CN110585216A - Application of lupeol in preparation of medicine for preventing or treating liver injury - Google Patents
Application of lupeol in preparation of medicine for preventing or treating liver injury Download PDFInfo
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- CN110585216A CN110585216A CN201910828894.6A CN201910828894A CN110585216A CN 110585216 A CN110585216 A CN 110585216A CN 201910828894 A CN201910828894 A CN 201910828894A CN 110585216 A CN110585216 A CN 110585216A
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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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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Abstract
The invention discloses an application of lupeol in preparing a medicine for preventing or treating liver injury, and the application shows that in a zebra fish liver injury model induced by LPS, lupeol can increase the liver area of zebra fish caused by LPS, and H & E staining shows that the lupeol can relieve liver cell degeneration and swelling; TUNEL staining showed lupeol to reduce hepatocyte apoptosis; in an LPS stimulated RAW264.5 in vitro cell model, lupeol shows anti-inflammatory activity, has functions of reducing INOS level and reducing TNF-alpha level; thus lupeol restored LPS-induced liver damage.
Description
Technical Field
The invention belongs to the field of medicines, and relates to application of lupeol in preparation of a medicine for preventing or treating liver injury.
Background
Acute Liver Injury (ALI) threatens human health. Infiltration of mononuclear phagocytes into the liver is associated with the severity of liver injury during which resident macrophages, known as Kupffer Cells (KCs), participate in the initial stress response and are widely recognized as one of the major cell types responsible for ALI, releasing various metabolites that cause cell damage, including superoxide radicals, nitric oxide, eicosanoids, proteases, and pro-inflammatory cytokines, among others. Lipopolysaccharide (LPS) can cause KCs to significantly secrete pro-inflammatory mediators and ultimately endotoxin-induced liver damage.
Lupeol (Lupeol) belongs to the pentacyclic terpene type triterpenes, which are present in edible vegetables, fruits and many plants. Molecular formula C30H50O, molecular weight 426.72, structural formula as follows:
many studies have shown that lupeol has many beneficial pharmacological activities, including antioxidant, anti-inflammatory, antihyperglycemic, antilipemic and anti-cancer effects. From various animal models targeting diseases, these reports indicate that lupeol has antidiabetic, antiasthmatic, antiarthritic, cardioprotective, hepatoprotective, nephroprotective, neuroprotective and anticancer effects in various routes of administration such as topical, oral, subcutaneous, intraperitoneal.
Disclosure of Invention
The invention aims to provide application of lupeol in preparation of a medicine for preventing or treating liver injury.
The purpose of the invention is realized as follows: application of lupeol in preparing medicine for preventing or treating liver injury is provided.
The liver injury is liver injury caused by lipopolysaccharide.
The medicine consists of active ingredient lupeol and pharmaceutic adjuvant.
The dosage form of the medicine is oral dosage form or injection dosage form.
Use of lupeol in the manufacture of a medicament for reducing the level of INOS expression and the level of TNF-a expression in macrophages.
The invention shows that in a zebra fish liver injury model induced by LPS, lupeol can increase the liver area of zebra fish caused by LPS, and H & E staining shows that lupeol can relieve hepatocyte degeneration and enlargement; TUNEL staining showed lupeol to reduce hepatocyte apoptosis; in an in vitro cell model of RAW264.5 stimulated by LPS, lupeol shows anti-inflammatory activity, has functions of reducing INOS level and reducing TNF-alpha level, so that lupeol recovers liver injury caused by LPS and can be used as a medicine for treating liver injury.
Drawings
FIG. 1 shows the liver morphology of the zebrafish embryo Control group (Control), Model group (Model) and administration group of example 1.
FIG. 2 shows the results of H & E pathological staining of the zebra fish embryo of example 1 in the Control group (Control), Model group (Model), DMSO group, and administration group.
FIG. 3 shows the TUNEL staining results of the zebrafish embryo Control (Control), Model (Model) and administration groups of example 1.
FIGS. 4 and 5 are graphs showing the effect of lupeol from example 1 on INOS expression in RAW264.7 cells.
FIG. 6 is the effect of lupeol from example 1 on ITNF- α expression in RAW264.7 cells.
Detailed Description
The invention relates to an application of lupeol in preparing a medicine for preventing or treating liver injury. Preferably, the liver damage is liver damage caused by LPS. The medicine consists of active ingredient lupeol and pharmaceutic adjuvant. The medicament can be prepared into oral dosage forms or injection dosage forms. Preferably, lupeol is used in the manufacture of a medicament for reducing the level of INOS expression and TNF-a expression in macrophages.
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1
1. Experimental materials:
lupeol (purchased from Shanghai leaf company, purity is more than or equal to 98%), transgenic zebra fish [ Tg (lfabp10 alpha-eGFP) ] zebra fish, AB strain wild adult zebra fish, RAW264.7 macrophage line, TUNEL reagent detection kit
2. The experimental method comprises the following steps:
2.1 in vivo zebrafish model: selecting and using wild fishes of AB strain in advance according to a male and female ratio of 1:1 or 2:1, respectively pairing in a mating box, isolating by using a partition plate, and taking out the partition plate for mating on the next day; the following day, fish eggs were collected, and the collected 10hpf fish eggs were randomly divided into a control group, a model group, a DMSO group, a low concentration administration group, a medium concentration administration group, and a high concentration administration group, and placed in six-well plates with 30 eggs/well for each group. Lupeol was dissolved in DMSO, and lupeol was added to each of the low concentration administration group, the medium concentration administration group, and the high concentration administration group in an amount of 25 μ M, 50 μ M, and 100 μ M, respectively, and after 1 hour of administration, LPS was added to each of the model group, DMSO group, the low concentration administration group, the medium concentration administration group, and the high concentration administration group in an amount of 10 μ g/mL, and cultured in a constant temperature incubator at 28.5 ℃ to 72hpf, thereby completing molding and collecting zebra fish embryos.
And respectively collecting the transgenic zebra fish [ Tg (lfabp10 alpha-eGFP) ] zebra fish embryos of the normal liver specifically expressing fluorescent protein without being processed by the LPS in the control group and the zebra fish embryos in the model group, photographing by using a fluorescence microscope with the model of OLYMPUS U-HGLGPS, and comparing the sizes of the livers in the blank group and the model group.
Collecting normal zebra fish embryos not subjected to LPS treatment in a control group and zebra fish embryos in a model group respectively, fixing the normal zebra fish embryos not subjected to LPS treatment in a Paraformaldehyde (PFA) with the mass concentration of 4%, collecting transgenic zebra fish [ Tg (lfabp10 alpha-eGFP) ] zebra fish embryos specifically expressing fluorescent protein in normal livers not subjected to LPS treatment in the control group and the zebra fish embryos in the model group respectively overnight in a refrigerator at 4 ℃, taking pictures by using a fluorescent microscope with the model of OLYMPUS U-HGLGPS, and comparing the sizes of the livers in the blank group and the model group. . Dehydrating with 80% ethanol for 2 hours, dehydrating with 90% ethanol for 2 hours, standing with 95% ethanol overnight the next day, dehydrating with 100% ethanol I for 0.5 hour, dehydrating with 100% ethanol II for 0.5 hour, dehydrating with 100% ethanol III for 0.5 hour, respectively processing with xylene for 0.5 hour, soaking with paraffin I for 0.5 hour, soaking with paraffin II for 0.5 hour, and soaking with paraffin III for 1 hour. The zebrafish embryos were taken out of paraffin, placed in an embedding box, poured into paraffin, embedded, and then sectioned on a microtome with the slice thickness set to 4 μm. The cut thin sheet was gently placed in a water bath at 60 ℃ and, after being completely developed, it was transferred to a coated slide glass and dried in a thermostat at 60 ℃ for about 2 hours.
H & E staining: the paraffin sections prepared above were dewaxed for 3 minutes respectively according to xylene I, II and III, absolute ethyl alcohol I, II for 2 minutes respectively, 95% ethyl alcohol, 90% ethyl alcohol and 80% ethyl alcohol for 1 minute respectively, hematoxylin staining for 10 minutes, after water washing, 1% hydrochloric acid alcohol was differentiated for 15 seconds, immersed in water for 10 minutes, eosin staining for 3 minutes, 80% ethyl alcohol and 90% ethyl alcohol for 10 seconds respectively, 95% ethyl alcohol for 1 to 2 minutes, absolute ethyl alcohol I, II and III for 3 minutes respectively, xylene I, II and III for 3 minutes respectively, finally, neutral gum was sealed, and photographed under a microscope.
TUNEL staining: the paraffin sections prepared above were first dewaxed (8 minutes each for xylene I, II, 5 minutes each for anhydrous ethanol I, II, 3 minutes each for 95% ethanol, 90% ethanol, 80% ethanol), washed 3 times with PBS, 5 minutes each, spin-dried, incubated in an oven at 37 ℃ for 1 hour in the dark at 1:10TUNEL, washed 3 times with PBS, 5 minutes each, dyed with DAPI for 10 minutes, washed 3 times with PBS, 5 minutes each, and finally sealed with an anti-fluorescence quencher and photographed under a fluorescence microscope.
2.2 in vitro cell model: the macrophage line RAW264.7 was cultured in high-sugar medium containing 10% FBS,100U/mL streptomycin and 100U/mL penicillin at 37 ℃ with 5% CO2And (5) incubation in an incubator. RAW264.5 cells were seeded in a six-well plate and divided into a control group, a model group, a DMSO group, a low concentration administration group, a medium concentration administration group and a high concentration administration group, and the low concentration administration group, the medium concentration administration group and the high concentration administration group were added with lupeol at 25. mu.M, 50. mu.M and 100. mu.M in this order, respectively, and administered for 6 hours. Model group, DMSO group, low concentration administration group, medium concentration administration group and high concentration administration group, wherein 1 μ g/mL LPS was added to the low concentration administration group, the medium concentration administration group and the high concentration administration group in this order, and 5% CO was added at 37 deg.C2And (5) continuously incubating for 18 hours in the incubator, finishing molding and collecting cells.
The 6 groups of cells were collected, washed 3 times with PBS for 5 minutes each, and the cells were fixed with 4% paraformaldehyde overnight at 4 ℃. The following day, PBS wash 3 times, 5 minutes each time, 0.1% Triton-X100 PBS through 10 minutes, PBS wash 3 times, 5 minutes each time, 5% goat serum (PBS + 5% goat serum + 0.1% Triton-X100) blocked for 2 hours at room temperature, incubated with 1:200INOS antibody/1: 200TNF- α antibody, respectively, and overnight at 4 ℃. The next day, PBS wash 3 times, 5 minutes each time, 1:400 fluorescent secondary antibody incubation for 2 hours at normal temperature, PBS wash 3 times, 5 minutes each time, DAPI staining for 5 minutes, PBS wash 3 times, 5 minutes each time, anti-fluorescent extraction and sterilization agent sealing, and fluorescence microscope photographing.
The 6 groups of cells were collected, washed 3 times with PBS for 5 minutes each, lysed with 2% phosphatase inhibitor and 2% protease inhibitor for 30 minutes on ice, scraped, centrifuged at 12000rpm at 4 ℃ for 15 minutes. Taking supernatant, carrying out protein quantification by BCA, adding 5X Loading Buffering, carrying out denaturation at 98 ℃ for 10 minutes, and storing in a refrigerator at 80 ℃.
Western blot analysis: taking 10 mu L of sample, carrying out gel electrophoresis step by step and transferring the sample to a PVDF membrane; subsequently, the membrane was blocked with 5% skim milk at room temperature for 2 hours; the target protein on the PVDF membrane and the primary antibody are incubated at 4 ℃ overnight; after washing three times with TBS-T buffer, the PVDF membrane was subsequently incubated with rabbit Ig G antibody as secondary antibody, and after 2 hours washed three times with TBS-T buffer.
3. The experimental results are as follows:
FIG. 1 is a graph showing the effect of lupeol on the liver of zebrafish according to the invention. The result shows that the reduction of the zebra fish embryonic liver caused by LPS can be reduced.
FIG. 2 shows the H & E pathological staining results of the zebra fish embryo control group, the model group and the administration group. The result shows that the liver tissue structure of the zebra fish in the control group is normal, and the liver cells are arranged regularly; the LPS treatment group can see structural disorder of liver tissues, and the liver cells can see swelling, degeneration and a small amount of vacuoles. The liver tissue structure of the zebra fish in the medium and high concentration administration groups is normal, and the liver cells are arranged regularly. And the preferred concentration of lupeol is 100 μ M.
FIG. 3 shows TUNEL staining of blank groups and model groups of TUNEL staining results of a zebra fish embryo control group, a model group and a dosing group, and shows that the apoptosis of zebra fish liver cells of the model group is obviously increased compared with the apoptosis of zebra fish liver cells of the control group, and the apoptosis of zebra fish liver cells of a medium-concentration and high-concentration dosing groups is obviously reduced. And the preferred concentration of lupeol is 100 μ M.
FIGS. 4 and 5 show the effect of lupeol on INOS expression in RAW264.7 cells. The result shows that compared with a normal control group, the expression of INOS of the cells in the model group is obviously increased, and the expression level of INOS can be obviously reduced in a medium-concentration administration group and a high-concentration administration group.
FIG. 6 shows the effect of lupeol on ITNF- α expression in RAW264.7 cells. The results show that compared with the normal group, the expression of the tumor necrosis factor (TNF-a) of the cells in the model group is obviously increased, and the expression level of the TNF-a can be obviously reduced by the RAW264.7 cells treated by lupeol. Normal levels of TNF-a in the body can modulate immune responses, resist infection, etc., but when expressed in large quantities, it can disrupt the immune balance of the body, producing various pathological lesions with other inflammatory factors. Lupeol significantly reduced the elevated expression of TNF-a by LPS, and the preferred concentration of lupeol was 100 μ M.
Claims (5)
1. Application of lupeol in preparing medicine for preventing or treating liver injury is provided.
2. The use of claim 1, wherein: the liver injury is liver injury caused by lipopolysaccharide.
3. The use of claim 1, wherein: the medicine consists of active ingredient lupeol and pharmaceutic adjuvant.
4. The use of claim 1, wherein: the dosage form of the medicine is oral dosage form or injection dosage form.
5. Use of lupeol in the manufacture of a medicament for reducing the level of INOS expression and the level of TNF-a expression in macrophages.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910828894.6A CN110585216A (en) | 2019-09-03 | 2019-09-03 | Application of lupeol in preparation of medicine for preventing or treating liver injury |
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| CN201910828894.6A CN110585216A (en) | 2019-09-03 | 2019-09-03 | Application of lupeol in preparation of medicine for preventing or treating liver injury |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111000849A (en) * | 2019-12-19 | 2020-04-14 | 淮阴师范学院 | Application and product of lupeol DYC-6 |
| CN112076237A (en) * | 2020-09-21 | 2020-12-15 | 海南医学院 | Extraction process, optimization method and application of triterpenoids in Eyeichhornia crassipes |
| CN115607554A (en) * | 2022-12-01 | 2023-01-17 | 贵州中医药大学 | Application of lupulone in preparation of product for preventing and treating heart, liver and spleen injuries |
-
2019
- 2019-09-03 CN CN201910828894.6A patent/CN110585216A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| SO-JIN KIM等: "Protective effects of lupeol against D-galactosamine and lipopolysaccharide-induced fulminant hepatic failure in mice", 《JOURNAL OF NATURAL PRODUCTS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111000849A (en) * | 2019-12-19 | 2020-04-14 | 淮阴师范学院 | Application and product of lupeol DYC-6 |
| CN112076237A (en) * | 2020-09-21 | 2020-12-15 | 海南医学院 | Extraction process, optimization method and application of triterpenoids in Eyeichhornia crassipes |
| CN112076237B (en) * | 2020-09-21 | 2022-03-11 | 海南医学院 | Extraction process, optimization method and application of triterpenoids in Eyeichhornia crassipes |
| CN115607554A (en) * | 2022-12-01 | 2023-01-17 | 贵州中医药大学 | Application of lupulone in preparation of product for preventing and treating heart, liver and spleen injuries |
| CN115607554B (en) * | 2022-12-01 | 2023-09-05 | 贵州中医药大学 | Application of lupeone in preparation of products for preventing and treating heart, liver and spleen injuries |
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Application publication date: 20191220 |