CN114848821A - Application of human beta 3 adrenergic receptor and inhibitor thereof in preparation of products for preventing and treating alopecia and leukotrichia - Google Patents
Application of human beta 3 adrenergic receptor and inhibitor thereof in preparation of products for preventing and treating alopecia and leukotrichia Download PDFInfo
- Publication number
- CN114848821A CN114848821A CN202210439551.2A CN202210439551A CN114848821A CN 114848821 A CN114848821 A CN 114848821A CN 202210439551 A CN202210439551 A CN 202210439551A CN 114848821 A CN114848821 A CN 114848821A
- Authority
- CN
- China
- Prior art keywords
- adrb3
- stem cells
- hair follicle
- inhibitor
- hair
- 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.)
- Granted
Links
- 201000004384 Alopecia Diseases 0.000 title claims abstract description 38
- 239000003112 inhibitor Substances 0.000 title claims abstract description 38
- 101000780539 Homo sapiens Beta-3 adrenergic receptor Proteins 0.000 title claims abstract description 34
- 231100000360 alopecia Toxicity 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims description 30
- 210000000130 stem cell Anatomy 0.000 claims abstract description 125
- 108060003355 ADRB3 Proteins 0.000 claims abstract description 109
- 102000017918 ADRB3 Human genes 0.000 claims abstract description 109
- 210000003780 hair follicle Anatomy 0.000 claims abstract description 81
- 210000002752 melanocyte Anatomy 0.000 claims abstract description 67
- 210000004209 hair Anatomy 0.000 claims abstract description 32
- 230000035755 proliferation Effects 0.000 claims abstract description 24
- 230000004069 differentiation Effects 0.000 claims abstract description 21
- 230000005012 migration Effects 0.000 claims abstract description 15
- 238000013508 migration Methods 0.000 claims abstract description 15
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 13
- 230000008929 regeneration Effects 0.000 claims abstract description 11
- 238000011069 regeneration method Methods 0.000 claims abstract description 11
- 230000001737 promoting effect Effects 0.000 claims abstract description 8
- 230000001939 inductive effect Effects 0.000 claims abstract description 4
- XTBQNQMNFXNGLR-MKSBGGEFSA-N (2s)-1-(2-ethylphenoxy)-3-[[(1s)-1,2,3,4-tetrahydronaphthalen-1-yl]amino]propan-2-ol;oxalic acid Chemical group OC(=O)C(O)=O.CCC1=CC=CC=C1OC[C@@H](O)CN[C@@H]1C2=CC=CC=C2CCC1 XTBQNQMNFXNGLR-MKSBGGEFSA-N 0.000 claims description 33
- 102000046995 human ADRB3 Human genes 0.000 claims description 30
- 230000022131 cell cycle Effects 0.000 claims description 13
- 238000009472 formulation Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 150000001875 compounds Chemical class 0.000 claims description 8
- 208000024963 hair loss Diseases 0.000 claims description 5
- 230000003676 hair loss Effects 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 17
- 230000001105 regulatory effect Effects 0.000 abstract description 5
- 206010049865 Achromotrichia acquired Diseases 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 101150090657 ADRB3 gene Proteins 0.000 abstract description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract 4
- 210000004027 cell Anatomy 0.000 description 36
- 210000000442 hair follicle cell Anatomy 0.000 description 22
- 108010077544 Chromatin Proteins 0.000 description 19
- 210000003483 chromatin Anatomy 0.000 description 19
- 238000004220 aggregation Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 108060008724 Tyrosinase Proteins 0.000 description 8
- 108010050345 Microphthalmia-Associated Transcription Factor Proteins 0.000 description 7
- 102000013760 Microphthalmia-Associated Transcription Factor Human genes 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 102000003425 Tyrosinase Human genes 0.000 description 7
- 210000002721 hair follicle melanocyte Anatomy 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 102000017919 ADRB2 Human genes 0.000 description 6
- 101000959437 Homo sapiens Beta-2 adrenergic receptor Proteins 0.000 description 6
- 102000011782 Keratins Human genes 0.000 description 6
- 108010076876 Keratins Proteins 0.000 description 6
- 238000011529 RT qPCR Methods 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 210000004940 nucleus Anatomy 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 239000000049 pigment Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 4
- 101150058971 KRT10 gene Proteins 0.000 description 4
- 101150040052 KRT14 gene Proteins 0.000 description 4
- 101150037996 KRT5 gene Proteins 0.000 description 4
- 101150023976 Krt19 gene Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000012292 cell migration Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000000284 resting effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 3
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000002981 blocking agent Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000005059 dormancy Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000002889 sympathetic effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 101710163881 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 2
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 101150017554 LGR5 gene Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- GEHJBWKLJVFKPS-UHFFFAOYSA-N bromochloroacetic acid Chemical compound OC(=O)C(Cl)Br GEHJBWKLJVFKPS-UHFFFAOYSA-N 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 239000002458 cell surface marker Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000001973 epigenetic effect Effects 0.000 description 2
- 230000003325 follicular Effects 0.000 description 2
- 238000013115 immunohistochemical detection Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000011278 mitosis Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 230000002087 whitening effect Effects 0.000 description 2
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 108060003345 Adrenergic Receptor Proteins 0.000 description 1
- 102000017910 Adrenergic receptor Human genes 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101001033280 Homo sapiens Cytokine receptor common subunit beta Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 208000009795 Microphthalmos Diseases 0.000 description 1
- 101100322708 Mus musculus Adrb3 gene Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000010428 chromatin condensation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000003661 hair follicle regeneration Effects 0.000 description 1
- 230000003659 hair regrowth Effects 0.000 description 1
- 102000055647 human CSF2RB Human genes 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000008099 melanin synthesis Effects 0.000 description 1
- 201000010478 microphthalmia Diseases 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000008035 nerve activity Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229960002748 norepinephrine Drugs 0.000 description 1
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- -1 small molecule compound Chemical class 0.000 description 1
- 210000000603 stem cell niche Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
-
- 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
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/18—Sulfonamides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/286—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against neuromediator receptors, e.g. serotonin receptor, dopamine receptor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Neurology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Cosmetics (AREA)
Abstract
The invention provides a new application of a human beta 3 adrenergic receptor (ADRB3) and an inhibitor thereof in preparing products for preventing and treating alopecia and white hair, and the specific application comprises the following steps: inducing differentiation of Hair Follicle Stem Cells (HFSCs) and melanocyte stem cells (MeSCs), and promoting regeneration of hair containing melanin. According to the research of the invention, ADRB3 is distributed in HFSCs and MeSCs in a proliferation stage, the proliferation, differentiation and migration of the HFSCs and/or the MeSCs are regulated to influence the regeneration of melanin-containing hair, and the significant effect on preventing and treating alopecia and white hair is achieved by reducing the expression of ADRB3 gene, reducing the level of ADRB3 protein in hair follicle tissues and inhibiting the activity of ADRB 3. Therefore, the ADRB3 inhibitor has good application value in the aspect of preparing and developing products for preventing and treating alopecia and poliosis. The invention provides a new application of an ADRB3 inhibitor in the aspect of preventing and treating alopecia and leukotrichia, and also provides a new strategy and technical means for preventing and treating alopecia and leukotrichia.
Description
Technical Field
The invention belongs to the technical field of medicines. More particularly, relates to application of human beta 3 adrenergic receptor (ADRB3) and inhibitor thereof in preparing preparation for preventing and treating alopecia and leukotrichia.
Background
There are two types of stem cells in human hair follicles: hair Follicle Stem Cells (HFSCs) and melanocyte stem cells (MeSCs), both of which are normally dormant, are activated into the cell cycle at the same time only during the early hair growth phase, and undergo mitosis to produce 2 progeny cells, respectively, which will all exit the cell cycle. One of the progeny cells enters the resting stage, the other differentiates into mature cells, such as HFSCs, into hair follicle cells, MeSCs differentiate into melanocytes, which determine the formation of hair, and the color of hair is determined by melanocytes. Therefore, the ability of the progeny HFSCs and MeSCs to exit the cell cycle and differentiate to mature is a key factor in determining pigment hair regrowth. If the progeny cells fail to exit the cell cycle, they will interfere with dormancy and differentiation, causing hair loss and hair whitening.
In the prior art of hyperactive of systemic neurons drive depletion of melanocyte stem cells, Zhang et al, in 2020, disclosed that sympathetic nerve activity can stimulate human β 2 adrenoceptors (ADRB2) expressed by human melanocyte stem cells (MeSCs), allowing the migration of MeSCs from the hair follicle, ultimately leading to white hair. The prior art discloses that ADRB2 is related to migration of human melanocyte stem cells, but whether and what effects ADRB2 have on cell proliferation and differentiation and chromatin de-aggregation of human melanocyte stem cells is unknown, and whether or not ADRB2 has an effect on preventing and treating human leukotrichia is unknown for a while. In addition, no relationship between ADRB2 and hair follicle stem cells has been reported, and no effect of ADRB2 on preventing and treating alopecia has been reported.
Disclosure of Invention
The invention aims to solve the technical problem of exploring a new technical means capable of preventing and treating alopecia and leukotrichia and providing a brand new preparation source for preventing and treating alopecia and leukotrichia.
The invention aims to provide application of an ADRB3 inhibitor in preparation of a preparation for preventing and treating alopecia and/or white hair.
The above purpose of the invention is realized by the following technical scheme:
based on a great deal of research and research, the ADRB3 expression levels of HFSCs and MeSCs in the proliferation stage are obviously increased compared with the resting stage, and the ADRB3 promotes chromatin de-aggregation, prevents cells in the proliferation stage from exiting the cell cycle, and inhibits newly-grown HFSCs and MeSCs from entering the resting stage and the differentiation stage, so that the number of HFSCs and MeSCs in the resting stage is obviously reduced, and hair follicle regeneration and melanocyte regeneration cannot be maintained; on the other hand, the terminal differentiation of HFSCs and MeSCs is disturbed, and the hair follicle cells and the melanocytes cannot be developed into mature hair follicle cells and melanocytes, and finally alopecia and white hair are caused.
The stem cell niches of HFSCs and MeSCs are regulated by sympathetic nerves, and we find that the sympathetic nerves release norepinephrine, activate ADRB3 of HFSCs and MeSCs in the proliferation stage, and enable cells newly born after mitosis to not exit the cell cycle by virtue of ADRB 3-mediated chromatin de-aggregation function, so that differentiation and dormancy are interfered, and generation of hair follicle cells and melanocytes is prevented. By using the ADRB3 retarder SR59230A, activation of ADRB3 can be blocked, newborn HFSCs and MeSCs can be promoted to withdraw from the cell cycle, the newborn HFSCs and the MeSCs are induced to be differentiated into hair follicle cells and melanocytes, regeneration of pigment hair is guaranteed, and clues are provided for preparing preparations for preventing and treating alopecia and white hair.
The invention discloses the role of ADRB3 in regulating the physiological functions of HFSCs and MeSCs in epigenetics, proliferation, differentiation, migration and pigmentation of hair and in the pathological mechanism of alopecia and leukotrichia. ADRB3 is disclosed as an epigenetic mark that is transferred to the progeny cells of HFSCs and MeSCs, ADRB3 prevents neogenetic HFSCs and MeSCs from exiting the cell cycle by promoting chromatin de-aggregation, preventing the differentiation fate of the neogenetic cells from differentiating into the hair follicle cells and melanocytes necessary for growth of pigmented hair; the dormancy of the new cells can be prevented, and the dormant stem cell reserve library which is lost due to the activation of HFSCs and MeSCs can not be supplemented; in addition, ADRB3 promotes migration of proliferating HFSCs and MeSCs out of the hair follicle by remodeling the cytoskeleton, causing permanent depletion of HFSCs and MeSCs at the follicular site, resulting in hair loss and hair whitening.
The invention discloses an ADRB3 inhibitor which can induce HFSCs and MeSCs to exit the proliferation phase to differentiate into hair follicle cells and melanocytes by reducing the activity of ADRB3, maintain the normal dormant state to supplement the reduced stem cells, inhibit the migration of the HFSCs and the MeSCs out of hair follicles, prevent the exhaustion of the HFSCs and the MeSCs in the hair follicles, stimulate the regeneration of the hair follicles and the melanocytes and prevent and treat alopecia and leukotrichia.
Therefore, based on the above findings, the present invention claims:
use of an ADRB3 inhibitor for the manufacture of a formulation for inducing differentiation of hair follicle stem cells and/or melanocyte stem cells.
Use of an ADRB3 inhibitor for the manufacture of a formulation for inhibiting migration of hair follicle stem cells and/or melanocyte stem cells out of a hair follicle.
Use of an ADRB3 inhibitor for the preparation of a formulation for promoting the regeneration of hair follicle stem cells and/or melanocyte stem cells.
Use of an ADRB3 inhibitor for the preparation of a formulation for inhibiting the proliferation of hair follicle stem cells and/or melanocyte stem cells.
Use of an ADRB3 inhibitor for the preparation of a formulation for promoting the withdrawal of hair follicle stem cells and/or melanocyte stem cells from the cell cycle.
Application of ADRB3 inhibitor in preparation of preparation for preventing and treating alopecia and/or canities.
A preparation for preventing and treating alopecia and/or canities comprises an ADRB3 inhibitor.
The ADRB3 inhibitor useful in the present invention is not particularly limited, and all compounds, antibodies, polypeptides, short peptides, CRISPR-Cas9 gene editing techniques against ADRB3 gene, etc. capable of inhibiting or blocking ADRB3 activity, function and/or reducing ADRB3 protein level, concentration and amount are suitable for the present invention.
Alternatively, preferably, the ADRB3 inhibitor is an ADRB3 blocker or a human ADRB3 monoclonal antibody.
Examples of such ADRB3 blockers are (but not limited to): SR59230A (molecular formula: C23H29NO 6; CAS number: 174689-39-5), L748337 (molecular formula: C26H31N3O 5S; CAS number: 244192-94-7), and compounds using SR59230A and L748337 as mother cores are all suitable for the invention.
The dosage form and preparation method of the ADRB3 inhibitor of the present invention are not particularly limited, and the ADRB3 inhibitor can be prepared into various dosage forms such as tablets, capsules, sustained-release preparations, injections, external preparations and the like by conventional and general preparation methods in the art.
The invention innovatively discovers a mechanism for regulating and controlling two stem cells in hair follicles by ADRB3, provides a compound with remarkable effects on preventing and treating alopecia and canities on the basis of the mechanism, and has important clinical significance for continuously developing more preparations for preventing and treating alopecia and canities.
In particular, in terms of experiments:
1. successfully isolate human hair follicle stem cells and human melanocyte stem cells with stem cell characteristics. The separated cells are identified by a flow cytometer that the masculinity rate of the hair follicle stem cell surface marker Lgr5 is 91% and the masculinity rate of the Lgr6 is 92%. The melanocyte stem cells are mostly bipolar or polygonal, L-DOPA staining is brownish black positive reaction, and flow cytometry and Western blot analysis results show that the obtained cells express MITF, TYR and TRP1 proteins. The results prove that the separated cells have stronger proliferation potential, can generate obvious clone colonies from single cells and accord with the characteristics of stem cells.
2. The role of ADRB3 in chromatin de-condensation was demonstrated by immunofluorescence assays measuring the levels of ADRB3 in different cell division phases for two stem cells. The ADRB3 protein was expressed in early G1 (Ki67 negative) and proliferative (Ki67 positive) human hair follicle stem cells (FIG. 1) and human melanocyte stem cells (FIG. 2). Cells strongly positive for ADRB3 had larger nuclei and looser chromatin compared to those weakly positive for ADRB 3. When SR59230A (10uM) or human ADRB3 monoclonal antibody (5ug/ml) was administered for 24 hours, the nuclei of both hair follicle stem cells and melanocyte stem cells were significantly condensed and chromatin was dense (fig. 3), indicating that ADRB3 promoted chromatin de-condensation.
3. The mRNA expression of the hair follicle cell markers of keratin Krt5, Krt10, Krt14 and Krt19 is detected by real-time quantitative PCR, and compared with the control group, the mRNA expression of the hair follicle stem cells of the ADRB3 retarder and antibody group, namely the keratin Krt5, Krt10, Krt14 and Krt19, is obviously increased by 2-4 times (FIG. 4), thereby indicating that the hair follicle stem cells are differentiated into hair follicle cells. Real-time quantitative PCR detection of mRNA expression of melanocyte differentiation protein Tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF) showed that mRNA expression of hair follicle stem cell keratin TYR and MITF in ADRB3 blocking agent and antibody group was significantly increased compared with control group (FIG. 5). When observed under an inverted microscope, the control cells were free of melanin production, while the melanocytes of the ADRB3 blocker and antibody groups were gray or gray black, indicating that the differentiation of human melanocyte stem cells into melanocytes was induced.
4. In vitro MTT experiments using blockers SR59230A, L748337 against ADRB3 and human ADRB3 monoclonal antibodies demonstrated that blocking ADRB3 and reducing ADRB3 activity using small molecule compound blockers or large molecule inhibitory antibodies would inhibit proliferation of human hair follicle stem cells and human melanocyte stem cells (fig. 6, 7).
5. In vitro Transwell cell migration experiments, both the SR 59230A-treated group and the human ADRB3 monoclonal antibody-treated group showed significantly reduced numbers of stem cell migration (P <0.01) compared to the control group. It was shown that ADRB3 promotes the migration of HFSCs and MeSCs. Inhibition of ADRB3 activity will retain HFSCs and MeSCs in the hair follicle, preventing reduction in numbers or depletion.
6. In animal experiments, by means of intraperitoneal injection of SR59230A and human ADRB3 monoclonal antibody groups into a plurality of aged mice which already suffer from alopecia and leukotrichia and comparison with a control group, the aged mice in the experimental group are black and bright, the hairs are thick, the alopecia and the leukotrichia are obviously reduced (figure 8), the protein positive rate of hair follicle cells ADRB3 is 20% and 10%, respectively, while the hair of the aged mice in the control group is sparse and white, and the positive rate of ADRB3 in hair follicle bulge region tissues is 100% (figure 9). Compared with the control group of ADRB3 positive hair follicle cells, the SR59230A and human ADRB3 monoclonal antibody group of ADRB3 positive hair follicle cells have smaller nuclei and more compact chromatin, indicating that ADRB3 promotes chromatin de-aggregation.
The above technical scheme shows the application of the ADRB3 inhibitor in the preparation for inducing the differentiation of hair follicle stem cells and/or melanocyte stem cells; the technical scheme of the invention can support the realization of the primary purpose of the invention.
The invention also provides application of the ADRB3 inhibitor in preparing a preparation for inhibiting hair follicle stem cells and/or melanocyte stem cells from migrating out of hair follicles, and the technical scheme of the invention can support realization of another purpose of the invention.
The invention also provides application of the ADRB3 inhibitor in promoting regeneration of hair follicle stem cells and/or melanocyte stem cells, and the technical scheme of the invention can support achievement of another purpose of the invention.
The invention also provides application of the ADRB3 inhibitor in inhibiting proliferation of hair follicle stem cells and/or melanocyte stem cells, and the technical scheme of the invention can support achievement of another purpose of the invention.
The invention also provides application of the ADRB3 inhibitor in promoting hair follicle stem cells and/or melanocyte stem cells to exit from a cell cycle, and the technical scheme of the invention can support achievement of another purpose of the invention.
Meanwhile, the application of the ADRB3 inhibitor in preparing the preparation for preventing and treating alopecia and/or white hair is also supported by the technical scheme. The specific experiment comprises three substances, namely SR59230A, L748337 and human ADRB3 monoclonal antibody, and real-time quantitative PCR experiments prove that the differentiation of human hair follicle stem cells and human melanocyte stem cells can be promoted by respectively adopting a small molecular compound retarder or a macromolecular inhibitory antibody to block ADRB3 and reduce the activity of ADRB3 (figures 4 and 5). In vitro MTT experiments show that the inhibition of ADRB3 and the reduction of the activity of ADRB3 by respectively adopting small molecular compound blockers or large molecular inhibitory antibodies can inhibit the proliferation of human hair follicle stem cells and human melanocyte stem cells (figures 6 and 7). In animal experiments, the aged mice injected with SR59230A and human ADRB3 monoclonal antibody are dark and bright, the hairs are dense, the alopecia and the white hairs are obviously reduced (figure 8), the protein positive rate of hair follicle cells ADRB3 is 20% and 10%, while the aged mice in the control group have sparse and white hairs, and the positive rate of ADRB3 in hair follicle bulge region tissues is 100% (figure 9). Nuclear transformation of hair follicle cells positive for SR59230A and human ADRB3 monoclonal antibody group ADRB3, compared to control ADRB3 positive hair follicle cellsSmall, more compact chromatin, thus demonstrating that ADRB3 promotes chromatin de-aggregation. By inhibiting ADRB 3-mediated chromatin de-aggregation, post-mitotic neogenetic cells can exit the cell cycle, and HFSCs and MeSCs can smoothly differentiate and hibernate, thereby producing hair follicle cells and melanocytes. By using ADRB3 inhibitors SR59230A, L748337 and human ADRB3 monoclonal antibodies, activation of ADRB3 is inhibited, chromatin de-aggregation function mediated by ADRB3 is inhibited, newborn HFSCs and MeSCs are promoted to withdraw from cell cycle, and are induced to be differentiated into hair follicle cells and melanocytes, and regeneration of pigment hair is guaranteed.Illustrate the invention The application of the ADRB3 inhibitor provided by the invention in the preparation of the preparation for preventing and treating alopecia and/or white hair can be the subject of the technical scheme And (4) maintaining.
ADRB3 is a key receptor of hair follicle stem cells and melanocyte stem cells, and ADRB 3-mediated signal pathways regulate the processes of proliferation, differentiation, migration, hair follicle formation, melanocyte regeneration and other pigment hair formation of hair follicle stem cells and melanocyte stem cells. Excessive activation of ADRB3 will lead to hair loss and white hair. Inhibitors such as ADRB3 blocking agent and neutralizing antibody can specifically bind and block the activity of ADRB3, and can be used for preventing and treating alopecia and canities.
The invention has the following beneficial effects:
the invention discloses application of ADRB3 in HFSCs and MeSCs in regulation of the epigenetics, proliferation, differentiation, migration and physiological functions in forming pigment hair of cells and an ADRB3 inhibitor in a pathological mechanism of alopecia and leukotrichia, discloses the effect of ADRB3 and the inhibitor thereof in regulation of HFSCs and MeSCs, and provides a brand-new preparation source for preventing and treating alopecia and leukotrichia.
Drawings
FIG. 1 shows immunofluorescence detection of human hair follicle stem cell ADRB3 protein.
FIG. 2 shows immunofluorescence assay of human melanocyte stem cell ADRB3 protein.
FIG. 3 is a DAPI staining test of human melanocyte stem cell nucleosum and chromatin condensation.
FIG. 4 shows the expression of the genes related to differentiation of hair follicle cells detected by real-time quantitative PCR of human hair follicle stem cells.
FIG. 5 shows the expression of melanocyte differentiation-associated genes detected by real-time quantitative PCR of human melanocyte stem cells.
FIG. 6 shows that SR59230A, L748337 dose-dependently inhibited the proliferation of human hair follicle stem cells and human melanocyte stem cells.
FIG. 7 shows that the human ADRB3 monoclonal antibody dose-dependently inhibited the proliferation of human hair follicle stem cells and human melanocyte stem cells.
FIG. 8 shows that SR59230A and human ADRB3 monoclonal antibody both significantly reduced hair loss and white hair in aged C57BL/6J mice.
FIG. 9 shows immunohistochemical detection of ADRB3 protein in hair follicles of C57BL/6J mice.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
SR59230A used in the following examples has the molecular formula C23H29NO6 and CAS number 174689-39-5 and the structural formula is as follows:
the molecular formula of L748337 used in the following examples is C26H31N3O5S, CAS number 244192-94-7, and the structural formula is as follows:
the human ADRB3 monoclonal antibody used in the following examples was an antibody produced by 5D9 hybridoma cells as disclosed in patent ZL 201710183354.8.
Example 1 isolation, culture and characterization of human Hair follicle Stem cells and human melanocyte Stem cells
Micro-separating hair follicle bulge area tissue of human head skin specimen, digesting dermal tissue with 0.1% type I collagenase at 37 deg.C for 1-2 hr, and repeatedly blowing to free hair follicle from dermis. Centrifuging at 1500rpm for 5min to obtain hair follicle, digesting hair follicle with 0.25% pancreatin at 37 deg.C for 1-5 min to obtain hair follicle source stem cell, digesting the exfoliated cell type human melanocyte stem cell, and collecting the hair follicle stem cells for use. The two stem cells were inoculated into a Matrigel-coated culture plate and cultured. After the cells are emigrated, subculture is carried out when the cell fusion reaches 70% -80%. Taking the stem cells after subculture for 2 weeks to perform L-DOPA staining, flow cytometry analysis and clone forming capacity determination, and identifying the phenotype and proliferation potential of the hair follicle stem cells and the melanocyte stem cells.
As a result: the hair follicle stem cells obtained by separation culture grow like paving stones, have large cell nucleus and higher nuclear-to-cytoplasmic ratio. The positive rate of the hair follicle stem cell surface marker Lgr5 is 91% and the positive rate of the Lgr6 is 92% through flow cytometry. The melanocyte stem cells are mostly bipolar or polygonal, L-DOPA staining is brownish black positive reaction, and flow cytometry and Western blot analysis results show that the obtained cells express MITF, TYR and TRP1 proteins. The cell proliferation capacity test result shows that the hair follicle stem cells and the melanocyte stem cells have stronger proliferation potential, obvious clone colonies can be generated by single cells, and the characteristics of the stem cells are met.
The results show that the human hair follicle stem cells and the human melanocyte stem cells are successfully separated and cultured in vitro.
Example 2 immunofluorescence assay for ADRB3 in human Hair follicle Stem cells and human melanocyte Stem cells
The human hair follicle stem cell and the human melanocyte stem cell are respectively 10 5 And inoculating the cells in a 6-well plate, placing a sterile cover glass in the plate, incubating until cell patches are formed, fixing the cells for 10min by 4% paraformaldehyde, and washing the cells for 3 times by PBS. 0.1% TritonX-100 was permeabilized for 10min, 3% BSA blocked for 1h, Ki67 antibody and human ADRB3 monoclonal antibody (1: 100) were added dropwise, and incubated overnight at 4 ℃. FITC-labeled secondary antibody (anti-rabbitt, 1: 800) and PE-labeled secondary antibody (A)anti-mouse, 1: 800), incubating at room temperature for 1h, DAPI 0.5 mu g/ml, 3min, PBS rinsing for 5min multiplied by 3, 50% glycerol/PBS sealing, observing under a laser confocal microscope, randomly observing 5-7 visual fields and shooting, and measuring the average red fluorescence intensity and the average green fluorescence intensity of cells by Fluorchem8900 software.
As a result: the ADRB3 protein was expressed in early G1 (Ki67 negative) and proliferative (Ki67 positive) human hair follicle stem cells (FIG. 1) and human melanocyte stem cells (FIG. 2). Cells strongly positive for ADRB3 had larger nuclei and looser chromatin compared to those weakly positive for ADRB 3. When SR59230A (10uM) or human ADRB3 monoclonal antibody (5ug/ml) was administered for 24 hours, respectively, the nuclei of both hair follicle stem cells and melanocyte stem cells were significantly condensed and the chromatin was dense (fig. 3), indicating that ADRB3 promoted chromatin decondensation.
Example 3 real-time quantitative PCR detection of hair follicle cells, melanocyte differentiation marker genes.
(1) The adherent human hair follicle stem cells and human melanocyte stem cells in logarithmic growth phase are respectively digested by pancreatin, RPMI l640 culture medium containing 10% calf serum is prepared into 5000/ml cell suspension, and the cell suspension is inoculated in a 6-hole culture plate at 37 ℃ and 5% CO 2 And culturing for 24 h.
(2) The experimental group is replaced by a new culture medium containing SR59230A, L748337 and human ADRB3 monoclonal antibodies with different concentrations, the control group is replaced by a culture medium containing an equal volume of solvent or murine IgG, each group is provided with 3-5 parallel holes, and the culture lasts for 4-5 days.
(3) Extracting cell RNA by a TRlzol method, performing reverse transcription by using a random primer and a reverse transcriptase to form cDNA, and detecting the expression level of hair follicle cell markers including keratin Krt5, Krt10, Krt14 and Krt19 and the expression level of melanocyte differentiation related gene Tyrosinase (TYR) and microphthalmia-related transcription factor (MITF) by real-time fluorescence quantitative PCR.
As a result: compared with the control group, the mRNA expression of the hair follicle stem cell keratins Krt5, Krt10, Krt14 and Krt19 of the ADRB3 retarder and antibody group are obviously increased by 2-4 times (P <0.01, figure 4). mRNA expression of both TYR and MITF, hair follicle stem cell keratins, was significantly elevated for the ADRB3 blocker and antibody groups (. sp. <0.01, fig. 5).
Example 4MTT assay to detect SR59230A, L748337 and human ADRB3 monoclonal antibodies inhibit proliferation of human hair follicle stem cells and human melanocyte stem cells
(1) Respectively digesting adherent human hair follicle stem cells and human melanocyte stem cells in logarithmic growth phase by pancreatin, preparing 5000/ml cell suspension by using RPMI l640 culture medium containing 10% calf serum, inoculating into 96-well culture plate, inoculating 200ul of cell suspension in each well, 37 ℃, and 5% CO 2 And culturing for 24 h.
(2) The experimental group was replaced with a new medium containing different concentrations of SR59230A, L748337 and human ADRB3 monoclonal antibody, the control group was replaced with a medium containing an equal volume of solvent or murine IgG, each group was provided with 3-5 parallel wells at 37 deg.C and 5% CO 2 Culturing for 4-5 days.
(3) The supernatant was discarded and 200. mu.l of freshly prepared serum-free medium containing 0.5mg/ml MTT was added to each well. The culture was continued at 37 ℃ for 4 h. Adding 200 μ l DMSO, mixing with ultrasonic oscillator, and measuring optical density with microplate reader at test wavelength of 570nm and reference wavelength of 450 nm.
As a result: SR59230A, L748337 inhibited proliferation of human hair follicle stem cells and human melanocyte stem cells dose-dependently, with P <0.05 and # P <0.01 (fig. 6, ordinate is cell proliferation inhibition rate, and concentration unit of blocking agent is umol/L) compared to control group. The human ADRB3 monoclonal antibody inhibited proliferation of human hair follicle stem cells and human melanocyte stem cells dose-dependently, with P <0.05 and P <0.01 (FIG. 7) compared to control group.
The results of examples 3 and 4 show that ADRB3 is highly related to the proliferation and differentiation of human hair follicle stem cells and human melanocyte stem cells, and the inhibition of ADRB3 and the reduction of the activity of ADRB3 by using a small molecular compound blocker or a large molecular inhibitory antibody can inhibit the proliferation and differentiation of human hair follicle stem cells and human melanocyte stem cells.
Example 5Transwell cell migration assay to examine the Effect of SR59230A and human ADRB3 monoclonal antibodies on migration of human hair follicle stem cells and human melanocyte stem cells
The gel was polymerized by diluting with Matrigel1:8 from BD, coating the upper face of the bottom membrane of the Transwell chamber and leaving the membrane at 37 ℃ for 30 min. Digesting cells and regulating their finenessCell density to 5X 10 5 And/ml. 100. mu.l of the cell suspension was taken and added to a Transwell chamber. 600 μ l of medium containing SR59230A or human ADRB3 monoclonal antibody was added to the lower chamber of the 24-well plate. Culturing for 24-48 h. The Transwell chamber was removed, washed 2 times with calcium-free PBS and fixed in methanol for 30 min. 0.1% crystal violet stain for 20min, gently wipe off the upper non-migrated cells with a cotton swab, wash 3 times with PBS. Cells were observed in five fields immediately under 400-fold microscope and counted.
As a result: compared with the migration number of human hair follicle stem cells and human melanocyte stem cells of a control group [ (21.3 +/-3.4) × 10 4 ，(24.1±3.6)×10 4 ]The number of stem cell migration in the SR 59230A-treated group was [ (9.2. + -. 1.4). times.10% 4 ，(7.5±1.2)×10 4 ]Are all obviously reduced (P)<0.01), number of migration of two stem cells of human ADRB3 monoclonal antibody-treated group [ (8.6. + -. 1.7). times.10) 4 ，(8.3±1.3)×10 4 ]Are all obviously reduced (P)<0.01). Indicating that ADRB3 is highly correlated with migration of HFSCs and MeSCs, inhibition of ADRB3 activity will retain HFSCs and MeSCs in the hair follicle preventing numbers reduction or depletion.
Example 6SR59230A and human ADRB3 monoclonal antibody for treating alopecia and canities in geriatric mice
1. Experiment grouping
All of 30 male 15-month-old C57BL/6J mice developed alopecia and poliosis. Randomized into 3 groups:
1. control group; the control group was given the same volume of solvent.
SR59230A group, 100nmol SR59230A per mouse was intraperitoneally injected every 3 days for 5 weeks;
3 O. human ADRB3 monoclonal antibody group, human ADRB3 monoclonal antibody was intraperitoneally injected every 3 days at 5mg/kg for 5 weeks.
2. The experimental observation shows the occurrence of alopecia and poliosis.
3. And collecting the skin of the head of the mouse, and performing immunohistochemical detection on the expression condition of ADRB3 in hair follicles.
The experimental steps are as follows: paraffin section: the thickness was 4 μm. 65 ℃ for 3 h. Dewaxed to water. PH8.0TRIS-EDTA repair liquid is used for high-pressure repair, and the air is naturally cooled after 3min from the beginning of air exhaust of the exhaust valve. Incubate with 3% hydrogen peroxide for 10 min. Blocking 10% goat serum for 10 min. Primary antibody (1: 100) was kept at 4 ℃ overnight. The secondary antibody was incubated at room temperature for 30 min. And (5) DAB color development. (the ratio of the solution A to the solution B is 1: 50), and the expression is proper and the background is clear when the solution A and the solution B are observed under a microscope, so that the expression can be stopped. Counter staining with hematoxylin for 1min, and bluing with warm water for 15 min. Dewatering with gradient alcohol, and sealing with neutral gum. And (3) judging the dyeing intensity: 6-8 high-power visual fields are observed at random on each tissue point, wherein weak positive is faint yellow, medium positive is tan, and strong positive is tan. And (3) judging the dyeing positive rate: 3 fields with different staining intensities are selected to be interpreted under a high power microscope, 100 cells are randomly recorded in each field, the percentage of positive cells in the 100 cells is recorded as X1%, the percentage of positive cells in the other 2 fields is also viewed according to the principle, then X2% and X3% are recorded, and finally the tissue point staining positive rate is averaged by X1%, X2% and X3%.
3. The experimental results are as follows:
compared with the control group, the mice in the SR59230A and human ADRB3 monoclonal antibody groups are black and bright in hair, thick in hair, and remarkably reduced in alopecia and white hair, and the SR59230A and human ADRB3 monoclonal antibodies can promote the regeneration of the pigmented hair of the aged C57BL/6J mice (figure 8). Cells positive for control mouse ADRB3 protein were predominantly located in the follicular bulge region, which is where HFSCs and MeSCs were enriched. The positive rate of ADRB3 in the hair follicle bulge tissue was 100% (10/10) (fig. 9). The SR59230A or human ADRB3 monoclonal antibody obviously reduces the positive rate of the hair follicle cell ADRB3 protein, the positive rate is 20% and 10% respectively, and is obviously lower than that of a control group (P < 0.05). Compared with control group ADRB3 positive hair follicle cells, SR59230A and human ADRB3 monoclonal antibody group ADRB3 positive hair follicle cells were smaller in nucleus and denser in chromatin, indicating that ADRB3 is likely to act as an epigenetic factor to promote chromatin de-aggregation.
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)
- Use of an ADRB3 inhibitor for the preparation of a formulation for inducing differentiation of hair follicle stem cells and/or melanocyte stem cells.
- Use of an ADRB3 inhibitor for the preparation of a formulation for inhibiting migration of hair follicle stem cells and/or melanocyte stem cells out of a hair follicle.
- Use of an ADRB3 inhibitor for the preparation of a formulation for promoting the regeneration of hair follicle stem cells and/or melanocyte stem cells.
- Use of an ADRB3 inhibitor for the preparation of a formulation for inhibiting the proliferation of hair follicle stem cells and/or melanocyte stem cells.
- Use of an ADRB3 inhibitor for the preparation of a formulation for promoting the withdrawal of hair follicle stem cells and/or melanocyte stem cells from the cell cycle.
- Use of an ADRB3 inhibitor for the preparation of a formulation for the prevention and treatment of hair loss and/or white hair.
- 7. The use of any one of claims 1 to 6, wherein said inhibitor of ADRB3 is an ADRB3 blocker or a human ADRB3 monoclonal antibody.
- 8. The use of claim 7, wherein the ADRB3 blocker is SR59230A or a compound comprising SR59230A as a parent nucleus.
- 9. The use according to claim 7, wherein the ADRB3 blocker is L748337 or a compound comprising L748337 as a parent nucleus.
- 10. A preparation for preventing and treating alopecia and/or canities, comprising an ADRB3 inhibitor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210439551.2A CN114848821B (en) | 2022-04-25 | 2022-04-25 | Application of human beta 3 adrenergic receptor and inhibitor thereof in preparation of hair loss prevention and treatment products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210439551.2A CN114848821B (en) | 2022-04-25 | 2022-04-25 | Application of human beta 3 adrenergic receptor and inhibitor thereof in preparation of hair loss prevention and treatment products |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114848821A true CN114848821A (en) | 2022-08-05 |
| CN114848821B CN114848821B (en) | 2024-01-02 |
Family
ID=82633307
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210439551.2A Active CN114848821B (en) | 2022-04-25 | 2022-04-25 | Application of human beta 3 adrenergic receptor and inhibitor thereof in preparation of hair loss prevention and treatment products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114848821B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130197091A1 (en) * | 2012-01-30 | 2013-08-01 | Shuguang LIN | Use of adrenergic beta-e-receptor blockers in cancer treatment |
| CN107271675A (en) * | 2017-03-24 | 2017-10-20 | 郑猛 | Anti-human ADRB3 monoclonal antibodies and its application in medical diagnosis on disease and treatment |
| CN112822948A (en) * | 2018-07-26 | 2021-05-18 | 应用生物学公司 | TAAR receptor agonists for the treatment of alopecia |
-
2022
- 2022-04-25 CN CN202210439551.2A patent/CN114848821B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130197091A1 (en) * | 2012-01-30 | 2013-08-01 | Shuguang LIN | Use of adrenergic beta-e-receptor blockers in cancer treatment |
| CN107271675A (en) * | 2017-03-24 | 2017-10-20 | 郑猛 | Anti-human ADRB3 monoclonal antibodies and its application in medical diagnosis on disease and treatment |
| CN112822948A (en) * | 2018-07-26 | 2021-05-18 | 应用生物学公司 | TAAR receptor agonists for the treatment of alopecia |
Non-Patent Citations (2)
| Title |
|---|
| ZHANG ET AL.: "Hyperactivation of Sympathetic Nerves Drives Melanocyte Stem Cell Depletion", vol. 577, pages 676, XP037000703, DOI: 10.1038/s41586-020-1935-3 * |
| 郭彦青等: "β肾上腺素受体研究进展", vol. 15, no. 12, pages 1331 - 1332 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114848821B (en) | 2024-01-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Commo et al. | Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study | |
| Rousseau et al. | RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration | |
| JP6923927B2 (en) | Compositions for suppressing or ameliorating hair loss and whitening and their use | |
| Dong et al. | The effects of NB-UVB on the hair follicle-derived neural crest stem cells differentiating into melanocyte lineage in vitro | |
| KR102072783B1 (en) | Molecular signature of cutaneous pigmentary spots, associated with the extracellular matrix | |
| Ganier et al. | Fibroblast heterogeneity in healthy and wounded skin | |
| Wang et al. | Human amniotic stem cells-derived exosmal miR-181a-5p and miR-199a inhibit melanogenesis and promote melanosome degradation in skin hyperpigmentation, respectively | |
| Fukunaga-Kalabis et al. | UV-Induced Wnt7a in the Human Skin Microenvironment Specifies the Fate of Neural Crest–Like Cells via Suppression of Notch | |
| WO2022062197A1 (en) | Medical use of honokiol | |
| Wang et al. | Bach2 regulates autophagy to modulate UVA-induced photoaging in skin fibroblasts | |
| Epperlein et al. | Xanthophores in chromatophore groups of the premigratory neural crest initiate the pigment pattern of the axolotl larva | |
| KR20130056955A (en) | Composition for promoting mature melanocyte migration comprising chemokine, detection kit for skin-active ingredients comprising chemokine gene and the method for detecting skin-active ingredients by using chemokine gene | |
| CN114848821A (en) | Application of human beta 3 adrenergic receptor and inhibitor thereof in preparation of products for preventing and treating alopecia and leukotrichia | |
| Sun et al. | Autophagy induces hair follicle stem cell activation and hair follicle regeneration by regulating glycolysis | |
| TWI450714B (en) | Use of compound for inhibiting breast tumor cell proliferation | |
| KR101032271B1 (en) | Composition of regenerating skin cell, method of manufacturing the same, method of regenerating skin cell and cosmetic composition | |
| Li et al. | Ultrasmall Prussian blue nanoparticles attenuate UVA-induced cellular senescence in human dermal fibroblasts via inhibiting the ERK/AP-1 pathway | |
| Klingel et al. | Coexpression of extracellular matrix glycoproteins undulin and tenascin in human autosomal dominant polycystic kidney disease | |
| WO2021197290A1 (en) | Novel application of acetylcholinesterase activity inhibitor | |
| Tiwari et al. | BMP6 regulates corneal epithelial cell stratification by coordinating their proliferation and differentiation and is upregulated in pterygium | |
| TW201110973A (en) | Methods for screening for anti-graying agents on the basis of AFF-4 | |
| Pang et al. | Endothelin-1 increases melanin synthesis in an established sheep skin melanocyte culture | |
| Chen et al. | Dedifferentiation of human epidermal melanocytes in vitro by long-term trypsinization | |
| Li et al. | Exploring the potential effect and mechanisms of protocatechuic acid on human hair follicle melanocytes | |
| US20160256419A1 (en) | Application of fluoxetine to treatment of depigmentation diseases |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |