Application of rhaponticin in preparation of medicine for preventing and treating acute pancreatitis

Abstract

The invention discloses an application of rhaponticin in preparation of a medicine for preventing and treating acute pancreatitis, and provides the application of rhaponticin in preparation of the medicine for preventing and treating acute pancreatitis for the first time, experiments prove that rhaponticin Rha can target acinus cells, pancreatic tissues damaged by AP are intervened through Rha, the serum enzyme level is reduced, and the application of rhaponticin in preparation of the medicine for preventing and treating acute pancreatitis is promoted. Pancreatic tissue inflammation damage and tissue NETS formation amount of mice with acute pancreatitis are obviously reduced, and cytotoxicity is effectively reduced through Rha intervention. Therefore, rhaponticin is expected to be developed into an effective medicine for preventing and treating acute pancreatitis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of rhubarb glycoside in the preparation of medicines for preventing and treating acute pancreatitis. Background Art

[0002] Acute pancreatitis (AP) is a sudden inflammatory disease. Due to various causes, pancreatic enzymes are activated in the pancreas, causing pancreatic tissue self-digestion, edema, bleeding and even necrosis, leading to an inflammatory response of the pancreatic tissue. Its clinical manifestations are severe pain that can extend to the back and shoulders, accompanied by nausea, vomiting, fever and other symptoms. AP is very dangerous, with an annual incidence of 13-80 / 100,000. It is the second most common cause of hospitalization in the gastroenterology department and the fifth leading cause of death in the hospital. Studies have shown that in the early stages of AP, activated immune cells capture microorganisms and release bactericidal enzymes to relieve pancreatic inflammatory reactions; if the inflammatory response related to immune cells is not cleared in time, it will aggravate pancreatic damage and cause systemic inflammatory reactions. Therefore, finding drugs and methods suitable for the prevention and treatment of acute pancreatitis is a top priority.

[0003] Rhaponticin (Rha) is a natural compound belonging to the flavonoids class, which is often found in some plants, such as rhubarb and honeysuckle. It has antioxidant, anti-inflammatory, and antibacterial effects. Previous studies have shown that Rha has shown good therapeutic effects in a variety of diseases, such as: Rha can effectively prevent pulmonary fibrosis by inhibiting the TGF-β / Smad pathway; Rha effectively inhibits (lipopolysaccharide) LPS-induced inflammation in endothelial cells by downregulating the iNOS / OX2 / NFκB / MAPK signaling pathway; Rha targets RANKL-induced NFATc1 and ROS activity to inhibit osteoclast formation and reabsorption, thereby achieving the effect of treating osteoporosis. However, the therapeutic effect of Rha in acute pancreatitis is still unclear, and there are no reports or patents indicating the relevant preventive and therapeutic effects of Rha in acute pancreatitis. Summary of the invention

[0004] Technical problem to be solved: In response to the above technical problem, the present invention provides the use of rhubarb glycoside in the preparation of drugs for preventing and treating acute pancreatitis. Rhubarb glycoside Rha can target acinar cells, intervene in pancreatic tissue damaged by AP through Rha, reduce serum enzyme levels, significantly reduce pancreatic tissue inflammatory damage and tissue NETS formation in mice with acute pancreatitis, and effectively reduce cytotoxicity through Rha intervention.

[0005] Technical solution: Application of rhubarb glycoside in the preparation of medicines for preventing and treating acute pancreatitis.

[0006] Preferably, the drug comprises rhubarb glycoside and pharmaceutically acceptable excipients and / or auxiliary ingredients.

[0007] Furthermore, the drug is a drug for administration via the gastrointestinal tract.

[0008] Furthermore, the dosage form of the drug is granules, tablets, powders, oral liquids, pills or capsules.

[0009] Preferably, the rhaponticin can be replaced by a pharmaceutically acceptable derivative or salt of rhaponticin.

[0010] Beneficial effects: The present invention proposes for the first time the use of rhubarb glycoside in the preparation of drugs for the prevention and treatment of acute pancreatitis, and proves through experiments that rhubarb glycoside Rha can target acinar cells, intervene in AP-damaged pancreatic tissue through Rha, reduce serum enzyme levels, significantly reduce pancreatic tissue inflammatory damage and tissue NETS formation in mice with acute pancreatitis, and effectively reduce cytotoxicity through Rha intervention. Therefore, rhubarb glycoside is expected to be developed as an effective drug for the prevention and treatment of acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a schematic diagram of the treatment time of mice in different groups, wherein A is the structural formula of rhubarb glycoside, and B is a schematic diagram of the treatment time of mice in different groups; Figure 2 This is the result of using an amylase kit to detect the amylase content in the peripheral serum of mice in different groups; Figure 3 This is the HE staining result of pancreatic tissue of mice in different groups; Figure 4 This is the result of immunofluorescence staining of pancreatic tissues of mice in different groups; Figure 5 This is a diagram showing the results of cytotoxicity testing of mouse primary acinar cells after different groups of treatment; Figure 6 This is the result of staining mouse primary acinar cells with Calcein-AM / PI cell double staining kit after being treated with different groups. DETAILED DESCRIPTION

[0012] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments: The ICR mice used were purchased from the Model Animal Center of Yangzhou University; The caerulein (Cae) used was purchased from NJPeptide Company with the catalog number 22-09-26; The cholecystokinin 8 (CCK8) used was purchased from ECHELON BIOSCIENCES with a catalog number of 25126-32-3. Other reagents can be purchased conventionally. The quantitative experiments involved in the examples were repeated at least three times, and the results were averaged.

[0013] Example 1: Application of intraperitoneal injection of rhacoside (Rha) in the prevention / treatment of mice with acute pancreatitis induced by Cae Experimental animals: male ICR mice (weight 20-25 g); ICR mice were randomly divided into blank control group, model group and drug intervention group; Blank control group (NC group): ICR mice without special treatment; Model group (Cae group): intraperitoneal injection of Cae (200 μg / kg), once every hour, for a total of 8 injections; Drug intervention group (Cae+RHA group): Rha (structural formula: Figure 1 The control group and the AP group were gavaged with the same volume of normal saline.

[0014] All mice in the groups were killed 12 h after the first intraperitoneal injection of Cae ( Figure 1 As shown in B), after the mouse was intraperitoneally injected with anesthetic, one eyeball was removed, blood was collected to retain the peripheral serum, and the amylase content in the mouse peripheral serum was detected using an amylase kit. The results are shown in Figure 2 Shown: Using the Cae-induced AP model, serum amylase at a dose of 200 mg / kg Rha significantly reduced the serum enzyme levels of AP mice.

[0015] Pancreatic tissue was then obtained and fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin blocks, and sliced. The slices were stained with hematoxylin and eosin. According to the following criteria, the pancreatic histological score of AP mice was mainly based on the following four indicators based on H&E staining: edema, inflammation, infiltration, and acinar necrosis, and the severity was evaluated by 0-4 points. Histological analysis was performed in a blinded manner, and the results are shown in Figure 3 As shown: The pancreatic tissue of the AP model was diffusely edematous and there was scattered acinar cell necrosis. After the intervention of rhubarb glycosides, a dose of 200 mg / kg of rhubarb glycosides effectively alleviated the pathological damage of the pancreatic tissue.

[0016] The sliced ​​tissue was subjected to immunofluorescence staining. Figure 4As shown in the figure: Compared with the control group, the immunofluorescence intensity of Amylase in the pancreatic tissue of AP induced by Cae decreased significantly, while after the intervention of 200 mg / kg of Rha, the immunofluorescence intensity of Amylase increased to a certain extent. This result shows that Rha effectively alleviates the damage caused by Cae to pancreatic tissue, repairs the damaged pancreatic tissue, and has a certain repair and protection effect.

[0017] Example 2: In vitro administration of rhacoside (Rha) helps to resist CCK8-induced cell damage Primary acinar cells: Primary acinar cells were extracted from mouse pancreas, resuspended in 5% FBS Hepes medium, and seeded on 96-well plates for stable culture at 37°C and 5% CO2. CCK8 (2.5 μM) was given to acinar cells for 6 h to induce cell damage.

[0018] The specific groups are as follows: Control group: cultured in Hepes containing 5% FBS for 6 hours; CCK8 group: 2.5 μM CCK8 was added to each well and cultured for 6 hours; Rha+CCK8 group: Different concentration gradients of Rha (10, 5, and 2.5 μM) were added to each well in advance. After the cells were incubated with Rha for half an hour, 2.5 μM CCK8 was added to each well and cultured for 6 hours.

[0019] Cells from each group were collected and tested for cytotoxicity using the lactate dehydrogenase cytotoxicity test kit (LDH). Figure 5 As shown: 5 μM Rha has the strongest resistance to CCK8-induced cell damage, where * represents a significant difference P < 0.05, ** represents a significant difference P < 0.01, *** represents a significant difference P < 0.001, and **** represents a significant difference P < 0.0001.

[0020] Example 3: Administration of rhagain (Rha) in vitro alleviates CCK8-induced cell damage Primary acinar cells: Acinar cells were extracted from the pancreas of mice, resuspended in 5% FBS Hepes medium, and seeded on 12-well plates for stable culture at 37°C and 5% CO2. CCK8 (2.5 μM) was given to acinar cells for 3 h to induce cell damage.

[0021] Control group: cultured in Hepes containing 5% FBS for 3 hours; CCK8 group: DMSO equal to Rha was added to each well in advance. After the cells were incubated with DMSO for half an hour, 2.5 μM CCK8 was added to each well and cultured for 3 hours; Rha+CCK8 group: 5 μM Rha was added to each well in advance. After the cells were incubated with Rha for half an hour, 2.5 μM CCK8 was added to each well and cultured for 3 hours.

[0022] After the incubation, the cells in each group were collected, stained with Calcein-AM / PI cell double staining kit, and then observed under a microscope. Figure 6 As shown: 5 μM Rha significantly reduced the cell damage caused by CCK8.

[0023] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of rhubarb glycoside in the preparation of medicines for preventing and treating acute pancreatitis.

2. The use of rhubarb glycoside according to claim 1 in the preparation of a medicament for preventing and treating acute pancreatitis, characterized in that: The medicine comprises rhubarb glycoside and pharmaceutically acceptable adjuvants and / or auxiliary components.

3. The use of rhubarb glycoside according to claim 2 in the preparation of a medicament for preventing and treating acute pancreatitis, characterized in that: The drug is a drug for administration via the gastrointestinal tract.

4. The use of rhubarb glycoside according to claim 3 in the preparation of a medicament for preventing and treating acute pancreatitis, characterized in that: The dosage form of the medicine is granules, tablets, powders, oral liquids, pills or capsules.

5. The use of rhubarb glycoside according to claim 1 or 2 in the preparation of a medicament for preventing and treating acute pancreatitis, characterized in that: The rhaponticin can be replaced by a pharmaceutically acceptable derivative or salt of rhaponticin.

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