CN111840578A - Application of ELOVL6 enzyme inhibitor in acute graft-versus-host disease - Google Patents
Application of ELOVL6 enzyme inhibitor in acute graft-versus-host disease Download PDFInfo
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- CN111840578A CN111840578A CN202010716261.9A CN202010716261A CN111840578A CN 111840578 A CN111840578 A CN 111840578A CN 202010716261 A CN202010716261 A CN 202010716261A CN 111840578 A CN111840578 A CN 111840578A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0325—Animal model for autoimmune diseases
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Abstract
The invention discloses application of an ELOVL6 enzyme inhibitor in acute graft-versus-host disease (aGVHD). The effect of the enzyme inhibitor ELOVL6 on the generation of aGVHD after allogeneic hematopoietic stem cell transplantation of mice receiving high stearic acid diet is studied by using mice receiving high stearic acid diet for 4 weeks as study objects and using a mouse aGVHD model, and the result shows that the ELOVL6 enzyme inhibitor can obviously reduce the incidence rate and the severity of acute graft-versus-host disease (aGVHD) of the mice receiving high stearic acid diet.
Description
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to application of an ELOVL6 enzyme inhibitor in acute graft-versus-host disease (aGVHD).
Background
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most effective methods for clinically treating hematological malignancies such as leukemia at present, but occurrence of acute Graft-versus-Host Disease (aGVHD) after transplantation seriously affects survival and prognosis of patients. Abnormal lipid metabolism is closely related to the immune system and may affect the development of aGVHD. Stearic acid and palmitic acid are two saturated fatty acids common in the human body, the former being octadecanoic acid and the latter hexadecanoic acid. Palmitic acid is converted into stearic acid in our body by supersaturated fatty acid elongases, stearic acid being the principal substance of change. The enzyme, the very long-chain fatty acid elongase 6 (ELOVL 6), is a common important enzyme involved in fatty acid synthesis and belongs to one of the members of the endoplasmic reticulum enzyme family that is highly conserved and involved in the formation of long-chain fatty acids. Therefore, the research on the influence of abnormal lipid metabolism on the generation and development of aGVHD has important scientific significance and clinical value.
Disclosure of Invention
The invention aims to provide application of an ELOVL6 enzyme inhibitor in acute graft-versus-host disease (aGVHD), which takes a mouse receiving high-stearic acid diet for 4 weeks as a research object, and utilizes a mouse aGVHD model to research the influence of the ELOVL6 enzyme inhibitor on the generation of aGVHD after the mouse receiving high-stearic acid diet is subjected to allogeneic hematopoietic stem cell transplantation.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of an ELOVL6 enzyme inhibitor in a mouse acute graft-versus-host disease (aGVHD) model receiving high-stearic acid diet.
Preferably, the application comprises the application of an ELOVL6 enzyme inhibitor in improving the generation of aGVHD of mice fed with high stearic acid, reducing the mortality rate of aGVHD and simultaneously reducing the damage of the target organs of the mice caused by the aGVHD.
Preferably, the ELOVL6 enzyme inhibitor is administered at a dose of 30 mg/kg/d.
Preferably, the mice receive a high stearic acid diet beginning 4 weeks prior to allogeneic hematopoietic stem cell transplantation and continuing until the end of aGVHD model observation, and the ELOVL6 enzyme inhibitor begins to intervene until the day of transplantation, beginning 3 weeks prior to allogeneic hematopoietic stem cell transplantation.
Preferably, the formula of the high stearic diet/kg is: 287.5g of corn starch, 200g of casein, 132g of dextrin corn starch, 100g of cane sugar, 150g of lard, 30g of soybean oil, 50g of fiber, 35g of mixed mineral, 10g of mixed vitamin, 3g of L-cystine, 2.5g of choline tartrate and 4492.9kcal of energy.
Preferably, the mouse acute graft versus host disease (aGVHD) model is established by: BALB/c (H-2)d) The recipient mice received 650cGy total body lethal irradiation, 4 hours later C57BL/6 (H-2) was infused by tail vein injectionb) Bone marrow cells and whole spleen cells from mice establish a model of acute graft-versus-host disease (aGVHD) of the mice.
Preferably, the whole splenocytes can also be from IL-27-/-(H-2b) A mouse.
Preferably, each mouse is infused with 1 × 107Bone marrow cells and 5X 106And (4) all splenocytes.
More preferably, the concentration of the bone marrow cells is 1X 107The concentration of the whole spleen cells is 5X 10 per 100 μ l6One by 100. mu.l.
The invention also provides application of the ELOVL6 enzyme inhibitor in preparing a medicament for treating acute graft-versus-host disease (aGVHD).
The invention has the following beneficial effects:
according to the invention, through a mouse aGVHD model, researches show that after an ELOVL6 enzyme inhibitor is drenched, the generation of the mouse aGVHD with high stearic acid diet can be obviously improved, the ELOVL6 enzyme inhibitor shows that the death rate of the aGVHD is reduced, and the effect of reducing the damage of the mouse target organ caused by the aGVHD is simultaneously relieved, so that the model can be used as a potential medicament for improving the generation rate and the severity of the aGVHD caused by the high stearic acid diet.
Drawings
FIG. 1 is a graph showing the survival curve of mice after receiving allogeneic hematopoietic stem cell transplantation in the example of the present invention.
FIG. 2 weight and aGVHD score of mice after receiving allogeneic hematopoietic stem cell transplantation in the examples of the invention. A. Body weight, b.agvhd score.
FIG. 3 pathological changes in target organs that occur in aGVHD after mice have been transplanted with allogeneic hematopoietic stem cells in an example of the invention.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Examples
Materials and methods
1.1 Experimental animals
6-8 week-old SPF grade C57BL/6 mouse (H-2)bC, 6-8 week old SPF-grade BALB/c mouse (H-2)dAnd (female), the weight is 16-25 g, and the certificate number SCXK (Shanghai Si) 2003-0003 is provided by Shanghai Si Laike experimental animal center. Mouse feed and padding are all in the irradiation center channel of Suzhou university60Co 30 kilo Gy is irradiated and sterilized, and the drinking water is sterilized at high temperature and high pressure. All experimental mice were housed in isolation cages at SPF (specific pathogen-free facility) level. The animals are raised for at least two weeks before the experiment, and gentamicin is added into the sterilized drinking water for one week before the experiment.
1.2 instruments and reagents
1.2.1 Experimental reagents
1X PBS (self-contained in the laboratory)
The formula is as follows:
sodium chloride-8 g
Potassium chloride-0.2 g
Disodium hydrogen phosphate-1.44 g
Potassium dihydrogen phosphate 0.24g
Adding double distilled water to a constant volume of 1 liter, adjusting the pH value to 7.2-7.4, and sterilizing at high temperature and high pressure.
Erythrocyte lysate (self-prepared in the laboratory)
The formula is as follows:
ammonium chloride-8.26 g
Potassium bicarbonate 1g
Disodium ethylenediamine tetraacetate-0.037 g
And (5) adding double distilled water to a constant volume of 1 liter, and sterilizing at high temperature and high pressure.
The ELOVL6 enzyme inhibitor is structurally referred to by Shimamura K, Nagumo A, Miyamoto Y, equivalent, discovery and characterization of a novel potential, selective and organic inhibitor for a mammarian ELOVL6[ J ]. European Journal of Pharmacology,2010,630(1-3):34-41.
1.2.2 Main instrumentation
1.3 Experimental design and methods
1.3.1 preparation of bone marrow cells and spleen cells
Killing 4 donor C57BL/6 mice by cervical dislocation method, soaking in 75% alcohol for 5min, taking out mouse spleen in a super clean bench by using ophthalmological scissors and ophthalmological forceps, placing in a dish poured with RPMI-1640 culture medium, fully grinding with glass slides, sucking the culture medium with a 1ml pipette gun, filtering through a 200 mesh nylon membrane to 50ml centrifuge tube, adding fresh RPMI-1640 culture medium to the dish, rinsing and filtering to the centrifuge tube.
Simultaneously, taking out the femur and tibia of a mouse by using an ophthalmic scissors and an ophthalmic forceps in a clean bench, cutting into 2-3 sections, and placing in a mortar poured with RPMI-1640 culture medium; and then stripping and taking out the spinal column of the mouse, cutting into 5-6 sections, placing the sections into a mortar, grinding leg bones and spinal bones by using a grinding rod, sucking the culture medium by using a 1ml pipette gun, filtering the culture medium through a 200-mesh nylon membrane into a 50ml centrifuge tube, adding a fresh RPMI-1640 culture medium into the mortar, grinding, filtering the mixture into the centrifuge tube, and repeating the steps for 3-5 times.
The prepared bone marrow cells and whole spleen cells were centrifuged (1200rpm, 8min), the supernatant was discarded, resuspended in 5ml of erythrocyte lysate, allowed to stand at room temperature for 5min, 10ml of RPMI-1640 medium was added, centrifuged (1200rpm, 8min), the supernatant was discarded, resuspended in 5ml of PBS, centrifuged (1200rpm, 8min), the supernatant was discarded, resuspended in PBS, and counted on a hemocytometer.
Finally, resuspending the cells in PBS to adjust the concentration of bone marrow cells to 1X 107Per 100. mu.l, whole spleen cell concentration 5X 106A total of 22 recipient mice were given in this study, each recipient mouse being infused caudally with 200. mu.l of mixed cells (100. mu.l bone marrow cells + 100. mu.l whole spleen cells).
1.3.2 high stearic acid diet
The formulation is shown in table 1:
TABLE 1 ingredient Table for high stearic diet/kg in the examples of the invention
| Item | Each 1 kilogram (kg) |
| Energy of | 4492.9kcal |
| Corn starch | 287.5g |
| Casein protein | 200g |
| Dextrin corn starch | 132g |
| Sucrose | 100g |
| Lard oil | 150g |
| Soybean oil | 30g |
| Fiber | 50g |
| Mixed minerals (AIN-93G-MX) | 35g |
| Mixed vitamin (AIN-93G-VX) | 10g |
| L-cystine | 3g |
| Tartaric acid choline | 2.5g |
The recipient mice are bred in a common cage by using less than 5-6 mice per cage, normal feed and high stearic acid feed are respectively placed in the breeding cages of the mice of the corresponding diet group from 4 weeks before bone marrow transplantation according to different feeds, the mice freely drink water and eat food, and cleaning padding is periodically replaced. Because the high-stearic acid feed has high fat content and is easy to deteriorate, the feed is stored at the temperature of-20 ℃ daily and completely replaced every 2-3 days, and the replacement is continued until the observation end point after transplantation.
1.3.3 establishment of mouse model for transplanting aGVHD by allogeneic transplantation
BALB/c(H-2d) The recipient mice received 650cGy total body lethal irradiation, 4 hours later C57BL/6 (H-2) was infused by tail vein injectionb) Mouse-derived 1X 107Bone marrow cells and 5X 106Whole spleen cells, or C57BL/6 (H-2)b) Mouse-derived 1X 107Bone marrow cells and IL-27-/- (H-2)b) Mouse origin 5X 106Whole splenocytes, total volume 200. mu.l, establish aGVHD model.
Mice on a high-stearic acid diet and normal diet recipient began intervention 3 weeks prior to allogeneic hematopoietic stem cell transplantation until the day of transplantation, specifically, the ELOVL6 inhibitor dried group received 200 μ l of ELOVL6 inhibitor (30mg/Kg body weight/day) in 0.5% methylcellulose, and the control group was given an equivalent amount of sterile PBS intragastric (vehicle).
TABLE 2 statistical table of treatment quantity and mode of control group and experimental group in the examples of the present invention
| Group of | Number of mice | Treatment method |
| Group of |
5 | Gavage with 200. mu.l sterile PBS |
| Normal diet + ELOVL6 enzyme inhibitor group | 6 | Gavage with 200. mu.l of ELOVL6 inhibitor |
| High stearic |
5 | Gavage with 200. mu.l sterile PBS |
| High stearic acid diet + ELOVL6 enzyme inhibitor group | 6 | Gavage with 200. mu.l of ELOVL6 inhibitor |
1.3.4 mouse aGVHD score criteria
After transplantation, mice were observed daily for survival, and their weights were recorded every 3 days, observed for weight loss, posture change, mobility, body hair texture and skin integrity and scored for aGVHD. The scoring criteria are referenced in table 3.
TABLE 3 standard table of mouse aGVHD clinical score after allogeneic hematopoietic stem cell transplantation
1.3.5 histological examination of aGVHD pathological sections
On day 4 after the allogeneic hematopoietic stem cell transplantation, the liver, lung, small intestine and skin of each group of mice were collected and fixed in 10% paraformaldehyde. The tissue was water-displaced with increasing alcohol concentration, paraffin-embedded and cut into 5 μm thick sections, deparaffinized with xylene, gradient alcohol hydrated, hematoxylin-eosin stained, and gum-encapsulated. Pictures of HE sections were taken using an olympus upright fluorescence microscope (tokyo, japan) at 200-fold or 400-fold magnification. The main target organs of aGVHD are liver, lung, small intestine and skin, and the damage degree of each target organ is observed by pathological tissue sections.
1.4 statistical analysis
The survival curve data for the aGVHD mice were statistically analyzed using the log-rank method. Comparisons between groups were performed using Student's two-tailed t-test (Student's t test), and data were analyzed using GraphPad Prism 6(GraphPad software, San Diego, CA) software, with significant differences when P < 0.05.
Second, result in
2.1 high stearic diet aggravates aGVHD in mice, and ELOVL6 enzyme inhibitor significantly improves aGVHD in mice with high stearic diet
Four groups of BABL/C mice received whole body lethal irradiation (X-Ray, 6.5Gy) divided into two times3.25Gy each time, and the interval of two times of irradiation is 4 hours, so as to relieve the gastrointestinal irradiation toxicity. Each group of mice was transplanted with 1X 10 mice derived from wild type C57BL/6 mice7Bone marrow cells and 5X 106And (4) spleen cells. Mice in the high stearic diet died within 5 days, with aGVHD being the most severe; whereas 50% of mice in the group of high stearic acid diet + ELOVL6 enzyme inhibitor survived for more than 5 days, the longest survival time was 11 days, the survival time of mice in the normal diet group was significantly longer than that of mice in the high stearic acid diet group, and the use of ELOVL6 enzyme inhibitor had no significant effect on aGVHD in mice in the normal diet group (FIG. 1).
2.2 Effect of ELOVL6 enzyme inhibitors on body weight and score of aGVHD mice
Mice in the high stearic acid diet died within 5 days, and were unable to collect weight and score data; there was no significant difference in body weight and score after aGVHD development in the normal diet group of mice with the addition of ELOVL6 enzyme inhibitor and without the addition of ELOVL6 enzyme inhibitor (figure 2).
2.3 pathological lesions of aGVHD
Pathological damage to aGVHD mainly affects the liver, lungs, intestines and skin. Severe aGVHD damage in the liver is characterized by inflammatory cell involvement of more than 15% of blood vessels, infiltration of parenchymal hepatic cells and large necrosis; severe aGVHD in the lung causes proliferative infiltration of a large number of inflammatory cells in the alveoli, leading to thickening of the lung interstitium; the severe aGVHD injury in the intestinal tract is pathologically represented by crypt cell necrosis, inflammatory cells infiltrate an inherent layer, and even obvious ulcer appears; severe aGVHD damage in the skin is pathologically characterized by lymphocyte infiltration into the basal layer of the skin, which results in damage to the skin structure.
From fig. 3, it can be seen that the ELOVL6 enzyme inhibitor significantly improved liver, lung, intestinal and skin damage in the mice in the high stearic acid diet group with a lower histopathological score (fig. 3).
Claims (10)
- Use of an ELOVL6 enzyme inhibitor in a mouse model of acute graft versus host disease, aGVHD, on a high stearic diet.
- 2. The use of claim 1, comprising use of an ELOVL6 enzyme inhibitor for ameliorating the development of aGVHD in mice fed with high stearic acid, reducing the mortality of aGVHD, and reducing damage to the mouse target organs caused by aGVHD.
- 3. The use of claim 1 or 2, wherein the ELOVL6 enzyme inhibitor is administered at a dose of 30 mg/kg/d.
- 4. The use of claim 1, wherein the mouse begins to receive a high stearic acid diet 4 weeks prior to allogeneic hematopoietic stem cell transplantation and continues to the aGVHD model observation endpoint, and the ELOVL6 enzyme inhibitor begins to intervene 3 weeks prior to allogeneic hematopoietic stem cell transplantation until the day of transplantation.
- 5. Use according to claim 1 or 4, characterized in that the formula of the high stearic diet/kg is: 287.5g of corn starch, 200g of casein, 132g of dextrin corn starch, 100g of cane sugar, 150g of lard, 30g of soybean oil, 50g of fiber, 35g of mixed mineral, 10g of mixed vitamin, 3g of L-cystine, 2.5g of choline tartrate and 4492.9kcal of energy.
- 6. The use of claim 1, wherein the mouse model of acute graft versus host disease, aGVHD, is established by: BALB/c H-2dThe recipient mice received 650cGy total body lethal irradiation, 4 hours later infused with C57 BL/6H-2 by tail vein injectionbBone marrow cells and whole spleen cells from mice establish a mouse acute graft-versus-host disease aGVHD model.
- 7. The use of claim 6, wherein said whole splenocytes are also derived from IL-27-/-(H-2b) A mouse.
- 8. The use of claim 6 or 7, wherein each mouse is infused with 1 x 107Bone marrow cells and 5X 106And (4) all splenocytes.
- 9. The use of claim 8, wherein the concentration of bone marrow cells is 1 x 107Per 100. mu.l, whole splenocytes concentration 5X 106/100μl。
- Use of an ELOVL6 enzyme inhibitor for the manufacture of a medicament for the treatment of acute graft versus host disease.
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| CN114191544A (en) * | 2022-01-13 | 2022-03-18 | 苏州大学 | Application of deubiquitinase in preparation of medicine for preventing or treating acute graft-versus-host disease and graft-versus-leukemia |
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