CN116999537A - Application of fibroblast growth factor 9 in treating testicular hypofunction - Google Patents
Application of fibroblast growth factor 9 in treating testicular hypofunction Download PDFInfo
- Publication number
- CN116999537A CN116999537A CN202311221131.8A CN202311221131A CN116999537A CN 116999537 A CN116999537 A CN 116999537A CN 202311221131 A CN202311221131 A CN 202311221131A CN 116999537 A CN116999537 A CN 116999537A
- Authority
- CN
- China
- Prior art keywords
- fgf
- testicular
- growth factor
- fibroblast growth
- cells
- 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.)
- Pending
Links
- 108090000367 Fibroblast growth factor 9 Proteins 0.000 title claims abstract description 120
- 102000003957 Fibroblast growth factor 9 Human genes 0.000 title claims abstract description 36
- 230000002381 testicular Effects 0.000 title abstract description 51
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims abstract description 136
- 229960003604 testosterone Drugs 0.000 claims abstract description 68
- 239000003814 drug Substances 0.000 claims abstract description 58
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 36
- 210000002966 serum Anatomy 0.000 claims abstract description 26
- 230000004069 differentiation Effects 0.000 claims abstract description 25
- 230000001965 increasing effect Effects 0.000 claims abstract description 19
- 230000001939 inductive effect Effects 0.000 claims abstract description 13
- 201000003585 eunuchism Diseases 0.000 claims abstract description 11
- 208000037106 male hypogonadism Diseases 0.000 claims abstract description 11
- 208000011580 syndromic disease Diseases 0.000 claims abstract description 10
- 230000014509 gene expression Effects 0.000 claims description 64
- 210000001550 testis Anatomy 0.000 claims description 62
- 108090000623 proteins and genes Proteins 0.000 claims description 36
- 210000002332 leydig cell Anatomy 0.000 claims description 29
- 108010084976 Cholesterol Side-Chain Cleavage Enzyme Proteins 0.000 claims description 28
- 102100027516 Cholesterol side-chain cleavage enzyme, mitochondrial Human genes 0.000 claims description 25
- 230000037361 pathway Effects 0.000 claims description 25
- 102000004169 proteins and genes Human genes 0.000 claims description 23
- 239000003098 androgen Substances 0.000 claims description 20
- 238000003786 synthesis reaction Methods 0.000 claims description 20
- 230000015572 biosynthetic process Effects 0.000 claims description 19
- -1 scarb1 Proteins 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 15
- 230000035755 proliferation Effects 0.000 claims description 14
- 102100036009 5'-AMP-activated protein kinase catalytic subunit alpha-2 Human genes 0.000 claims description 10
- 101000783681 Homo sapiens 5'-AMP-activated protein kinase catalytic subunit alpha-2 Proteins 0.000 claims description 10
- 101150092219 LHCGR gene Proteins 0.000 claims description 9
- 102100031455 NAD-dependent protein deacetylase sirtuin-1 Human genes 0.000 claims description 9
- 102100022679 Nuclear receptor subfamily 4 group A member 1 Human genes 0.000 claims description 9
- 101710092553 Nuclear receptor subfamily 4 group A member 1 Proteins 0.000 claims description 9
- 108010041191 Sirtuin 1 Proteins 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- 102100036506 11-beta-hydroxysteroid dehydrogenase 1 Human genes 0.000 claims description 8
- 101000928753 Homo sapiens 11-beta-hydroxysteroid dehydrogenase 1 Proteins 0.000 claims description 8
- 101001039035 Homo sapiens Lutropin-choriogonadotropic hormone receptor Proteins 0.000 claims description 8
- 101000896517 Homo sapiens Steroid 17-alpha-hydroxylase/17,20 lyase Proteins 0.000 claims description 8
- 102100040788 Lutropin-choriogonadotropic hormone receptor Human genes 0.000 claims description 8
- 102100021719 Steroid 17-alpha-hydroxylase/17,20 lyase Human genes 0.000 claims description 8
- 101150051357 CYP17A1 gene Proteins 0.000 claims description 6
- 102100039082 3 beta-hydroxysteroid dehydrogenase/Delta 5->4-isomerase type 1 Human genes 0.000 claims description 5
- 101000744065 Homo sapiens 3 beta-hydroxysteroid dehydrogenase/Delta 5->4-isomerase type 1 Proteins 0.000 claims description 5
- 101150044508 key gene Proteins 0.000 claims description 5
- 230000000694 effects Effects 0.000 abstract description 30
- 229940079593 drug Drugs 0.000 abstract description 6
- 231100000331 toxic Toxicity 0.000 abstract description 5
- 230000002588 toxic effect Effects 0.000 abstract description 5
- 102100037665 Fibroblast growth factor 9 Human genes 0.000 description 94
- 210000004027 cell Anatomy 0.000 description 43
- 241000700159 Rattus Species 0.000 description 33
- 208000002197 Ehlers-Danlos syndrome Diseases 0.000 description 25
- 210000002863 seminiferous tubule Anatomy 0.000 description 22
- 239000001963 growth medium Substances 0.000 description 21
- 239000000463 material Substances 0.000 description 18
- 239000002609 medium Substances 0.000 description 18
- 238000011282 treatment Methods 0.000 description 13
- 101001027380 Homo sapiens Fibroblast growth factor 9 Proteins 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 102000009151 Luteinizing Hormone Human genes 0.000 description 11
- 108010073521 Luteinizing Hormone Proteins 0.000 description 11
- 229940040129 luteinizing hormone Drugs 0.000 description 11
- 108091005981 phosphorylated proteins Proteins 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 238000012258 culturing Methods 0.000 description 9
- 230000006870 function Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 108010051779 histone H3 trimethyl Lys4 Proteins 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000001737 promoting effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 101150027061 RPS16 gene Proteins 0.000 description 6
- 239000002552 dosage form Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 101000711846 Homo sapiens Transcription factor SOX-9 Proteins 0.000 description 5
- 206010058359 Hypogonadism Diseases 0.000 description 5
- 108091054455 MAP kinase family Proteins 0.000 description 5
- 102000043136 MAP kinase family Human genes 0.000 description 5
- 102100034204 Transcription factor SOX-9 Human genes 0.000 description 5
- 239000002775 capsule Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 230000002062 proliferating effect Effects 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 4
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 4
- 108010033040 Histones Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 201000010063 epididymitis Diseases 0.000 description 4
- 239000008187 granular material Substances 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102100022585 17-beta-hydroxysteroid dehydrogenase type 3 Human genes 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 101001045211 Homo sapiens 17-beta-hydroxysteroid dehydrogenase type 3 Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- 229940125829 fibroblast growth factor receptor inhibitor Drugs 0.000 description 3
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 210000002536 stromal cell Anatomy 0.000 description 3
- 102100031571 40S ribosomal protein S16 Human genes 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 101000706746 Homo sapiens 40S ribosomal protein S16 Proteins 0.000 description 2
- 101000861327 Homo sapiens Cholesterol side-chain cleavage enzyme, mitochondrial Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 229940030486 androgens Drugs 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000000022 bacteriostatic agent Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 239000007474 bm medium Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 210000000918 epididymis Anatomy 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 102000057240 human FGF9 Human genes 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000007951 isotonicity adjuster Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000013222 sprague-dawley male rat Methods 0.000 description 2
- 230000009469 supplementation Effects 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 210000005239 tubule Anatomy 0.000 description 2
- 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
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 206010002261 Androgen deficiency Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000008137 Bone Morphogenetic Protein 4 Human genes 0.000 description 1
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 1
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 1
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 1
- 239000011547 Bouin solution Substances 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 101100477411 Dictyostelium discoideum set1 gene Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 108091008794 FGF receptors Proteins 0.000 description 1
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 206010024419 Libido decreased Diseases 0.000 description 1
- 240000000233 Melia azedarach Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010027940 Mood altered Diseases 0.000 description 1
- 208000008601 Polycythemia Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 101100480944 Rattus norvegicus Tes gene Proteins 0.000 description 1
- 206010041277 Sodium retention Diseases 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 229940112869 bone morphogenetic protein Drugs 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 230000008451 emotion Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000001856 erectile effect Effects 0.000 description 1
- RYSRRQRKJUEFRT-UHFFFAOYSA-N ethane;methanesulfonic acid Chemical compound CC.CS(O)(=O)=O.CS(O)(=O)=O RYSRRQRKJUEFRT-UHFFFAOYSA-N 0.000 description 1
- 230000008713 feedback mechanism Effects 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002710 gonadal effect Effects 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 210000001596 intra-abdominal fat Anatomy 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007510 mood change Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 231100000989 no adverse effect Toxicity 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 210000003899 penis Anatomy 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Reproductive Health (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Pregnancy & Childbirth (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Gynecology & Obstetrics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Endocrinology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
Abstract
The invention relates to the technical field of medicines, in particular to application of fibroblast growth factor 9 in treating testicular hypofunction. The medicament for treating male hypogonadism syndrome prepared by using the fibroblast growth factor 9 has the effects of increasing the content of serum testosterone and inducing differentiation of testicular mesenchymal stem cells, is safe to use, has no obvious toxic or side effect, and can be used for treating testicular hypofunction.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of fibroblast growth factor 9 in treating testicular hypofunction.
Background
Hypogonadism (hypogonadism) is a multiple testis hypofunction in men and is mainly characterized by hyposexuality and erectile quality, mood changes with reduced mental and spatial orientation, reduced muscle strength, reduced body hair and skin changes, reduced bone density, and increased visceral fat [ NatRev Dis primers.2019May 30;5 (1): 38 ]. Hypogonadism generally occurs well in the ages of 45-55 years, and can be as early as 40 years or as late as 65 years, and the onset of the hypogonadism is related to hypothalamic-pituitary-testicular shafting hypofunction and testicular interstitial cell (LCs) decline [ Nat Commun.2022jul11;13 4020.
Testicular stromal cells are cells with testosterone synthesizing and secreting functions, and are the most important source of androgens in males. Testosterone (T) in human serum is produced by stimulation of Testosterone cells by pituitary-secreted Luteinizing Hormone (LH) and is regulated by a series of negative feedback mechanisms. Clinical studies have shown that male hypothalamic-pituitary shaft function gradually decreases with age, resulting in a decrease in the magnitude of LH pulse release, ultimately affecting testicular interstitial cell synthesis and secretion of androgens [ Proc Natl Acad Sci U S a.2006feb 21;103 2719 to 24; proc Natl Acad Sci U S a.2016mar8; 113 (10) 2666-71; endocrRev.2020Feb 1;41 And (1) bnz013.
Currently, clinically treating hypogonadal syndromes is mainly by testosterone supplementation therapy, however, this therapy has significant safety issues in addition to the need for regular testosterone injections. First, chronic quantitative supplementation of testosterone can predispose patients to acne and erythrocytosis; secondly, the concentration of the testosterone in the serum is easy to fluctuate greatly, and obvious fluctuation of symptoms of the patients with the symptoms of the low-grade gonadal function of the emotion and the tardive is caused; again, patients are prone to adverse reactions such as water and sodium retention, abnormal erection of penis, difficult urination and the like, and even diseases such as impaired liver and kidney functions and initiation of prostate cancer [ Endocr rev.2020feb1;41 (1) 22-32 ]. Therefore, a medicament for treating male hypogonadism syndrome with good treatment effect and no obvious toxic or side effect is urgently needed.
Disclosure of Invention
In order to solve the problems, the invention provides application of fibroblast growth factor 9 (Fibroblast Growth Factor-9, FGF-9) in preparing medicaments for treating male hypogonadism syndrome. The medicine for treating male hypogonadism syndrome prepared by the fibroblast growth factor 9 has the effects of increasing testosterone content in serum and testes and inducing proliferation and differentiation of testicular mesenchymal stem cells, and has the advantages of safe administration and no obvious toxic or side effect.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of fibroblast growth factor 9 in preparing a medicament for treating male hypogonadism syndrome.
The invention provides application of fibroblast growth factor 9 in preparing a medicament for inducing proliferation and/or differentiation of testicular mesenchymal stem cells.
The invention provides application of fibroblast growth factor 9 in preparing a medicament for improving testosterone content in serum and/or testis.
The invention provides application of fibroblast growth factor 9 in preparing a medicament for improving the number of testicular interstitial cells in testes.
The invention provides the use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of expression of a key gene in the androgen synthesis pathway in testosterone cells, said key gene comprising one or more of Lhcgr, scarb1, cyp11a1, hsd b1, cyp17a1 and Hsd b 3.
The present invention provides the use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of expression of a key protein in the androgen synthesis pathway in testosterone cells, said key protein comprising one or more of LHCGR, CYP11A1, CYP17A1, HSD3B1 and HSD11B 1.
The invention provides the use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of key proteins in testicular stromal cells, including one or more of pAMPK, AMPK, pMAPK, pPI3K, pCREB, SIRT1, NUR77 and H3K4me 3.
The beneficial effects are that:
the invention provides application of fibroblast growth factor 9 in preparing a medicament for treating male hypogonadism syndrome. The medicine prepared by using fibroblast growth factor 9 (Fibroblast Growth Factor-9, FGF-9) for treating male hypogonadism has the effects of increasing the content of serum testosterone and inducing differentiation of testicular mesenchymal stem cells, and has the advantages of safe administration and no obvious toxic or side effect. The results of the examples show that: the fibroblast growth factor 9 can obviously improve the testosterone content in serum of an EDS model rat, and the weight and the testis wet weight of the rat have no obvious change after continuous administration for 14 days, so that the administration safety performance is high; meanwhile, the fibroblast growth factor 9 also has the effect of inducing proliferation and differentiation of the testicular mesenchymal stem cells, can up-regulate the expression level of key genes Lhcgr, scarb1, cyp11A1, hsd B1, cyp17A1 and Hsd B3 in the androgen synthesis pathway, up-regulate the expression level of key proteins LHCGR, CYP11A1, CYP17A1, HSD3B1 and HSD11B1 in the androgen synthesis pathway in the testicular mesenchymal cells, up-regulate the expression level of key proteins pAMPK and AMPK in the testicular mesenchymal cells; levels of pMAPK, ppri 3K, pCREB, SIRT1 and NUR77 expression; up-regulating the expression level of key protein H3K4me3 in testicular interstitial cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the effect of FGF-9 on testosterone, LH and FSH levels in serum of EDS model rats in example 1; wherein A is the operation scheme of the EDS model, B is the content of testosterone in serum after the EDS model rat is dosed, C is the content of LH in serum after the EDS model rat is dosed, and D is the content of FSH in serum after the EDS model rat is dosed;
FIG. 2 is a graph showing the effect of FGF-9 on the number of cells positive for the CYP11A1 in the testicular interstitial cells of an EDS model rat in example 1; wherein A is the state of CYP11A1 expression by cells after staining of cells of a control group (0 ng/testis FGF-9), B is the state of CYP11A1 expression by cells after staining of cells of a 10 ng/testis FGF-9, C is the state of CYP11A1 expression after staining of 100 ng/testis FGF-9, and D is the number of cells expressing CYP11A1 after staining of 0 ng/testis, 10 ng/testis and 100 ng/testis FGF-9; e is the state of expressing SOX9 by cells after staining of 0 ng/testis FGF-9 group, F is the state of expressing SOX9 after staining of 10 ng/testis FGF-9 group, G is the state of expressing SOX9 after staining of 100 ng/testis FGF-9 group, F is the number of cells expressing SOX9 after staining of 0 ng/testis, 10 ng/testis and 100 ng/testis FGF-9 group;
FIG. 3 shows the results of FGF-9 stimulating proliferation of mesenchymal stem cells in example 1; wherein A is the state of PCNA (proliferating cell nucleus), CYP11A1 (testicular interstitial cell marker for localization) and DAPI (cell nucleus) expressed by cells after staining of testis sections in groups 0 ng/testis, 10 ng/testis and 100 ng/testis FGF-9, and B is the effect of each treatment in example 1 on the number of cells expressing PCNA;
FIG. 4 shows the effect of FGF-9 on androgen synthesis pathway-related gene expression in testicular interstitial cells in example 1 (Rps 16 is an internal reference gene);
FIG. 5 shows the effect of FGF-9 on the expression of a protein involved in the androgen synthesis pathway in testicular interstitial cells of an EDS model rat in example 1; wherein A is gel strip; B-F are results of measuring the expression levels of LHCGR, CYP11A1, CYP17A1, HSD17B3 and HSD11B1 in rat testis interstitial cells by WB after EDS administration and administration of FGF-9 (0, 10 and 100 ng/testis) at different concentrations (RPS 16 is an internal reference protein);
FIG. 6 shows the effect of FGF-9 on expression levels of phosphorylated proteins and total proteins thereof in testicular interstitial cells after administration of an EDS model rat in example 1; wherein A is gel strip; b is the result of giving FGF-9 (0, 10 and 100 ng/testis) with different concentrations after EDS administration, and WB detects the expression level of AMPK and phosphorylated proteins thereof, MAPK and phosphorylated proteins thereof, PI3K and phosphorylated proteins thereof, CREB and phosphorylated proteins thereof in rat testicular interstitial cells (RPS 16 is an internal reference protein); c is the result of measuring SIRT1 protein expression level in rat testicular interstitial cells by administering FGF-9 (0, 10 and 100 ng/testis) at different concentrations after EDS administration (RPS 16 is an internal reference protein); d is the result of measuring the expression level of NUR77 protein in rat testicular interstitial cells (RPS 16 is an internal reference protein) by administering FGF-9 (0, 10 and 100 ng/testis) at different concentrations after EDS administration;
FIG. 7 is a graph showing the effect of FGF-9 on the expression level and localization of histone H3K4me3 and H3K9me3 proteins in testicular interstitial cells after administration of an EDS model rat in example 1; wherein A is a gel strip of H3K4me3, and RPS16 is an internal reference protein; b is the expression level of FGF-9 (0, 10 and 100 ng/testis), H3K4me3 and CYP11A1 double-stained, localized expression and expression level in testis, CYP11A1 (red) localized testicular interstitial cells, expression level of H3K4me3 (green) in testis and expression level on testicular interstitial cells with different concentrations after EDS administration; c is a gel band of H3K9me3, and RPS16 is an internal reference protein; d is FGF-9 (0, 10 and 100 ng/testis) with different concentrations, H3K9me3 and CYP11A1 double-dyeing, positioning expression and expression quantity in testis, CYP11A1 (red) positioning testicular interstitial cells, and expression position of H3K9me3 (green) in testis and expression quantity on testicular interstitial cells after EDS administration;
FIG. 8 shows the proliferation of FGF-9 pair-stimulated mesenchymal stem cells in example 3; wherein A is a scheme for treating the seminiferous tubules; B-E are states of testicular mesenchymal stem cells proliferated on the surface of the seminiferous tubule, B is 0ng/mL FGF9 treatment group, C is 10ng/mL FGF9 treatment group, and D is FGFR inhibitor PD (1 mu M) treatment group; e is the effect on proliferation of mesenchymal stem cells after co-culture with addition of FGF9 (10 ng/mL) and PD (1. Mu.M); f is the effect of each treatment group on the number of cells proliferating testicular mesenchymal stem cells in example 3; g is the effect on the number of cells proliferating mesenchymal stem cells after addition of FGF receptor inhibitor PD in example 3; h is the effect of each treatment (0, 1,10 and 100 ng/mL) in example 3 on testosterone levels (proliferation);
FIG. 9 is the effect of FGF-9 treatments (0, 1 and 10 ng/mL) on testosterone content (differentiation) in example 4; and the effect of each treatment (0, 1 and 10 ng/mL) on androgen synthesis pathway-associated gene expression in testosterone cells;
wherein, relative to the control group or the corresponding group, P <0.05, P < 0.01, and P < 0.005.
Detailed Description
The invention provides application of fibroblast growth factor 9 in preparing a medicament for treating male hypogonadism syndrome. The effective component of the medicine for treating male hypogonadism syndrome is fibroblast growth factor 9. The medicine for treating male hypogonadism prepared by using FGF-9 has the effects of increasing the content of serum testosterone and inducing proliferation and differentiation of testicular mesenchymal stem cells, so that the aim of relieving or treating the hypogonadism is fulfilled, and meanwhile, the medicine has no obvious toxic or side effect after administration of FGF-9 and has good safety performance.
In the present invention, the FGF-9 is preferably obtained by isolation from a natural organism, chemical synthesis of a polypeptide, expression of a prokaryotic microorganism genetically engineered bacterium followed by purification or expression by an animal cell or the like and purification. FGF-9 in the examples of the present invention is recombinant human FGF-9 (Catalog #: 273-F9) from R & D company.
The FGF-9 according to the present invention preferably also comprises a polymer, i.e.an aggregate of several FGFs-9 assembled together in an automated manner (e.g.by physical adsorption), in which case it is understood that a natural phenomenon of biological origin is observed, or that several FGF-9 are genetically linked.
The FGF-9 provided by the present invention preferably further comprises a linker, i.e. the interconnecting of FGF-9 is effected in a manually operated manner using a biological or chemical linking technique (BioConjugate Chemistry), in which case it is understood that it is distinct from the natural phenomenon of living beings, but is effected in the case of human intervention.
The invention has no special requirement on the dosage form of the medicine, adopts the conventional dosage form in the field, and is not limited to aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder fog agent, sustained release agent, controlled release agent and the like.
The invention has no special requirement on auxiliary materials of the medicine, adopts the conventional auxiliary materials in the field, and is not limited to isotonic agent, buffer solution, corrigent, excipient, filler, adhesive, disintegrating agent, lubricant and the like; may also be selected for adaptation to the substance, such as: the auxiliary materials can effectively improve the stability and the solubility of the compounds contained in the composition or change the release rate, the absorption rate and the like of the compounds, thereby improving the metabolism of various compounds in organisms and further enhancing the administration effect of the composition. In addition, the auxiliary materials can also be used for realizing specific administration purposes or modes, such as: sustained release administration, controlled release administration, pulse administration, etc., and used auxiliary materials such as gelatin, albumin, chitosan, polyether and polyester type high molecular materials, polyethylene glycol, polyurethane, polycarbonate, copolymers thereof, etc.
When the medicament is an aqueous solution injection, the auxiliary materials preferably comprise an isotonic agent, a buffer solution, an emulsifying agent, a solubilizer and a bacteriostatic agent. In addition, it is also preferable to include other pharmaceutically acceptable pharmaceutical excipients such as: antioxidants, pH adjusters, analgesics, and the like.
When the medicament is an oral liquid preparation, the auxiliary materials preferably comprise a solvent, a flavoring agent, a bacteriostatic agent, an emulsifying agent and a coloring agent.
When the medicament is a tablet, the auxiliary materials are preferably fillers, binders, disintegrants and lubricants.
When the medicament is an emulsion, the auxiliary materials are preferably water, oil, emulsifying agents, necessary preservatives and flavoring agents.
When the medicament is a granule, the auxiliary materials are similar to tablets in kind, but the granulation process is different.
When the medicine is in the form of capsule, the auxiliary materials are similar to granules, and the prepared granules and the glidant are mixed and then are filled into capsules to obtain the capsule.
The invention provides application of fibroblast growth factor 9 in preparing a medicament for inducing proliferation and/or differentiation of testicular mesenchymal stem cells. The active ingredient of the medicament for inducing the proliferation and/or differentiation of the testicular mesenchymal stem cells is FGF-9, and the medicament has no special requirement on the source of FGF-9 and can be prepared by adopting common commercial products. The invention has no special requirement on the dosage form of the medicament for inducing the proliferation and/or differentiation of the testicular mesenchymal stem cells, and can be used by adopting the conventional dosage form in the field. The invention has no special requirement on the auxiliary materials of the medicament for inducing the proliferation and/or differentiation of the testicular mesenchymal stem cells, and the auxiliary materials are adopted by the conventional auxiliary materials in the field. The medicament for inducing the proliferation and/or differentiation of the testicular mesenchymal stem cells prepared by using the FGF-9 can increase the number of the CYP11A1 positive testicular mesenchymal stem cells, and can remarkably promote the differentiation of the testicular mesenchymal stem cells, thereby promoting the secretion of testosterone and improving the testosterone level.
The invention provides application of fibroblast growth factor 9 in preparing a medicament for improving testosterone content in serum and/or testis. The active ingredient of the medicine for improving the testosterone content in serum and/or testis is FGF-9, the source of the FGF-9 is not specially required, and common commercial products are adopted. The invention has no special requirement on the dosage form of the medicament for improving the testosterone content in serum and/or testis, and can be used in the conventional dosage form in the field. The invention has no special requirement on the auxiliary materials of the medicament for improving the testosterone content in serum and/or testis, and the conventional auxiliary materials in the field are adopted. The medicine prepared by FGF-9 and containing testosterone in serum and/or testis has the function of obviously improving the testosterone level in serum and testis.
The invention provides application of fibroblast growth factor 9 in preparing a medicament for improving the number of testicular interstitial cells in testes. The effective component of the medicament for improving the number of testicular interstitial cells in the testis is FGF-9. The medicine for improving the number of the testicular mesenchymal cells in the testis prepared by using FGF-9 has the effect of obviously improving the number of the testicular mesenchymal cells in the testis.
The invention provides the use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of expression of a key gene in the androgen synthesis pathway in testosterone cells, said key gene comprising one or more of Lhcgr, scarb1, cyp11a1, hsd b1, cyp17a1 and Hsd b 3. The FGF-9 is used for preparing the medicine for up-regulating the expression level of the key genes in the testosterone synthesis pathway of the testicular mesenchymal cells, and the medicine can play a role in promoting the differentiation of the testicular mesenchymal stem cells through the Lhcgr, scarb1, cyp11a1, hsd1b1, cyp17a1 and Hsd b3 pathways in the testicular mesenchymal cells.
The present invention provides the use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of expression of a key protein in the androgen synthesis pathway in testosterone cells, said key protein comprising one or more of LHCGR, CYP11A1, CYP17A1, HSD3B1 and HSD11B 1. The invention utilizes FGF-9 to prepare medicines for up-regulating the contents of key proteins LHCGR, CYP11A1, CYP17A1, HSD3B1 and HSD11B1 in the testosterone synthesis pathway of testicular mesenchymal cells, and the medicines can pass through pAMPK and AMPK in the testicular mesenchymal cells; the pMAPK, pPI3K, pCREB, SIRT1 and NUR77 pathways function to promote differentiation of testicular mesenchymal stem cells.
The invention provides the use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of key proteins in testicular stromal cells, including one or more of pAMPK, AMPK, pMAPK, pPI3K, pCREB, SIRT1, NUR77 and H3K4me 3.
For further explanation of the present invention, the use of the fibroblast growth factor 9 provided by the present invention in the treatment of testicular hypofunction is described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Therapeutic action of FGF-9 on EDS-treated testosterone deficiency
18 male Sprague-Dawley rats (90 days old, 260.+ -.20 g/rat purchased from the university of Etsuzhou medical laboratory animal center) were selected as subjects. The serum testosterone-free model was prepared by intraperitoneally injecting 75mg/kg of ethane dimethanesulfonic acid (EDS, as prepared, dissolved in DMSO: normal saline=1:3 (V: V) solution) per kg of rat body weight) to kill testicular interstitial cells, and randomly divided into 3 groups of 6, specific groupings and treatments were as follows:
1) FGF-90 ng/testis/day (0.9% saline control); 2) FGF-910 ng/testis/day (FGF-9 in 0.9% saline); 3) FGF-9100 ng/testis/day (FGF-9 in 0.9% saline); each rat was administered daily by intratesticular injection at a volume of 50 μl for 14 days after EDS treatment (testosterone is zero) and daily for a continuous period of 14-28 d after EDS treatment.
After the end of the experiment, serum and testes were collected. Serum was used to detect testosterone (T), LH and FSH levels; preserving the testis at the side of the testis at the temperature of minus 80 ℃ for extracting RNA (Trizol method) to carry out a real-time fluorescence quantitative polynucleotide chain reaction (qPCR) and extracting protein to carry out Western Blotting (WB), and detecting the expression of related genes (detection primers shown in Table 1) and protein on the androgen synthesis pathway and detecting the expression of rat testis mesenchymal cell phosphorylate protein; the other testis was fixed in Bouin's fluid for immunohistological examination of paraffin sections and examined for the effect of FGF-9 on the number of mesenchymal cells. qPCR was performed using SYBR Green qPCR kit (Roche, basel, switzerland) to detect the expression level of the relevant gene on the testosterone synthetic pathway. For detection methods, reference is made to [ Regulation ofseminiferous tubule-associated stem Leydig cells in adult rat tes.Li X, wang Z, jiang Z, guo J, zhang Y, li C, chung J, folmer J, liu J, lian Q, ge R, zirkin BR, chen H.Proc Natl Acad Sci U S A.2016Mar 8;113 (10) 2666-71.Doi:10.1073/pnas.1519395113.Epub 2016Feb 29.PMID:26929346 and [ Bone morphogenetic protein 4. 4inhibits rat stem/progenitor Leydig cell development and regeneration via SMAD-dependent and SMAD-independent signaling.Li X, fang Y, chen L, quan H, wang Y, ge RS.cell Death Dis.2022Dec13;13 (12) 1039.doi:10.1038/s41419-022-05471-8.PMID:36513649 ], wherein the preparation steps of the Bouin's fixative solution are as follows: 2 clean and dry 100mL glass bottles were prepared; weighing 100mL of double distilled water, adding 2g of picric acid, and fully and uniformly mixing to prepare a saturated picric acid solution; 75mL of saturated picric acid solution, 25mL of formaldehyde and 5mL of glacial acetic acid are measured, uniformly mixed and stored at room temperature for later use.
TABLE 1 qPCR detection primers for different genes
qPCR was detected as follows:
(1) putting the sample on ice in advance to melt, vibrating and centrifuging;
(2) the reaction mixture Mix was prepared as in table 2.
TABLE 2 reaction mixture Mix
| Reagent(s) | Volume required per well (μl) |
| SYBR GREEN | 7.5 |
| Upstream primer (10. Mu.M) | 0.75 |
| Downstream primer (10. Mu.M) | 0.75 |
| DEPC water | 4 |
(3) Sequentially adding 2 mu L of standard substance and sample into a 96-well plate, adding 13 mu L of Mix into each well, and simultaneously setting up multiple wells to obtain an average value to reduce errors;
(4) after the sample is added, sealing each hole by using film sealing paper, centrifuging for 2 minutes at 4 ℃ at 3000 rpm on a centrifuge, vibrating to fully mix the mixture, and centrifuging for 2 minutes at 4 ℃ at 3000 rpm;
(5) the 96-well plate was placed in a qPCR instrument according to the procedure: the cycle was carried out for 40 cycles at 95℃for 2 minutes to 95℃for 10 seconds to 60℃for 40 seconds.
(6) After the operation is completed, the melting curve and the data of each sample are checked, a standard curve is manufactured according to the Ct value of the standard substance, and then the data of each sample is brought into the standard curve, so that the gene expression level of each sample is calculated.
(7) The relative levels of the remaining gene mRNAs were normalized to the expression level of housekeeping gene Rps 16.
The detection results are shown in FIGS. 1 to 7.
From the results in FIG. 1, FGF-9 significantly increases testosterone content in serum of EDS model rats, without significantly affecting LH and FSH in serum. FGF-9 has the effect of improving the testosterone level in serum of an EDS model rat, can be used for treating testosterone hyposymptoms caused by testes, and has no influence on the functions of pituitary and hypothalamus.
From the results of FIG. 2, FGF-9 increases the number of CYP11A1 positive cells without changing the number of SOX9 positive cells, indicating that FGF-9 increases androgen (testosterone) levels by increasing the number of CYP11A1 positive cells.
From the results of FIG. 3, FGF-9 increased the number of CYP11A 1-positive and PCNA-positive cells, indicating that FGF-9 increased the number of cells, both PCNA-positive newly-proliferating cells and CYP11A 1-expressing cells, indicating that these newly-proliferating cells were testicular mesenchymal stem cells, thereby increasing androgen (testosterone) levels.
From the results of FIG. 4, FGF-9 significantly up-regulates the expression levels of the genes Lhcgr, scarb1, cyp11a1, hsd11b1, cyp17a1 and Hsd b3 without affecting the expression levels of the other androgen synthesis associated genes Star and Hsd b 1. The FGF-9 is shown to selectively up-regulate the expression level of a critical gene in the testosterone pathway synthesized by the testicular mesenchymal cells and promote the differentiation of the testicular mesenchymal stem cells.
From the results of FIG. 5, FGF-9 was found to significantly up-regulate the expression levels of LHCGR, CYP11A1, CYP17A1, HSD17B3 and HSD11B1 proteins, as compared to the control group, consistent with their corresponding changes in mRNA levels, as seen from the results of the WB assay.
From the results of FIG. 6, FGF-9 can up-regulate the expression level of AMPK protein; at the same time, the expression level of AMPK phosphorylated protein (pAMPK) was up-regulated; the expression level of the total protein of MAPK and PI3K is regulated down, but the expression level of the phosphorylated protein of MAPK (pMAPK) and the phosphorylated protein of PI3K (pPI 3K) is regulated up; at the same time, FGF-9 also up-regulates the expression level of CREB phosphorylated protein (pCREB); in addition, FGF-9 also functions by up-regulating SIRT1 and NUR77 expression levels. FGF-9 is shown to function by AMPK, MAPK, PI, 3, K, CREB, SIRT1, NUR77, etc.
From the results of FIG. 7, FGF-9 can up-regulate the expression level of histone H3K4 trimethylation (H3K 4me 3) (FIG. 7A), down-regulate the expression level of histone H3K9 trimethylation (H3K 9me 3) (FIG. 7B); meanwhile, the immunofluorescence results of CYP11A1 and the two histones show that H3K4me3 and H3K9me3 are expressed on rat testicular interstitial cells, and as the concentration of FGF-9 is increased, the expression of H3K4me3 is increased, the expression of H3K9me3 is reduced, and the results are consistent with the results of WB.
Example 2
Safety performance investigation
Experimental protocol:
the in vivo dosing regimen was the same as in example 1. At the end of the experiment, rats were weighed, dissected, and the bilateral testes and bilateral epididymis were taken, wet weighed and recorded, and the detection results are shown in table 3.
TABLE 3 Effect of FGF-9 on rat weight, testis and epididymal weight
Mean±se, n=6. There was no significant difference between the body weight of the rats, the testis weight and the epididymal weight in the FGF 9-dosed group compared to the control group.
From the results in Table 3, it can be seen that: after 14 days of continuous administration, the weight and testis wet weight of each group of rats are compared, and the statistical significance (P is greater than 0.05) is not achieved, so that FGF-9 does not affect the weight of the rats, the weight of the testis and the epididymis, and the administration of FGF-9 is safe and has no adverse effect on the rats.
From the results of examples 1 and 2, FGF-9 can significantly increase the testosterone content in serum of EDS model rats; simultaneously, FGF-9 also increases the number of CYP11A1 positive testicular interstitial cells, up-regulates the expression level of key genes Lhcgr, scarb1, cyp11A1, hsd B1, cyp17A1 and Hsd B3 and key proteins LHCGR, CYP11A1, CYP17A1, HSD17B3 and HSD11B1 on the androgen synthesis pathway, and the effect can be exerted through AMPK, MAPK, PI3K, CREB total protein and phosphorylated proteins thereof, SIRT1, NUR77 and other pathways; it was also found that FGF-9 can also be used by histone (H3K 4me3 and H3K9me 3) modifications; in addition, the continuous administration of 14d does not change the body weight and the wet weight of the testes of the rats significantly, and the administration safety performance is high.
Example 3
Proliferation promoting effect of FGF-9 on rat testicular mesenchymal stem cells
Materials: corning 12 well plate (available from Corning, inc., cat# 3336), testosterone radioimmunoassay kit (available from Zhejiang national medicine, stock # L2KTW 6), recombinant human FGF-9 (R & D (Catalog # 273-F9)), insulin-transferrin-sodium selenite medium additive (ITS, available from sigma, stock # I1884), luteinizing hormone (LH, available from sigma, stock # L9773), ethane dimethanesulfone (EDS, synthesized by Beijing Shikon medical science, inc.), DMEM/F12 (available from sigma, stock # D2906-10X 1L), bio-Rad cDNA synthesis kit and Bio-Rad SYBR fluorescent dye (available from Bio-Rad, stock # 170-8890, 170-8880), edU kit (available from Invitrogen, carlsbad, C10337).
Male Sprague-Dawley rats (purchased from the university of medical science animal experiment center, etsu, 90 days old, 260+ -20 g/day) were subjected to intraperitoneal injection of EDS (75 mg/kg) 7 days before the experiment, after carbon dioxide sacrifice, testes were removed, placed in phosphate buffer at 4℃to trim off the capsule, and after separation of the seminiferous tubules into individual pieces, divided into 12 well plates, and subjected to the following 2-part experiment:
a first part: testicular mesenchymal stem cell proliferation assay (EdU kit assay).
control group: culturing the seminiferous tubules in common medium (BM) for 1 week, and replacing the medium every 3.5 days; culturing until day 7, adding solution A in EdU kit, culturing in dark for 24 hr, and detecting the number of testis stem cells proliferated on seminiferous tubule with EdU kit;
FGF-9 dosing group: FGF-9 (0, 10 and 100 ng/mL) at various concentrations and inhibitor PD of FGF9 (PD 173074, purchased from Selleck, cat No. S1264, final concentration 1 μm) were added to a common medium (BM), the seminiferous tubules were cultured in this medium for 1 week, the culture and the drug were changed every 3.5 days, and when cultured until day 7, liquid a in the EdU kit was added, and the culture was conducted in the dark for 24 hours, and then the number of testicular stem cells proliferated on the seminiferous tubules was detected with the EdU kit;
a second part: testicular mesenchymal stem cell proliferation assay (testosterone level assay).
control group: culturing the seminiferous tubules in common medium (BM) for 1 week, and replacing the medium every 3.5 days; culturing until day 7, then changing culture medium of the seminiferous tubules into differentiation promoting culture medium (LDM), changing culture medium every 3.5 days, and detecting testosterone level in the culture medium until day 21, wherein the detection method is described in [ Bone morphogenetic protein 4inhibits rat stem/progenitor Leydig cell evelopment and regeneration via SMAD-dependent and SMAD-independent signaling.Li X, fang Y, chen L, quan H, wang Y, ge RS.cell Death Dis.2022Dec13;13 (12) 1039.doi:10.1038/s 41419-022-05471-8.PMID:36513649;
FGF-9 dosing group: FGF-9 (0, 1,10 and 100 ng/mL) with different concentrations was added to a common medium (BM), the seminiferous tubules were cultured in this medium for 1 week, with the culture and the drug being changed every 3.5 days until day 7, then the medium of the seminiferous tubules was changed to a differentiation promoting medium (LDM), with the medium being changed every 3.5 days, and the testosterone levels in the medium being detected until day 21;
the preparation steps of the LDM culture medium are as follows:
adding luteinizing hormone (LH, final concentration of 5 ng/mL) and lithium chloride (Li) into BM + Final concentration is 5 mmol/L), and uniformly mixing for later use.
After the experiment is finished, collecting the culture medium to measure testosterone level; collecting the seminiferous tubules for EdU staining (labeling of proliferating testicular mesenchymal stem cells):
first part of the experimental procedure: the fixative was washed off with PBS, blocked with 50% Glycerol (Glycerol: PBS=1:1, V/V), observed under a mirror and photographed, and the number of blue-violet cells (differentiated cells) at the periphery of the seminiferous tubules was recorded as the number of cells/tubule area (cm) 2 ) The percentage of differentiation of testicular stem cells was calculated by the ratio of (3). The detection results are shown in FIG. 8.
A second part: the culture medium was collected and testosterone levels in the culture medium were measured, and the measurement results are shown in fig. 8.
As can be seen from the results of fig. 8, the number of EdU-positive testicular mesenchymal stem cells was not large after 1 week of culture in BM medium (B in fig. 8); in the group of FGF9 added with 10ng/mL in BM, the production of EdU-positive testicular mesenchymal stem cells was induced (C in fig. 8), and as the FGF9 concentration increased, the number of EdU-positive testicular mesenchymal stem cells increased (F in fig. 8); addition of FGFR inhibitor PD to BM for culture, induced numbers of EdU positive testicular mesenchymal stem cells were almost as high as control (B in fig. 8) (D and G in fig. 8); however, after addition of FGFR inhibitor PD to FGF9 and incubation, the number of proliferating testicular mesenchymal stem cells was found to be reduced compared to C in fig. 8 (E and G in fig. 8).
From the results of FIG. 8, it was also found that testosterone levels were very low after 3 weeks of culture of the seminiferous tubules in BM medium (H in FIG. 8). However, testosterone can be produced after BM has been incubated with FGF9 at different concentrations for 2 weeks; and 1ng/mL FGF-9 significantly increased testosterone production compared to BM groups, but there was no significant difference in testosterone levels produced by 10ng/mL and 100ng/mL FGF9 (H in fig. 8). This suggests that FGF-9 stimulates differentiation of testosterone stem cells into testosterone cell lines, thereby inducing testosterone production. The same letter indicates no difference between groups (p > 0.05) and the different letters indicate a significant difference (p < 0.05).
Example 4
Action of FGF-9 on differentiation promotion of rat testis mesenchymal stem cells (testosterone level detection and qPCR detection of Gene level expression)
control group: culturing the seminiferous tubules in common medium (BM) for 1 week, and replacing the medium every 3.5 days; culturing until day 7, then changing culture medium of the seminiferous tubules into differentiation promoting culture medium (LDM), changing culture medium every 3.5 days, and detecting testosterone level in the culture medium until day 21; extracting the RNA of the seminiferous tubules, and detecting the expression level of the related gene on the testosterone synthetic pathway by qPCR (quantitative polymerase chain reaction), wherein the detection method is the same as that of the example 1;
FGF-9 dosing group: culturing the seminiferous tubules in common medium (BM) for 1 week, and replacing the medium every 3.5 days; culturing until day 7, then changing the culture medium of the seminiferous tubules into differentiation promoting culture medium (LDM), adding FGF-9 (0, 1 and 10 ng/mL) with different concentrations into the LDM culture medium, changing the culture medium every 3.5 days, and detecting the testosterone level in the culture medium until day 21; extracting the RNA of the seminiferous tubules, and detecting the expression level of the related gene on the testosterone synthetic pathway by qPCR (quantitative polymerase chain reaction), wherein the detection method is the same as that of the example 1;
collecting the culture medium, and detecting the testosterone level in the culture medium; collecting the seminiferous tubules, removing the culture medium, washing with PBS for 2 times, extracting RNA by using a method of extracting RNA by using trizol, and detecting the expression level of related genes on a testosterone synthetic pathway by using a qPCR method. The detection results are shown in FIG. 9.
From the results of fig. 9, testosterone levels were very low after 3 weeks of culture of the seminiferous tubules in BM and DM medium (a in fig. 9). However, testosterone can be produced after 3 weeks of BM and DM culture with FGF9 at different concentrations; and 1ng/mL and 10ng/mL FGF-9 significantly increased testosterone production compared to the BM group (A in FIG. 9). At the same time, FGF-9 significantly upregulates the expression levels of genes Lhcgr, star, cyp a1 and Hsd b1 without affecting the expression levels of the other androgen synthesis associated genes Cyp17a1, hsd17b3 and Hsd11b 1. The FGF-9 is shown to selectively up-regulate the expression level of a critical gene in the testosterone pathway synthesized by the testicular mesenchymal cells and promote the differentiation of the testicular mesenchymal stem cells.
From the results of the above examples, FGF-9 significantly increased the number of EdU positive testicular mesenchymal stem cells in vitro, increased testosterone levels in the culture medium, and likewise FGF-9 up-regulated the expression levels of the key genes Lhcgr, star, cyp a1 and Hsd b1 on the androgen synthesis pathway. This effect is substantially consistent with the results of in vivo experiments.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
1. Application of fibroblast growth factor 9 in preparing medicament for treating male hypogonadism syndrome.
2. Use of fibroblast growth factor 9 in the manufacture of a medicament for inducing proliferation and/or differentiation of mesenchymal stem cells.
3. Use of fibroblast growth factor 9 in the manufacture of a medicament for increasing testosterone levels in serum and/or testes.
4. Application of fibroblast growth factor 9 in preparing medicament for increasing number of testicular interstitial cells in testis.
5. Use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of expression of a key gene in the androgen synthesis pathway in testicular interstitial cells, including one or more of Lhcgr, scarb1, cyp11a1, hsd b1, cyp17a1 and Hsd b 3.
6. Use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the level of expression of a key protein in the androgen synthesis pathway in testicular interstitial cells, including one or more of LHCGR, CYP11A1, CYP17A1, HSD3B1 and HSD11B 1.
7. Use of fibroblast growth factor 9 in the manufacture of a medicament for upregulating the expression level of a key protein in testicular interstitial cells, including one or more of pAMPK, AMPK, pMAPK, pPI3K, pCREB, SIRT1, NUR77 and H3K4me 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311221131.8A CN116999537A (en) | 2023-09-20 | 2023-09-20 | Application of fibroblast growth factor 9 in treating testicular hypofunction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311221131.8A CN116999537A (en) | 2023-09-20 | 2023-09-20 | Application of fibroblast growth factor 9 in treating testicular hypofunction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116999537A true CN116999537A (en) | 2023-11-07 |
Family
ID=88572974
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311221131.8A Pending CN116999537A (en) | 2023-09-20 | 2023-09-20 | Application of fibroblast growth factor 9 in treating testicular hypofunction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116999537A (en) |
-
2023
- 2023-09-20 CN CN202311221131.8A patent/CN116999537A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cheng et al. | Methyl ferulic acid attenuates liver fibrosis and hepatic stellate cell activation through the TGF-β1/Smad and NOX4/ROS pathways | |
| Folgueira et al. | Hypothalamic dopamine signalling regulates brown fat thermogenesis | |
| Zhang et al. | Artesunate promotes the proliferation of neural stem/progenitor cells and alleviates Ischemia-reperfusion Injury through PI3K/Akt/FOXO-3a/p27kip1 signaling pathway | |
| WO2021032213A1 (en) | Anti-aging medicine d/s targeting aging cells in tissue microenvironment and use thereof | |
| Jang et al. | Coffee consumption promotes skeletal muscle hypertrophy and myoblast differentiation | |
| Yuan et al. | Isoflurane post-conditioning down-regulates expression of aquaporin 4 in rats with cerebral ischemia/reperfusion injury and is possibly related to bone morphogenetic protein 4/Smad1/5/8 signaling pathway | |
| CN107375910B (en) | Application of PTHrP in preparation of medicine for treating male hypogonadism syndrome | |
| CN111358789B (en) | Application of NSC228155 in preparation of medicine for preventing and treating chronic renal fibrosis | |
| Li et al. | AMPK activates FOXO3a and promotes neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia | |
| Wei et al. | Homer1a attenuates endoplasmic reticulum stress-induced mitochondrial stress after ischemic reperfusion injury by inhibiting the PERK pathway | |
| Alasmari et al. | Effects of ampicillin on cystine/glutamate antiporter and glutamate transporter 1 isoforms as well as ethanol drinking in male P rats | |
| Petrie et al. | Neurotensin activates GABAergic interneurons in the prefrontal cortex | |
| He et al. | Exploring the pathogenesis of type 2 diabetes mellitus intestinal damp-heat syndrome and the therapeutic effect of Gegen Qinlian Decoction from the perspective of exosomal miRNA | |
| CN100446772C (en) | Treatment of diabetes | |
| Yapislar et al. | The melatonin MT2 receptor is involved in the anti-apoptotic effects of melatonin in rats with type 2 diabetes mellitus | |
| CN112656808A (en) | Application of heparin oligosaccharide in preparation of antitumor drugs | |
| EP3738581A1 (en) | Rectal mucosal administration preparation of pulsatilla chinensis (bge.) regel saponin b4 and preparation method therefor | |
| CN116999537A (en) | Application of fibroblast growth factor 9 in treating testicular hypofunction | |
| Rakovska et al. | Physiological release of striatal acetylcholine (in vivo): effect of somatostatin on dopaminergic–cholinergic interaction | |
| Kumar et al. | Oxalate disrupts monocyte and macrophage cellular function via Interleukin-10 and mitochondrial reactive oxygen species (ROS) signaling | |
| CN111991408B (en) | Application of quercetin-3-O-robioside as inhibitor of calcium ion channel | |
| Gu et al. | Dexamethasone enhances adenosine 5′-triphosphate-sensitive potassium channel expression in the blood–brain tumor barrier in a rat brain tumor model | |
| Bender et al. | SERUM TRYPTOPHAN BINDING IN CHLORPROMAZINE‐TREATED CHRONIC SCHIZOPHRENICS | |
| Telles et al. | Lateral hypothalamic serotonin is not stimulated during central leptin hypophagia | |
| CN112316121A (en) | Application of glucagon-like peptide-1 in preparing medicine for treating male hypogonadism syndrome |
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 |