CN115531370A - A method for inhibiting hepatocyte aging and promoting liver regeneration - Google Patents
A method for inhibiting hepatocyte aging and promoting liver regeneration Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to a method for inhibiting hepatocyte aging and promoting hepatocyte regeneration, which belongs to the technical field of hepatocyte regeneration, regulates a miRNA-155-5p/SIRT1/VDAC1 positive feedback loop through dihydromyricetin to promote hepatocyte regeneration and relieve hepatocyte aging, and comprises the following steps: establishing a liver injury model of a mouse; treating liver-injured mice with active dihydromyricetin to change key protein expression, and feeding for seven weeks; mice and livers were tested. The action mechanism and the molecular mechanism of the dihydromyricetin for blocking the miR-155-5p/SIRT1/VDAC1 positive feedback loop to promote the liver cell to repair and regenerate at the molecular, cell and animal levels are clarified, a new treatment target and a new strategy are provided for the research of the liver regeneration promoting medicine, and the problem that the molecular mechanism for promoting the liver regeneration to be deep by using the traditional Chinese medicine in the prior art is difficult to clarify is solved.
Description
Technical Field
The invention belongs to the technical field of liver regeneration, and particularly relates to a method for inhibiting hepatocyte aging and promoting liver regeneration and a preparation process thereof.
Background
The incidence of liver diseases is increasing year by year, and the liver diseases become an important public health problem in the world today due to alcoholism, diabetes, high blood fat, heavy weight, poor diet and habit of work and rest. Research shows that many liver disease patients have liver regeneration dysfunction, but the pathogenesis of liver regeneration involves multiple links and a complex signal regulation network, and dihydromyricetin, also called as ampicillin, is flavanonol flavone found in ampere, and the content of the flavanonol flavone in ampelopsis grossedentata can reach 30%. The active ingredients of the medicine are proved to have various pharmacological effects, have good promotion effect on liver regeneration, include anti-tumor, anti-inflammatory, anti-oxidation, anti-alcohol, anti-pathogen and blood fat regulation activities and the like, have the effect characteristics of multi-target point, multi-path, multi-level, multi-system and multi-time limit overall dynamic fine adjustment and early regulation, meet the requirements of regulating and controlling liver regeneration in various aspects and complexity, regenerate and repair the injury of viscera tissues by utilizing the natural healing capacity of the viscera tissues, and reconstruct and restore the structure and the function of the viscera tissues. However, liver regeneration is a complex biological process, the research on the regulation mechanism of liver regeneration under pathological conditions is slow, and the deep molecular mechanism of dihydromyricetin for regulating liver regeneration is difficult to elucidate.
Disclosure of Invention
The invention aims to provide a method for inhibiting hepatocyte aging and promoting liver regeneration, wherein a miRNA-155-5p/SIRT1/VDAC1 positive feedback loop is regulated and controlled by dihydromyricetin to promote liver regeneration and relieve hepatocyte aging; the operation method comprises the following steps: establishing a liver injury model of the mouse, then treating the liver injury mouse with active dihydromyricetin to change the expression of the key protein, feeding for seven weeks, and detecting the liver of the mouse. Solves the problem that the molecular mechanism of using traditional Chinese medicine to promote the deep regeneration of the liver is difficult to be clarified in the prior art.
The purpose of the invention can be realized by the following technical scheme:
a method for inhibiting hepatocyte aging and promoting liver regeneration, which regulates miRNA-155-5p/SIRT1/VDAC1 positive feedback loop through dihydromyricetin to promote liver regeneration and relieve hepatocyte aging, comprises the following steps:
(1) Establishing a liver injury model of a mouse;
weighing C57BL/6J male mice at 8-10 weeks, and pairing and grouping according to body weight, wherein each group comprises 6 mice; firstly, all the control liquid feeds are given for adaptation for 5 days; on the 6 th day, the model building group is replaced by liquid feed containing 5% alcohol solution, after 5 days of culture, the model building group is filled with 5g/kg of ethanol, and a control group is filled with equal-calorie equal-volume dextrin; after 9h, sacrifice and harvest samples; during the experiment, liquid feed was freely drunk.
(2) The liver injury mice are taken and fed with dihydromyricetin with the mass of 75 mg/kg and 150mg/kg respectively, the mice are intervened by the same dose every day, and the mice are fed for 7 weeks.
(3) Mice and livers were tested:
a1, injecting 10% chloral hydrate into abdominal cavities of mice treated by different doses of dihydromyricetin for anesthesia, opening the abdominal cavities, taking livers, weighing, calculating liver indexes, taking a part of livers, storing the part of livers in fresh methanol saline, cutting the parts into slices with the thickness of about 2mm by using a single-side cutter, dehydrating the slices by using 70%, 95% and 100% gradient ethanol solutions, and replacing the slices in a tissue cleanser for 3 times every 4 hours until the slices are transparent. Then immersing the slices in melted paraffin for embedding to obtain paraffin sections;
a2, taking blood from the abdominal aorta, carrying out water bath at 37 ℃, preserving heat for 30min, taking out the abdominal aorta, placing the abdominal aorta in a refrigerator at 4 ℃ for 30min, centrifuging the abdominal aorta for IOmin, and separating serum for determination;
a3, washing the tissue by using precooled PBS, weighing and shearing the tissue into pieces; placing the minced tissue and PBS buffer solution into a glass homogenizer, adding protease inhibitor, and grinding on dry ice; carrying out ultrasonic disruption on the homogenate, centrifuging for 20 minutes, taking a supernatant, and measuring the tissue homogenate;
further, the detection indexes for detecting the mouse liver are as follows: biochemical and case indexes, cell aging indexes, liver regeneration indexes and miRNA-155-5p/SIRT1/VDAC1 positive feedback loop mechanism indexes.
Further, in the step A3, the mass-to-volume ratio of the minced tissue to the PBS buffer is 1g.
Further, in the step A3, the mass ratio of the protease inhibitor to the minced tissue is 1 μ L:1g.
The invention has the beneficial effects that:
in the technical scheme of the invention, an alcohol liquid feed is adopted to prepare a mouse liver injury model, biochemical and case indexes, a cell aging index, a liver regeneration index and miRNA-155-5p/SIRT1/VDAC1 positive feedback loop mechanism indexes are measured, the action mechanism and the molecular mechanism of the dihydromyricetin blocking miR-155-5p/SIRT1/VDAC1 positive feedback loop promoting liver cell repair and regeneration in the levels of molecules, cells and animals are systematically elucidated through serology, histopathology and molecular biology by combining methods such as in-vitro cell experiments and bifluorescin reporter genes, and a new treatment target and strategy are provided for liver regeneration medicine research promotion.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method of inhibiting hepatocyte senescence and promoting liver regeneration, comprising the steps of:
(1) Establishing a liver injury model of a mouse:
weighing C57BL/6J male mice at 8-10 weeks, and pairing and grouping according to body weight, wherein each group comprises 6 mice; firstly, all the control liquid feeds are given for adaptation for 5 days; on the 6 th day, the model building group is replaced by liquid feed containing 5% alcohol solution, after 5 days of culture, the model building group is filled with 5g/kg of ethanol, and a control group is filled with equal-calorie equal-volume dextrin; after 9h, sacrifice and harvest samples; during the experiment, liquid feed was freely drunk.
(2) The liver injury mice are taken and fed with dihydromyricetin with the mass of 75 mg/kg and 150mg/kg respectively, the mice are intervened by the same dose every day, and the mice are fed for 7 weeks.
(3) The method for detecting the mouse and the liver comprises the following steps:
a1, injecting 10% chloral hydrate into abdominal cavities of mice treated by different doses of dihydromyricetin for anesthesia, opening the abdominal cavities, taking livers, weighing, calculating liver indexes, taking a part of livers, storing the part of livers in fresh methanol saline, cutting the parts into slices with the thickness of about 2mm by using a single-side cutter, dehydrating the slices by using 70%, 95% and 100% gradient ethanol solutions, and replacing the slices in a tissue cleanser for 3 times every 4 hours until the slices are transparent. Then immersing the slices in melted paraffin for embedding to obtain paraffin sections;
a2, taking blood from an abdominal aorta, carrying out water bath at 37 ℃, preserving heat for 30min, taking out a refrigerator at 4 ℃, standing for 30min, centrifuging for IOmin, and separating serum for determination;
a3, washing the tissue by using precooled PBS, weighing and then shearing the tissue into pieces; placing 2g of minced tissue and 20ml of PBS buffer solution together into a glass homogenizer, adding 2. Mu.L of protease inhibitor, and grinding on dry ice; carrying out ultrasonic disruption on the homogenate, centrifuging for 20 minutes, taking a supernatant, and measuring the tissue homogenate;
example 2
A method of inhibiting hepatocyte senescence and promoting liver regeneration, comprising the steps of:
(1) Establishing a liver injury model of a mouse:
weighing C57BL/6J male mice at 8-10 weeks, and pairing and grouping according to body weight, wherein each group comprises 6 mice; firstly, all the control liquid feeds are given for adaptation for 5 days; on the 6 th day, the model building group is replaced by liquid feed containing 5% alcohol solution, after 5 days of culture, the model building group is perfused with stomach and is given 5g/kg of ethanol, and the control group is given equal-calorie equal-volume dextrin; after 9h, sacrifice and sample collection; during the experiment, liquid feed was freely drunk.
(2) The liver injury mice are taken and fed with dihydromyricetin with the mass of 150mg/kg respectively, the mice are intervened by the same dose every day, and the mice are raised for 7 weeks.
(3) The method for detecting the mouse and the liver comprises the following steps:
a1, injecting 10% chloral hydrate into abdominal cavities of mice treated by different doses of dihydromyricetin for anesthesia, opening the abdominal cavities, taking livers, weighing, calculating liver indexes, taking a part of livers, storing the part of livers in fresh methanol saline, cutting the parts into slices with the thickness of about 2mm by using a single-side cutter, dehydrating the slices by using 70%, 95% and 100% gradient ethanol solutions, and replacing the slices in a tissue cleanser for 3 times every 4 hours until the slices are transparent. Then immersing the slices in melted paraffin for embedding to obtain paraffin sections;
a2, taking blood from an abdominal aorta, carrying out water bath at 37 ℃, preserving heat for 30min, taking out a refrigerator at 4 ℃, standing for 30min, centrifuging for IOmin, and separating serum for determination;
a3, washing the tissue by using precooled PBS, weighing and shearing the tissue into pieces; placing 2g of minced tissue together with 20ml of PBS buffer into a glass homogenizer, adding 2. Mu.L of protease inhibitor, and grinding on dry ice; carrying out ultrasonic crushing on the homogenate, centrifuging for 20 minutes, taking supernatant, and measuring the tissue homogenate;
comparative example 1
A method of inhibiting hepatocyte senescence and promoting liver regeneration, comprising the steps of:
(1) Establishing a liver injury model of a mouse:
weighing C57BL/6J male mice at 8-10 weeks, and grouping by pairs according to body weight, wherein each group contains 6 mice; firstly, control liquid feed is given for 5 days; on the 6 th day, the model building group is replaced by liquid feed containing 5% alcohol solution, after 5 days of culture, the model building group is filled with 5g/kg of ethanol, and a control group is filled with equal-calorie equal-volume dextrin; after 9h, sacrifice and harvest samples; during the experiment, liquid feed was freely drunk.
(2) Liver-injured mice were taken, treated with deionized water, and fed for 7 weeks.
(3) The method for detecting the mouse and the liver comprises the following steps:
a1, mice treated by different doses of dihydromyricetin are respectively subjected to intraperitoneal injection of 10% chloral hydrate for anesthesia, the abdominal cavity is opened, livers are taken out, the liver indexes are calculated, a part of the livers are taken out, stored in fresh methanol saline, cut into slices with the thickness of about 2mm by using a single-sided scalpel, dehydrated by using 70%, 95% and 100% gradient ethanol solutions, and replaced for 3 times every 4 hours in a tissue cleanser until the slices are transparent. Then immersing the slices in melted paraffin for embedding to obtain paraffin sections;
a2, taking blood from an abdominal aorta, carrying out water bath at 37 ℃, preserving heat for 30min, taking out a refrigerator at 4 ℃, standing for 30min, centrifuging for IOmin, and separating serum for determination;
a3, washing the tissue by using precooled PBS, weighing and then shearing the tissue into pieces; placing 2g of minced tissue and 20ml of PBS buffer solution together into a glass homogenizer, adding 2. Mu.L of protease inhibitor, and grinding on dry ice; carrying out ultrasonic crushing on the homogenate, centrifuging for 20 minutes, taking supernatant, and measuring the tissue homogenate;
comparative example 2
A method of inhibiting hepatocyte senescence and promoting liver regeneration, comprising the steps of:
(1) Establishing a liver injury model of a mouse:
weighing C57BL/6J male mice at 8-10 weeks, and pairing and grouping according to body weight, wherein each group comprises 6 mice; firstly, control liquid feed is given for 5 days; on the 6 th day, the model building group is replaced by liquid feed containing 5% alcohol solution, after 5 days of culture, the model building group is filled with 5g/kg of ethanol, and a control group is filled with equal-calorie equal-volume dextrin; after 9h, sacrifice and harvest samples; during the experiment, liquid feed was freely drunk.
(2) Liver-injured mice were treated with 30% ethanol and fed for 7 weeks.
(3) The method for detecting the mouse and the liver comprises the following steps:
a1, injecting 10% chloral hydrate into abdominal cavities of mice treated by different doses of dihydromyricetin for anesthesia, opening the abdominal cavities, taking livers, weighing, calculating liver indexes, taking a part of livers, storing the part of livers in fresh methanol saline, cutting the parts into slices with the thickness of about 2mm by using a single-side cutter, dehydrating the slices by using 70%, 95% and 100% gradient ethanol solutions, and replacing the slices in a tissue cleanser for 3 times every 4 hours until the slices are transparent. Then immersing the slices in melted paraffin for embedding to obtain paraffin sections;
a2, taking blood from the abdominal aorta, carrying out water bath at 37 ℃, preserving heat for 30min, taking out the abdominal aorta, placing the abdominal aorta in a refrigerator at 4 ℃ for 30min, centrifuging the abdominal aorta for IOmin, and separating serum for determination;
a3, washing the tissue by using precooled PBS, weighing and then shearing the tissue into pieces; placing 2g of minced tissue and 20ml of PBS buffer solution together into a glass homogenizer, adding 2. Mu.L of protease inhibitor, and grinding on dry ice; carrying out ultrasonic crushing on the homogenate, centrifuging for 20 minutes, taking supernatant, and measuring the tissue homogenate;
the sera, tissues and homogenates of the livers of the mice in examples 1-2 and comparative examples 1-2 were now examined.
The levels of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and total cholesterol (TG) in the mouse serum were determined using a full-automatic biochemical analyzer. The results are shown in table 1:
from the results in table 1, dihydromyricetin can significantly reduce the content of ATL, AST and TG in the serum of liver-injured mice, and keep the liver healthy.
The degree of senescence of the liver cells in examples 1-2 and comparative examples 1-2 was now tested. Fresh liver tissues are fixed in 4% paraformaldehyde for 4 hours, then placed in a staining box, added with beta-galactosidase staining solution, shaken in a shaking table at 37 ℃ at a low speed for 24 hours, then the tissues are embedded with paraffin, sliced by a slicer, baked, dewaxed by xylene, dehydrated by gradient alcohol, stained with eosin staining solution containing 0.2% glacial acetic acid for 2 minutes, dehydrated, transparent, mounted, and the activity of the beta-galactosidase in the hepatocytes is observed under a microscope.
The results are shown in table 2:
| group of | Proportion of senescent cells (%) |
| Example 1 | 0.28 |
| Example 2 | 0.32 |
| Comparative example 1 | 0.21 |
| Comparative example 2 | 0.22 |
From the results in Table 2, it can be seen that senescent cells can express beta-galactosidase, alcohol can cause the reduction of the division ability of hepatocytes, and dihydromyricetin can inhibit the senescence of hepatocytes by comparison with that after dihydromyricetin treatment. The cell counting plate finds that the sample treated by the dihydromyricetin has more cell increment, and can promote the growth of the liver cells, thereby promoting the liver regeneration.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is illustrative and explanatory only of the present invention, and it is intended that the present invention cover modifications, additions, or substitutions by those skilled in the art, without departing from the spirit of the invention or exceeding the scope of the claims.
Claims (7)
1. A method for inhibiting hepatocyte aging and promoting liver regeneration is characterized in that a miRNA-155-5p/SIRT1/VDAC1 positive feedback loop is regulated and controlled by dihydromyricetin to promote liver regeneration and relieve hepatocyte aging, and comprises the following steps:
(1) Establishing a liver injury model of a mouse;
(2) Treating liver injury mice with active dihydromyricetin to change key protein expression, and feeding for 7 weeks;
(3) Mice and livers were tested.
2. The method of claim 1, wherein the liver injury model of the mouse is established by:
weighing C57BL/6J male mice at 8-10 weeks, and pairing and grouping according to body weight, wherein each group comprises 6 mice; firstly, all the control liquid feeds are given for adaptation for 5 days; on the 6 th day, the model building group is replaced by liquid feed containing 5% alcohol solution, after 5 days of culture, the model building group is perfused with stomach and is given 5g/kg of ethanol, and the control group is given equal-calorie equal-volume dextrin; after 9h, sacrifice and sample collection; during the experiment, liquid feed was freely drunk.
3. The method of inhibiting hepatocyte senescence and promoting liver regeneration according to claim 1, wherein: liver-injured mice were treated with active dihydromyricetin to alter key protein expression, and the specific procedure for seven weeks of feeding was:
the liver injury mice are taken and fed with dihydromyricetin with the mass of 75 mg/kg and 150mg/kg respectively, the mice are intervened by the same dose every day, and the mice are fed for 7 weeks.
4. The method of claim 1, wherein the liver of the mouse is tested by the following test indexes: biochemical and case indexes, cell aging indexes, liver regeneration indexes and miRNA-155-5p/SIRT1/VDAC1 positive feedback loop mechanism indexes.
5. The method for inhibiting aging of liver cells and promoting liver regeneration as claimed in claim 4, wherein the mouse and liver are tested by:
a1, mice treated by different doses of dihydromyricetin are respectively subjected to intraperitoneal injection of 10% chloral hydrate for anesthesia, the abdominal cavity is opened, livers are taken out, the liver indexes are calculated, a part of the livers are taken out, stored in fresh methanol saline, cut into slices with the thickness of about 2mm by using a single-sided scalpel, dehydrated by using 70%, 95% and 100% gradient ethanol solutions, and replaced for 3 times every 4 hours in a tissue cleanser until the slices are transparent. Then immersing the slices in melted paraffin for embedding to obtain paraffin sections;
a2, taking blood from an abdominal aorta, carrying out water bath at 37 ℃, preserving heat for 30 minutes, taking out a refrigerator at 4 ℃, standing for 30 minutes, and centrifuging for IOmin to obtain mouse serum;
a3, washing the tissue by using precooled PBS, weighing and shearing the tissue into pieces; placing the minced tissue and PBS buffer solution into a glass homogenizer, adding protease inhibitor, and grinding on dry ice; and (3) carrying out ultrasonic disruption on the homogenate, centrifuging for 20 minutes, and taking a supernatant to obtain a tissue homogenate.
6. The method for inhibiting aging of liver cells and promoting regeneration of liver as claimed in claim 5, wherein in the step A3, the mass volume ratio of the minced tissue to the PBS buffer is 1g.
7. The method of claim 5, wherein the ratio of protease inhibitor to minced tissue in step A3 is 1 μ L:1g.
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| CN103751173A (en) * | 2014-01-06 | 2014-04-30 | 广东医学院附属医院 | Application of dihydromyricetin in preparation of liver regeneration medicine |
| CN111494359A (en) * | 2020-04-29 | 2020-08-07 | 上海爱启医药技术有限公司 | Dihydromyricetin with alcohol effect dispelling function |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103751173A (en) * | 2014-01-06 | 2014-04-30 | 广东医学院附属医院 | Application of dihydromyricetin in preparation of liver regeneration medicine |
| CN111494359A (en) * | 2020-04-29 | 2020-08-07 | 上海爱启医药技术有限公司 | Dihydromyricetin with alcohol effect dispelling function |
Non-Patent Citations (1)
| Title |
|---|
| 苏东林等: "二氢杨梅素的急性毒理学评价及对酒精性 肝损伤的防治效果", 《湖南农业科学》, no. 11, pages 90 - 93 * |
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