Application of IQGAP3 in preparation of medicine for preventing and treating atherosclerosis

Abstract

The invention belongs to the technical field of atherosclerosis treatment, and discloses application of IQGAP3 in preparation of a medicine for preventing and treating atherosclerosis. In an in-vitro experiment, the IQGAP3 and the GRD structural domain of the IQGAP3 can inhibit the expression of an adhesion factor caused by oxidized low-density lipoprotein (ox-LDL), and can relieve the breakage of connection between endothelial cells and the adhesion of mononuclear cells. And through endothelial specific overexpression of IQGAP3-GRD, the plaque area, caused by high fat diet, of the aorta root of a mouse can be reduced, and expression of an adhesion factor ICAM1 / VCAM1 in vascular tissue is reduced. The invention develops a new application field of IQGAP3 and GRD structural domains thereof, and provides a significant reference for preventing and treating endothelial cell injury and atherosclerosis and improving vascular disease conditions.

Description

Technical Field

[0001] The present invention belongs to the technical field of atherosclerosis treatment, and specifically relates to the application of IQGAP3 in the prevention and / or treatment of atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is the main cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Lipid metabolism disorders are the pathological basis of atherosclerosis. Its characteristic is that the lesions of the affected arteries start from the intima, generally with lipid and complex carbohydrate accumulation, bleeding and thrombosis, followed by fibrosis and calcification, and gradual degeneration and calcification of the middle layer of the artery, leading to thickening and hardening of the arterial wall and narrowing of the vascular lumen. The lesions often involve large and medium muscular arteries. Once they develop to block the arterial lumen, the tissues or organs supplied by the artery will be ischemic or necrotic, which seriously endangers human health. However, its pathogenesis is not yet fully understood, and there is no effective and specific drug treatment target in clinical practice. Therefore, in-depth exploration of the molecular mechanism of AS and finding corresponding intervention targets have become a hot topic in scientific research at home and abroad, which is of great significance for the early diagnosis, prevention and drug treatment of AS.

[0003] Endothelial dysfunction is a key factor affecting the occurrence and development of atherosclerosis. Endothelial dysfunction is manifested by impaired endothelium-dependent vasodilation, enhanced oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Under the stimulation of pathological factors, endothelial cell junctions are destroyed, permeability is increased, and the expression of adhesion factors and chemotactic molecules is increased, which promotes the recruitment of circulating monocytes into the intima to become foam cells, and can induce the proliferation and migration of adjacent vascular smooth muscle cells, thereby promoting or aggravating AS. Therefore, clarifying the mechanism of endothelial dysfunction will help to find new solutions to vascular diseases from the direction of preventing vascular endothelial damage, and provide new ideas for the prevention and treatment of vascular diseases such as atherosclerosis.

[0004] IQ motif-containing GTPase activating protein 3 (IQGAP3) is a member of the IQGAP family. It regulates cell proliferation, adhesion, migration, cytoskeleton dynamics, etc. by binding to its target proteins. In addition, IQGAP3 is a major regulator of gastric epithelial tissue stability and repair, and IQGAP3 is also a key regulator of mitotic progression and genomic stability. RasGTPase activating protein-related domain (GRD) is an important domain in IQGAP3, which can selectively bind to active GTP-Cdc42 and GTP-Rac1, thereby stabilizing the active state of GTP-binding proteins. However, there has been no relevant research and reports on whether IQGAP3 can improve endothelial cell damage and atherosclerosis and its mechanism. Summary of the invention

[0005] The purpose of the present invention is to provide an application of IQGAP3 in the preparation of treatment for endothelial cell damage and / or atherosclerosis. During the research, technicians injected IQGAP3 into the tail vein of atherosclerosis model mice to reduce the plaque area of ​​the aortic root of the mice and reduce the expression of adhesion factor ICAM1 / VCAM1 in vascular tissue.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention claims protection for at least one use of an IQGAP3 protein or a GRD domain of an IQGAP3 protein in (a1) and (a2):

[0008] (a1) Use in the preparation of drugs for preventing and / or treating atherosclerosis;

[0009] (a2) Application in the preparation of drugs for treating endothelial cell damage.

[0010] The amino acid sequence of the IQGAP3 protein is shown in SEQ ID NO.3;

[0011] The amino acid sequence of the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.2.

[0012] In a second aspect, the present invention claims protection for at least one application of a biomaterial related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein in the following (a1) and (a2):

[0013] (a1) Use in the preparation of drugs for preventing and / or treating atherosclerosis;

[0014] (a2) Application in the preparation of drugs for treating endothelial cell damage.

[0015] The biological material related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein is at least one of the following (b1)-(b2):

[0016] (b1) a gene encoding the IQGAP3 protein;

[0017] (b2) a gene encoding the GRD domain of the IQGAP3 protein;

[0018] (b3) an adenovirus comprising the coding gene described in (b1);

[0019] (b4) An adenovirus comprising the coding gene described in (b2).

[0020] Furthermore, the NCBI sequence number of the nucleotide sequence of the gene encoding the IQGAP3 protein is 128239; the nucleotide sequence of the gene encoding the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.1.

[0021] Furthermore, the active ingredient of the drug in the above application includes IQGAP3 protein or the GRD domain of IQGAP3 protein; or includes biological materials related to IQGAP3 protein or the GRD domain of IQGAP3 protein.

[0022] In a third aspect, the present invention seeks to protect a drug for preventing and / or treating atherosclerosis or treating endothelial cell damage, wherein the active ingredient of the drug includes an IQGAP3 protein or a GRD domain of an IQGAP3 protein, or a biomaterial related to an IQGAP3 protein or a GRD domain of an IQGAP3 protein.

[0023] Furthermore, the amino acid sequence of the IQGAP3 protein, the amino acid sequence of the GRD domain of the IQGAP3 protein, and the biological material related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein are as described above.

[0024] The present invention studies the medical use of GTPase activating protein 3 containing an IQ motif and its Ras GTPase activating protein related domain (IQGAP3-GRD), and specifically relates to the use of GTPase activating protein 3 containing an IQ motif and the GRD domain of IQGAP3 protein in the prevention and treatment of atherosclerosis.

[0025] Beneficial effects of the present invention:

[0026] In in vitro experiments, IQGAP3 and its GRD domain can inhibit the expression of adhesion factors caused by oxidized low-density lipoprotein (ox-LDL), and can reduce the breakage of endothelial cell connections and the adhesion of monocytes. And through endothelial-specific overexpression of IQGAP3-GRD, the plaque area of ​​the aortic root of mice caused by a high-fat diet can be reduced, and the expression of adhesion factors ICAM1 / VCAM1 in vascular tissue can be reduced. The present invention has opened up a new application field for IQGAP3 and its GRD domain, and provides a meaningful reference for preventing and treating endothelial cell damage and atherosclerosis, and improving vascular lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The schematic diagrams for constructing full-length IQGAP3, IQGAP3 with GRD domain removed, and adenovirus with GRD domain as well as the overexpression efficiency diagrams of the three adenoviruses.

[0028] Figure 2Adenovirus transfection control (Ad-GFP), full-length IQGAP3 (Ad-IQGAP3), and IQGAP3 with GRD domain removed (Ad-ΔGRD) were added to human umbilical vein endothelial cells for 24 hours, and then treated with ox-LDL (100μM, 24 hours). A is the expression of endothelial cell adhesion factors ICAM1, VCAM1 and IQGAP3 detected by western bolt, and B is the immunofluorescence detection of monocyte adhesion, *<0.05.

[0029] Figure 3 After adding transfection control (Ad-GFP) and (IQGAP3-GRD) to human umbilical vein endothelial cells, ox-LDL (100μM, 24h) was given. A is the expression of endothelial cell adhesion factors ICAM1 and VCAM1 detected by western bolt, and B is the immunofluorescence detection of monocyte adhesion, *<0.05.

[0030] Figure 4 Six-week-old male ApoE- / - mice were injected with endothelial-specific adeno-associated virus (AAVendo) containing control (GFP) and IQGAP3-GRD domain (IQGAP3-GAD) through the tail vein. Two weeks later, they were given a high-fat diet (HFD). After eight weeks of feeding, samples were collected. Oil red O staining was used to detect gross plaque formation in the aorta.

[0031] Figure 5 The aortic root was paraffin-embedded and sectioned and histologically stained. A is a schematic diagram of the morphology of aortic root plaques detected by H&E tissue staining; B is a Masson tissue staining to detect collagen deposition in aortic root plaques; C is an Oil Red O staining to detect lipid deposition in aortic root.

[0032] Figure 6 Aortic tissue protein and RNA were extracted, and the expression of adhesion factors (icam1, vcam1, sele, selp) and chemotactic molecules (ccl2, cxcl2) in aortic tissue was detected by western bolt and qPCR, *<0.05. DETAILED DESCRIPTION

[0033] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0034] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The reagents and materials used in the following examples are all commercially available products. The full-length IQGAP3, IQGAP3 with GRD domain removed, and adenovirus with GRD domain of IQGAP3 used in the examples were purchased from GeneCare, and the sequences are as follows: IQGAP3-GRD encoding gene sequence (933-1286aa)

[0035] ATGGTTCTGGACAAGCAGAAGGGTTTTAAAGTCGCTGAGCAAAGAGAAACGGCAGAA

[0036] ACTAGAAGCATACCAACACCTCTTCTACCTGCTCCAGACTCAGCCCATCTACCTGGCC

[0037] AAGCTGATCTTTCAGATGCCACAGAACAAAACCACCAAGTTCATGGAGGCAGTGATT

[0038] TTCAGCCTGTACAACTATGCCTCCAGCCGCCGAGAGGCCTATCTCCTGCTCCAGCTGT

[0039] TCAAGACAGCACTCCAGGAGGAAATCAAGTCAAAGGTGGAGCAGCCCCAGGACGTG

[0040] GTGACAGGCAACCCAACAGTGGTGAGGCTGGTGGTGAGATTCTACCGTAATGGGCGG

[0041] GGACAGAGTGCCCTGCAGGAGATTCTGGGCAAGGTTATCCAGGATGTGCTAGAAGAC

[0042] AAAGTGCTCAGCGTCCACACAGACCCTGTCCACCTCTATAAGAACTGGATCAACCAG

[0043] ACTGAGGCCCAGACAGGGCAGCGCAGCCATCTCCCATATGATGTCACCCCGGAGCAG

[0044] GCCTTGAGCCACCCCGAGGTCCAGAGACGACTGGACATCGCCCTACGCAACCTCCTC

[0045] GCCATGACTGATAAGTTCCTTTTAGCCATCACCTCATCTGTGGACCAAATTCCGTATGG

[0046] GATGCGATATGTGGCCAAAGTCCTGAAGGCAACTCTGGCAGAGAAATTCCCTGACGC

[0047] CACAGACAGCGAGGTCTATAAGGTGGTCGGGAACCTCCTGTACTACCGCTTCCTGAA

[0048] CCCAGCTGTGGTGGCTCCTGACGCCTTCGACATTGTGGCCATGGCAGCTGGTGGAGC

[0049] CCTGGCTGCCCCCCAGCGCCATGCCCTGGGGGCTGTGGCTCAGCTCCTACAGCACGC

[0050] TGCGGCTGGCAAGGCCTTCTCTGGGCAGAGCCAGCACCTACGGGTCCTGAATGACTA

[0051] TCTGGAGGAAACACACCTCAAGTTCAGGAAGTTCATCCATAGAGCCTGCCAGGTGCC

[0052] AGAGCCAGAGGAGCGTTTTGCAGTGGACGAGTACTCAGACATGGTGGCTGTGGCCAAACCCATGGTGTACATCACCGTGGGGGAGCTG(SEQ ID NO.1)。

[0053] IQGAP3 - GRD amino acid sequence:

[0054] MMVLDKQKGLKSLSKEKRQKLEAYQHLFYLLQTQPIYLAKLIFQMPQNKTTKFMEAVIF

[0055] SLYNYASSRREAYLLLQLFKTALQEEIKSKVEQPQDVVTGNPTVVRLVVRFYRNGRGQS

[0056] ALQEILGKVIQDVLEDKVLSVHTDPVHLYKNWINQTEAQTGQRSHLPYDVTPEQALSHP

[0057] EVQRRLDIALRNLLAMTDKFLLAITSSVDQIPYGMRYVAKVLKATLAEKFPDATDSEVY

[0058] KVVGNLLYYRFLNPAVVAPDAFDIVAMAAGGALAAPQRHALGAVAQLLQHAAAGKAFSGQSQHLRVLNDYLEETHLKFRKFIHRACQVPEPEERFAVDEYSDMVAVAKPMVYI(SEQ ID NO.2).

[0059] Amino acid sequence of IQGAP3 protein

[0060] MERRAAGPGWAAYERLTAEEMDEQRRQNVAYQYLCRLEEAKRWMEACLKEELPSPVE

[0061] LEESLRNGVLLAKLGHCFAPSVVPLKKIYDVEQLRYQATGLHFRHTDNINFWLSAIAHIG

[0062] LPSTFFPETTDIYDKKNMPRVVYCIHALSLFLFRLGLAPQIHDLYGKVKFTAEELSNMASE

[0063] LAKYGLQLPAFSKIGGILANELSVDEAAVHAAVLAINEAVERGVVEDTLAALQNPSALLE

[0064] NLREPLAAVYQEMLAQAKMEKAANARNHDDRESQDIYDHYLTQAEIQGNINHVNVHG

[0065] ALEVVDDALERQSPEALLKALQDPALALRGVRRDFADWYLEQLNSDREQKAQELGLVE

[0066] LLEKEEVQAGVAAANTKGDQEQAMLHAVQRINKAIRRRVAADTVKELMCPEAQLPPVY

[0067] PVASSMYQLELAVLQQQQGELGQEELFVAVEMLSAVVLINRALEARDASGFWSSLVNPA

[0068] TGLAEVEGENAQRYFDALLKLRQERGMGEDFLSWNDLQATVSQVNAQTQEETDRVLA

[0069] VSLINEALDKGSPEKTLSALLLPAAGLDDVSLPVAPRYHLLLVAAKRQKAQVTGDPGAVL

[0070] WLEEIRQGVVRANQDTNTAQRMALGVAAINQAIKEGKAAQTERVLRNPAVALRGVVPD

[0071] CANGYQRALESAMAKKQRPADTAFWVQHDMKDGTAYYFHLQTFQGIWEQPPGCPLNT

[0072] SHLTREEIQSAVTKVTAAYDRQQLWKANVGFVIQLQARLRGFLVRQKFAEHSHFLRTWL

[0073] PAVIKIQAHWRGYRQRKIYLEWLQYFKANLDAIKIQAWARMWAARRQYLRRRLHYFQK

[0074] NVNSIVKIQAFFRARKAQDDYRILVHAPHPPLSVVRRFAHLLNQSQQDFLAEAELLKLQE

[0075] EVVRKIRSNQQLEQDLNIMDIKIGLLVKNRITLQEVVSHCKKLTKRNKEQLSDMMVLDK

[0076] QKGLKSLSKEKRQKLEAYQHLFYLLQTQPIYLAKLIFQMPQNKTTKFMEAAVIFSLYNYAS

[0077] SRREAYLLLQLFKTALQEEIKSKVEQPQDVVTGNPTVVRLVVRFYRNGRGQSALQEILG

[0078] KVIQDVLEDKVLSVHTDPVHLYKNWINQTEAQTGQRSHLPYDVTPEQALSHPEVQRRL

[0079] DIALRNLLAMTDKFLLAITSSVDQIPYGMRYVAKVLKATLAEKFPDATDSEVYKVVGNL

[0080] LYYRFLNPAVVAPDAFDIVAMAAGGALAAPQRHALGAVAQLLQHAAAGKAFSGQSQHL

[0081] RVLNDYLEETHLKFRKFIHRACQVPEPEERFAVDEYSDMVAVAKPMVYITVGELVNTHRL

[0082] LLEHQDCIAPDHQDPLHELLEDLGELPTIPDLIGESIAADGHTDLSKLEVSLTLTNKFEGLE

[0083] ADADDSNTRSLLSTKQLLADIIQFHPGDTLKEILSLSASREQEAAHKQLMSRRQACTAQ

[0084] TPEPLRRHRSLTAHSLLPLAEKQRRVLRNLRRLEALGLVSARNGYQGLVDELAKDIRNQH

[0085] RHRHRRKAELVKLQATLQGLSTKTTFYEEQGDYYSQYIRACLDHLAPDSKSSGKGKKQP

[0086] SLHYTAAQLLEKGVLVEIEDLPASHFRNVIFDITPGDEAGKFEVNAKFLGVDMERFQLHYQDLLQLQYEGVAVMKLFNKAKVNVNLLIFLLNKKFLRK(SEQ ID NO.3)

[0087] NCBI sequence number of IQGAP3 gene nucleotide sequence: 128239

[0088] Example 1: Inhibitory effect of IQGAP3 on endothelial cell damage induced by ox-LDL

[0089] To explore the effect of IQGAP3 on ox-LDL-induced endothelial cell injury, we first added adenovirus transfected with full-length IQGAP3 (Ad-IQGAP3), IQGAP3 with GRD domain removed (Ad-ΔGRD), and IQGAP3 with GRD domain only (Ad-IQGAP3-GRD) to human umbilical vein endothelial cells for 24 hours to detect the overexpression efficiency. Then, adenovirus transfected with control (Ad-GFP), full-length IQGAP3 (Ad-IQGAP3), IQGAP3 with GRD domain removed (Ad-ΔGRD), or IQGAP3 with GRD domain only (Ad-IQGAP3-GRD) was added to human umbilical vein endothelial cells for 24 hours, and then ox-LDL (100 μM, 24 hours) was treated. The expression of endothelial cell adhesion factors ICAM1 and VCAM1 was detected by western bolt, and the adhesion of monocytes was detected by immunofluorescence.

[0090] like Figure 1 As shown, the schematic diagram of constructing full-length IQGAP3, IQGAP3 with GRD domain removed, and adenovirus with GRD domain ( Figure 1 A), immunofluorescence images show that three adenoviruses can successfully overexpress human umbilical vein endothelial cells ( Figure 1 B).

[0091] like Figure 2 As shown, western bolt and immunofluorescence results showed that overexpression of IQGAP3 could inhibit the increased expression of adhesion factors and increased monocyte adhesion caused by ox-LDL, while overexpression of IQGAP3-ΔGRD did not have an improvement effect.

[0092] like Figure 3 As shown, western bolt and immunofluorescence results showed that overexpression of IQGAP3-GRD could inhibit the increased expression of adhesion factors and increased monocyte adhesion caused by ox-LDL.

[0093] Example 2: Inhibitory effect of IQGAP3 on atherosclerotic lesions

[0094] To explore the effects of IQGAP3 and its GRD domain on atherosclerotic lesions, we selected 6-week-old SPF male ApoE - / -Mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were injected with control (GFP) and IQGAP3-GRD domain endothelial-specific adeno-associated virus (AAVendo) with a Flag tag (purchased from Suzhou Jimai Gene Pharmaceutical Biotechnology Co., Ltd.) through the tail vein. After 2 weeks, they were fed with a normal diet (NC) or a high-fat diet (HFD) for 8 weeks. The aortic gross vessels were collected for Oil Red O staining, and the aortic root was collected for tissue staining, and the aortic tissue protein and RNA were collected.

[0095] like Figure 4 As shown, by collecting the mouse aorta and staining with Oil Red O, it was found that the aortic plaque area of ​​the mice injected with IQGAP3-GRD by the tail vein was significantly smaller than that of the mice injected with the control virus by the tail vein.

[0096] like Figure 5 As shown, the aortic root of mice was collected, and paraffin-embedded sections were performed and H&E staining was performed to observe the morphological structure of plaques, Masson staining was used to detect collagen deposition in plaques, and Oil Red O staining was used to detect the size of plaques and lipid deposition. The results showed that the aortic plaque area and lipid deposition of mice injected with IQGAP3-GRD by tail vein were significantly smaller than those of mice injected with control virus by tail vein, while the collagen deposition content was significantly higher than that of the control group.

[0097] like Figure 6 As shown, by collecting mouse aortic tissue proteins and RNA, western bolt and qPCR results showed that the expression of adhesion factors and chemotactic molecules in the aortic tissue of mice injected with IQGAP3-GRD through the tail vein was significantly lower than that of mice injected with the control virus through the tail vein.

Claims

1. Use of an IQGAP3 protein or a GRD domain of an IQGAP3 protein in at least one of the following (a1) and (a2): (a1) Use in the preparation of drugs for preventing and / or treating atherosclerosis; (a2) Application in the preparation of drugs for treating endothelial cell damage. The amino acid sequence of the IQGAP3 protein is shown in SEQ ID NO.3; The amino acid sequence of the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.

2.

2. Use of a biomaterial related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein according to claim 1 in at least one of the following (a1) and (a2): (a1) Use in the preparation of drugs for preventing and / or treating atherosclerosis; (a2) Application in the preparation of drugs for treating endothelial cell damage. The biological material related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein is at least one of the following (b1)-(b2): (b1) a gene encoding the IQGAP3 protein; (b2) a gene encoding the GRD domain of the IQGAP3 protein; (b3) an adenovirus comprising the coding gene described in (b1); (b4) An adenovirus comprising the coding gene described in (b2).

3. The use according to claim 3, characterized in that: The NCBI sequence number of the nucleotide sequence of the gene encoding the IQGAP3 protein is 128239; the nucleotide sequence of the gene encoding the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.

1.

4. The use according to claim 1, characterized in that: The active ingredient of the drug includes IQGAP3 protein or the GRD domain of IQGAP3 protein.

5. The use according to claim 2, characterized in that: The active ingredient of the drug includes biological materials related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein.

6. A drug, characterized in that The active ingredient of the drug includes IQGAP3 protein or the GRD domain of the IQGAP3 protein, or biological materials related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein.

7. The drug according to claim 6, characterized in that The amino acid sequence of the IQGAP3 protein is shown in SEQ ID NO.3; The amino acid sequence of the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.2; The biological material related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein is at least one of the following (b1)-(b2): (b1) a gene encoding the IQGAP3 protein; (b2) a gene encoding the GRD domain of the IQGAP3 protein; (b3) an adenovirus comprising the coding gene described in (b1); (b4) An adenovirus comprising the coding gene described in (b2).

8. The drug according to claim 7, characterized in that The NCBI sequence number of the nucleotide sequence of the gene encoding the IQGAP3 protein is 128239; the nucleotide sequence of the gene encoding the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.1.

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