CN117503936A - Application of NEU3 in preparation of medicines for preventing and treating vascular endothelial inflammatory injury - Google Patents
Application of NEU3 in preparation of medicines for preventing and treating vascular endothelial inflammatory injury Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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Abstract
The invention discloses an application of NEU3 in preparing a medicament for preventing and treating vascular endothelial inflammatory injury, and belongs to the technical field of biological medicines. According to the invention, NEU3 is used as a target point to mainly down regulate sialic acid level after NEU3 gene silencing, so that endothelial inflammatory injury is inhibited, and an important gene in endothelial inflammatory injury is provided, so that a new treatment method is conveniently developed, the treatment effect of endothelial inflammatory injury is fundamentally improved, or the purpose of eradicating endothelial inflammatory injury is realized.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of NEU3 serving as a target spot in medicaments for preventing and/or treating vascular endothelial inflammatory injury.
Background
Atherosclerosis (AS) is a chronic inflammatory disease that results from the interaction of inflammatory cell infiltration, endothelial dysfunction, and the like. The vascular endothelial integrity damage is considered AS a starting link of the occurrence and development of atherosclerosis, so that the exploration and control of vascular endothelial injury mechanism provides more theoretical basis for perfecting AS progressive theory and provides more data support for AS diagnosis and treatment and targeted drug development.
In long-term studies of atherosclerosis, it was found that levels of circulating metabolites in the blood in contact with the endothelium were altered, with significant increases in sialic acid (sialic acid) content, and the literature reports that elevated sialic acid was able to promote the progression of atherosclerosis by inducing inflammatory lesions of the endothelium.
NEU3 (sialidase 3) is a member of the sialyltransferase family responsible for performing the function of cleaving glycoprotein terminal sialic acids, while the free sialic acid produced enters the blood for its physiological role. However, functional applications of NEU3 in regulating sialic acid to participate in endothelial inflammatory injury process have not been reported yet.
Disclosure of Invention
The invention aims to provide a novel strategy for treating endothelial inflammatory injury and application of NEU3 in preparing medicaments for preventing and treating vascular endothelial inflammatory injury.
The above object of the present invention is achieved by the following technical solutions:
the present inventors have found that abnormal upregulation of NEU3 expression is detected after inflammatory injury relative to normal HUVEC. In silencing NEU3, sialic acid and its induced endothelial inflammatory injury level can be reduced, which indicates that NEU3 can regulate sialic acid level to participate in endothelial inflammatory injury process, and can further reduce sialic acid level by inhibiting NEU3 expression (including but not limited to knockout, silencing, down-regulation and the like), thereby preventing and/or treating vascular endothelial inflammatory injury.
Thus, the present invention provides the following new applications for NEU 3:
application of NEU3 in regulation of sialic acid level
Application of NEU3 in preparation of medicines for preventing and/or treating vascular endothelial inflammatory injury
Preferably, the endothelial inflammatory injury model is constructed by TNF- α (tumor necrosis factor).
Preferably, the marker protein of endothelial inflammatory injury level is VCAM-1, ICAM-1.
Preferably, the sialic acid level is detected by liquid chromatography-mass spectrometry (LC-MS).
Preferably, the inhibition of NEU3 expression is the addition of siRNA.
Preferably, the siRNA has a sense sequence of 5'-GGUUGACCUAGGUAUCUAU-3', an antisense sequence of 5'-AUAGAUACCUAGGUCAACC-3' or a sense sequence of 5'-CGCCUUUGCUUCAUCUACA-3', an antisense sequence of 5'-UGUAGAUGAAGCAAAGGCG-3' or a sense sequence of 5'-CACCAUGGUAGACUCAUUA-3', and an antisense sequence of 5'-UAAUGAGUCUACCAUGGUG-3'.
Preferably, the siRNA has a sense sequence of 5'-GGUUGACCUAGGUAUCUAU-3' and an antisense sequence of 5'-AUAGAUACCUAGGUCAACC-3'.
Preferably, the siRNA can reduce endothelial inflammatory injury by inhibiting NEU3, down-regulating sialic acid levels. Thereby realizing the function of preventing and treating vascular endothelial inflammation.
The application provides application of NEU3 serving as a target point in preparing medicines for treating endothelial inflammatory injury, and after NEU3 gene silencing, sialic acid level is mainly regulated down so as to inhibit endothelial inflammatory injury.
Drawings
FIG. 1 is a graph comparing the up-regulation of NEU3 in an embodiment of the invention. (A) NEU3mRNA levels in endothelial inflammatory lesions induced with varying concentrations of TNF- α. (B) NEU3 protein levels in endothelial inflammatory lesions induced with varying concentrations of TNF- α. (C) Sialic acid levels in endothelial inflammatory lesions were induced using different concentrations of TNF- α.
FIG. 2 is a graph showing a comparison of NEU3 expression level decrease after verification of NEU3siRNA addition according to one embodiment of the present invention. (A) NEU3mRNA levels after treatment with siRNA of different sequences. (B) NEU3 protein levels after treatment with siRNA of different sequences.
FIG. 3 is a graph showing comparison of reduced sialic acid levels after validation of NEU3siRNA in accordance with an embodiment of the present invention.
FIG. 4 is a graph showing a comparison of reduced endothelial inflammatory injury levels after the addition of NEU3siRNA in accordance with one embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Application of NEU3 in preparation of medicines for preventing and treating vascular endothelial inflammatory injury
The NEU3 is collectively referred to as neuroaminidase 3. Gene encoded protein number:
NP_001354789 1mrpadlpprp meespasssa pteteepgss aevmeevttc sfnsplfrqe ddrgityrip
61allyipptht flafaekrst rrdedalhlv lrrglrigql vqwgplkplm eatlpghrtm
121npcpvweqks gcvflfficv rghvterqqi vsgrnaarlc fiysqdagcs wsevrdltee
181vigselkhwa tfavgpghgi qlqsgrlvip aytyyipswf fcfqlpcktr phslmiysdd
241lgvtwhhgrl irpmvtvece vaevtgragh pvlycsartp nrcraealst dhgegfqrla
301lsrqlcepph gcqgsvvsfr pleiphrcqd ssskdaptiq qsspgsslrl eeeagtpses
361wllyshptsr kqrvdlgiyl nqtpleaacw srpwilhcgp cgysdlaale eeglfgclfe
421cgtkqeceqi afrlfthrei lshlqgdcts pgrnpsqfks n
the drug is an siRNA which inhibits the up-regulation of NEU3 expression.
The agent down regulates sialic acid levels, thereby inhibiting endothelial inflammatory injury
The present application is illustrated below in connection with specific embodiments:
preparing an experiment:
a establishment of endothelial inflammatory injury model
In one embodiment, the cells used are HUVECs (human umbilical vein endothelial cells) cultured in DMEM hyperglycemic medium (containing 10% fetal bovine serum and 1% antibiotics) at 37℃and 5% CO 2.
Cells were seeded in 6-well plates and after cells had attached and grown to 70% density, cells were harvested after 12 hours of treatment with TNF- α (tumor necrosis factor α) addition.
B cell transfection
Planting cells in a 12-well plate, and transfecting the cells until the cells grow to 60-80% of the culture area; the transfection reagent is Lipofectamine 3000, the cells are subjected to liquid exchange treatment before operation, and a serum-free culture medium is exchanged; after 6h of liquid exchange, preparing a mixed solution (hereinafter, the dosage of one hole of a 12-hole plate), diluting 4 mu L of Lipo3000 with 100 mu L of Opti-MEM, fully and uniformly mixing, and waiting for 5min; diluting 2. Mu.L of siRNA with 100. Mu.L of Opti-MEM, fully and uniformly mixing the siRNA with the mixed solution, and waiting for 20min; adding the mixed solution into a pore plate, continuously culturing for 48h and 72h respectively, collecting cells,
example 1: NEU3 upregulation upon endothelial inflammatory injury
Step one: protein extraction and immunoblotting validation:
the collected cells were lysed using RIPA lysis buffer containing 2% protease inhibitor, and the supernatant was collected after centrifugation at 12000rpm for 10 minutes at 4 ℃. Adding 5x loading buffer solution, mixing well, boiling at 100 ℃ for 10 minutes to obtain a protein sample. Proteins were resolved by SDS-PAGE and electrotransferred to PVDF membrane. The PVDF membrane was then blocked with TBST containing 5% skim milk for 1.5h at room temperature, washed for 5min and incubated overnight with diluted primary antibody. After 7 min four TBST washes, incubation with specific secondary antibodies was performed for 1h at room temperature, and after three TBST washes the strips were visualized with a biological imaging system using an enhancing luminophore. ACTIN was used as a protein reference.
Step two: RNA extraction and RT-PCR validation
Extraction of RNA
(1) RNA was extracted from cells cultured in 6-well plates, 1mL of Trizol was added to each well, and the mixture was allowed to stand on ice for 10min
min, gently scraping the cells with a cell scraper, transferring to an enzyme-free EP tube;
(2) mixing with vortex instrument, adding 200 μl chloroform, mixing, and standing on ice for 10min;
(3) pre-cooling the refrigerated centrifuge in advance, centrifuging at 4 ℃ and 12000rpm for 15min, and tapping the supernatant to about 300
Mu L, volume recorded at each time;
(4) adding isopropanol with the same volume into an enzyme-free EP tube, uniformly mixing and standing for 10min;
(5) centrifugation at 12000rpm at 4deg.C for 15min, discarding supernatant, adding 1mL of 75% ethanol (75% ethanol here is prepared with anhydrous ethanol and anhydrous water);
(6) centrifuging at 7500rpm at 4deg.C for 5min, discarding supernatant (soft action), and air drying for 3min;
(7) DEPC water was added for dilution (10-20. Mu.L), and after measuring the RNA concentration, the sample was stored at-80 ℃.
Reversion of RNA
The above proposed RNA was reverse transcribed into cDNA by a reverse transcription kit using 10. Mu.L reaction system as follows:
5xRTase Reaction Buffer MixⅡ2μL,Evo M-MLV RTase Enzyme Mix 0.5μL,Oligo dT(18T)Primer(50μM)0.5μL,Random 6mers Primer(100μM)0.5μL,Total RNA-RNase free water Up to10μL。
reaction conditions: 15min at 37 ℃, 5sec at 85 ℃,4 ℃.
(III) PCR reaction
Reverse transcription was performed on a gradient PCR apparatus using 10 μl reaction system as follows:
2XSYBR green qPCR Mix 5μL,Primer F 0.5μL,Primer R 0.5μL,DEPC Water 3μL,cDNA 1μL。
the reaction conditions were 1min 1 cycle at 95℃and 40 cycles at 10s 60℃10-15s total. The primer is designed and synthesized by Beijing qingke biotechnology Co., ltd, the internal reference is GAPDH, and the primer sequence is as follows:
GAPDH sense sequence: 5'-GCACCGTCAAGGCTGAGAAC-3', antisense sequence: 5'-TGGTGAAGACGCCAGTGGA-3'; NEU3 sense sequence: 5'-AAGTGACAACATGCTCCTTCAA-3', antisense sequence: 5'-TCTCCTCGTAGAACGCTTCTC-3'.
Referring to FIGS. 1A and B, FIG. 1A shows NEU3mRNA levels in the induction of inflammatory lesions of the endothelium using varying concentrations of TNF- α. FIG. 1B shows NEU3 protein levels in the induction of endothelial inflammatory lesions using different concentrations of TNF- α. It can be seen that NEU3 was significantly up-regulated at the time of the dermatitis lesions, compared to the control group.
Step three: sialic acid concentration determination
After extracting the collected cell culture medium with acetonitrile (1:3), taking supernatant, volatilizing the supernatant in a vacuum dryer for 1.5 hours, redissolving ammonium acetate, and measuring sialic acid concentration by using LC-MS.
Referring to FIG. 1C, FIG. 1C shows sialic acid levels in endothelial inflammatory lesions induced by varying concentrations of TNF- α. It can be seen that the sialic acid content was significantly up-regulated at the time of endothelial inflammatory injury compared to the control group.
Example 2: NEU3siRNA addition reduced NEU3 expression levels
Step one, NEU3mRNA level is reduced after NEU3siRNA is added
Step one in this example is similar to step two in example 1, except that: the cells in this example are endothelial cells transfected with NEU3 siRNA.
Referring to FIG. 2A, FIG. 2A shows NEU3mRNA levels after siRNA treatment with different sequences. It can be seen that NEU3mRNA levels were significantly reduced after NEU3siRNA addition compared to the control.
Step two: NEU3 protein levels were reduced upon addition of NEU3siRNA
Step two in this example is similar to step one in example 1, except that: the cells in this example are endothelial cells transfected with NEU3 siRNA.
Referring to FIG. 2B, FIG. 2B shows NEU3 protein levels after siRNA treatment with different sequences. It can be seen that NEU3 protein levels were significantly reduced after NEU3siRNA addition compared to the control.
Example 3: reduced sialic acid levels following NEU3siRNA addition
This example is similar to step three of example 1, except that: the cells in this example were endothelial cells treated with TNF- α (50 ng/mL) following transfection of NEU3siRNA using NEU3siRNA having a sense sequence of 5'-GGUUGACCUAGGUAUCUAU-3' and an antisense sequence of 5'-AUAGAUACCUAGGUCAACC-3'.
Referring to fig. 3, fig. 3 shows sialic acid levels after siRNA addition treatment. It can be seen that the reduction in sialic acid levels was significant after addition of NEU3siRNA compared to the control group.
Example 4: reduced endothelial inflammatory injury levels following NEU3siRNA addition
This example is similar to step two of example 2, except that the cells in this example are endothelial cells treated with TNF- α (50 ng/mL) following transfection of NEU3siRNA and verification of VCAM-1, ICAM-1 protein.
Referring to fig. 4, fig. 4 shows the endothelial inflammatory injury level after siRNA addition treatment. It can be seen that the endothelial inflammatory injury level was significantly reduced after the addition of NEU3siRNA compared to the control group.
Claims (7)
- Application of NEU3 in preparing medicine for preventing and treating vascular endothelial inflammation injury.
- 2. The use according to claim 1, characterized by the steps of:s1.NEU3 upregulation at endothelial inflammatory injuryS2, after NEU3siRNA is added, NEU3 expression level is reducedS3, after NEU3siRNA is added, the sialic acid level is reducedS4, after NEU3siRNA is added, the endothelial inflammatory injury level is reduced.
- 3. The use according to claim 2, wherein the model of endothelial inflammatory injury in S1 is constructed by TNF- α (tumor necrosis factor).
- 4. The use according to claim 2, wherein the NEU3siRNA in S2 has a sense sequence 5'-GGUUGACCUAGGUAUCUAU-3' and an antisense sequence 5'-AUAGAUACCUAGGUCAACC-3'.
- 5. The use according to claim 2, wherein the sialic acid level in S3 is detected by liquid chromatography-mass spectrometry (LC-MS).
- 6. The use according to claim 2, wherein the marker protein of endothelial inflammatory injury level in S4 is VCAM-1, icam-1.
- 7. The use of claim 2, wherein NEU3 is used as a target in the preparation of a medicament for treating endothelial inflammatory injury, wherein after gene silencing by adding NEU3siRNA, sialic acid levels are predominantly down-regulated, thereby inhibiting endothelial inflammatory injury.
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