CN115634230B - Application of sodium mannite in preparation of medicine for preventing and treating severe acute pancreatitis - Google Patents
Application of sodium mannite in preparation of medicine for preventing and treating severe acute pancreatitis Download PDFInfo
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Abstract
The invention discloses an application of sodium mannite in preparing a medicine for preventing and treating severe acute pancreatitis. The research of the invention shows that the mannite sodium has good effect of resisting severe acute pancreatitis, can obviously reduce the pancreatic volume of severe acute pancreatitis mice, and obviously improve the pancreatic histopathological score of the severe acute pancreatitis mice and the infiltration of neutrophils and mononuclear macrophages of pancreatic tissues; and simultaneously, the serum amylase, lipase and IL-6 levels of severe acute pancreatitis mice are obviously reduced. The mannitol sodium studied by the invention has wide pharmacological activity, obvious treatment effect, small toxic and side effect and high in vivo safety, and can replace the application of octreotide in treating severe acute pancreatitis.
Description
Technical Field
The invention belongs to the technical field of biological medicine. More particularly relates to the application of sodium mannite in preparing a medicine for preventing and treating severe acute pancreatitis.
Background
Acute Pancreatitis (AP) is the most common gastrointestinal disorder, an inflammatory reaction of various etiologies that causes self-digestion, edema, hemorrhage and even necrosis of pancreatic tissue after activation of pancreatic enzymes in the pancreas, requires acute hospitalization, and causes great pain and socioeconomic burden. The incidence of AP worldwide is 4.9 to 80 per 100000 people per year. According to the revised atlanta classification, acute pancreatitis is classified into Mild Acute Pancreatitis (MAP), moderate acute pancreatitis (MSAP), and Severe Acute Pancreatitis (SAP). The prognosis of AP patients of different severity varies widely. For example MAP patients need supportive care such as fluid and analgesia only and recover within a few days. Severe Acute Pancreatitis (SAP) is a severe acute pancreatitis, with organ failure lasting more than 48 hours, which is dangerous and can affect one or more organs with mortality rates as high as 30%.
Chemotherapy is an important means for the prevention and treatment of current severe acute pancreatitis. The currently preferred drug for the treatment of SAP in clinic is octreotide, and prior studies report that octreotide treatment of SAP can alleviate SAP and reduce the number of hospitalization days. However, there are also a number of limitations to the use of octreotide, the most common side effects reported in clinical trials of this product are diarrhea, abdominal pain, nausea, bloating, headache, gallstones, hyperglycemia and constipation, while other common side effects include dizziness, local pain, bile turbidity, thyroid dysfunction (e.g. reduction of thyrotropin), changes in total T4 and free T4, impaired glucose tolerance, vomiting, fatigue and hypoglycemia etc. The side effects of octreotide are a major problem in the work of treatment of severe acute pancreatitis. Meanwhile, as octreotide is repeatedly used for a long time and on a large scale, the organism is easy to generate tolerance to the octreotide, so that the search for an effective drug which has high safety, small side effect and good treatment effect and can replace the octreotide is urgent.
Disclosure of Invention
The invention provides an application of sodium mannite in preparing a medicine for preventing and treating severe acute pancreatitis.
The invention aims to provide a new application of sodium mannite.
The above object of the present invention is achieved by the following technical scheme:
in-vivo experiments are carried out by establishing a mouse model of heavy acute pancreatitis induced by ranpirin and lipopolysaccharide, and the mannitol sodium has good effect of resisting the heavy acute pancreatitis. The mannite sodium can obviously reduce the pancreatic volume of the severe acute pancreatitis mice, and obviously improve the pancreatic histopathological score of the severe acute pancreatitis mice and the infiltration of neutrophils and mononuclear macrophages of pancreatic tissues; meanwhile, the serum amylase, lipase and IL-6 levels of the severe acute pancreatitis mice are obviously reduced, the compound has wide pharmacological activity and obvious treatment effect, has small toxic and side effects and high safety, and can replace the application of octreotide in treating severe acute pancreatitis.
The mannite sodium adopted by the invention is low molecular acid oligosaccharide compound, i.e. oligosaccharide, which is prepared by taking marine brown algae extract as a raw material. The mannite sodium has wide pharmacological activity, has proved to have good in vivo safety and small toxic and side effects, and does not find any toxic effect or death in 400mg/kg of mannite sodium infused in the stomach of mice. The structural formula of the mannite sodium is shown as the following formula (I):
therefore, the following applications of the sodium mannite are all within the scope of the invention:
the application of sodium mannite in preparing medicine for preventing and treating severe acute pancreatitis.
Application of sodium mannite in preparation of medicine for relieving severe acute pancreatitis
The application of sodium mannite in preparing a medicament for reducing the pancreatic volume of a severe acute pancreatitis mouse.
Use of sodium mannite in the manufacture of a medicament for improving neutrophil infiltration caused by severe acute pancreatitis.
Further, the improvement is to reduce the expression level of MPO.
The application of sodium mannite in preparing medicine for improving mononuclear macrophage infiltration caused by severe acute pancreatitis.
Further, the improvement is to reduce the expression level of CD 68.
Use of sodium mannite for the preparation of a medicament for reducing serum amylase, lipase and IL-6 levels in severe acute pancreatitis mice.
Further, the reduction of severe acute pancreatitis is reduction of pancreatic tissue edema, acinar necrosis, hemorrhage, fat necrosis, and perivascular infiltration of inflammatory cells.
Preferably, the dosage form of the medicament is capsules, tablets, oral preparations, microcapsule preparations or injection.
The invention has the following beneficial effects:
the invention provides a new application of sodium mannite in preparing a medicine for preventing and treating severe acute pancreatitis, and researches show that the sodium mannite has good effect of resisting severe acute pancreatitis, and in vivo experimental results prove that the sodium mannite can obviously reduce the pancreatic volume of a severe acute pancreatitis mouse, obviously improve the pancreatic histopathological score and the MPO and CD68 levels of the severe acute pancreatitis mouse, and simultaneously obviously reduce the serum amylase, lipase and IL-6 levels of the severe acute pancreatitis mouse.
The mannite sodium adopted by the invention has wide pharmacological activity, small toxic and side effects, the mannite sodium of 400mg/kg of mice lavage does not find any toxic effect or death, and the mannite sodium has better safety and smaller toxic and side effects than octreotide, can replace the application of octreotide in treating severe acute pancreatitis, and provides more possibility and treatment means for avoiding clinical tolerance.
Drawings
FIG. 1 shows the volume of the pancreas of mice with severe acute pancreatitis after the action of sodium mannite (A: the size of the pancreas of mice; B: statistical graphs of the volumes of the pancreas of each group).
FIG. 2 is a graph of H & E staining of the pancreas of a mice with severe acute pancreatitis after the action of sodium mannite (A: pathological damage of pancreas tissue of the mice; B: statistical graph of the result of pancreatic histopathological scoring).
FIG. 3 is a chart showing the staining of the pancreas CD68 of a severe acute pancreatitis mouse after the action of sodium mannite ((A: CD68 expression in the pancreas of the mouse; B: statistical chart of the CD68 results of the pancreas IHC of the mouse).
FIG. 4 is a graph showing the IHC staining of the pancreatic MPO of a mouse with severe acute pancreatitis after the action of sodium mannite (A: MPO expression in the pancreas of the mouse; B: MPO result statistics of the pancreatic IHC of the mouse).
FIG. 5 is a statistical plot of serum amylase (A), lipase (B) and IL-6 in severe acute pancreatitis mice after action of sodium mannite (C).
The quantitative results in the figures are expressed as mean ± Standard Deviation (SD), all data were statistically analyzed using GraphPad Prism 9.0 (GraphPad Software, san Diego, USA), the differences between groups were analyzed using one-way analysis of variance (ANOVA), all histograms were plotted using GraphPad Prism 9.0, P <0.05, P <0.01, P <0.001, P <0.0001, P <0.05 have statistical significance.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The C57BL/6J mice used in this example were purchased from the Experimental animals Co., ltd. In Beijing, and the mannite sodium (GV-971) was purchased from Shanghai Lugu pharmaceutical Co., ltd; ranpirin (caerulein) is available from Shanghai Siberian cypress biotechnology limited; lipopolysaccharide (LPS) was purchased from Sigma Aldrich (Shanghai) trade Limited.
EXAMPLE 1 Effect of Mante sodium
The mice obtained by purchase are randomly grouped into a normal control group (control), a modeling group (SAP) and a treatment group, wherein the treatment group is treated by using sodium mannite prepared by 0.9 percent physiological saline, 100mg/kg,200mg/kg and 400mg/kg of sodium mannite are respectively infused into the mice every day, and the treated mice are correspondingly marked as 100mg/kg of sodium mannite treatment group (GV-971 (100 mpk) +SAP), 200mg/kg of sodium mannite treatment group (GV-971 (200 mpk) +SAP) and 400mg/kg of sodium mannite treatment group (GV-971 (400 mpk) +SAP); 8 mice per group.
The treatment group was perfused with sodium mannite, and the modeling group (SAP) and the normal control group (control) were perfused with the same volume of physiological saline for 7 days per day for 1 time.
EXAMPLE 2 establishment of severe acute pancreatitis mouse model
Using each group of mice 7 days after the gastric lavage in example 1, the remaining mice were continuously injected intraperitoneally with ranpirin (caerulein) 50. Mu.g/kg/h once/h 11 times, except for the normal control group. Immediately after the 11 th injection of ranpirin, lipopolysaccharide (LPS, 10 mg/kg) was injected intraperitoneally by the same volume of physiological saline as the normal control group.
After lipopolysaccharide injection for 12 hours, the weight of the mice was measured, then the mice were anesthetized with isoflurane gas, and then the orbital blood was collected, the blood was allowed to stand at room temperature for more than one hour, centrifuged at 5000rpm for 20min at 4 ℃, and serum was collected. Then, the mice of different treatment groups are taken for cervical dislocation treatment, the pancreas of the mice is carefully dissected from the joints of the mice with the stomach, the duodenum and the spleen, redundant fat and tissues are trimmed off, and the pancreas is collected and photographed. Fresh pancreas of 3 mice was randomly picked from each group, fixed overnight with 4% paraformaldehyde, rinsed with tap water for 2 hours, gradient dehydrated with ethanol, paraffin embedded and cut into 5 μm sections. Sections were dewaxed in xylene, hydrated by upgraded ethanol solution and stained with hematoxylin and eosin (H & E staining). The pancreas was scored for histopathology under a light microscope at 200 x magnification, with reference to the pancreas's histopathology scoring criteria.
The morphological results of the pancreas of each group of mice are shown in FIG. 1A, with the pancreas volume of the modeled group (SAP) mice increased, and the pancreas volume was significantly reduced in the groups treated with 200mg/kg of sodium mannite and 400mg/kg of sodium mannite, with 400mg/kg of sodium mannite being the most pronounced. Meanwhile, as shown in fig. 1B, in order to correspond to the statistics of the pancreatic volumes of each group in fig. 1A, it is shown that the pancreatic volume of the model building (SAP) mice is increased, and the pancreatic volume of the group treated with 200mg/kg of sodium mannite and the group treated with 400mg/kg of sodium mannite is obviously reduced, wherein the group treated with 400mg/kg of sodium mannite is most obvious, and the difference has a statistical significance, which indicates that sodium mannite can effectively reduce the pancreatic volume of severe acute pancreatitis.
The pathological damage condition of the pancreatic tissue of the mice is observed through H & E staining, and the result is shown in figure 2A, so that the pancreatic microscopic structure of the mice in the normal group is complete, and oedema, acinar necrosis, hemorrhage, fat necrosis and perivascular infiltration of inflammatory cells are not seen. Whereas the pancreas after infection by the modeling group (SAP) was seen as oedema, acinar necrosis, hemorrhage and fat necrosis, with massive inflammatory cell infiltration around the blood vessels; edema, acinar necrosis, hemorrhage, fat necrosis, perivascular infiltration of inflammatory cells were significantly reduced in the three concentrations of mannitol treatment compared to the modeling group, with 400mg/kg mannitol treatment being most pronounced. Fig. 2B is a statistical score obtained after scoring the pancreatic histopathological sections of each group of mice in fig. 2A according to the pancreatic histopathological scoring criteria. Compared with a normal control group, after the pancreas is infected by a building block (SAP), edema, acinar necrosis, hemorrhage and fat necrosis can be seen, a large number of inflammatory cells infiltrate around blood vessels, the corresponding score is obviously increased according to the pancreatic histopathological scoring standard, and the difference has statistical significance; the edema, acinar necrosis, hemorrhage, fat necrosis and perivascular infiltration of inflammatory cells of the three concentration mannite sodium treatment groups are obviously reduced compared with the modeling group, wherein 400mg/kg mannite sodium treatment group is most obvious, and the difference has statistical significance, so that the mannite sodium can effectively reduce pancreatic lesions of severe acute pancreatitis.
EXAMPLE 3 therapeutic Effect of Mante sodium
This example uses 3 random different pancreatic tissue samples from each treatment group of mice in example 2 for immunohistochemical experiments, and uses anti-Myeloperoxidase (MPO) antibodies (Abcam, ab 9535) and anti-Cluster of Differentiation (CD 68) antibodies (Servicebio, GB 113109) to evaluate the infiltration of neutrophils and mononuclear macrophages, respectively. Fresh pancreas of 3 mice was randomly picked from each group, fixed overnight with 4% paraformaldehyde, rinsed with tap water for 2 hours, gradient dehydrated with ethanol, paraffin embedded and cut into 5 μm sections. The sections were dewaxed in xylene and hydrated by upgraded ethanol solution. With 3% H 2 O 2 After blocking endogenous peroxidase activity for 8 minutes, the cells were incubated overnight with diluted (1:200) anti-MPO antibody and (1:200) anti-CD 68 antibody, developed with 3, 3-Diaminobenzidine (DAB) solution, counterstained with hematoxylin, then dehydrated, followed by scanning the slides with a pathology slide scanner to make a picture, and finally counting the area ratio of the brown-yellow staining in the picture using ImageJ software to assess MPO and CD68 positive cell mass. Serum amylase, lipase and IL6 levels were detected using the alpha-Amylase (AMS) kit (Jiancheng Biotech, C016-1), the Lipase (LPS) kit (Jiancheng Biotech, A054-2) and the mouse interleukin-6 detection kit (Neobiosccience, EMC 004.96) simultaneously, the detection steps being described in the specification.
The results are shown in fig. 3A, which shows that the proportion of yellowish brown areas in the pancreas of mice in the modeling group is significantly increased compared with the normal control group, indicating that CD68 molecule expression is significantly increased; and after the action of the mannite sodium, the brown yellow area proportion in the pancreas of the mice in the three-concentration mannite sodium treatment group is obviously reduced, which indicates that the expression of CD68 molecules is obviously reduced. Meanwhile, a statistical graph of CD68 results of IHC of the pancreas of each group of mice is shown in FIG. 3B, and the pancreas of the mice in the modeling group has a large amount of CD68 molecules expressed; and after the mannite sodium acts, the expression of the CD68 molecules of the mannite sodium treatment group with three concentrations is obviously reduced, and the difference has statistical significance, which proves that the mannite sodium can effectively improve the infiltration of mononuclear macrophages of severe acute pancreatitis.
In fig. 4A, the proportion of yellowish brown areas in the pancreas of mice in the modeling group was significantly increased compared to the normal control group, indicating that MPO molecule expression was significantly increased; and after the action of the mannite sodium, the brown yellow area proportion in the pancreas of the mice of the three-concentration mannite sodium treatment group is obviously reduced, which indicates that the MPO molecule expression is obviously reduced. Meanwhile, the MPO result statistical graph of the IHC of the pancreas of each group of mice is shown as figure 4B, and the pancreas of the modeling group of mice has a large number of MPO molecules expressed; and after the mannite sodium acts, the MPO molecular expression of the mannite sodium treatment group with three concentrations is obviously reduced, and the difference has statistical significance, so that the mannite sodium can effectively improve neutrophil infiltration of severe acute pancreatitis. Meanwhile, the study of the invention shows that 400mg/kg of mannite sodium infused in the stomach of the mice does not find any toxic effect or death.
Results for the serum biomarker levels of SAP (amylase (FIG. 5A), lipase (FIG. 5B) and IL-6 (FIG. 5C) for each group of mice are shown in FIG. 5, and the serum biomarker levels of amylase, lipase and IL-6 in the mice in the modeling group are increased compared with the serum biomarker levels in the normal control group, and the serum biomarker levels in the 400mg/kg mannite sodium treatment group are significantly decreased, so that the difference has statistical significance, and the fact that the mannite sodium can effectively improve the serum biomarker levels of severe acute pancreatitis is shown.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The application of sodium mannite in preparing medicine for treating severe acute pancreatitis.
2. The application of sodium mannite in preparing medicine for relieving severe acute pancreatitis.
3. The use according to claim 1 or 2, characterized in that the use of sodium mannite for the preparation of a medicament for reducing pancreatic volume.
4. The use according to claim 1 or 2, wherein the medicament treats or reduces severe acute pancreatitis by improving or modulating neutrophil infiltration.
5. The use according to claim 4, wherein the improvement is a reduction in the expression level of MPO.
6. The use according to claim 1 or 2, wherein the medicament treats or reduces severe acute pancreatitis by improving or modulating mononuclear macrophage infiltration.
7. The use of claim 6, wherein the improvement is a reduction in the expression level of CD 68.
8. The use according to claim 1 or 2, wherein the medicament treats or reduces severe acute pancreatitis by reducing serum amylase, lipase and IL-6 levels in severe acute pancreatitis mice.
9. The use according to claim 2, wherein the medicament reduces severe acute pancreatitis by reducing pancreatic tissue oedema, acinar necrosis, bleeding and fat necrosis, inflammatory cell perivascular infiltration.
10. The use according to claim 1 or 2, wherein the medicament is in the form of a capsule, tablet or injection.
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| WO2022067430A1 (en) * | 2020-09-30 | 2022-04-07 | The Governing Council Of The University Of Toronto | Blood-brain barrier-penetrating nanotheranostics for acute and chronic neurodegenerative diseases and the like |
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| WO2019178490A1 (en) * | 2018-03-15 | 2019-09-19 | Evelo Biosciences, Inc. | Compositions and methods for treating cancer and inflammation using klebsiella oxytoca |
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| WO2022067430A1 (en) * | 2020-09-30 | 2022-04-07 | The Governing Council Of The University Of Toronto | Blood-brain barrier-penetrating nanotheranostics for acute and chronic neurodegenerative diseases and the like |
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