CN113499338A - Application of dieckol as and/or in preparation of iron death inhibitor - Google Patents
Application of dieckol as and/or in preparation of iron death inhibitor Download PDFInfo
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- CN113499338A CN113499338A CN202110672217.7A CN202110672217A CN113499338A CN 113499338 A CN113499338 A CN 113499338A CN 202110672217 A CN202110672217 A CN 202110672217A CN 113499338 A CN113499338 A CN 113499338A
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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
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
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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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- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of medicines, and discloses application of dieckol as an iron death inhibitor and/or in preparation of the iron death inhibitor. The invention discloses the application of the disparl or the derivative thereof in serving as and/or preparing the iron death inhibitor for the first time, and is based on the discovery that the disparl or the derivative thereof can inhibit iron accumulation, active oxygen and lipid active oxygen accumulation, the expression of iron death-related proteins PTGS2 and TF, and promote the expression of SLC7A11, so that the disparl or the derivative thereof can be used as the iron death inhibitor and used for preventing and/or treating iron death-related diseases.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of dieckol as and/or in preparation of an iron death inhibitor.
Background
Iron death (Ferroptosis) is a newly discovered mode of cell death distinct from apoptosis and necrosis, triggered by iron-dependent lipid peroxidation accumulation, and was first used to describe the small molecule Erastin-induced form of cell death, primarily manifested by cell volume contraction, increased mitochondrial membrane density, and no typical manifestations of apoptosis and necrosis. Iron death has been reported to be associated with a variety of diseases, and has played an important role in cancer, inflammatory diseases, neurodegenerative diseases, and diabetes.
Inflammatory reaction is an important physiological process of the body, the essence of inflammation is the defense reaction of the body to various injurious stimuli, moderate inflammatory reaction is beneficial to the body, and excessive inflammatory reaction can cause damage to the body. At present, there is increasing evidence that iron death plays a central role in inflammation and is involved in the development and progression of a variety of inflammatory diseases. Some compounds have shown anti-inflammatory activity as iron death inhibitors in experimental models of certain diseases. For example, it has been found that inflammatory responses associated with iron death can occur in a mouse model of acute lung injury induced by Lipopolysaccharide (LPS), and that inhibition of iron death can be effective in reducing disease in mice with acute lung injury. Therefore, iron death inhibitors may be a promising drug for the treatment of inflammatory diseases.
Brown algae, which is industrially used as a source of alginic acid, is also widely used as a food in many countries. Dicranol is a root tannin widely found in brown algae. At present, the effect of the diaspore phenol as the iron death inhibitor on inflammation is not reported at home and abroad.
Disclosure of Invention
The first aspect of the invention aims to provide application of the diacrinol or the derivatives thereof in serving as and/or preparing the iron death inhibitor.
The second aspect of the invention aims to provide application of the swamp palm phenol or the derivatives thereof in serving as and/or preparing the iron accumulation inhibitor.
The third aspect of the invention aims to provide the application of the diaspore phenol or the derivative thereof in serving as and/or preparing the active oxygen inhibitor.
The fourth aspect of the invention aims to provide the application of the diaspore phenol or the derivatives thereof in serving as and/or preparing the iron death-related protein inhibitor.
The fifth aspect of the invention aims to provide the application of the diaspore phenol or the derivative thereof in serving as and/or preparing the SLC7A11 accelerant.
The sixth aspect of the invention aims to provide the application of the swamp palm phenol or the derivatives thereof in preparing anti-inflammatory drugs.
The seventh aspect of the invention aims to provide the application of the dioxetaneol or the derivative thereof in serving as and/or preparing COX-2 inhibitors.
The eighth aspect of the invention aims to provide application of the dieselol or the derivatives thereof in preparing and/or using iNOS inhibitors.
The ninth aspect of the invention aims to provide application of the swamp cabbage phenol or the derivatives thereof in preparing medicines for preventing and/or treating diseases related to iron death.
It is an object of a tenth aspect of the present invention to provide a medicament.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided the use of dicamba or a derivative thereof as an iron death inhibitor and/or in the preparation of an iron death inhibitor.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
In a second aspect of the invention, there is provided the use of dicranol or a derivative thereof as an iron accumulation inhibitor and/or in the preparation of an iron accumulation inhibitor.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the iron accumulation inhibitor is a ferrous ion accumulation inhibitor.
In a third aspect of the invention, the application of the diaspore phenol or the derivative thereof as an active oxygen inhibitor and/or in the preparation of the active oxygen inhibitor is provided.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the active oxygen inhibitor is an inhibitor that inhibits the production and accumulation of active oxygen.
Preferably, the active oxygen is a lipid active oxygen.
In a fourth aspect of the invention, the application of the diacrinol or the derivative thereof in serving as and/or preparing the iron death-related protein inhibitor is provided.
Preferably, the iron death-related protein is prostaglandin endoperoxide synthase 2(PTGS2) and/or Transferrin (TF).
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the inhibitor of iron-death-related protein is an inhibitor that inhibits the expression of iron-death-related protein.
In a fifth aspect of the present invention, there is provided the use of dicamba or a derivative thereof as and/or in the preparation of a SLC7a11 promoter.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the SLC7a11 promoter is a promoter that promotes expression of SLC7a 11.
Preferably, the SLC7a11 promotes glutathione synthesis by mediating cystine uptake and glutamate release, protects cells from oxidative stress, maintains the redox balance of cells, prevents lipid peroxidation-induced cell death.
In a sixth aspect of the invention, the invention provides the use of dicranol or its derivatives in the preparation of anti-inflammatory drugs.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the pharmaceutically acceptable adjuvant is at least one of a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant and a lubricant.
Preferably, the dosage form of the drug is at least one of a solid formulation, a liquid formulation and a semi-solid formulation.
Preferably, the solid formulation includes tablets, granules, powders and capsules.
Preferably, the liquid formulation comprises an injection.
Preferably, the semi-solid formulation comprises an ointment and a cream.
In a seventh aspect of the invention, there is provided the use of dicranol or a derivative thereof as a COX-2 inhibitor and/or in the preparation of a COX-2 inhibitor.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the COX-2 inhibitor is an inhibitor that inhibits COX-2 expression.
In an eighth aspect of the invention, there is provided the use of dicamba or a derivative thereof as and/or in the preparation of an iNOS inhibitor.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the iNOS inhibitor is an inhibitor that inhibits the expression of iNOS.
In a ninth aspect of the invention, the invention provides application of the diacrinol or the derivatives thereof in preparing medicines for preventing and/or treating diseases related to iron death.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the pharmaceutically acceptable adjuvant is at least one of a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant and a lubricant.
Preferably, the dosage form of the drug is at least one of a solid formulation, a liquid formulation and a semi-solid formulation.
Preferably, the solid formulation includes tablets, granules, powders and capsules.
Preferably, the liquid formulation comprises an injection.
Preferably, the semi-solid formulation comprises an ointment and a cream.
Preferably, the iron death-related diseases include acute lung injury, stroke, brain trauma, organ fibrosis, ischemia reperfusion injury, neurodegenerative disease, liver and kidney failure, and heart disease.
In a tenth aspect of the invention, there is provided a medicament comprising the following components:
(1) dicranol and/or its derivatives; and
(2) pharmaceutically acceptable adjuvants.
The molecular formula of the diacrinol is C36H22O18The molecular weight is 742.55, the CAS number is 88095-77-6, and the chemical structural formula is shown in formula (I).
Preferably, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs of disparlacel.
Preferably, the pharmaceutically acceptable adjuvant is at least one of a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant and a lubricant.
Preferably, the dosage form of the drug is at least one of a solid formulation, a liquid formulation and a semi-solid formulation.
Preferably, the solid formulation includes tablets, granules, powders and capsules.
Preferably, the liquid formulation comprises an injection.
Preferably, the semi-solid formulation comprises an ointment and a cream.
The invention has the beneficial effects that:
the invention discloses the application of the disparl or the derivative thereof in the preparation and/or the application of the disparl or the derivative thereof as the iron death inhibitor for the first time, which is based on the discovery that the disparl or the derivative thereof can inhibit the iron accumulation, the accumulation of active oxygen and lipid active oxygen, the expression of iron death related proteins PTGS2 and TF and promote the expression of SLC7A11, wherein the iron accumulation is a necessary condition for the occurrence of iron death, iron ions are mainly involved in the occurrence of iron death by catalyzing the lipid peroxidation process, the accumulation of lipid active oxygen is an important mark for inducing iron death, the iron death related proteins PTGS2 and TF can promote the occurrence of iron death, the SLC7A11 can promote the synthesis of glutathione by mediating cystine uptake and glutamic acid release, protect cells from oxidative stress, maintain the redox balance of the cells and prevent the cell death induced by lipid peroxidation, therefore, the disparl or the derivative thereof can be used as the iron death inhibitor, and is useful for the prevention and/or treatment of iron death-related diseases.
The invention also discloses application of the dieckol or the derivatives thereof in preparing anti-inflammatory drugs, which is based on the discovery that the dieckol or the derivatives thereof can inhibit the expression of COX-2 and iNOS, thereby inhibiting the generation of inflammatory mediators such as NO and playing a role in resisting inflammation.
Drawings
FIG. 1 is a graph of the effect of dicranol on COX-2 and iNOS expression in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells: wherein A is an immunoblot of the effect of dieckol on COX-2 and iNOS expression in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; b is a graph of statistical results of the effect of dicranol on COX-2 expression in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; c is a statistical result chart of the effect of diaspore phenol on iNOS expression in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; wherein, P represents the lipopolysaccharide group compared to the blank control group, P < 0.001; denotes lipopolysaccharide group vs blank control group, P < 0.01; the # # indicates that the lipopolysaccharide + swamp cabbage phenol group compared with the lipopolysaccharide group, P < 0.001.
FIG. 2 is an immunofluorescence plot of the effect of dicranol on COX-2 expression in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells.
FIG. 3 is a graph of the effect of dicranol on nitric oxide production in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; wherein, P represents lipopolysaccharide group compared to blank control group, P < 0.0001; the # # # # indicates that the lipopolysaccharide + swamp cabbage phenol group is compared with the lipopolysaccharide group, and P is less than 0.0001.
FIG. 4 is a graph showing the effect of dicranol on the accumulation of ferrous ions in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells.
FIG. 5 is a graph of the effect of dicranol on reactive oxygen species in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells: wherein A is a flow cytogram of the effect of swamp cabbage phenol on active oxygen in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; b is a graph of statistical results of the effect of diaspore on reactive oxygen species in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; wherein, P represents lipopolysaccharide group compared with blank control group, P < 0.01; # denotes that P is <0.05 for the lipopolysaccharide plus swainsonol group compared to the lipopolysaccharide group; the # # indicates that the lipopolysaccharide + swamp cabbage phenol group compared with the lipopolysaccharide group, P < 0.001.
FIG. 6 is a graph of the effect of dicranol on lipid reactive oxygen species in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells: wherein A is a flow cytogram of the effect of swamp cabbage phenol on lipid reactive oxygen species in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; b is a graph of the statistical results of the effect of diaspore on lipid reactive oxygen species in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; wherein, P represents lipopolysaccharide group compared with blank control group, P < 0.01; # denotes that the LPS + Diptersocyanol group compared to the LPS group, P < 0.05.
FIG. 7 is a graph of the effect of dicranol on SLC7A11, PTGS2 and TF expression in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells: wherein A is an immunoblot of the effect of diaquintusol on the expression of SLC7A11, PTGS2 and TF in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; b is a graph of statistical results of the effect of diaspore on SLC7A11 expression in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; c is a statistical plot of the effect of diaspore on the expression of PTGS2 in Lipopolysaccharide (LPS) -stimulated RAW264.7 cells; d is a graph of statistical results of the effect of diaspore on TF expression in RAW264.7 cells stimulated by Lipopolysaccharide (LPS); wherein, P is <0.05 when comparing the lipopolysaccharide group with the blank control group; denotes lipopolysaccharide group vs blank control group, P < 0.01; denotes lipopolysaccharide group vs blank control group, P < 0.001; # denotes that P is <0.05 for the lipopolysaccharide plus swainsonol group compared to the lipopolysaccharide group; and # indicates that the lipopolysaccharide + swamp cabbage phenol group is compared with the lipopolysaccharide group, and P is less than 0.01.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The starting materials used in the examples were prepared by conventional means or purchased from commercial sources, except as otherwise specified.
The reagents or methods of preparation of the reagents in this example were as follows:
preparing a diel palmate cresol storage liquid: weighing the dicranol powder by using an electronic balance, placing the dicranol powder into a sterile centrifuge tube, adding dimethyl sulfoxide (DMSO) for dissolving, and preparing a storage solution with the concentration of 10 mg/mL.
Example 1 Diphenicol inhibits expression of COX-2 and iNOS and nitric oxide production in LPS-stimulated RAW264.7 cells
1) Western Blot for detecting expression of COX-2 and iNOS
RAW264.7 cells were placed in DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2Culturing in a 37 deg.C incubator, and culturing RAW264.7 cells at 2 × 105The cells were seeded in 12-well plates at a density of one mL, 1mL per well, and treated as follows: blank Control group (Control, adding 1mL of DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin), Lipopolysaccharide (LPS) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS as solute, the final concentration of LPS is 250ng/mL), lipopolysaccharide + Palmal (LPS + Dieckol) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS and Dieckol as solute, the final concentration of LPS is 250ng/mL, and the final concentrations of Palmal (Dieckol) are 12.5, 25 and 50 μ g/mL respectively), and each treating 2 multiple wells specifically as follows: the LPS + administration group is pretreated by using diacrinol for half an hour, LPS is used for co-stimulation for 24 hours in the LPS group and the LPS + Dieckol group after half an hour, the cells are washed twice by using precooled PBS after 24 hours, and then the cells are cracked by using RIPA lysate, and the cell protein is collected. And (3) carrying out SDS-PAGE electrophoresis on the collected protein samples, and placing the protein samples in a membrane transfer instrument for membrane transfer. After membrane transfer, primary antibodies (COX-2 antibody (Cell Signaling Technology (CST), 12282S); iNOS antibody (CST, 13120S)) were incubated overnight at 4 deg.C, respectively; the next day, after washing the membrane, a diluted (1: 4000) secondary Antibody (Anti-rabbitIgG, HRP-linked Antibody, CST, 7074S) was added and incubated for 1h at room temperature; after the secondary antibody incubation is finished, developing by a developing instrument; finally, the blot was quantitatively analyzed by densitometry using Image J software. The results are shown in FIG. 1: dipulfop can inhibit the expression of COX-2 and iNOS in RAW264.7 cells stimulated by LPS.
2) Immunofluorescence method for detecting expression of COX-2 protein
RAW264.7 cells were plated at 2X 105The cells were plated in 6-well plates at 37 ℃ with 5% CO2Incubate overnight and add sterile square coverslips to the 6-well plate before plating. Respectively carried out the next dayThe following treatment is carried out: blank Control group (Control, adding 1mL of DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin), Lipopolysaccharide (LPS) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS as solute, the final concentration of LPS is 250ng/mL), lipopolysaccharide + dipalmitols (LPS + Dieckol) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS and Dieckol as solute, the final concentration of LPS is 250ng/mL, and the final concentration of dipalmitols (Dieckol) is 50 μ g/mL), each of which is processed for 3 replicate wells, as follows: pretreating LPS + Dieckol group with diclol for half an hour, co-stimulating LPS in LPS group and LPS + Dieckol group for 24 hours after half an hour, removing culture medium after 24 hours, washing with PBS for three times, and adding 4% Paraformaldehyde (PFA) to fix at room temperature for 20 min; PFA was discarded, and the column was rinsed 3 times with PBS for 5min each time at room temperature; then adding 500 μ L0.5% Triton X-100 (PBS) for membrane rupture at room temperature for 15min, and then washing with PBS for 3 times, each time for 5 min; then sealing with goat serum for 0.5 h; removing the blocking solution, adding PBS, and soaking for 3 times, each for 5 min; adding corresponding primary antibody (anti-COX-2 antibody (CST, 12282S)), diluent (5% BSA diluted 1:300), standing at 4 deg.C for overnight in refrigerator; recovering primary antibody the next day, adding PBS, and soaking for 3 times, each time for 5 min; then adding corresponding fluorescent secondary antibody diluent ((Thermo Fisher Scientific, 35552, PBS diluted 1:1000)) and keeping out of the sun for 1h at room temperature; washing with PBS in dark for 3 times, each for 5 min; adding DAPI staining solution (PBS diluted 1:2000) and keeping out of the sun at room temperature for 10 min; removing DAPI, adding PBS, and washing for 5min each time in dark for 3 times; finally, prepare the slide, and each sample with 50 u L anti quenching mounting fluid, the slide will be reversed on the mounting fluid, 4 degrees C light protection storage. The observation is carried out under a laser confocal microscope, and the result is recorded by photographing, and the result is shown in figure 2: dipterdol can inhibit COX-2 expression in RAW264.7 cells stimulated by LPS.
3) Detection of nitric oxide content in cell culture solution supernatant
RAW264.7 cells were placed in DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2Culturing in 37 deg.C incubator, and culturing RAW264.7 cellsAt 2X 105The cells were seeded in 12-well plates at a density of 1mL per well volume and were treated as follows: blank Control group (Control, adding 1mL of DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin), Lipopolysaccharide (LPS) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS as solute, the final concentration of LPS is 250ng/mL), lipopolysaccharide + Palmal (LPS + Dieckol) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS and Dieckol as solute, the final concentration of LPS is 250ng/mL, and the final concentrations of Palmal (Dieckol) are 12.5, 25 and 50 μ g/mL respectively), each treating 3 multiple wells, specifically as follows: the LPS + Dieckol group is pretreated by using diaspore phenol for half an hour, LPS is used for co-stimulation for 24 hours after half an hour in the LPS group and the LPS + Dieckol group, cell culture solution supernatant is collected after 24 hours, detection is carried out according to a nitric oxide detection kit (Biyunyan, Shanghai), and finally the nitric oxide concentration is calculated according to a standard curve. The results are shown in FIG. 3: dipterdol can inhibit the production of nitric oxide in RAW264.7 cells stimulated by LPS.
In conclusion, the swamp palm phenol has an inhibiting effect on LPS-induced RAW264.7 cell inflammation.
Example 2 inhibition of ferrous ion accumulation, production of active oxygen and lipid active oxygen, and expression of iron death-related protein in RAW264.7 cells stimulated by LPS by Diphenna
1) Detecting the content of ferrous ions in cells: RAW264.7 cells were plated at 2X 105one/mL density was seeded in 6-well plates at 37 ℃ with 5% CO2Culturing in an incubator overnight, and respectively carrying out the following treatments on the next day: blank Control group (Control, adding 1mL DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin), Lipopolysaccharide (LPS) group (adding 1mL solution with 10% fetal bovine serum and 1% penicillin/streptomycin in DMEM high-sugar medium as solvent and LPS as solute, and LPS final concentration of 250ng/mL), lipopolysaccharide + Palmal phenol (LPS + Dieckol) group (adding 1mL solution with 10% fetal bovine serum and 1% penicillin/streptomycin DMEM high-sugar medium as solvent and LPS and Dieckol as solute,the final concentration of LPS was 250ng/mL and the final concentration of diclol (Dieckol) was 50. mu.g/mL), 3 replicates of each treatment were as follows: LPS + Dieckol group was pretreated with diclol for half an hour, LPS was CO-stimulated with LPS for 24 hours after half an hour, LPS + Dieckol group, and serum-free medium containing Ferro orange (1. mu. mol/ml, Dojindo, Japan) was added to the cells 24 hours later at 37 ℃ with 5% CO2Incubate in the incubator for 30 minutes, and finally observe the cells under a fluorescent confocal microscope (FV3000, Olympus, Japan). The results are shown in FIG. 4: under the stimulation of LPS, the content of ferrous ions in RAW264.7 cells is increased, the content of the ferrous ions in the cells is obviously reduced after the cells are pretreated by the swamp palm phenol, the iron accumulation is a necessary condition for the occurrence of iron death, and the iron ions mainly participate in the occurrence of the iron death through the process of catalyzing lipid peroxidation; the result shows that the swamp palm phenol can effectively inhibit the accumulation of ferrous ions in RAW264.7 cells caused by LPS stimulation.
2) Flow cytometry for detecting intracellular active oxygen and lipid active oxygen
The fluorescent probe DCFH-DA (Biyuntian, Shanghai) detects the formation of active oxygen in cells: RAW264.7 cells were plated at 2X 105The density of/mL was seeded in 12-well plates at 1mL per well volume in 5% CO2The culture was carried out overnight in an incubator at 37 ℃ and the following treatments were carried out on the following day: blank Control group (Control, adding 1mL of DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin), Lipopolysaccharide (LPS) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS as solute, the final concentration of LPS is 250ng/mL), lipopolysaccharide + Palmal (LPS + Dieckol) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS and Dieckol as solute, the final concentration of LPS is 250ng/mL, and the final concentrations of Palmal (Dieckol) are 12.5, 25 and 50 μ g/mL respectively), each treating 3 multiple wells, specifically as follows: the LPS + administration group is pretreated with dicranol for half an hour, CO-stimulated with LPS for 24 hours after half an hour in LPS group and LPS + Dieckol group, and 24 hours later, the cells are mixed with active oxygen fluorescent probe (DCFH-DA) with final concentration of 10 μ M at 37 deg.C and 5% CO2Incubation in incubator30 min; finally, the cells were washed with PBS, resuspended, and then fluorescence detected and analyzed on a flow cytometer.
Fluorescent probe C11-BODIPY 581/591 (Ebola, Wuhan) detects the formation of intracellular lipid reactive oxygen species: RAW264.7 cells were plated at 2X 105The density of/mL was seeded in 12-well plates at 1mL per well volume in 5% CO2The culture was carried out overnight in an incubator at 37 ℃ and the following treatments were carried out on the following day: blank Control group (Control, adding 1mL of DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin), Lipopolysaccharide (LPS) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS as solute, the final concentration of LPS is 250ng/mL), lipopolysaccharide + Palmal (LPS + Dieckol) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS and Dieckol as solute, the final concentration of LPS is 250ng/mL, and the final concentrations of Palmal (Dieckol) are 12.5, 25 and 50 μ g/mL respectively), each treating 3 multiple wells, specifically as follows: the LPS + administration group is pretreated with dicranol for half an hour, CO-stimulated with LPS for 24 hr in LPS group and LPS + administration group after half an hour, and 24 hr later, the cells are mixed with lipid peroxidation fluorescent probe (C11-BODIPY 581/591) with final concentration of 10 μ M at 37 deg.C and 5% CO2Incubate in incubator for 1 hr. After the incubation was completed, the cells were washed twice with PBS to remove excess dye; cells were then trypsinized and resuspended in 5% PBS and finally fluorescence was detected by flow cytometry and analyzed.
The results are shown in fig. 5 and 6: under the stimulation of LPS, the content of active oxygen and lipid active oxygen in RAW264.7 cells is increased, the content of active oxygen and lipid active oxygen in cells is reduced after the pretreatment of the swamp palm phenol, and the accumulation of the lipid active oxygen is an important mark for inducing iron death, which indicates that the swamp palm phenol can effectively inhibit the accumulation of the active oxygen and lipid active oxygen in RAW264.7 cells caused by the stimulation of LPS and inhibit the occurrence of iron death.
3) Western Blot for detecting expression of SLC7A11, PTGS2 and TF
RAW264.7 cells were plated in a cell culture containing 10% fetal bovine serum and 1% penicillin/streptomycinDMEM high-sugar medium containing mycin in 5% CO2Culturing in a 37 deg.C incubator, and culturing RAW264.7 cells at 2 × 105The cells were seeded in 12-well plates at a density of/mL and treated as follows: blank Control group (Control, adding 1mL of DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin), Lipopolysaccharide (LPS) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS as solute, the final concentration of LPS is 250ng/mL), lipopolysaccharide + Palmal (LPS + Dieckol) group (adding 1mL of solution with the DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as solvent and LPS and Dieckol as solute, the final concentration of LPS is 250ng/mL, and the final concentrations of Palmal (Dieckol) are 12.5, 25 and 50 μ g/mL respectively), and each treating 2 multiple wells specifically as follows: LPS + Dieckol group is pretreated by swan palm phenol for half an hour, LPS is used for co-stimulation for 24 hours after half an hour in LPS group and LPS + Dieckol group, precooled PBS is used for washing cells twice after 24 hours, then RIPA lysate is used for lysis, and cell protein is collected. And (3) carrying out SDS-PAGE electrophoresis on the collected protein samples, and placing the protein samples in a membrane transfer instrument for membrane transfer. After membrane transfer, primary antibodies (SLC7A11(ABClonal, A13685); PTGS2(CST, 12282S); TF (ABClonal, A1448)) were incubated overnight at 4 ℃; the next day, after washing the membrane, a diluted (1: 4000) secondary Antibody (Anti-rabbitIgG, HRP-linked Antibody, CST, 7074S) was added and incubated for 1h at room temperature; after the secondary antibody incubation is finished, developing by a developing instrument; finally, the blot was quantitatively analyzed by densitometry using Image J software. The results are shown in FIG. 7: the swamp palm phenol can inhibit the expression of iron death related proteins PTGS2 and TF, and increase the expression of SLC7A11(SLC7A11 promotes the synthesis of glutathione by mediating cystine uptake and glutamate release, protects cells from oxidative stress, maintains the redox balance of cells, and prevents cell death induced by lipid peroxidation).
In conclusion, the swamp palm phenol can inhibit the iron death of RAW264.7 cells caused by LPS stimulation and can be used as an iron death inhibitor.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. Application of dicranol or derivatives thereof in serving as and/or preparing iron death inhibitors.
2. Application of dicranol or its derivatives in preparation of iron accumulation inhibitor.
3. Application of dicranol or its derivatives in preparation of active oxygen inhibitor is provided.
4. The application of the dicranol or the derivatives thereof in serving as and/or preparing the iron death related protein inhibitor is characterized in that: the iron death-related protein is prostaglandin endoperoxide synthase 2 and/or transferrin.
5. Application of dicranol or its derivatives in preparing and/or promoting SLC7A11 is provided.
6. Application of dicranol or its derivatives in preparing antiinflammatory medicine is provided.
7. Application of dicranol or its derivatives in preparing COX-2 inhibitor and/or its derivatives is provided.
8. Application of dicranol or derivatives thereof in serving as and/or preparing iNOS inhibitors.
9. Application of dicranol or derivatives thereof in preparing medicines for preventing and/or treating iron death related diseases.
10. A medicament comprising the following components:
(1) dicranol and/or its derivatives; and
(2) pharmaceutically acceptable adjuvants.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115093356A (en) * | 2022-06-21 | 2022-09-23 | 广东医科大学 | Preparation method and application of iron death inducer |
| CN115093356B (en) * | 2022-06-21 | 2024-02-23 | 广东医科大学 | Preparation method and application of iron death inducer |
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