Application of NANS in preparation of medicine for preventing and treating vascular endothelial inflammation injury

Abstract

The invention discloses application of NANS in sialic acid induced vascular endothelial injury, and belongs to the technical field of biological medicine. The research shows that NANS is used as a key protease in endogenous synthesis of sialic acid. After the NANS gene is silenced, sialic acid level is reduced by inhibiting sialic acid endogenous synthesis, so that endothelial inflammation injury is relieved. The invention provides a new target for endothelial inflammation injury, so that a new treatment method is provided for development of drugs for treating endothelial injury vascular diseases, the treatment effect of the endothelial injury vascular diseases is improved from inhibition of sialic acid endogenous synthesis, and the purpose of eradicating the endothelial inflammation injury is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of NANS as a target in drugs for preventing and / or treating vascular endothelial inflammatory damage. Background Art

[0002] Atherosclerosis (AS) is the initial step in the development of cardiovascular disease and the main cause of high morbidity and mortality of cardiovascular disease. Endothelial dysfunction and inflammatory cell infiltration are important causes of atherosclerosis. Therefore, exploring the mechanism of regulating endothelial damage will provide more theoretical basis for improving the AS progression theory and provide more data support for the diagnosis and treatment of AS and the development of targeted drugs.

[0003] In long-term studies on AS, it was found that in addition to known causes, the level of metabolites in the circulation is also an important factor in inducing endothelial inflammatory damage. The level of metabolites affects the normal function of the endothelium and causes endothelial dysfunction. In recent years, a large number of studies have shown that sialic acid is a biomarker of AS and is significantly increased in the serum of AS patients. High concentrations of sialic acid aggravate the process of atherosclerosis by inducing endothelial inflammatory damage.

[0004] NANS is a protease that converts ManNAc-6-P to Neu5Ac-9-P in the sialic acid synthesis pathway and plays an important role in the endogenous synthesis of sialic acid. By analyzing the carotid artery tissues of normal patients and patients with atherosclerosis, it was found that the expression of NANS in the tissues of patients with atherosclerosis was significantly increased. However, there have been no reports on the functional application of NANS in regulating the process of sialic acid synthesis-mediated endothelial inflammatory damage. Summary of the invention

[0005] The purpose of the present invention is to provide a new strategy for treating endothelial inflammatory damage and the application of NANS in preparing drugs for preventing and treating vascular endothelial inflammatory damage.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention has found that, compared with normal HUVEC, abnormal NANS expression is detected to be increased after inflammatory damage occurs. After silencing NANS with siRNA, sialic acid levels can be reduced and endothelial inflammatory damage can be alleviated, indicating that NANS plays an important role in regulating sialic acid levels and participating in the process of endothelial inflammatory damage. Endogenous sialic acid synthesis can be reduced by inhibiting NANS expression (including but not limited to knockdown, silencing, drug inhibition, etc.), thereby preventing or treating vascular endothelial inflammatory damage. Therefore, the present invention provides the following new applications of NANS:

[0008] Application of NANS in regulating endogenous sialic acid synthesis.

[0009] Application of NANS in the preparation of drugs for preventing or treating vascular endothelial inflammatory damage.

[0010] Preferably, the endothelial inflammatory injury model is constructed by TNF-α (tumor necrosis factor α).

[0011] Preferably, the marker proteins of endothelial inflammatory damage level are ICAM-1 and VCAM-1.

[0012] Preferably, the sialic acid level is detected by a sialic acid detection kit.

[0013] Preferably, the inhibition of NANS expression is by adding siRNA.

[0014] Preferably, the sense sequence of the siRNA is 5'-AUGAAACAGGCAUAGCGAUTT-3', and the antisense sequence is 5'-AUCGCUAUGCCUGUUUCAUTT-3'.

[0015] Preferably, the siRNA can inhibit NANS expression, downregulate sialic acid levels, and alleviate endothelial inflammatory damage, thereby achieving the effect of preventing and treating vascular endothelial inflammation.

[0016] The present application provides the use of NANS as a target in drugs for preventing vascular injury diseases. After NANS gene silencing, sialic acid synthesis is inhibited, sialic acid levels are reduced, and endothelial inflammatory damage is inhibited. The present application provides important genes in endothelial inflammatory damage, improves the therapeutic effect of endothelial inflammatory damage from the endogenous metabolic pathway, and provides a new treatment method for the development of endothelial injury drugs, thereby achieving the purpose of cardiovascular disease inflammatory damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Comparison diagrams verifying the upregulation of NANS in endothelial inflammatory injury in the embodiments of the present invention. (A) NANS mRNA level after inflammatory injury of human umbilical vein endothelial cells (HUVEC) induced by 100 ng / mL TNF-α. (B) NANS protein level after inflammatory injury of HUVEC induced by 100 ng / mL TNF-α. (C) HUVEC sialic acid level after inflammatory injury of HUVEC induced by 100 ng / mL TNF-α. Figure 2The following are comparison diagrams to verify the decrease in NANS expression level after adding NANS siRNA in the embodiments of the present invention. (A) NANS mRNA expression level after HUVECs were treated with NANS siRNA at different concentrations. (B) NANS protein level after HUVECs were treated with NANS siRNA at different concentrations.

[0018] Figure 3 This is a comparison chart verifying the reduction of HUVEC sialic acid level after adding NANS siRNA in the embodiment of the present invention.

[0019] Figure 4 This is a comparison chart verifying that the level of HUVEC inflammatory damage is reduced after adding NANS siRNA in the embodiment of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments.

[0021] Application of NANS in the preparation of drugs for preventing and treating vascular endothelial inflammatory damage.

[0022] The full name of NANS is N-acetylneuraminic acid synthase. Protein number encoded by the gene: NP_0618191mplelelcpg rwvggqhpcf iiaeigqnhq gdldvakrmi rmakecgadc akfqkselef

[0023] 61kfnrkalerp ytskhswgkt ygehkrhlef shdqyrelqr yaeevgifft asgmdemave

[0024] 121flhelnvpff kvgsgdtnnf pylektakkg rpmvissgmq smdtmkqvyq ivkplnpnfc

[0025] 181flqctsaypl qpedvnlrvi seyqklfpdi pigysghetg iaisvaaval gakvlerhit

[0026] 241ldktwkgsdh saslepgela elvrsvrlve ralgsptkql lpcemacnek lgksvvakvk

[0027] 301ipegtiltmd mltvkvgepk gyppedifnl vgkkvlvtve eddtimeelv dnhgkkiks

[0028] The drug is siRNA that inhibits the up-regulation of NANS expression.

[0029] The drug inhibits sialic acid synthesis and downregulates sialic acid levels, thereby alleviating endothelial inflammatory damage

[0030] The present application is explained below in conjunction with specific embodiments.

[0031] Prepare the experiment:

[0032] A Construction of Endothelial Inflammation Injury Model

[0033] In one embodiment, the cells used are HUVEC (human umbilical vein endothelial cells), which are cultured in DMEM high glucose medium (containing 10% fetal bovine serum and 1% antibiotics) at 37°C and 5% CO2. The cells are seeded in a 6-well plate, and when the cells adhere to the wall and grow to 70% density, 100 ng / mL TNF-α (tumor necrosis factor α) is added and treated for 24 hours before the cells are collected.

[0034] B cell transfection

[0035] Plant the cells in a 6-well plate, wait for the cells to adhere to the wall and reach a density of 50% before transfection; the transfection reagent is Lipofectamine 2000. Taking 10nM as an example, dilute 1μL of siRNA storage solution and 5μL of Lipo2000 with 50μL of serum-free medium, respectively, and let stand for 5 minutes. Mix the two thoroughly and let stand for 20 minutes, then add the mixture to the well plate containing 1500μL of medium without antibodies containing cells, mix gently, wait for 6 hours, and replace with 2mL of fresh medium containing antibodies. Continue to culture for 48h to collect RNA, and culture for 72h to collect protein.

[0036] Example 1: NANS is upregulated during endothelial inflammatory injury

[0037] Step 1: Protein extraction and immunoblotting verification:

[0038] Protein extraction: Remove the plate and place it on ice. After discarding the culture medium, wash it with PBS three times, and then lyse the cells with 80μL of RIPA lysis buffer containing 1% PMSF. Collect the lysate in a 1.5mL Eppendorf tube, centrifuge at 4℃, 12000rpm, and collect the supernatant. Add 20μL of 5× Loading buffer to each tube, mix well, place in a 100℃ metal bath for 10min, take out and cool to obtain the protein sample.

[0039] Immunoblotting verification: After the proteins were separated by SDS-PAGE, they were transferred to the membrane at 300 mA for 90 minutes, and then the PVDF membrane was blocked with TBST containing 5% skim milk powder at room temperature for 1 hour, and the bands were incubated with the corresponding primary antibody overnight. The next day, the bands were washed with TBST 3 times, each for 10 minutes. The bands were incubated with the corresponding secondary antibody and then washed with TBST 3 times, each for 10 minutes. The bands were observed with a bioimaging system using an enhanced luminescence agent. β-actin was used as a protein internal reference.

[0040] Step 2: RNA extraction and RT-PCR verification

[0041] RNA extraction: Take out the 6-well plate and place it on ice. After washing with PBS, add 1 mL of Trizol to each well. After standing on ice for 15 min, scrape the cells with a cell scraper and collect them in a 1.5 mL enzyme-free EP tube.

[0042] Add 200 μL of chloroform to each tube, mix thoroughly and place on ice for 10 min. After stratification is observed, centrifuge at 4°C, 12,000 rpm for 15 min, and collect 500 μL of supernatant in a new enzyme-free EP tube.

[0043] Add 500 μL of isopropanol to a new EP tube, mix thoroughly by inversion, and place on ice for 10 min. Centrifuge at 4°C, 12,000 rpm for 15 min, and observe a flocculent precipitate.

[0044] The supernatant was discarded, and the precipitate was retained. 1 mL of 75% ethanol was added, and the mixture was mixed by inversion and allowed to stand for 5 min. The mixture was centrifuged at 4°C and 12,000 rpm for 5 min, and the supernatant was discarded, and the precipitate was retained.

[0045] Add 1 mL of anhydrous ethanol, mix thoroughly by inversion, let stand for 5 min, centrifuge at 4°C, 12,000 rpm for 5 min, discard the supernatant, retain the precipitate, and air-dry the remaining ethanol at room temperature.

[0046] After adding 10 μL DEPC water to each tube, measure the RNA concentration and store at -80°C for future use.

[0047] Reverse transcription of RNA: The above RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0048] 5x RTase Reaction Buffer MixII (2μL), Evo M-MLV RTase Enzyme Mix (0.5μL), Oligo dT (18T) Primer (50μM) (0.5μL), Random 6mers Primer (100μM) (0.5μL), TotalRNA-RNase free water Up to 10μL.

[0049] Reaction conditions: 37℃15min, 85℃5sec, 4℃∞.

[0050] PCR reaction: Reverse transcription was performed on a gradient PCR instrument using a 10 μL reaction system as follows: 2XSYBR green qPCR Mix 5 μL, Primer F 0.5 μL, Primer R 0.5 μL, DEPC Water 3 μL, and cDNA 1 μL.

[0051] Reaction conditions: 1 cycle at 95°C for 1 min, 40 cycles at 95°C for 10 s and 60°C for 10-15 s. Primers were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd., and the internal reference was β-Actin. The primer sequences are as follows: β-actin sense sequence: 5′-TTGCCGACAGGATGCAGAA-3′, antisense sequence: 5′-GCCGATCCACACGGAGTACT-3′; NANS sense sequence: 5'-AACACCCGTGCTTCATCATTG-3', antisense sequence: 5'-GGATCATGCGCTTGGCTAC-3'.

[0052] See also Figure 1 A and 1B, where Figure 1 A is the expression level of NANS protein in endothelial inflammatory damage induced by 100nM concentration of TNF-α. Figure 1 B is the NANS mRNA level in endothelial inflammatory injury induced by 100 nM TNF-α. It can be seen that compared with the control group, NANS was significantly upregulated in endothelial inflammatory injury.

[0053] Step 3: Sialic acid concentration determination

[0054] The 6-well plate was taken out and placed on ice. After washing with PBS three times, 60 μL of RIPA lysis buffer was added to each well and the plate was placed on ice for 10 min. The cell suspension was collected in a 1.5 mL EP tube using a cell scraper.

[0055] Take 2 μL of cell suspension and measure protein concentration using BCA method

[0056] Take another 50 μL of cell suspension into a 1.5 mL EP tube, add 500 μL of reagent 2 into the tube, vortex mix, and place in a 100°C water bath for 15 min (the boiling water level is higher than the reagent liquid level in the EP tube).

[0057] After taking out, cool with running water, centrifuge at 2325×g for 10 min. Take 200 μL of the supernatant after centrifugation and add it to the ELISA plate, and measure the OD value at 560 nm on the ELISA reader.

[0058] The protein sialic acid concentration was calculated by comparing the OD value △A560 after detection with the standard fitting curve (y=a1x+b1) using the following formula: Sialic acid concentration (mmol / gprot)=(△A560-b1) / (a1*C pr )(△A560 is the sample OD value minus the blank OD value; a1, b1: standard fitting curve parameters; C pr : protein concentration of the sample to be tested).

[0059] See also Figure 1 C, Figure 1 C is the sialic acid level in endothelial inflammatory damage induced by 100 nM TNF-α. It can be seen that compared with the control group, the sialic acid content was significantly upregulated in endothelial inflammatory damage.

[0060] Example 2: After adding NANS siRNA, the NANS expression level is reduced

[0061] Step 1: After adding NANS siRNA, the NANS mRNA level decreased.

[0062] Step 1 in this embodiment is similar to step 2 in Example 1, except that the cells in this embodiment are endothelial cells transfected with NANS siRNA.

[0063] See also Figure 2 A, Figure 2 A shows the NANS mRNA level after treatment with different concentrations of siRNA. It can be seen that compared with the control group, the NANS mRNA level was significantly reduced after the addition of NANS siRNA.

[0064] Step 2: After adding NANS siRNA, the NANS protein level decreases. Step 2 in this example is similar to step 1 in Example 1, except that the cells in this example are endothelial cells transfected with NANS siRNA.

[0065] See also Figure 2 B, Figure 2B is the NANS protein level after treatment with different concentrations of NANS siRNA. It can be seen that compared with the control group, the NANS protein level was significantly reduced after the addition of NANS siRNA.

[0066] Based on the comprehensive PCR experimental results and protein immunoblotting verification results, 10nM was finally selected as the experimental concentration for subsequent experiments.

[0067] Example 3: Sialic acid levels decreased after addition of NANS siRNA

[0068] This example is similar to step 3 in Example 1, except that the cells in this example are endothelial cells treated with TNF-α (100 ng / mL) after being transfected with NANS siRNA, the sense sequence of NANS siRNA used is 5'-AUGAAACAGGCAUAGCGAUTT-3', and the antisense sequence is 5'-AUCGCUAUGCCUGUUUCAUTT-3'.

[0069] See also Figure 3 , Figure 3 Figure 2 shows the sialic acid level after adding NANS siRNA. It can be seen that compared with the control group, TNF-α significantly upregulated the sialic acid level, and after adding NANS siRNA, the increase in sialic acid induced by TNF-α was significantly inhibited.

[0070] Example 4: After adding NANS siRNA, the level of endothelial inflammatory damage is reduced

[0071] This example is similar to step 2 in Example 2, except that the cells in this example are HUVECs treated with TNF-α (100 ng / mL) after transfection with NANS siRNA, and the proteins of VCAM-1 and ICAM-1 are verified.

[0072] See also Figure 4 , Figure 4 The endothelial inflammatory damage level after adding NANS siRNA treatment. It can be seen that compared with the control group, TNF-α significantly upregulated the level of inflammatory factors, and after adding NANS siRNA, the expression level of endothelial inflammatory factors was significantly reduced.

Claims

1. Application of NANS in the preparation of drugs for preventing inflammatory damage to vascular endothelium.

2. The use according to claim 1, characterized in that: The following steps are involved: S1 and NANS are upregulated during endothelial inflammatory injury S2. After adding NANS siRNA, NANS expression was reduced S3. After adding NANS siRNA, sialic acid levels decreased S4. After adding NANS siRNA, endothelial inflammatory damage was alleviated.

3. The application according to claim 2, characterized in that: The endothelial inflammatory injury model in S1 was constructed by TNF-α (tumor necrosis factor α).

4. The application according to claim 2, characterized in that: The sense sequence of NANS siRNA in S2 is 5'-AUGAAACAGGCAUAGCGAUTT-3', and the antisense sequence is 5'-AUCGCUAUGCCUGUUUCAUTT-3'.

5. The application according to claim 2, characterized in that: The sialic acid level in the S3 is detected by a sialic acid detection kit.

6. The use according to claim 2, characterized in that ,The marker proteins of endothelial inflammatory damage levels in the S4 are ICAM-1 and VCAM-1.

7. The use according to claim 2, characterized in that NANS is used as a target in the preparation of drugs for the treatment of endothelial inflammatory damage. After adding NANS siRNA to silence its gene, sialic acid levels are downregulated, thereby inhibiting endothelial inflammatory damage.

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