CN114469968B - 一种红景天苷的应用 - Google Patents
一种红景天苷的应用 Download PDFInfo
- Publication number
- CN114469968B CN114469968B CN202210141773.6A CN202210141773A CN114469968B CN 114469968 B CN114469968 B CN 114469968B CN 202210141773 A CN202210141773 A CN 202210141773A CN 114469968 B CN114469968 B CN 114469968B
- Authority
- CN
- China
- Prior art keywords
- mitochondrial
- salidroside
- polarization
- macrophage
- macrophages
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 title claims abstract description 76
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 210000002540 macrophage Anatomy 0.000 claims abstract description 57
- 230000002438 mitochondrial effect Effects 0.000 claims abstract description 50
- 230000010287 polarization Effects 0.000 claims abstract description 47
- 230000013632 homeostatic process Effects 0.000 claims abstract description 21
- 210000001700 mitochondrial membrane Anatomy 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 15
- 108010001946 Pyrin Domain-Containing 3 Protein NLR Family Proteins 0.000 claims abstract description 13
- 101000605835 Homo sapiens Serine/threonine-protein kinase PINK1, mitochondrial Proteins 0.000 claims abstract description 12
- 102100038376 Serine/threonine-protein kinase PINK1, mitochondrial Human genes 0.000 claims abstract description 12
- 102000017946 PGC-1 Human genes 0.000 claims abstract description 10
- 108700038399 PGC-1 Proteins 0.000 claims abstract description 10
- 101150092416 tom20 gene Proteins 0.000 claims abstract description 10
- 210000003690 classically activated macrophage Anatomy 0.000 claims abstract description 6
- 102000000874 Pyrin Domain-Containing 3 Protein NLR Family Human genes 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 34
- 230000014509 gene expression Effects 0.000 claims description 26
- 102100037850 Interferon gamma Human genes 0.000 claims description 7
- 108010074328 Interferon-gamma Proteins 0.000 claims description 7
- 101150050341 Mfn2 gene Proteins 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 230000001737 promoting effect Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 230000002829 reductive effect Effects 0.000 abstract description 14
- 201000001320 Atherosclerosis Diseases 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 238000011160 research Methods 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 9
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 230000002757 inflammatory effect Effects 0.000 abstract description 7
- 230000001105 regulatory effect Effects 0.000 abstract description 7
- 229940079593 drug Drugs 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 102100033703 Mitofusin-2 Human genes 0.000 abstract description 3
- 101001018717 Homo sapiens Mitofusin-2 Proteins 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 19
- 239000002158 endotoxin Substances 0.000 description 18
- 229920006008 lipopolysaccharide Polymers 0.000 description 13
- 101000686031 Homo sapiens Proto-oncogene tyrosine-protein kinase ROS Proteins 0.000 description 12
- 108090000193 Interleukin-1 beta Proteins 0.000 description 12
- 102100023347 Proto-oncogene tyrosine-protein kinase ROS Human genes 0.000 description 12
- 102100022691 NACHT, LRR and PYD domains-containing protein 3 Human genes 0.000 description 11
- 102000003777 Interleukin-1 beta Human genes 0.000 description 9
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 9
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 101001123331 Homo sapiens Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Proteins 0.000 description 5
- 102100028960 Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Human genes 0.000 description 5
- 244000042430 Rhodiola rosea Species 0.000 description 5
- 235000003713 Rhodiola rosea Nutrition 0.000 description 5
- 210000003470 mitochondria Anatomy 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 230000000879 anti-atherosclerotic effect Effects 0.000 description 3
- 230000004900 autophagic degradation Effects 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- IKEOZQLIVHGQLJ-UHFFFAOYSA-M mitoTracker Red Chemical compound [Cl-].C1=CC(CCl)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 IKEOZQLIVHGQLJ-UHFFFAOYSA-M 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102100032543 Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN Human genes 0.000 description 2
- 101710132081 Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN Proteins 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- -1 mfn2 Proteins 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- 125000004203 4-hydroxyphenyl group Chemical group [H]OC1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 101100190541 Caenorhabditis elegans pink-1 gene Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 108050004120 Mitofusin-2 Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 108010016731 PPAR gamma Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102000012132 Peroxisome proliferator-activated receptor gamma Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 238000009012 ROS assay kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- ILRCGYURZSFMEG-RKQHYHRCSA-N Salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RKQHYHRCSA-N 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- SAZUGELZHZOXHB-UHFFFAOYSA-N acecarbromal Chemical compound CCC(Br)(CC)C(=O)NC(=O)NC(C)=O SAZUGELZHZOXHB-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004094 calcium homeostasis Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000003081 coactivator Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000002884 effect on inflammation Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 125000003563 glycoside group Chemical group 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000008437 mitochondrial biogenesis Effects 0.000 description 1
- 230000006676 mitochondrial damage Effects 0.000 description 1
- 230000021125 mitochondrion degradation Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004202 respiratory function Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
-
- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/41—Crassulaceae (Stonecrop family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Cardiology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Alternative & Traditional Medicine (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明属于生物医药技术领域,具体涉及一种红景天苷的应用。发明人在研究过程中发现,红景天苷可起到调节巨噬细胞M1极化的线粒体稳态的作用,有助于进一步拓展红景天苷的药用价值。红景天苷干预巨噬细胞M1极化后,可通过升高线粒体膜电位,降低ROS水平,抑制炎性因子,并对线粒体稳态相关的MFN2、PINK1、Tom20、PGC‑1α及NLRP3等蛋白进行调控,从而抑制M1巨噬细胞极化,维持巨噬细胞极化后线粒体稳态,继而干预动脉粥样硬化。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种红景天苷的应用。
背景技术
线粒体稳态是线粒体生物发生和降解之间的稳态平衡,主要由线粒体分裂和融合,线粒体嵴重构,线粒体生物合成,Ca2+稳态和线粒体自噬等动态过程的调节,一旦线粒体稳态被破坏,会导致一系列心血管等疾病的发生。
红景天苷(Salidroside,SAL),化学名称是2-(4-羟基苯基)乙基-B-D-葡萄糖苷,是藏药红景天的主要有效成分,具有抗炎、抗氧化、清除自由基等多种药理作用。研究表明SAL具有抗动脉粥样硬化的作用,且对巨噬细胞的M1极化具有抑制作用。
发明内容
发明人在研究过程中发现,红景天苷可起到调节巨噬细胞M1极化的线粒体稳态作用,有助于进一步拓展红景天苷的药用价值。
为了实现上述目的,本发明提供如下技术方案:一种红景天苷的应用,红景天苷在调节巨噬细胞M1极化的线粒体稳态中的应用。
优选地,红景天苷在制备调节巨噬细胞M1极化的线粒体稳态的药物中的应用。
优选地,红景天苷在制备通过调节巨噬细胞M1极化的线粒体稳态来改善动脉粥样硬化的药物中的应用。
优选地,红景天苷在促进巨噬细胞M1极化的Mfn2、Tom20和PGC-1α中的至少一种的表达中的应用。
优选地,红景天苷在升高巨噬细胞M1极化的线粒体膜电位和/或线粒体质量中的应用。
优选地,红景天苷在抑制巨噬细胞M1极化的线粒体稳态蛋白NLRP3和/或PINK1的表达中的应用。
优选地,红景天苷在降低巨噬细胞M1极化中炎症基因IL-1β、iNOS和ROS中的至少一种的表达中的应用。
优选地,所述巨噬细胞M1极化通过对RAW264.7细胞进行极化诱导后制备得到。
优选地,所述巨噬细胞M1极化通过采用LPS和/或IFN-γ对RAW264.7细胞进行极化诱导后得到。
优选地,所述巨噬细胞M1极化通过采用LPS和IFN-γ对RAW264.7细胞进行极化诱导后得到,所述LPS的浓度为1-10μg·ml-1,所述IFN-γ的浓度为50-100ng·ml-1。
优选地,所述红景天苷的浓度为25-400μM。
优选地,所述红景天苷的浓度为400μM。
有益效果:红景天苷还可起到干预巨噬细胞M1极化的线粒体稳态作用,有助于进一步拓展红景天苷的药用价值。
红景天苷干预巨噬细胞M1极化后,可通过升高线粒体膜电位,降低ROS水平,抑制炎性因子,并对线粒体稳态相关的MFN2、PINK1、Tom20、PGC-1α及NLRP3等蛋白进行调控,从而抑制巨噬细胞M1极化,维持巨噬细胞极化后线粒体稳态。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。其中:
图1为本发明实施例提供的SAL对RAW264.7细胞增殖活性的影响的测试结果图;
图2为本发明实施例提供的SAL对M1型巨噬细胞IL-1β、iNOS、TNF-α的影响的测试结果图;
图3为本发明实施例提供的SAL对M1型巨噬细胞线粒体稳态的影响的测试结果图;
图4为本发明实施例提供的SAL对M1型巨噬细胞线粒体膜电位和线粒体质量的影响的测试结果图(´ 20);
图5为本发明实施例提供的SAL对M1型巨噬细胞内的ROS水平的影响的荧光显微镜下观察到的图片(´ 20);
图6为本发明实施例提供的SAL对M1型巨噬细胞内的ROS水平的影响的平均荧光强度测试结果图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员所获得的所有其他实施例,都属于本发明保护的范围。
下面将结合实施例来详细说明本发明。需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。
本发明针对目前存在的红景天苷的药用价值有待进一步拓展的问题,提供一种红景天苷的应用,红景天苷在调节巨噬细胞M1极化的线粒体稳态中的应用;
本发明优选实施例中,红景天苷在制备调节巨噬细胞M1极化的线粒体稳态的药物中的应用。
本发明优选实施例中,红景天苷在制备通过调节巨噬细胞M1极化的线粒体稳态来改善动脉粥样硬化的药物中的应用。
本发明优选实施例中,红景天苷在促进巨噬细胞M1极化的Mfn2、Tom20和PGC-1α中的至少一种的表达中的应用。
本发明优选实施例中,红景天苷在升高巨噬细胞M1极化的线粒体膜电位和/或线粒体质量中的应用。
本发明优选实施例中,红景天苷在抑制巨噬细胞M1极化的线粒体稳态蛋白NLRP3和/或PINK1的表达中的应用。
本发明优选实施例中,红景天苷在抑制巨噬细胞M1极化中炎症基因IL-1β、iNOS和ROS中的至少一种的表达中的应用。
本发明优选实施例中,所述巨噬细胞M1极化通过对RAW264.7细胞进行极化诱导后制备得到。
本发明优选实施例中,所述巨噬细胞M1极化通过采用LPS和/或IFN-γ对RAW264.7细胞进行极化诱导后得到。
本发明优选实施例中,所述巨噬细胞M1极化通过采用LPS和IFN-γ对RAW264.7细胞进行极化诱导后得到,所述LPS的浓度为1-10μg·ml-1(例如,1μg·ml-1、3μg·ml-1、5μg·ml-1、8μg·ml-1或10μg·ml-1),所述IFN-γ的浓度为50-100ng·ml-1(例如,50ng·ml-1、80ng·ml-1或100ng·ml-1)。
本发明优选实施例中,所述红景天苷的浓度为25-400μM(例如,25μM、50μM、100μM、200μM、300μM或400μM)。
本发明优选实施例中,所述红景天苷的浓度为400μM。
下面通过具体实施例对本发明的红景天苷的应用进行详细说明。
下面实施例中:
所采用的药品及相应来源分别如下:脂多糖(LPS)(absin,货号abs42020800);重组小鼠(Recombinant Murine)IFN-γ(PEPROTECH,货号315-05);红景天苷(Salidroside,SAL)(MCE,货号HY-N0109);
所采用的药品及相应来源分别如下:Cell Counting Kit-8(DOJIDO,批号CT04);PGC-1α Rabbit mAb(Millipore,货号AB3242),NLRP3 Rabbit mAb(CST,货号15101),Mitofusin-2 (D2D10)Rabbit mAb(CST, 9482S),Rabbit Anti-PINK1(CST,货号ab23707),Rabbit Anti-Tom20(ProteintechGroup,货号11802-1-AP),JC-1线粒体膜电位检测试剂盒(碧云天,批号C2006);Mito-Tracker Red CMXRos(碧云天,批号C1049);活性氧检测试剂盒(碧云天,批号S0033S);Trizol裂解液(美国Invitrogen,批号15596018);RNA逆转录试剂盒(TaKaRa,RR047A);实时荧光定量PCR试剂盒(TaKaRa,RR820A)
所采用的药品及相应来源分别如下:SpectraMax Plus384酶标仪(美国MolecularDevices) ,Western Blot设备(美国Bio-Rad),QuantStudio™ 6 Flex实时荧光定量PCR系统(ThermoFisher),Ts2倒置荧光显微镜(Nikon)
1.1 RAW264.7细胞的培养与M1极化模型的建立
RAW264.7细胞购于北纳创联生物技术有限公司;将冻存的RAW264.7在37℃水浴锅中60s内快速解冻,200G离心5min,弃去上清,用完全培养基(含10%FBS、1%青链霉素双抗的高糖DMEM)重悬后移入培养皿中,于37℃,5%CO2培养箱中培养。
1.2 分组及给药
实验分为Mφ、M1、红景天苷组(400M1),其中:
Mφ组:将2×105个细胞接种于6孔板内,生长至70%左右。
M1组:细胞生长至70-80%后用1μg·ml-1LPS、50ng·ml-1IFN-γ刺激Mφ极化为M1型巨噬细胞(极化的巨噬细胞),24h后提取RNA进行验证。
红景天苷组(400M1),具体如下:将2×105个细胞接种于6孔板内,生长至70%左右,以400μM浓度的红景天苷孵育2小时,然后用1μg·ml-1LPS、50ng·ml-1IFN-γ诱导Mφ极化为M1巨噬细胞,24小时后收取基因或者蛋白,以备后续实验。
1.3 CCK8测定SAL的细胞毒性
Mφ组:将2×105个细胞接种于6孔板内,生长至70%左右。
M1组:细胞生长至70-80%后用1μg·ml-1LPS、50ng·ml-1IFN-γ刺激Mφ极化为M1型巨噬细胞(极化的巨噬细胞),24h后提取RNA进行验证。
红景天苷组(400M1):接种100μL细胞悬液(5000个/孔)于96孔板,在37℃、5%CO2的培养箱中培养24小时,加入10μL不同浓度的SAL(0、25μM、50μM、100μM、200μM、400μM)到孔内,在培养箱中培养24h后,在每孔加入10μL CCK-8试剂,培养箱内培养1h后,在450nm波长处测定吸光值,参比波长为600nm或以上。
1.4 IL-1β、iNOS、TNF-α基因表达水平
按照TAKARA试剂盒,使用trizol法提取细胞(按照“1.2 分组及给药”部分的分组得到的Mφ组、M1组和红景天苷组细胞)的总RNA,逆转录后,使用Real-time PCR测定RAW264.7细胞的基因表达水平。引物由金唯智生物科技有限公司合成。qPCR程序为:95℃30s;95℃ 5s,55℃ 30s,72℃30s,40个循环。PCR经内参基因GAPDH归一化处理后,以Mφ组作为参照,采用 2-△△Ct法计算各组基因的相对表达水平。引物序列见表1。
表1 实时荧光定量PCR引物序列
1.5 线粒体NLRP3、Mfn2、PGC-1α、PINK1、Tom20蛋白表达水平的测定
收集细胞(按照“1.2 分组及给药”部分的分组得到的Mφ组、M1组和红景天苷组细胞),RIPA裂解,BCA测定蛋白浓度。收集的蛋白进行SDS-PAGE凝胶电泳、转膜后于5%脱脂牛奶中封闭1h,4℃过夜孵育一抗NLRP3(1:1000)、Mfn2(1:1000)、PGC-1α(1:500)、PINK1(1:1000)、Tom20(1:1000),TBST溶液中每10min洗3次,将膜置于稀释好的二抗GAPDH(1:5000)中孵育1h,TBST洗涤每10min洗3次;ECL发光液曝光,用ImageJ对条带进行分析。
1.6 化学荧光法测定细胞ROS的水平
Mφ组:将2×105个细胞接种于6孔板内,生长至70%左右。
M1组:细胞生长至70-80%后用1μg·ml-1LPS、50ng·ml-1IFN-γ刺激Mφ极化为M1型巨噬细胞(极化的巨噬细胞),24h后提取RNA进行验证。
红景天苷组(400M1):6孔板内细胞长至50%时,加入SAL,2h后加入LPS/IFN-γ,24h后按照碧云天ROS检测试剂盒说明测定ROS。具体如下:去除细胞培养液,加入适当体积稀释好的DCFH-DA,37℃细胞培养箱内孵育20min。用无血清细胞培养液洗涤细胞三次,以充分去除未进入细胞内的DCFH-DA,在荧光显微镜下观察。
1.7 化学荧光法测定细胞线粒体膜电位及线粒体质量
1.7.1 化学荧光法测定细胞线粒体膜电位
Mφ组:将2×105个细胞接种于6孔板内,生长至70%左右。
M1组:细胞生长至70-80%后用1μg·ml-1LPS、50ng·ml-1IFN-γ刺激Mφ极化为M1型巨噬细胞(极化的巨噬细胞),24h后提取RNA进行验证。
红景天苷组(400M1):6孔板内细胞长至50%时,加入SAL,2h后加入LPS/IFN-γ, 24h后按照碧云天线粒体膜电位检测试剂盒(JC-1法)测定线粒体膜电位。具体如下:去除细胞培养液,PBS洗涤2次后加入1ml细胞培养液,同时加入1ml配制好的JC-1工作液,37℃孵育20min后,去除上清,用已配制好的JC-1Buffer(1X)洗涤2次,加入2ml培养液在荧光显微镜下观察。
1.7.1 化学荧光法测定细胞线粒体质量
Mφ组:将2×105个细胞接种于6孔板内,生长至70%左右。
M1组:细胞生长至70-80%后用1μg·ml-1 LPS、50ng·ml-1 IFN-γ刺激Mφ极化为M1型巨噬细胞(极化的巨噬细胞),24h后提取RNA进行验证。
红景天苷组(400M1):6孔板内细胞长至50%时,加入SAL,2h后加入LPS/IFN-γ,24h后按照碧云天线粒体膜电位检测试剂盒(mito-tracker Red法)测定线粒体质量。具体如下:.去除细胞培养液,加入配制好的Mito-TrackerRed CMXRos工作液和Hoechst 33342染色液,37℃孵育15-30min,去除上清,加入2ml培养液在荧光显微镜下观察。
1.8 统计学分析
用GraphPad Prism 8软件进行统计分析,两组之间比较采用组间t检验,多组之间比较采用One-way ANOVA检验,以P<0.05表示差异有统计学意义。
2.结果
2.1 SAL对RAW264.7细胞增殖活性的影响
CCK8检测细胞活力,结果发现在24小时内所需的浓度对细胞活力没有影响,见图1。
2.2 SAL对M1型巨噬细胞的影响
RAW264.7极化为M1后,SAL干预并在0、12h、24h、48h检测IL-1β的基因表达量发现,与24h相比12h、48h表达量均显著降低(P<0.01,P<0.05),见图2A;SAL干预M1极化后24h检测IL-1β、iNOS、TNF-α的基因表达量发现,与正常组Mφ相比,M1组显著升高(P<0.001、P<0.05、P<0.001),见图2B、2C、2D;与模型组M1相比,IL-1β、TNF-α的表达量,400M1组显著降低(P<0.05、P<0.01),见图2B、2D;iNOS的表达量:400M1组与M1组无显著差异,见图2C。
2.3SAL对M1型巨噬细胞中线粒体稳态的影响
SAL干预M1极化后24h检测NLRP3、Mfn2、Tom20、PGC-1α和PINK1的蛋白表达量发现,NLRP3、PINK1表达量:M1组显著高于Mφ(P<0.001、P<0.001),400M1组显著低于M1组(P<0.01、P<0.001),见图3A、3E;Mfn2、Tom20、PGC-1α表达量:M1组显著低于Mφ(P<0.001、P<0.05、P<0.001),400M1组显著高于M1组(P<0.01、P<0.05、P<0.01),见图3B、3C、3D。
2.4 SAL对M1型巨噬细胞中线粒体膜电位的影响
SAL干预M1极化后24h检测线粒体膜电位及其质量,发现:与Mφ相比,M1膜电位和线粒体质量明显降低(P<0.01,P<0.001);与M1相比,400M1膜电位和线粒体质量有所升高(P<0.05,P<0.01);线粒体膜电位的检测结果详见图4A和4C,线粒体质量的检测结果详见图4B和4D。
2.5 SAL对M1型巨噬细胞活性氧的影响
SAL干预M1极化后24h检测细胞内ROS水平,发现:M1明显高于Mφ(P<0.001),400M1明显低于M1(P<0.01),见图5和图6。
3. 讨论
巨噬细胞是机体固有免疫系统的重要组成部分,可在不同微环境条件下极化为不同的亚型:经典活化巨噬细胞(M1)和选择性活化巨噬细胞(M2),在炎症、防御、修复、代谢等生理病理过程中起重要作用,参与动脉粥样硬化等疾病的发生发展。
线粒体是真核细胞中能量代谢的主要场所,其可为机体提供所需的能量。最近有研究发现线粒体稳态与动脉粥样硬化的发生发展具有密切关系。线粒体稳态是一个复杂的过程,主要包括通过蛋白酶体作用减少线粒体损伤,维持线粒体稳定;通过线粒体融合/分裂保证线粒体数量和形态;通过线粒体自噬选择性清除受损的线粒体。
研究表明,在动脉粥样硬化进展中,M1型巨噬细胞占据主导地位,M1具有促进动脉粥样硬化发展的作用。本研究采用LPS建立M1极化模型,LPS是革兰阴性细菌壁的主要成分,能够刺激巨噬细胞极化为M1型,且分泌大量的炎性因子TNF-α、IL-1β、iNOS,本研究结果显示LPS明显增促进RAW264.7的TNF-α、IL-1β、iNOS的mRNA的表达,与报道一致,建立模型成功;有研究表明NLRP3炎性小体介导M1巨噬细胞极化和IL-1β的产生,本研究在M1极化状态下发现炎症小体NLRP3大量表达,与报道一致;
研究表明,高ROS可以引起线粒体损伤,降低线粒体膜电位;线粒体受损后,稳态遭到破坏,磷酸酶及张力蛋白同源物(PTEN)诱导的激酶1(PINK1)高表达,启动线粒体自噬;Mfn2是线粒体融合蛋白,在线粒体受损后,融合/分裂异常,融合蛋白Mfn2表达降低;过氧化物酶体增殖物激活受体-γ共激活因子PGC-1α对于线粒体的生成以及氧化呼吸功能有着不可或缺的作用,在过度炎症下,PGC-1α合成降低,介导蛋白转运的线粒体外膜蛋白Tom20合成降低;
在M1极化模型下,本研究结果显示胞内ROS升高,膜电位降低,PINK1水平升高,融合蛋白Mfn2减少,功能蛋白PGC-1α、Tom20等合成降低,与报道一致,提示我们M1极化状态下,细胞线粒体稳态遭到破坏,这可能是M1促进动脉粥样硬化发展的原因。
研究发现,红景天苷(SAL)具有抗动脉粥样硬化作用。本团队前期研究也显示红景天苷对于动脉粥样硬化模型中内皮损伤具有抑制作用。本研究选择400μM浓度SAL进行研究,结果发现对于炎症具有明显的抑制作用,M1型巨噬细胞促炎基因IL-1β、TNF-α、iNOS明显降低;为了研究SAL是否对线粒体稳态具有保护作用,本研究检测了M1细胞内线粒体膜电位、ROS水平,以及使用Western Blot检测了自噬启动蛋白PINK1、融合蛋白Mfn2、功能蛋白PGC-1α、Tom20,结果发现ROS降低,膜电位升高,PINK1表达减少,Mfn2减少,以及NLRP3降低,功能蛋白PGC-1α、Tom20表达升高,这提示我们红景天苷可能通过对线粒体稳态的保护从而达到抗动脉粥样硬化的作用。
综上所述,本研究利用巨噬细胞M1模型,揭示了红景天苷对于线粒体稳态的保护作用,为红景天苷抗动脉粥样硬化的机制阐述进一步提供了理论依据。
以上所述仅为本发明的优选实施例,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.一种调节M1型巨噬细胞的线粒体稳态的实验方法,其特征在于,所述方法用于非疾病的诊断或治疗目的,所述方法包括下述步骤:
将2×105个RAW264.7细胞接种于6孔板内,生长至70%左右,以400μM浓度的红景天苷孵育2小时,然后用1μg·ml-1LPS、50ng·ml-1IFN-γ诱导Mφ极化为M1巨噬细胞,继续培养24小时;
所述红景天苷用于升高M1型巨噬细胞的线粒体膜电位和/或线粒体质量,促进巨噬细胞M1极化的Mfn2、Tom20和PGC-1α中的至少一种的表达和抑制巨噬细胞M1极化的线粒体稳态蛋白NLRP3和/或PINK1的表达。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210141773.6A CN114469968B (zh) | 2022-02-16 | 2022-02-16 | 一种红景天苷的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210141773.6A CN114469968B (zh) | 2022-02-16 | 2022-02-16 | 一种红景天苷的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114469968A CN114469968A (zh) | 2022-05-13 |
CN114469968B true CN114469968B (zh) | 2024-05-03 |
Family
ID=81480418
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210141773.6A Active CN114469968B (zh) | 2022-02-16 | 2022-02-16 | 一种红景天苷的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114469968B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115969723A (zh) * | 2023-01-09 | 2023-04-18 | 上海中医药大学 | 红景天苷在制备具有抗皱紧致功能化妆品中的应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111381050A (zh) * | 2020-04-21 | 2020-07-07 | 南通大学 | Reg3β/ HMGB1环路调控EAM小鼠巨噬细胞再编程的实验方法 |
-
2022
- 2022-02-16 CN CN202210141773.6A patent/CN114469968B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111381050A (zh) * | 2020-04-21 | 2020-07-07 | 南通大学 | Reg3β/ HMGB1环路调控EAM小鼠巨噬细胞再编程的实验方法 |
Non-Patent Citations (3)
Title |
---|
景虎通脉方及红景天苷介导巨噬细胞极化抗动脉粥样硬化的研究;周敏;中国优秀硕士学位论文全文数据库 医药卫生科技辑(第第2期期);第E057-552页 * |
红景天苷对小鼠腹腔巨噬细胞体外增殖、凋亡、吞噬、ROS和NO产生的影响;叶莎莎;曾耀英;尹乐乐;;细胞与分子免疫学杂志(03);全文 * |
红景天苷对巨噬细胞极化的调控及对巨噬细胞中脂质积累的影响;程仕强;中国优秀硕士学位论文全文数据库;摘要、结果、讨论 * |
Also Published As
Publication number | Publication date |
---|---|
CN114469968A (zh) | 2022-05-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Jacoby et al. | Antagonism of cholera enterotoxin by anti-inflammatory agents in the rat | |
US11110126B2 (en) | Method of expanding NK cell and composition for culturing | |
KR101723265B1 (ko) | mTOR/STAT3 신호억제제 처리된 면역조절능을 갖는 간엽줄기세포 및 이를 포함하는 면역질환의 예방 또는 치료용 세포치료제 조성물 | |
Wong et al. | Lipopolysaccharide regulation of toll-like receptor-4 and matrix metalloprotease-9 in human primary corneal fibroblasts | |
Wang et al. | Peritoneal M2 macrophage-derived extracellular vesicles as natural multitarget nanotherapeutics to attenuate cytokine storms after severe infections | |
WO2016127503A1 (zh) | 来源于粒细胞样髓源性抑制细胞的exosomes及其应用 | |
CN114469968B (zh) | 一种红景天苷的应用 | |
Yin et al. | Zinc oxide nanoparticles ameliorate collagen lattice contraction in human tenon fibroblasts | |
Lv et al. | Costunolide ameliorates colitis via specific inhibition of HIF1α/glycolysis-mediated Th17 differentiation | |
Yang et al. | Protection of bone marrow mesenchymal stem cells from acute lung injury induced by paraquat poisoning | |
Ding et al. | Roles of autophagy in rheumatoid arthritis | |
Chang et al. | From Hair to Colon: Hair Follicle‐Derived MSCs Alleviate Pyroptosis in DSS‐Induced Ulcerative Colitis by Releasing Exosomes in a Paracrine Manner | |
CN111904980B (zh) | 间充质干细胞与在治疗急性肺损伤、急性呼吸窘迫症或肺纤维化中的用途 | |
CN110075269B (zh) | Murabutide在制备电离辐射致骨髓、小肠和脾脏损伤防治药物中应用 | |
Liu et al. | Mesenchymal Stem Cell-Derived Exosomes as Drug Carriers for Delivering miRNA-29b to Ameliorate Inflammation in Corneal Injury Via Activating Autophagy | |
CN111920802B (zh) | 穿心莲内酯在制备防治成人t细胞白血病药物的应用 | |
BR112020016473A2 (pt) | Aplicação de (5r)-5-hidroxitriptolida na preparação de fármacos | |
TWI826635B (zh) | 包含單克隆幹細胞的組合物的用途、製備單克隆幹細胞的方法以及幹細胞的用途 | |
CN110279862B (zh) | 一种抗癌组合物及其在制备治疗骨肉瘤的药物中的应用 | |
CN111358804B (zh) | Cynanoside H在制备防治乳腺癌的药物中的应用 | |
CN113398140A (zh) | 一种酸枣仁皂苷抑制间充质干细胞氧化损伤并增强其免疫调节能力的用途 | |
CN114341346A (zh) | 用于治疗骨关节炎的治疗性凋亡细胞 | |
WO2017071379A1 (zh) | 一种用于治疗胃癌的肿瘤疫苗及其制备方法 | |
KR101336386B1 (ko) | 림프관 생성 유도제 | |
CN117899080B (zh) | 石蒜碱在抗鳜蛙虹彩病毒中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
CB03 | Change of inventor or designer information |
Inventor after: Lin Fei Inventor after: Wang Xiulong Inventor after: Zhao Guoan Inventor after: Chen Zhigang Inventor after: Li Dongxu Inventor before: Wang Xiulong Inventor before: Zhao Guoan Inventor before: Lin Fei Inventor before: Chen Zhigang Inventor before: Li Dongxu |
|
CB03 | Change of inventor or designer information | ||
GR01 | Patent grant | ||
GR01 | Patent grant |