damage in obese mice, reduce inflammatory cell infiltration and acinar cell death in pancreatic tissue, decrease the expression levels of pancreatitis markers (amylase) and pro-inflammatory factors (TNF-α, IL-6, IL-1β) in serum, and promote pancreatic regeneration and repair, as well as inhibit fibrosis, thus playing a role in the prevention and treatment of severe acute pancreatitis in obese mice.

[0048] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. Application of xanthine nucleoside in the preparation of drugs for the prevention and treatment of acute pancreatitis.

2. The application according to claim 1, characterized in that, The acute pancreatitis referred to is severe acute pancreatitis or obesity-related acute pancreatitis.

3. A drug for the prevention and treatment of acute pancreatitis, characterized in that, Including xanthine nucleosides.

4. The drug for preventing and treating acute pancreatitis according to claim 3, characterized in that, The acute pancreatitis referred to is severe acute pancreatitis or obesity-related acute pancreatitis.

5. The drug for preventing and treating acute pancreatitis according to claim 3, characterized in that, The drugs mentioned for the prevention and treatment of acute pancreatitis also include pharmaceutically acceptable carriers.

6. The drug for preventing and treating acute pancreatitis according to claim 5, characterized in that, The pharmaceutically acceptable carriers include one or more of the following commonly used medical fillers, binders, humectants, disintegrants, lubricants, and flavoring agents.

7. The drug for preventing and treating acute pancreatitis according to claim 5, characterized in that, The pharmaceutically acceptable carriers are DMSO, water, or physiological saline.

8. The drug for preventing and treating acute pancreatitis according to claim 3, characterized in that, The dosage forms of the drugs for the prevention and treatment of acute pancreatitis include, but are not limited to, tablets, capsules, solutions, granules, injections, powder for injection, or aerosols.

9. The drug for preventing and treating acute pancreatitis according to claim 3, characterized in that, When using the aforementioned medication for the prevention and treatment of acute pancreatitis, the pharmaceutically effective dose of xanthine nucleoside is 40-60 mg / kg.

10. The drug for preventing and treating acute pancreatitis according to claim 9, characterized in that, When using the aforementioned medication for the prevention and treatment of acute pancreatitis, the pharmaceutically effective dose of xanthine nucleoside is 50 mg / kg.

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