CN111175522B - Application of oxidized high-density lipoprotein - Google Patents
Application of oxidized high-density lipoprotein Download PDFInfo
- Publication number
- CN111175522B CN111175522B CN201811331617.6A CN201811331617A CN111175522B CN 111175522 B CN111175522 B CN 111175522B CN 201811331617 A CN201811331617 A CN 201811331617A CN 111175522 B CN111175522 B CN 111175522B
- Authority
- CN
- China
- Prior art keywords
- density lipoprotein
- oxidized high
- aortic valve
- hdl
- valve diseases
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010010234 HDL Lipoproteins Proteins 0.000 title claims abstract description 97
- 102000015779 HDL Lipoproteins Human genes 0.000 title claims abstract description 97
- 208000027896 Aortic valve disease Diseases 0.000 claims abstract description 53
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 239000003814 drug Substances 0.000 claims abstract description 20
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 18
- 238000012216 screening Methods 0.000 claims abstract description 17
- 239000003112 inhibitor Substances 0.000 claims abstract description 13
- 229940079593 drug Drugs 0.000 claims abstract description 12
- 239000003550 marker Substances 0.000 claims abstract description 9
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- 230000002308 calcification Effects 0.000 claims description 30
- 230000002401 inhibitory effect Effects 0.000 claims description 20
- 210000002966 serum Anatomy 0.000 claims description 20
- 210000002570 interstitial cell Anatomy 0.000 claims description 15
- 210000001765 aortic valve Anatomy 0.000 claims description 12
- 230000004069 differentiation Effects 0.000 claims description 12
- 230000002188 osteogenic effect Effects 0.000 claims description 10
- 229940126585 therapeutic drug Drugs 0.000 claims description 8
- 239000000032 diagnostic agent Substances 0.000 claims description 7
- 229940039227 diagnostic agent Drugs 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 5
- 229940124597 therapeutic agent Drugs 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 5
- 238000003759 clinical diagnosis Methods 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 26
- 230000001965 increasing effect Effects 0.000 description 20
- 238000010186 staining Methods 0.000 description 19
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 18
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 18
- 101150118570 Msx2 gene Proteins 0.000 description 15
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 14
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 14
- JKYKXTRKURYNGW-UHFFFAOYSA-N 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S(O)(=O)=O)=C2 JKYKXTRKURYNGW-UHFFFAOYSA-N 0.000 description 8
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 8
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 7
- 108010033266 Lipoprotein(a) Proteins 0.000 description 7
- 102000057248 Lipoprotein(a) Human genes 0.000 description 7
- 230000000903 blocking effect Effects 0.000 description 7
- 108091005995 glycated hemoglobin Proteins 0.000 description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 6
- 206010020751 Hypersensitivity Diseases 0.000 description 6
- 239000011575 calcium Substances 0.000 description 6
- 229910052791 calcium Inorganic materials 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 230000002596 correlated effect Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000007477 logistic regression Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 108010074051 C-Reactive Protein Proteins 0.000 description 5
- 102100032752 C-reactive protein Human genes 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000011164 ossification Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 201000001320 Atherosclerosis Diseases 0.000 description 4
- 206010020772 Hypertension Diseases 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 201000006800 aortic valve disease 1 Diseases 0.000 description 4
- 229940109239 creatinine Drugs 0.000 description 4
- 230000008021 deposition Effects 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 230000036542 oxidative stress Effects 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- -1 runx2 Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 3
- 108010007622 LDL Lipoproteins Proteins 0.000 description 3
- 102000007330 LDL Lipoproteins Human genes 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000036772 blood pressure Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 238000012937 correction Methods 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 206010050559 Aortic valve calcification Diseases 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- 108010023302 HDL Cholesterol Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 108010028554 LDL Cholesterol Proteins 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000033558 biomineral tissue development Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 2
- 230000009610 hypersensitivity Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000002536 stromal cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 2
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 208000025494 Aortic disease Diseases 0.000 description 1
- 208000034792 Aortic inflammatory disease Diseases 0.000 description 1
- 206010002915 Aortic valve incompetence Diseases 0.000 description 1
- 102000005666 Apolipoprotein A-I Human genes 0.000 description 1
- 108010059886 Apolipoprotein A-I Proteins 0.000 description 1
- 101150061927 BMP2 gene Proteins 0.000 description 1
- 206010004552 Bicuspid aortic valve Diseases 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000012192 Cystatin C Human genes 0.000 description 1
- 108010061642 Cystatin C Proteins 0.000 description 1
- 101100055841 Danio rerio apoa1 gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- MIJPAVRNWPDMOR-ZAFYKAAXSA-N L-ascorbic acid 2-phosphate Chemical compound OC[C@H](O)[C@H]1OC(=O)C(OP(O)(O)=O)=C1O MIJPAVRNWPDMOR-ZAFYKAAXSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 238000010818 SYBR green PCR Master Mix Methods 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 201000002064 aortic valve insufficiency Diseases 0.000 description 1
- 230000000923 atherogenic effect Effects 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000021654 bicuspid aortic valve disease Diseases 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 1
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000002586 coronary angiography Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000002592 echocardiography Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 208000018578 heart valve disease Diseases 0.000 description 1
- 229940126904 hypoglycaemic agent Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000002464 muscle smooth vascular Anatomy 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000004072 osteoblast differentiation Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004141 reverse cholesterol transport Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000011272 standard treatment Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000036262 stenosis Effects 0.000 description 1
- 208000037804 stenosis Diseases 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/775—Apolipopeptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/329—Diseases of the aorta or its branches, e.g. aneurysms, aortic dissection
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Urology & Nephrology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of medicines, and particularly relates to application of oxidized high-density lipoprotein. The oxidized high-density lipoprotein is used as a target or marker of the calcified aortic valve diseases, and the detection reagent of the oxidized high-density lipoprotein and/or the oxidized high-density lipoprotein can be used for preparing a diagnostic reagent of the calcified aortic valve diseases, monitoring the concentration of the oxidized high-density lipoprotein and predicting the occurrence and the process of the calcified aortic valve diseases; the inhibitor or blocker of oxidized high density lipoprotein can also be used for preparing medicine for treating calcific aortic valve diseases; the oxidized high-density lipoprotein and/or the detection reagent of the oxidized high-density lipoprotein can also be used for preparing a screening agent of drugs for treating calcified aortic valve diseases and screening drugs for treating calcified aortic valve diseases. The application of the oxidized high-density lipoprotein provides a new way and basis for clinical diagnosis and treatment of calcified aortic valve diseases.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of oxidized high-density lipoprotein.
Background
At present, calcific Aortic Valve Disease (CAVD) is the most common heart valvular disease worldwide and is characterized by progressive mineralization of the aortic valve. However, the specific cellular and molecular mechanisms of calcified aortic disease are not fully studied. Similar to the pathogenic mechanism of vascular atherosclerosis, increasing lipid infiltration and increasing oxidative stress levels can promote the development of CAVD.
Aortic Valve Interstitial Cells (AVICs) are the most abundant cell type in the valve leaflets and play an important role in maintaining aortic valve function. In normal aortic valves, AVICs are quiescent fibroblast-like cells (qVICs); however, under a series of changes (such as lipid infiltration, oxidative stress, inflammation and the like) induced by mechanical stress, disease states and the like, AVICs are activated and differentiated into osteoblast-like valve mesenchymal cells (oVICs), which are important for calcium deposition caused by CAVD.
High-density lipoprotein (HDL) can protect the arterial wall through multiple mechanisms to prevent atherosclerosis, for example, HDL can promote reverse cholesterol transport, inhibit low-density lipoprotein (LDL) oxidation and inflammatory reaction, and directly exert endothelial protection effect. In vitro studies have found that native human high density lipoprotein (native human HDL, N-HDL) can reduce the degree of calcification of Vascular Smooth Muscle (VSMCs) and valvular myofibroblasts by down-regulating inflammatory factor expression. However, the protective effects of HDL are quite unstable, e.g. in oxidative stress, HDL has an atherogenic effect after oxidative modification, ox-HDL can induce VSMC proliferation, cause Endothelial Cell (EC) and Endothelial Progenitor Cell (EPC) dysfunction, and promote the occurrence of oxidative stress and apoptosis in monocytes/macrophages. Recent studies have found that apolipoprotein A1 (a major protein component of apoproteina A1, apoA1, HDL) accumulates in valve tissue of CAVD patients, and apoA 1-derived amyloid extracts promote apoptosis of AVICs. However, it is not currently clear whether oxidative modification of HDL occurs in CAVD patients, and what role ox-HDL plays in the mineralization of AVICs.
The research discovers that HDL oxidation modification in CAVD patients generates oxidized high density lipoprotein (ox-HDL), the ox-HDL participates in the pathological process of aortic valve calcification by promoting osteoblast differentiation of AVICs, and discovers that the concentration of the ox-HDL is positively correlated with the risk of CAVD, thereby providing a new way for monitoring, diagnosing and treating CAVD diseases.
Disclosure of Invention
The invention aims to provide application of oxidized high-density lipoprotein.
Clinical researches of the application find that the content of oxidized high-density lipoprotein (ox-HDL) in the serum of a patient with Calcific Aortic Valve Diseases (CAVD) is obviously increased, and the concentration of the ox-HDL is positively correlated with the incidence rate of the calcific aortic valve diseases. In vitro researches show that the ox-HDL can increase ALP activity of alkaline phosphatase in a concentration-dependent manner, and/or increase the gene expression level of osteogenic factors such as BMP2, runx2, msx2 and the like, promote differentiation and/or calcification of Aortic Valve Interstitial Cells (AVICs) in an osteogenic direction, and increase the risk of onset of Calcific Aortic Valve Diseases (CAVD).
By monitoring the concentration of oxidized high-density lipoprotein in serum and the change thereof, the occurrence and the progress of calcified aortic valve diseases can be predicted, and effective diagnosis, monitoring and evaluation can be carried out. Therefore, oxidized high-density lipoprotein can be used as a target or marker for diagnosis and/or treatment of calcified aortic valve diseases.
The oxidized high-density lipoprotein or the detection reagent of the oxidized high-density lipoprotein can be used for preparing a calcified aortic valve disease diagnostic agent, and the concentration of the oxidized high-density lipoprotein in serum is detected by taking the oxidized high-density lipoprotein as a target or a marker.
The oxidized high-density lipoprotein is used as a target or a marker of the calcified aortic valve diseases, and a detection reagent of the oxidized high-density lipoprotein can be used for screening the medicines for treating the calcified aortic valve diseases.
The inhibitor or the blocker of the oxidized high-density lipoprotein can be used for preparing medicaments for treating calcified aortic valve diseases and inhibiting and/or reversing calcified aortic valve diseases by inhibiting and/or blocking the generation of the oxidized high-density lipoprotein in serum, reducing ALP activity of alkaline phosphatase, and/or inhibiting BMP2, runx2 and Msx2 osteogenic factor gene expression, and inhibiting and/or reversing differentiation and/or calcification of interstitial cells of the aortic valve in the osteogenesis direction.
A diagnostic agent for calcified aortic valve diseases contains oxidized high density lipoprotein and/or detection reagent. The detection reagent for the oxidized high-density lipoprotein can detect the concentration of the oxidized high-density lipoprotein in serum, predict the progress of calcified aortic valve diseases, and carry out effective diagnosis, monitoring and evaluation.
The diagnostic agent can be prepared into any pharmaceutically acceptable form with the functions of diagnosis and/or monitoring according to needs. The diagnostic agent also comprises any one or more pharmaceutically acceptable auxiliary diagnostic components and/or carriers.
The inhibitor or blocker of oxidized high-density lipoprotein can also be used for preparing medicine for treating calcific aortic valve diseases.
The inhibitor or blocker of oxidized high-density lipoprotein can inhibit and/or reverse differentiation and/or calcification of interstitial cells of aortic valve towards osteogenesis direction by inhibiting and/or blocking the production of oxidized high-density lipoprotein in serum, thereby inhibiting and/or reversing calcified aortic valve diseases.
The inhibitor or the blocker of oxidized high-density lipoprotein inhibits the ALP activity of alkaline phosphatase and/or inhibits the expression of genes related to the calcification of valve interstitial cells by inhibiting and/or blocking the generation of oxidized high-density lipoprotein in serum, thereby inhibiting and/or reversing the differentiation and/or calcification of the valve interstitial cells to the osteogenic direction.
The genes related to the calcification of the valve interstitial cells are BMP2, runx2 and Msx2.
A medicine for treating calcified aortic valve diseases contains inhibitor or blocker of oxidized high density lipoprotein.
The therapeutic drug can be prepared into any pharmaceutically acceptable dosage form with the functions of prevention and/or treatment according to the needs. The therapeutic drug also comprises any one or more pharmaceutically acceptable auxiliary therapeutic ingredients and/or carriers.
The oxidized high-density lipoprotein is used as a new target or marker for diagnosing the calcified aortic valve diseases, and can also be used for screening therapeutic drugs for the calcified aortic valve diseases. The oxidized high-density lipoprotein or the detection reagent of the oxidized high-density lipoprotein can be used for preparing a screening agent of a calcified aortic valve disease treatment drug, screening substances for inhibiting and/or blocking the generation of the oxidized high-density lipoprotein in serum, inhibiting and/or reversing differentiation and/or calcification of aortic valve interstitial cells to the osteogenesis direction, and treating calcified aortic valve diseases.
The oxidized high-density lipoprotein or the detection reagent of the oxidized high-density lipoprotein can be used for screening the therapeutic drugs for calcified aortic valve diseases, and the oxidized high-density lipoprotein is used as a target or a marker to screen substances which can inhibit or block the generation of the oxidized high-density lipoprotein.
The inhibitor or the blocker of the oxidized high-density lipoprotein is used as an active molecule for inhibiting and/or reversing calcified aortic valve diseases, and inhibiting and/or blocking the generation of the oxidized high-density lipoprotein in serum, thereby inhibiting and/or reversing differentiation and/or calcification of interstitial cells of the aortic valve towards osteogenesis.
The inhibitor or blocker of oxidized high-density lipoprotein can inhibit ALP activity of alkaline phosphatase and/or inhibit gene expression related to calcification of valve interstitial cells by inhibiting and/or blocking the production of oxidized high-density lipoprotein in serum, thereby inhibiting differentiation and/or calcification of valve interstitial cells towards osteogenesis direction.
The genes related to the calcification of the valve interstitial cells are BMP2, runx2 and Msx2.
A screening agent for a therapeutic agent for calcified aortic valve diseases contains a detection reagent for oxidized high-density lipoprotein and/or oxidized high-density lipoprotein. The detection reagent for the oxidized high-density lipoprotein can monitor and/or evaluate the therapeutic activity of the therapeutic drug for the calcified aortic valve diseases by detecting the concentration of the oxidized high-density lipoprotein, and screen the therapeutic drug for the calcified aortic valve diseases.
The screening agent can be prepared into any pharmaceutically acceptable form with diagnosis and/or monitoring effects according to needs. The screening agent also comprises any one or more pharmaceutically acceptable auxiliary screening components and/or carriers.
Compared with the prior art, the invention has the following advantages:
clinical researches of the application find that the content of oxidized high-density lipoprotein (ox-HDL) in the serum of a patient with Calcified Aortic Valve Disease (CAVD) is obviously increased, the incidence of CAVD is gradually increased along with the increase of the concentration of ox-HDL, and the concentration of ox-HDL is positively correlated with the incidence of CAVD. In vitro studies show that the ox-HDL can promote differentiation and/or calcification of Aortic Valve Interstitial Cells (AVICs) to the osteogenic direction by enhancing ALP activity of alkaline phosphatase and/or increasing the expression level of BMP2, runx2 and Msx2 genes.
Therefore, the oxidized high-density lipoprotein can be used as a new target or a marker for clinical diagnosis and treatment of the calcified aortic valve diseases, and the oxidized high-density lipoprotein and a detection reagent thereof can be used for preparing a diagnostic agent for the calcified aortic valve diseases, detecting the concentration of the oxidized high-density lipoprotein in serum, predicting the occurrence and the process of the calcified aortic valve diseases, and diagnosing, monitoring and evaluating the calcified aortic valve diseases.
Meanwhile, the inhibitor or the blocker of the oxidized high-density lipoprotein can also be used for preparing a therapeutic drug for calcified aortic valve diseases, can inhibit ALP activity of alkaline phosphatase and/or inhibit expression levels of BMP2, runx2 and Msx2 genes by inhibiting and/or blocking the generation of the oxidized high-density lipoprotein, further inhibit and/or reverse differentiation and/or calcification of Aortic Valve Interstitial Cells (AVICs) to the osteogenic direction, finally inhibit and/or reverse calcified aortic valve diseases, and is used for treating calcified aortic valve diseases.
The oxidized high-density lipoprotein and the detection reagent thereof can also be used for preparing a screening agent of calcified aortic valve disease treatment drugs, detecting the concentration of the oxidized high-density lipoprotein, predicting and/or evaluating the treatment activity of the calcified aortic valve disease treatment drugs and screening the calcified aortic valve disease treatment drugs.
In conclusion, the oxidized high-density lipoprotein is used as a new target or marker for clinical diagnosis and treatment of the calcified aortic valve diseases, so that a new way and basis are provided for clinical diagnosis and treatment of the calcified aortic valve diseases, and a new way and basis are provided for research and development of drugs for treating the calcified aortic valve diseases.
Drawings
FIG. 1 is a ROC curve for the evaluation of CAVD risk using ox-HDL as described in example 1.
FIG. 2 shows the results of alizarin Red S staining and alkaline phosphatase ALP activity assay after the calcification culture of AVICs in example 2. FIG. 2A is a graph showing alizarin Red S staining and alkaline phosphatase ALP activity staining for each experimental group (Con, 50. Mu.g/mL N-HDL, 10. Mu.g/mL ox-HDL, 50. Mu.g/mL ox-HDL), FIG. 2B is a graph showing ALP activity test results for each experimental group (Con, 50. Mu.g/mL N-HDL, 10. Mu.g/mL ox-HDL, 50. Mu.g/mL ox-HDL), and FIG. 2C is a graph showing calcium deposition level for each experimental group (Con, 50. Mu.g/mL N-HDL, 10. Mu.g/mL ox-HDL, 50. Mu.g/mL ox-HDL).
FIG. 3 is a graph showing the expression levels of the calcification-associated genes (BMP 2, runx2, msx 2) in each experimental group (Con, 50. Mu.g/mL N-HDL, 10. Mu.g/mL ox-HDL, 50. Mu.g/mL ox-HDL) in example 2.
Detailed Description
The statistical analysis method comprises the following steps: statistical data analysis was performed using SPSS 22.0 (usa, illinois) and MedCalc 11.4 (kelck, belgium, mary) software. Measure data to
And (4) showing. The metering data conforming to normal distribution is tested by an independent sample t; the non-normal distribution is tested using the ManWhitney rank sum test. The counting data is checked by chi-square. The difference between the mean values of the multiple samples was analyzed using variance.
Multivariate linear regression and Pearson's test were used to assess the correlation between ox-HDL concentration and hypertension, diabetes, glycated hemoglobin, triglycerides, total cholesterol, high density lipoprotein, low density lipoprotein, lipoprotein (a), hypersensitive C-reactive protein, creatinine, statin intake, and body mass index.
After correcting the incidence of CAD and the related risk factors of atherosclerosis, logistic regression was used to analyze whether the increase in ox-HDL level is an independent predictor of CAVD risk. Calculating ox-HDL by an ROC curve to evaluate the diagnostic value of CAVD suffering risk. The areas under the ROC curves between groups were compared by MedCalc. Differences were statistically significant with a two-sided P < 0.05.
Example 1
1.1 test population
A total of 2674 patients with myocardial ischemia were collected continuously in the Shanghai Rekin Hospital, and an echocardiogram and a coronary angiography were performed for each patient. Of these, 168 patients were diagnosed as CAVD patients by echocardiography and Doppler flow imaging and were enrolled in the CAVD cohort. The aortic valve in CAVD patients exhibits calcific degeneration and meets the criteria of leaflet opacity with mild focal thickening and leaflet stiffness (according to AHA guidelines). Each CAVD patient was age (+ -1 year) and gender matched to one of the remaining patients, and was included in the non-CAVD group.
The study did not incorporate patients with a history of rheumatic diseases, bicuspid aortic valve malformations, moderate to severe aortic regurgitation or inflammatory diseases to avoid confounding factors. The CAD is diagnosed when the diameter stenosis degree of the main coronary artery lumen is more than or equal to 50 percent. According to the ethical guidelines of Helsinki declaration, the study was approved by the ethical Committee of the medical college of Shanghai university of transportation, and all participants signed informed consent.
1.2 general data on subjects
Subject baseline data and risk factors are shown in table 1. The blood pressure, body mass index, hypersensitivity C-reactive protein and lipoprotein (a) of CAVD group patients are all obviously higher than those of non-CAVD group. And the CAVD group patients had lower ejection fraction compared to the non-CAVD group.
Between groups, fasting plasma glucose, glycated hemoglobin, calcium, phosphorus, renal function, diabetes, CAD and hyperlipidemia were not statistically different. Except that more CAVD patients receive the treatment of hypertension, the treatment conditions of the medicines in the two groups are equivalent.
TABLE 1 Baseline data and Biochemical evaluation data
| CAVD(n＝168) | non-CAVD (n = 168) | P value | |
| Age (year) | 70.0±7.4 | 69.9±7.3 | Matching |
| Male/female | 101/67 | 101/67 | Matching |
| Body mass index | 25.6±3.4 | 23.7±4.0 | <0.001 |
| Blood pressure (n,%) | 134(79.7) | 110(65.5) | 0.003 |
| Diabetes mellitus (n,%) | 56(33.3) | 53(31.5) | Is not significant |
| History of smoking (n,%) | 37(22.0) | 35(20.8) | Is not significant |
| History of drinking (n,%) | 56(33.3) | 50(29.8) | Is not significant |
| Coronary artery disease (n,%) | 129(76.8) | 123(73.2) | Is not significant |
| Triglyceride (mmol/L) | 1.67±1.14 | 1.60±1.82 | Is not significant |
| Total cholesterol (mmol/L) | 4.28±1.17 | 4.1±0.97 | Is not significant |
| High density lipoprotein cholesterol (mmol/L) | 1.10±0.22 | 1.10±0.26 | Is not significant |
| Low density lipoprotein cholesterol (mmol/L) | 2.42±0.93 | 2.30±0.94 | Is not significant |
| Lipoprotein (a) (g/L)) | 0.29±0.34 | 0.20±0.19 | 0.004 |
| Fasting blood glucose (mmol/L) | 5.62±1.52 | 5.60±1.62 | Is not significant |
| Glycated hemoglobin (%) | 6.33±1.11 | 6.16±0.99 | Is not significant |
| Hypersensitivity C-reactive protein (mg/L) | 2.20±2.14 | 1.74±1.95 | 0.038 |
| Creatinine (mu mol/L) | 82.77±18.6 | 81.21±19.95 | Is not significant |
| Blood urea nitrogen (mmol/L) | 6.08±1.99 | 5.81±1.40 | Is not significant |
| Glomerular filtration Rate (mL/min/1.73 m) 2 ) | 81.34±17.78 | 79.81±17.06 | Is not significant |
| Serum cystatin C (mg/L) | 1.17±0.28 | 1.13±0.31 | Is not significant |
| Calcium (mmol/L) | 2.21±0.10 | 2.20±0.11 | Is not significant |
| Phosphate (mmol/L) | 1.13±0.18 | 1.10±0.18 | Is not significant |
| Ejection fraction (%) | 63.19±7.38 | 66.07±5.16 | <0.001 |
| High density lipoprotein oxide (ng/mL) | 131.52±30.96 | 112.58±32.20 | <0.001 |
| Medical treatment | |||
| Statins (n,%) | 42(72.4) | 50(84.7) | Is not significant |
| Hypoglycemic agent (n,%) | 50(29.8) | 48(28.6) | Is not significant |
| Antihypertensive drug (n,%) | 130(77.4) | 105(62.5) | 0.003 |
1.3 Biochemical detection of clinical specimens
All participants collected blood samples before cardiac catheterization, and serum was stored in a-70 ℃ refrigerator after standard treatment. Peripheral blood biochemical assays were performed using a Hitachi 912Analyzer (Roche Diagnostics, germany) according to standard laboratory manual protocols. ELISA kit (Cell Biolabs, USA) is adopted to detect the concentration of ox-HDL in serum, and the specific operation is as follows: firstly, adding serum to a high-affinity ELISA plate pre-coated by an MDA antibody, then co-incubating with an anti-human apoA1 antibody, then adding horse radish peroxidase labeled streptavidin for detection, and finally reading the absorbance value of each sample with the wavelength of 450nm of a spectrophotometer.
1.4ox-HDL levels and risk of CAVD
Compared with a non-CAVD group, the serum ox-HDL concentration of the CAVD group patients is remarkably increased (131.52 +/-30.96 ng/mL vs.112.58 +/-32.20 ng/mL, P is less than 0.001); the rates of CAVD patients also increased progressively from lower quartiles (< 97.0 ng/mL) to upper quartiles (> 143.7 ng/mL) in serum ox-HDL concentrations (Table 2).
Compared with the quartile at the serum ox-HDL concentration, the CAVD risk is significantly increased by 3.06 (95%: 1.98-4.74) compared with the quartile at the ox-HDL concentration (Table 2).
The ROC analysis showed that the area under the curve for the ox-HDL predicted CAVD risk was 0.681 (95% CI 0.628-0.730, P-Ap-0.001), and the optimal screening value for the ox-HDL predicted CAVD risk was 106.8ng/mL. The ox-HDL at this concentration was 80.4% sensitive and 51.8% specific for CAVD.
TABLE 2 CAVD incidence based on ox-HDL concentration levels
| CAVD,n(％) | Probability (95% confidence interval) | |
| The first quartile (n =84,<97.0ng/mL) | 18(21.4) | 1 |
| second quartile (n =84,97.1-119.3 ng/mL) | 43(51.2) | 2.39(1.51–3.78) |
| Third quartile (n =84,119.4-143.6 ng/mL) | 52(62.0) | 2.89(1.86–4.50) |
| The fourth quartile (n =84,>143.7ng/mL) | 55(65.4) | 3.06(1.98–4.74) |
| quartile P value | <0.001 |
1.5 correlation between ox-HDL and aortic valve calcification independent of CAD
After correction for CAD incidence, logistic regression showed that ox-HDL levels were independently correlated with CAVD (OR 1.019,95% CI 1.012-1.027, P-Ap 0.001) (Table 3).
Similarly, after correction for atherosclerosis-related risk factors including hypertension, diabetes, glycated hemoglobin, lipid metabolism disorders, lipoprotein (a), body mass index, hypersensitive C-reactive protein, and creatinine, logistic regression showed that ox-HDL levels remained independently associated with CAVD risk (OR 1.027,95% CI 1.017-1.037, P < -0.001) (Table 4).
In addition, logistic regression showed that hypertension, body mass index, hypersensitive C reactive protein and lipoprotein (a) are determinants of CAVD (model 1). After inclusion of the ox-HDL concentration, the ox-HDL was independently correlated with CAVD risk (model 2). Furthermore, the addition of ox-HDL significantly increased the risk predictive effect (AUC 0.703 (0.651-0.751) vs.0.764 (0.715-0.808), P < 0.01) (fig. 1).
TABLE 3 Logistic regression analysis of CAVD patients
| Variables of | Probability (95% confidence interval) | P value |
| CAD | 1.058(0.631–1.772) | Is not significant |
| ox-HDL | 1.019(1.012–1.027) | <0.001 |
TABLE 4 Logistic regression analysis after correction of atherosclerotic Risk factors
| Variables of | OR(95％CI) | P value |
| Blood pressure | 2.013(1.126–3.599) | 0.018 |
| Diabetes mellitus | 0.684(0.395–1.187) | Is not significant |
| Glycated hemoglobin | 0.876(0.678–1.132) | Is not significant |
| Triglycerides | 0.901(0.730–1.113) | Is not significant |
| Total Cholesterol | 1.245(0.873–1.775) | Is not significant |
| High density lipoprotein cholesterol | 0.320(0.092–1.107) | Is not significant |
| Low density lipoprotein cholesterol | 1.001(0.670–1.495) | Is not significant |
| Lipoprotein (a) | 5.232(1.634–16.748) | 0.005 |
| Body mass index | 1.180(1.090–1.278) | <0.001 |
| Hypersensitive C response protein | 1.147(1.008–1.305) | 0.037 |
| Creatinine | 1.001(0.988–1.015) | Is not significant |
| Statins medicine | 1.473(0.588–3.691) | Is not significant |
| High density lipoprotein oxide | 1.027(1.017–1.037) | <0.001 |
1.6 factors affecting ox-HDL plasma levels
To further investigate potential factors that may affect ox-HDL concentration, general linear analysis and multiple linear regression analysis were employed. General linear correlation analysis showed a positive correlation between ox-HDL and HDL, glycated hemoglobin and lipoprotein (a), and multiple linear regression analysis showed independent correlation between ox-HDL and glycated hemoglobin levels, as shown in Table 5.
TABLE 5 influencing factors of the plasma ox-HDL level
Example 2
2.1 preparation of HDL and ox-HDL
Fresh fasting plasma of 10 healthy normal participants was collected and mixed, HDL (1.063-1.210 g/mL) was obtained by density gradient centrifugation, and the detailed procedures were as follows: firstly, placing the blood plasma in an ultracentrifuge, and centrifuging for 5 hours at 500000g and 4 ℃; then placing the HDL obtained by centrifugation in phosphate buffer solution (PBS, pH 7.4) for full dialysis; finally, the dialyzed HDL was filtered through a 0.22 μm pore size filter and stored at 4 ℃ in the dark until used (N-HDL). The concentration of the HDL was determined by determining the concentration of apoA-1 by SDS-PAGE.
Incubating HDL and copper sulfate solution (20 μmol/L) prepared from PBS at 37 deg.C for 24 hr, adding EDTA to stop oxidation reaction, and dialyzing in PBS for 48 hr to obtain ox-HDL for use. The degree of HDL oxidation, expressed as nanomolar amounts of TBARS per milligram of protein, was determined by the TBARS kit.
The TBARS value in ox-HDL is 25.3 +/-5.2 nmol/mg, and the TBARS value in N-HDL is less than 5nmol/mg, which indicates that the in vitro oxidation modification of the N-HDL to generate ox-HDL is successful and is used for the subsequent in vitro induction of AVICs calcification.
2.2 culture of Primary aortic valve stromal cells
Aortic valve leaflets (uncalcified normal leaflets) of 9 patients with heart transplantation were collected and cultured in primary AVICs, specifically: firstly, the valve leaflet is digested by collagenase to eliminate endothelial cells, and then the valve leaflet is cut into 1-2 mm 2 The mass of (2) was treated with DMEM F12 (1) (containing 20% fetal bovine serum, 2 mmol/L-glutamine, 100U/mL penicillin and 100. Mu.g/mL streptomycin) medium at 37 ℃ 5% 2 Culturing in an incubator.
2.3 in vitro Induction of AVICs calcification
To induce calcification, cell cultures were performed in osteogenic medium (containing 15% fetal bovine serum, 50mg/mL ascorbic acid-2-phosphate, 10nM dexamethasone, and 10mM beta-glycerophosphate) with DMEM.
When the cells cultured according to the "culture of 2.2 primary aortic valve stromal cells" method were confluent to 90%, they were grouped as follows: blanks (control, con), N-HDL (50. Mu.g/mL), ox-HDL (10. Mu.g/mL) and ox-HDL (50. Mu.g/mL) were sequentially stimulated with no additives, 50. Mu.g/mLN-HDL, 10. Mu.g/mL ox-HDL and 50. Mu.g/mL ox-HDL, respectively, for two weeks, and the medium was changed every two days.
The preparation method of N-HDL and ox-HDL is described in "2.1 preparation of HDL and ox-HDL".
2.4 detection of results of AVICs calcification
Alizarin Red S staining (Alzarin Red S): fixing the cells with 4% formaldehyde for 30min, washing with distilled water for 3 times, adding 2% alizarin red S (pH 4.1-4.3), and dyeing at room temperature for 30min, wherein the dyeing result is shown in FIG. 2A: alizarin red S positive staining is red/orange, blank (control, con) has obvious red staining, the number of positive staining cells in N-HDL (50 mu g/mL) group is reduced compared with that in blank group, the number of positive staining cells in ox-HDL (10 mu g/mL, 50 mu g/mL) group is increased compared with that in blank group, and the number of positive staining cells is gradually increased along with the increase of the concentration level of ox-HDL.
For quantitative analysis of alizarin red staining result, cells were incubated with cetylpyridinium chloride for 15min to release the dye in the cell matrix, absorbance values of each sample at 540nm wavelength of the spectrophotometer were read, and the quantitative analysis result is shown in fig. 2C: the amount of calcification was reduced in the N-HDL group (50. Mu.g/mL) compared to the control group (Con), resulting in reversal; the calcification amount of ox-HDL (10. Mu.g/mL, 50. Mu.g/mL) increased with the increase of the concentration of ox-HDL, and both were significantly higher than the blank group.
Alizarin red S staining shows that ox-HDL formed by oxidizing modification of N-HDL shows bone-promoting capacity and stimulates AVICs calcium deposition, and the calcification capacity of the ox-HDL is in positive correlation with the concentration of the ox-HDL; while N-HDL reverses AVICs calcium deposition and AVICs calcification.
Alkaline phosphatase ALP activity assay: the alkaline phosphatase activity was measured using a colorimetric kit (Biyuntian, china), and the staining results for alkaline phosphatase activity were obtained by Image-Pro Plus 6.0 photography, and are shown in FIG. 2A: ALP positive staining of alkaline phosphatase is purple, blank (control, con) has obvious purple staining, the number of positive staining cells of N-HDL (50 mu g/mL) group is reduced compared with that of blank group, the number of positive staining cells of ox-HDL (10 mu g/mL and 50 mu g/mL) group is increased compared with that of blank group, and the number of positive staining cells is gradually increased along with the increase of the concentration level of ox-HDL.
The activity of alkaline phosphatase ALP was quantitatively analyzed by p-nitrophenol level in AVICs, absorbance value at 405nm was read and substituted into a standard curve for calculation, and the quantitative analysis result is shown in FIG. 2B: reduction in ALP activity of alkaline phosphatase in the N-HDL group (50. Mu.g/mL) as compared with the control group (Con); ALP activity of ox-HDL group (10. Mu.g/mL, 50. Mu.g/mL) alkaline phosphatase increased with increasing ox-HDL concentration, and both were significantly higher than blank group.
Alizarin red S staining and alkaline phosphatase ALP activity assay showed: the ALP activity of the alkaline phosphatase is related to the calcification of AVICs, and the higher the ALP activity of the alkaline phosphatase, the more remarkable the calcification of the AVICs; ox-HDL can induce differentiation and/or calcification of AVICs in the osteogenic direction by enhancing ALP activity, and this effect is enhanced with increasing concentration of ox-HDL. N-HDL reverses calcification of AVICs by inhibiting ALP activity.
2.5AVICs Calcification-related Gene expression
Total RNA was first extracted with Trizol, after which 5. Mu.g of total RNA was reverse transcribed to cDNA by a reverse transcription kit (Promega, wisconsin, USA). PCR amplification was carried out using Power SYBR Green PCR Master Mix (Applied Biosystems, calif., USA) kit, and amplification was carried out using StepOne systems (Applied Biosystems). The oligonucleotides used in the real-time quantitative fluorescent PCR analysis are all listed in Table 6. The gene expression levels were analyzed using beta-actin as an endogenous reference and StepOne v2.1 software (Applied Biosystems), and the results are shown in FIG. 3.
TABLE 6real-time PCR primers
Compared with a blank control (Con), after adding ox-HDL (10 mu g/mL and 50 mu g/mL), BMP2, runx2 and Msx2 gene expression levels are obviously improved, and the BMP2, runx2 and Msx2 gene expression levels are positively correlated with the concentration of ox-HDL.
When N-HDL (50. Mu.g/mL) was added, BMP2, runx2 and Msx2 gene expression levels decreased and N-HDL inhibited BMP2, runx2 and Msx2 gene expression, compared to control (Con).
Combining alizarin red S staining results (2A, 2C), it is known that BMP2, runx2, and Msx2 gene expression levels are related to AVICs calcification, and the higher BMP2, runx2, and Msx2 gene expression levels are, the more significant AVICs calcification is. ox-HDL can induce differentiation and/or calcification of AVICs in the osteogenic direction by increasing BMP2, runx2 and Msx2 gene expression levels, and this effect is enhanced with increasing concentration of ox-HDL. N-HDL reverses AVICs calcification by inhibiting BMP2, runx2 and Msx2 gene expression levels.
Claims (9)
1. The application of oxidized high-density lipoprotein or the detection reagent of the oxidized high-density lipoprotein in the preparation of the calcified aortic valve disease diagnostic agent.
2. The use according to claim 1, wherein the oxidized high density lipoprotein detection reagent is used for detecting the concentration of oxidized high density lipoprotein in serum.
3. A diagnostic agent for calcific aortic valve diseases, comprising a detection reagent for oxidized high-density lipoprotein and/or oxidized high-density lipoprotein.
4. Use of a blocker or inhibitor of oxidized high density lipoprotein in the preparation of a medicament for the treatment of calcified aortic valve disorders.
5. The use according to claim 4, wherein the inhibitor and/or blocker of oxidized high-density lipoprotein as an active molecule for inhibiting and/or reversing calcified aortic valve disorders, inhibiting and/or reversing differentiation and/or calcification of interstitial cells of the aortic valve into osteogenic direction.
6. A drug for treating calcific aortic valve diseases, which comprises an inhibitor or a blocker of oxidized high-density lipoprotein.
7. The oxidized high-density lipoprotein or the detection reagent of the oxidized high-density lipoprotein is used for screening the therapeutic drugs for calcified aortic valve diseases.
8. The use according to claim 7, wherein the oxidized high density lipoprotein is used as a target or marker for screening an inhibitor or a blocker of the oxidized high density lipoprotein.
9. A screening agent for a therapeutic agent for calcified aortic valve diseases, which comprises a detection reagent for oxidized high-density lipoprotein and/or oxidized high-density lipoprotein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811331617.6A CN111175522B (en) | 2018-11-09 | 2018-11-09 | Application of oxidized high-density lipoprotein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811331617.6A CN111175522B (en) | 2018-11-09 | 2018-11-09 | Application of oxidized high-density lipoprotein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111175522A CN111175522A (en) | 2020-05-19 |
| CN111175522B true CN111175522B (en) | 2023-04-07 |
Family
ID=70647970
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811331617.6A Active CN111175522B (en) | 2018-11-09 | 2018-11-09 | Application of oxidized high-density lipoprotein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111175522B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725266B (en) * | 2021-01-28 | 2023-04-18 | 四川大学华西医院 | Method for establishing simulation type valve interstitial cell calcification model |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7780950B2 (en) * | 2002-01-02 | 2010-08-24 | The Cleveland Clinic Foundation | Systemic marker for monitoring anti-inflammatory and antioxidant actions of therapeutic agents |
| US7749729B2 (en) * | 2004-07-19 | 2010-07-06 | University Of Washington | Methods for assessing the risk for development of cardiovascular disease |
| WO2013185214A1 (en) * | 2012-06-11 | 2013-12-19 | Universite Laval | Pla2g7/lp-pla2 as biomarker and therapeutic target in the prevention and treatment of calcific aortic valve disease |
| WO2015127517A1 (en) * | 2014-02-27 | 2015-09-03 | Katholieke Universiteit Leuven | Oxidative stress and cardiovascular disease events |
| CN108379552B (en) * | 2018-04-12 | 2020-03-27 | 上海长海医院 | Application of reagent for improving CAB39 protein expression quantity in preparation of medicine for preventing or treating heart valve diseases |
-
2018
- 2018-11-09 CN CN201811331617.6A patent/CN111175522B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 董敏 等.氧化型高密度脂蛋白与动脉粥样硬化.《中国动脉硬化杂志》.2015,第第23卷卷(第第23卷期),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111175522A (en) | 2020-05-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Natorska et al. | Does diabetes accelerate the progression of aortic stenosis through enhanced inflammatory response within aortic valves? | |
| Kang et al. | Plasma concentration of visfatin is a new surrogate marker of systemic inflammation in type 2 diabetic patients | |
| Lu et al. | Interpretation of elevated plasma visfatin concentrations in patients with ST-elevation myocardial infarction | |
| Nakamura et al. | Serum levels of pigment epithelium‐derived factor (PEDF) are positively associated with visceral adiposity in Japanese patients with type 2 diabetes | |
| Dziedzic et al. | Association of vitamin D deficiency and degree of coronary artery disease in cardiac patients with type 2 diabetes | |
| Takahashi et al. | The expression of SPARC in adipose tissue and its increased plasma concentration in patients with coronary artery disease | |
| Shirpoor et al. | Vasoprotective effect of vitamin E: rescue of ethanol-induced atherosclerosis and inflammatory stress in rat vascular wall | |
| Zhang et al. | Undercarboxylated osteocalcin as a biomarker of subclinical atherosclerosis in non-dialysis patients with chronic kidney disease | |
| Sun et al. | Oxidized HDL, as a novel biomarker for calcific aortic valve disease, promotes the calcification of aortic valve interstitial cells | |
| Chen et al. | Race-specific associations of myeloperoxidase with atherosclerosis in a population-based sample: the Dallas Heart Study | |
| Sibal et al. | A study of endothelial function and circulating asymmetric dimethylarginine levels in people with Type 1 diabetes without macrovascular disease or microalbuminuria | |
| Ji et al. | Serum sclerostin as a potential novel biomarker for heart valve calcification in patients with chronic kidney disease. | |
| CN111175522B (en) | Application of oxidized high-density lipoprotein | |
| US20220341938A1 (en) | Use of soluble urokinase plasminogen activator receptor levels in the management of patients with cardiovascular disease | |
| Leoncini et al. | Combined use of urinary neutrophil gelatinase-associated lipocalin (uNGAL) and albumin as markers of early cardiac damage in primary hypertension | |
| Wu et al. | lncRNA FAS-AS1 served as a diagnostic biomarker of end-stage renal disease and mediated vascular calcification via regulating oxidative stress and inflammation | |
| Mendonça Coelho et al. | Is procalcitonin useful to differentiate rejection from bacterial infection in the early post‐operative period of liver transplantation in children? | |
| Li et al. | Potential ferroptosis key genes in calcific aortic valve disease | |
| Zhou et al. | Serum fatty acid binding protein 4 levels are associated with abdominal aortic calcification in peritoneal dialysis patients | |
| Celik et al. | Serum prolidase activity in systemic sclerosis | |
| Su et al. | The relationship between klotho and SIRT1 expression in renal aging related disease | |
| Suzuki et al. | Correlation of circulating dehydroepiandrosterone with activated protein C generation and carotid intima‐media thickness in male patients with Type 2 diabetes | |
| Mistrík et al. | Anti-inflammatory properties of high-density lipoprotein cholesterol in chronic hemodialysis patients: impact of intervention | |
| Nakamura et al. | Involvement of angiotensin II in intestinal cholesterol absorption | |
| Yang et al. | Advanced glycation end product-modified low-density lipoprotein promotes pro-osteogenic reprogramming via RAGE/NF-κB pathway and exaggerates aortic valve calcification in hamsters |
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 |