Medical use of tnf receptor associated protein 1 inhibitors

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to medical use of TNF receptor associated protein 1. In the cell and animal models related to atherosclerosis, it is found that interfering with TNF receptor associated protein 1 can inhibit the aging of smooth muscle cells and the occurrence of atherosclerosis; the application provides a new target for diagnosis and treatment of atherosclerosis related cardiovascular diseases, and opens up a new direction for preparation of atherosclerosis prevention and treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of substances that inhibit the activity of TNF receptor-associated protein 1 (TRAP1) in drugs for the prevention and treatment of atherosclerosis-related vascular diseases caused by aging. Background Technology

[0002] With an increasingly severe aging population, cardiovascular disease (CVD) has become a leading cause of death worldwide. Atherosclerosis, a chronic disease of the arterial walls characterized by lipid accumulation and the formation of atherosclerotic plaques, is one of the main causes of CVD. Deaths due to CVD are associated with cardiovascular complications resulting from plaque rupture, which can lead to thrombosis, acute coronary syndrome, or stroke. Therefore, timely prevention and treatment of cardiovascular diseases, including CVD, remains extremely important. Smooth muscle cells play a crucial role in the formation of CVD. Increasing research indicates that vascular smooth muscle cells (VSMCs) in advanced CVD exhibit premature aging characteristics, such as poor proliferation, significantly shortened telomerase, and expression of aging markers. VSMC aging leads to a decrease in VSMC content, affecting plaque repair after rupture and thus inducing plaque fragility. Therefore, VSMC aging is a key factor in the development and progression of CVD.

[0003] Cellular senescence is an irreversible physiological process characterized by cell cycle arrest, with metabolic disorders and changes in chromatin structure as its main features, leading to cellular dysfunction and homeostasis. In senescent smooth muscle cells, metabolic disturbances cause them to rely on aerobic glycolysis to produce ATP for energy. We have discovered a novel energy regulator, TNF receptor-associated protein 1 (TRAP1), involved in the regulation of smooth muscle senescence. TRAP1 is a major member of the heat shock protein HSP90 family in mitochondria and is a key regulator of metabolism and organelle homeostasis in various pathological states and healthy cells. Recent studies have found that mice lacking TRAP1 exhibit a reduced incidence of age-related diseases, while excessive TRAP1 expression leads to mitochondrial dysfunction. However, whether TRAP1 in smooth muscle cells is involved in the development of atherosclerosis has not yet been reported. Summary of the Invention

[0004] To address the aforementioned issues, this application provides a pharmaceutical use for inhibiting TNF receptor-associated protein 1.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] First, this invention provides the gene sequence and amino acid sequence of TRAP1.

[0007] TRAP1 gene sequence:

[0008] https: / / www.ncbi.nlm.nih.gov / nuccore / NC_000016.10?report=fasta&from =3658037&to=3717524&strand=true

[0009] >NC_000016.10:c3717524-3658037Homo sapiens chromosome 16,GRCh38.p14PrimaryAssembly.

[0010] The TRAP1 amino acid sequence is shown in SEQ ID NO.1:

[0011] https: / / www.uniprot.org / blast

[0012] >sp|Q12931|TRAP1_HUMAN Heat shock protein 75kDa,mitochondrial OS=Homo sapiens OX=9606GN=TRAP1 PE=1SV=3.

[0013] In a first aspect, the present invention protects the use of TRAP1 in the preparation of auxiliary diagnostic reagents for cardiovascular diseases.

[0014] Secondly, this invention protects the use of reagents for detecting TRAP1 in the preparation of auxiliary diagnostic reagents for cardiovascular diseases.

[0015] Thirdly, this invention protects the use of TRAP1 as a detection target in screening or assisting in the screening of drugs for the treatment of atherosclerosis.

[0016] Fourthly, this invention protects the use of TRAP1 as a target in the preparation of drugs for the prevention and treatment of cardiovascular diseases.

[0017] Fifthly, the present invention protects the use of substances that knock down or inhibit TRAP1 in the preparation of drugs for the prevention and treatment of cardiovascular diseases.

[0018] Sixthly, the present invention protects the use of substances that knock down or inhibit TRAP1 expression in the preparation of screening or auxiliary screening drugs for cardiovascular diseases.

[0019] As a preferred embodiment of the present invention, the substance that inhibits or knocks out TRAP1 expression is selected from TRAP1 siRNA, a gene editing system that specifically knocks out TRAP1, or other small molecule compounds that can specifically inhibit TRAP1.

[0020] As a preferred embodiment of the present invention, the siRNA is as shown in SEQ ID NO.2-3.

[0021] As a preferred embodiment of the present invention, the cardiovascular disease is a cardiovascular disease drug caused by aging.

[0022] As a preferred embodiment of the present invention, the cardiovascular disease caused by aging is atherosclerosis.

[0023] In a seventh aspect, the present invention protects a method for screening drugs for the treatment of atherosclerosis, which involves detecting TRAP1 levels before and after drug administration and assessing the efficacy of candidate drugs for the treatment of atherosclerosis by the degree of reduction in TRAP1 levels.

[0024] Apoe with smooth muscle cell-specific knockout of Trap1 KO mouse (Apoe) KO Trap1 SMCKO The construction of ) and its application in the preparation of treatments for atherosclerosis.

[0025] The beneficial effects of this invention are:

[0026] This invention, through experiments including Western blotting, RT-qPCR, and immunofluorescence staining, revealed increased TRAP1 expression in Ras-induced senescent smooth muscle cells. TRAP1 deficiency significantly improved smooth muscle cell senescence and metabolic disorders. Simultaneously, an Apoe cell-specific TRAP1 knockout assay was constructed. KO Mice: 8-week-old Apoe - / - Mice were randomly divided into a control group and a TRAP1 knockout group, and fed a high-fat diet for 16 weeks. Compared with the control group, the mice showed reduced plaque area, aging markers, and SASP. This invention is the first to clearly demonstrate the regulation of smooth muscle cell senescence by TRAP1, providing a new approach for drug development and a drug target for the diagnosis and treatment of atherosclerosis, and has significant medicinal value. Attached Figure Description

[0027] Figure 1 TRAP1 protein levels in Ras-induced human vascular smooth muscle cells: TRAP1 expression levels in normal and Ras-induced smooth muscle cells were detected by Western blotting.

[0028] Figure 2 To investigate the effect of TRAP1 knockout on the protein expression levels of P53, P21, and P16 in Ras-induced smooth muscle cells: smooth muscle cells were treated with siTRAP1 and then induced with Ras. Cell proteins were extracted, and the protein expression levels of P16, P21, and P53 were detected by Western Blot.

[0029] Figure 3 To detect smooth muscle senescence by β-galactosidase staining after TRAP1 knockout: Smooth muscle cells were treated with siTRAP1 and then induced with Ras. The senescence level of smooth muscle cells was detected by β-galactosidase staining.

[0030] Figure 4 To detect the expression levels of SASP (IL-6, IL-8, IL-1β, CCL2, ICAM-1, TNF-α) after TRAP1 knockout using real-time quantitative PCR (RT-qPCR): Smooth muscle cells were treated with siTRAP1 and then induced with Ras. RNA was extracted from the samples, and SASP expression was detected by RT-qPCR.

[0031] Figure 5 Apoe fed normal (NC) and high-fat (HFD) diets KO Trap1 WT Mice and Apoe KO Trap1 SMCKO Mouse blood vessel Oil Red O staining: Apoe KO Trap1 WT and Apoe KO Trap1 SMCKO Mice were fed a normal diet (NC) or a high-fat diet (HFD) for 16 weeks. Aortic blood vessels were extracted from mice, and plaque size was detected by Oil Red O staining.

[0032] Figure 6 Fluorescent staining of blood vessels for the aging marker P21: Apoe KO Trap1 WT and Apoe KO Trap1 SMCKO Mice were fed a normal diet (NC) or a high-fat diet (HFD) for 16 weeks. Aortic blood vessels were extracted from mice, embedded by OCT, and frozen sections were prepared. The expression of P21 and α-SMA was detected by fluorescent staining.

[0033] Figure 7 Apoe fed normal (NC) and high-fat (HFD) diets KO Trap1 WT Mice and Apoe KO Trap1 SMCKO Protein content of P16, P21 and P53 in mouse vascular smooth muscle cells: Eight-week-old mice were fed a high-fat diet for 16 weeks, and then aortic vascular smooth muscle cells were extracted. The protein expression of P21, P16 and P53 was detected by Western Blot.

[0034] Figure 8 Apoe fed normal (NC) and high-fat (HFD) dietsKO Trap1 WT Mice and Apoe KO Trap1 SMCKO SASP expression in mouse vascular smooth muscle cells: Eight-week-old mice were fed a high-fat diet for 16 weeks, and then aortic vascular smooth muscle cells were extracted. SASP expression was detected by RT-qPCR. Detailed Implementation

[0035] The following embodiments are intended to enable those skilled in the art to fully understand the present invention, but do not limit the invention in any way.

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The described embodiments are not all embodiments, but only a part of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Correlation assay of TRAP1 with smooth muscle cell senescence

[0038] To explore the level of the mitochondrial protein TRAP1 in atherosclerosis and to verify whether TRAP1 is involved in regulating cellular senescence and thus modulating atherosclerosis, this experiment used Western blot to detect the TRAP1 level in normal and Ras-induced smooth muscle cells.

[0039] To establish a smooth muscle cell senescence model: Smooth muscle cells were cultured in plates for 24 h, and then transfected with 20 μg of retroviral plasmid at 37 °C for 15 h. The virus-containing medium was filtered through a 0.45 μm filter, and the first supernatant was collected. The same procedure was repeated once, and the virus was collected again. At infection, an appropriate amount of the first supernatant was added to DMEM medium, and smooth muscle cells were cultured at 37 °C for 12 h. Then, cells were screened with 2 μg / mL puromycin for 3 days to establish a cell senescence model, while a control group (Con) with an equal amount of PBS was used.

[0040] Western Blot Detection of TRAP1 Protein Expression: Cellular protein was extracted, and the protein concentration was quantified using a BCA quantitative kit. The sample was then diluted and loaded, with gentle centrifugation before loading. After electrophoresis and transfer to a PVDF membrane, the sample was blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation with specific primary antibody at 4°C. After washing the membrane three times with TBST, it was incubated with secondary antibody. Protein bands were developed using ECL chromogenic buffer, and statistical quantification was performed using ImageJ software. The detection results are shown below. Figure 1As shown, after Ras stimulation, the expression level of TRAP1 protein in senescent smooth muscle cells significantly increased. Figure 1 ).

[0041] To further verify whether TRAP1 is involved in smooth muscle cell senescence, thereby leading to atherosclerosis, we constructed siTRAP1 smooth muscle cells and detected the expression of senescence biomarkers by Western blotting.

[0042] Small interfering RNA (siRNA) transfection of human smooth muscle cells: Prepare solutions A and B according to the transfection system (A: ECM 125μL + siRNA 6.25μL; B: ECM 125μL + lipo3000 3μL). Mix well and incubate at room temperature for 5 min. Then mix solutions A and B again, incubate at room temperature for 15-20 min, add to the dish, and distribute evenly. Finally, place the cells in a 37°C incubator and replace with fresh preheated culture medium after 4-6 h.

[0043] In this invention, siRNA is designed based on the TRAP1 gene sequence. The siRNA sequence is as follows:

[0044] Justice chain: 5'→3'GCUGCUGGUGGAUCAGAUAUA, as shown in SEQ ID NO.3;

[0045] Antisense chain: 3'→5'UAUCUGAUCCACCAGCAGCUG, as shown in SEQ ID NO.4.

[0046] Test results as follows Figure 2 As shown, compared with senescent smooth muscle cells, the Ras-induced increase in aging markers was reversed after TRAP1 loss. Simultaneously, a β-galactosidase staining assay (β-Gal staining assay) was performed.

[0047] β-Gal staining assay: Cells were seeded into six-well plates. After adhesion, the cell culture medium was aspirated, and the cells were washed three times with PBS. 1 mL of staining fixative was added, and the plates were fixed at room temperature for 15 min. The fixative was then aspirated, and the cells were washed three times with PBS for 3 min each time. PBS was then aspirated, and 1 mL of SA-β-gal staining solution was added to each well. The plates were incubated overnight at 37°C. The next day, the staining solution was discarded, the cells were washed with PBS, and the detection solution was added. The cells were then observed under a regular optical microscope.

[0048] Test results as follows Figure 3 As shown, compared with senescent smooth muscle cells, the number of Ras-induced positive staining cells was significantly reduced after TRAP1 deficiency. To further confirm that TRAP1 can regulate smooth muscle senescence and induce atherosclerosis, SASP expression was analyzed using RT-qPCR.

[0049] RT-qPCR analysis of SASP: Total RNA was extracted from cells according to the instructions in the Trizol kit.

[0050] (1) Remove the cells, discard the culture medium, wash once with 1×PBS, add 1mL Trizol, let stand for a while, and then use a pipette to blow the cells to completely detach them.

[0051] (2) Use a pipette to repeatedly blow and aspirate the lysis buffer until there is no obvious precipitate. Transfer all the lysis buffer to a labeled 1.5 mL EP tube and let it stand at room temperature for 5 min.

[0052] (3) Add 0.2 mL of chloroform to each EP tube, mix by inverting, and place on ice for 10 min to lyse;

[0053] (4) Centrifuge at 4℃ and 12000 rpm for 15 min;

[0054] (5) Transfer the supernatant to a new EP tube, add an equal volume of isopropanol to each tube, mix by inversion, and place on ice for 10 min.

[0055] (6) Centrifuge at 4℃, 12000rpm for 15min to form a gel-like precipitate on the side and bottom of the tube. Discard the supernatant and add 1mL of 75% ethanol to suspend the precipitate.

[0056] (7) Centrifuge at 4℃, 12000rpm for 15min, discard the supernatant, air dry the precipitate at room temperature for 5min, add 20μL DEPC water to dissolve the RNA, measure the RNA concentration with NanoDrop, and store in a -80℃ refrigerator for later use.

[0057] (8) Use The 1st Strand cDNA Synthesis Kit was used for reverse transcription. The total reaction volume was 20 μL, and the specific composition is shown below:

[0058] RNase-free ddH2O To 20 μL

[0059] Total RNA 1μg

[0060] II Buffer plus 4μL

[0061] (9) After mixing thoroughly, reverse transcription was performed using a PCR instrument:

[0062] 25℃ for 5 minutes

[0063] 42℃ for 30 minutes

[0064] 85℃ for 5 minutes

[0065] (10) After reverse transcription, the cDNA is diluted with 80 μL LEPC water at a ratio of 1:4 and stored at -20℃ for later use.

[0066] (11) Use qPCR SYBR Green Master Mix was used for relative quantification of the target gene. The PCR reaction system is as follows:

[0067]

[0068] (16) Divide into groups and calculate the system (add cDNA at the end);

[0069] (17) Seal the membrane, centrifuge the 384-well plate and place it in a real-time PCR instrument;

[0070] (18) The reaction was performed on a Bio-Rad 480II quantitative PCR instrument.

[0071] The primer sequences are as follows:

[0072] IL-6Forward:CTCCAGAACAGATTTGAGAG;

[0073] Reverse:GGGTCAGGGGTGGTTATTGC;

[0074] IL-8Forward:CTGAGGTGCCAGTGCATTAG;

[0075] Reverse:AGCACACCTCTCTTCCATCC;

[0076] IL-1βForward:TTGCCAGCCAGTGACACAAT;

[0077] Reverse:GAGAAGGTGGTTGTCTGGGAAT;

[0078] ICAM-1Forward:AGGTTGAACCCCACAGTCAC;

[0079] Reverse:TCTGAGACCTCTGGCTCGT;

[0080] CCL2 Forward:GATCTCAGTGCAGAGGCTCG;

[0081] Reverse:TCTGGGGAAAGCTAGGGGAA;

[0082] TNF-αForward:TAACAAGCCGGTAGCCCACG;

[0083] Reverse:TCTTGATGGCAGACAGGATG.

[0084] Test results as follows Figure 4 As shown, compared with senescent smooth muscle cells, SASP expression was significantly reduced after TRAP1 loss.

[0085] Example 2: Mouse Model Experiment

[0086] To further verify the effect of TRAP1 on atherosclerosis in vivo, this embodiment constructs smooth muscle-specific TRAP1 knockout mice: by using male TRAP1... flox / flox Mice hybridized with the Tagln promoter-driven Cre recombinase system (Tagln-Cre) produced Apoe KO Smooth muscle cell-specific Trap1-deficient mice (Apoe) in the background KO Trap1 SMCKO Apoe KO Trap1 WT Mice were used as control animals.

[0087] An atherosclerosis model was established by feeding 8-week-old mice with either a normal diet (NC) or a high-fat diet (HFD) for 16 weeks. Simultaneously, aortic blood vessels were collected from the mice, and Oil Red O staining was performed to detect plaque size.

[0088] Oil Red O staining of aortic root vessels:

[0089] (1) After the aortic tree was separated, it was placed in a clean six-well plate and fixed with 4% paraformaldehyde.

[0090] (2) Use micro forceps to take the aorta fixed with paraformaldehyde into a new six-well plate and rinse it with triple-distilled water for about 10 minutes.

[0091] (3) Use a pipette to remove the triple-distilled water from the six-well plate, add 60% isopropanol solution, and treat for 2 min.

[0092] (4) Use a pipette to remove the isopropanol from the six-well plate, add the pre-filtered Oil Red O staining solution, and place it on a horizontal shaker for 1 hour for staining.

[0093] (5) Use a pipette to remove the Oil Red O staining solution from the six-well plate, add 60% isopropanol solution to rinse for 1 min, repeat this process 3 times until the background of the blood vessels is no longer red.

[0094] (6) Carefully remove any remaining fat from the outer wall of the blood vessel under a microscope using micro-scissors.

[0095] (7) Finally, lay the stained aortic tree flat on a black anatomical wax plate and take a picture.

[0096] Test results as follows Figure 5 As shown, compared to Apoe fed a high-fat diet, KO Trap1 WT Mouse, Apoe KO Trap1 SMCKO The area of ​​the plaques in the mice was significantly reduced.

[0097] Further immunofluorescence assays were performed on blood vessels. Aortic tissues were extracted from mice in different groups after 16 weeks of high-fat feeding and embedded in optimal cutting temperature compound (OCT) frozen sections. Immunofluorescence was then used to detect the expression of p21 and α-SMA. Figure 6 As shown, red represents P21, green represents α-SMA, and the white arrow indicates the co-localized yellow area of ​​red and green. It can be seen that Apoe... KO After HFD administration, P21 expression in vascular smooth muscle cells significantly increased, and the co-localization of P21 with α-SMA was yellow; while Apoe KO Trap1 SMCKO After HFD administration to mice, the expression of P21 in vascular smooth muscle decreased, and the colocalization of P21 and α-SMA (yellow color) also decreased significantly. Simultaneously, proteins from vascular smooth muscle cells of different groups of mice were extracted, and aging markers such as P16, P21, and P53 were detected by Western blotting.

[0098] Extraction of mouse aortic vascular smooth muscle cells:

[0099] (1) Anesthetize mice by intraperitoneal injection of 2 mL of 10% chloral hydrate and immerse them in 75% ethanol;

[0100] (2) Open the thoracic and abdominal cavities under aseptic conditions to expose the heart;

[0101] (3) Cut off the thoracic and abdominal organs one by one until the aorta is exposed. Completely separate the aorta, place it in a 35mm sterile petri dish, add 1mL of sterile PBS, and quickly transfer it to a laminar flow hood.

[0102] (4) Rinse repeatedly with D-Hanks solution to remove residual blood. Carefully peel off the adipose tissue outside the aorta with tweezers and carefully peel off the outer membrane until the blood vessel is smooth and transparent.

[0103] (5) Place the aorta into DMEM solution (containing 4.5 g / L glucose, 10 mmol / L pyruvate, 2 mmol / L glutamine, 100 U / L penicillin and streptomycin, 20% fetal bovine serum), cut the aorta longitudinally with ophthalmic scissors, and gently scrape the intima 2-3 times with a blade to remove the intima.

[0104] (6) Transfer the dissected blood vessel to another 35mm sterile petri dish containing 3mL of 20% FBSDMEM / F12 culture medium, and cut it into small pieces with ophthalmic scissors, with the pieces being about 1mm × 1mm in size.

[0105] (7) Plant the tissue block evenly at the bottom of the culture flask, gently turn the culture flask up so that the bottom is facing up, and add 2 mL of fresh DMEM / F12 medium containing 20% ​​FBS;

[0106] (8) Place the tissue block in a 37℃, 5% CO2 cell culture incubator and let it stand for 4-5 hours to allow it to dry slightly. Once the tissue block is firmly attached to the bottom of the cell culture flask, gently turn the culture flask over so that the tissue block is completely immersed in the culture medium.

[0107] (9) Place it in a 37°C incubator containing 5% CO2 and incubate statically for 3 days. Observe and change the medium afterward.

[0108] (10) After the cells have grown to 80% of the bottom area of ​​the culture flask, they are digested and passaged with 0.25% trypsin. After passage for 2 hours, the supernatant is aspirated and seeded into another culture flask. After 24 hours, the non-adherent cell suspension is collected according to the cell adhesion status, centrifuged for 5 minutes, resuspended and seeded into a new culture flask. The differential adhesion method is repeated multiple times to purify the cells.

[0109] Western blotting was used to detect aging markers P16, P21, and P53. The results are as follows: Figure 7 As shown, compared to Apoe fed a high-fat diet, KO Trap1 WT Mouse, Apoe KO Trap1 SMCKO The number of aging smooth muscle cells in mice was significantly reduced.

[0110] Further RT-qPCR experiments were performed to detect SASP gene levels. The results are as follows: Figure 8 As shown, compared to Apoe fed a high-fat diet, KO Trap1 WT Mouse, Apoe KO Trap1 SMCKO SASP was significantly reduced in mouse smooth muscle cells.

[0111] As can be seen from the above examples, inhibiting or knocking out TRAP1 in patients with age-related atherosclerosis and in atherosclerosis-related cell and animal models can suppress cellular senescence and slow the progression of atherosclerosis. Therefore, substances that inhibit or knock out TRAP1 can be used to prepare drugs for treating cardiovascular diseases caused by cellular senescence, especially drugs for treating age-related atherosclerosis.

[0112] The above examples are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The use of substances that knock down or inhibit TRAP1 in the preparation of drugs for the prevention and treatment of atherosclerosis, wherein the atherosclerosis is aging-related atherosclerosis.

2. The application according to claim 1, characterized in that, The substances that knock down or inhibit TRAP1 include small interfering RNAs that knock down TRAP1 expression, gene editing systems that specifically knock out TRAP1, or other small molecule compounds that can specifically inhibit TRAP1.

3. The application according to claim 2, characterized in that, The small interfering RNA sequences that knock down TRAP1 expression are shown in SEQ ID NO.3-4.

Images