CN106344935B

CN106344935B - Function and application of Toll-like protein interacting protein in treating restenosis after vascular injury

Info

Publication number: CN106344935B
Application number: CN201610887742.XA
Authority: CN
Inventors: 李红良; 秦娟娟; 程文林
Original assignee: Wuhan University WHU
Current assignee: Wuhan huikangda Technology Co., Ltd
Priority date: 2016-10-11
Filing date: 2016-10-11
Publication date: 2020-01-07
Anticipated expiration: 2036-10-11
Also published as: CN106344935A

Abstract

The invention discloses a function and application of Toll-like protein interacting protein (Tollip) in treating restenosis after vascular injury. According to the invention, a Tollip knockout mouse and a wild mouse are taken as experimental objects, and the detection of mouse intimal neogenesis, vascular wall cell proliferation level and smooth muscle cell phenotype conversion is carried out through a vascular injury model. The results show that the Tollip gene knockout can obviously promote neointima and cell proliferation and promote the smooth muscle cell to be converted from contraction type to synthesis type. This suggests that the function of tollid in restenosis following vascular injury is mainly manifested in that tollid has the function of inhibiting intimal neogenesis and cell proliferation, as well as inhibiting the phenotypic transition of smooth muscle cells. Aiming at the functions of Tollip, the Tollip can be used as a drug target for screening drugs for preventing and treating the restenosis diseases after vascular injury, and can be used for preparing drugs for preventing and treating the restenosis diseases after vascular injury and arterial stents.

Description

Function and application of Toll-like protein interacting protein in treating restenosis after vascular injury

Technical Field

The invention belongs to the field of gene function and application, and particularly relates to application of Toll-like Protein interaction Protein (Toll interaction Protein) in preparation of a medicine for preventing, relieving and/or treating restenosis after vascular injury.

Background

With the continuous development of human socioeconomic, the people's living standard and dietary structure, the incidence of cardiovascular diseases keeps rising year by year, which seriously affects the public health and becomes one of the main causes of death in our country. Neointimal formation is accompanied in the development of vascular proliferative diseases such AS Atherosclerosis (AS), pulmonary hypertension, post-Percutaneous Coronary Intervention (PCI) Restenosis (RS), post-transplant arterial disease, and pulmonary hypertension. And at the present stage, after surgical operations such as balloon dilatation, stent implantation, artery bypass operations and the like capable of effectively dredging blocked arteries and improving blood supply are performed, the incidence rate of restenosis is high (30-60%) and the treatment effect is greatly influenced. Studies have shown that, during the formation of lesions, the hyperproliferation of neointima and media tissues, and the concomitant formation of extracellular matrix, are the main pathological basis responsible for restenosis following revascularization. Vascular Smooth Muscle Cell (VSMCs) phenotypic transformation plays an important role in neointimal formation. When VSMCs are transformed from a contractile type to a synthetic type, the proliferation and migration ability of the VSMCs is enhanced, and a large amount of extracellular matrix is secreted and synthesized, so that a neointima is formed, thereby causing the occurrence of a serious vascular proliferative disease. Therefore, at the present stage, it is very important to find new targets and therapeutic methods for effectively inhibiting vascular restenosis.

TLR receptors are key action molecules for activating the natural immune system, and the mediated signal pathways are involved in the occurrence and development of various cardiovascular diseases, including atherosclerosis, ischemia-reperfusion injury and myocardial remodeling [1-3 ]. Toll-like protein interacting proteins (Tollips), which are adaptor proteins of Toll-interleukin-1 receptor (IL-1R), can be expressed in a variety of tissues and cells, including heart, brain, smooth muscle cells [4], and the like. Meanwhile, as an endogenous protein for negatively regulating a TLR signaling pathway [5], Tollip can inhibit TLR2 and TLR 4-mediated cell signaling by directly regulating the TLR signaling pathway or inhibiting the activity of IL-1R-associated kinase (IRAK). Recent studies have found that in a model using IL-1 to induce cardiomyocyte hypertrophy, it was found that overexpression of Tollip can significantly inhibit the cardiomyocyte hypertrophy response by down-regulating the NF-Kb signaling pathway dependent on MyD88 [6 ]. Nevertheless, the influence of tollid on intimal hyperplasia, the mechanism of action, is currently poorly understood.

Reference documents:

1.Xu XH,Shah PK,Faure E,Equils O,Thomas L,Fishbein MC et al.Toll-like receptor-4 is expressed by macrophages in murine and human lipid-richatherosclerotic plaques and upregulated by oxidized ldl.Circulation 2001；104:3103–3108.

2.Arslan F,Smeets MB,O’Neill LA,Keogh B,McGuirk P,Timmers L et al.Myocardial ischemia/reperfusion injury is mediated by leukocytic Toll-likereceptor-2 and reduced by systemic administration of a novel anti-Toll-likereceptor-2 antibody.Circulation 2010；121:80–90.

3.Topkara VK,Evans S,ZhangW,Epelman S,Staloch L,BargerPMet al.Therapeutic targeting of innate immunity in the failing heart.J Mol CellCardiol 2011；51:594–599.

4.Nishimura M,Naito S.Tissue-specific mRNA expression profiles of human Toll-like receptors and related genes.Biol Pharm Bull 2005；28:886–892.

5.Burns K,Clatworthy J,Martin L,Martinon F,Plumpton C,Maschera B et al.Tollip,a new component of the IL-1RI pathway,links IRAK to the IL-1receptor.Nat Cell Biol 2000；2:346–351.

6.Hu Y,Li T,Wang Y,Li J,Guo L,Wu M et al.Tollipattenuated the hypertrophic response of cardiomyocytes induced by IL-1beta.Front Biosci2009；14:2747–2756

disclosure of Invention

To solve the above-mentioned drawbacks and deficiencies of the prior art, the present invention aims to determine the correlation between Tollip expression and restenosis after vascular injury, and to provide a novel use of a target gene Tollip for preventing, alleviating and/or treating restenosis after vascular injury.

The purpose of the invention is realized by the following technical scheme:

according to the invention, a wild type C57BL/6 mouse and a Tollip knockout mouse (Tollip-KO mouse) are taken as experimental objects, a mouse vascular injury model (vascular injury, VI) is obtained through carotid artery guide wire injury model induction, the vascular injury model (VI) is subjected to mouse intimal neogenesis determination, vascular wall cell proliferation level detection and smooth muscle cell phenotype detection research, and the results show that: compared with wild type C57BL/6 mice, Tollip knockout mice show that intimal neogenesis and cell proliferation are significantly greater than WT mice; the Tollip gene knockout can promote the expression of cell proliferation Nuclear Antigen (PCNA) and Cyclin D1, and can promote the proliferation of smooth muscle cells and intimal hyperplasia; the Tollip gene knockout can inhibit the expression of Smooth Muscle cell differentiation specific antigen (smoothenin), Smooth Muscle Actin (SMA) and Smooth Muscle22 alpha (Smooth Muscle22 alpha, SM22 alpha), and promote the phenotype transition of Smooth Muscle cells from contractile to synthetic, thereby promoting intimal hyperplasia. The results show that the knockout of the Tollip gene can promote the occurrence of restenosis after vascular injury, and Tollip can inhibit the formation of restenosis after vascular injury, thereby providing theoretical basis and clinical basis for researching new targets and new strategies for preventing, relieving and/or treating restenosis after vascular injury.

The research of the inventor proves that: in a carotid artery guide wire injury induced vascular injury model, tollid has the functions of inhibiting smooth muscle cell proliferation and intimal hyperplasia and inhibiting intimal hyperplasia.

A function of Tollip gene in restenosis after vascular injury is mainly embodied in that Tollip has the functions of inhibiting smooth muscle cell proliferation and intimal hyperplasia, inhibiting the phenotype conversion of smooth muscle cells from contraction type to synthesis type, and inhibiting restenosis after vascular injury.

Aiming at the function of inhibiting the occurrence of restenosis after vascular injury of Tollip, the application of Tollip in the preparation of a medicament for preventing, relieving and/or treating restenosis after vascular injury is provided.

A medicament for preventing, ameliorating and/or treating restenosis following vascular injury comprising tollid.

An arterial stent for the prevention, alleviation and/or treatment of restenosis following vascular injury, which is coated with tollid.

In the present invention, the vascular injury is primarily arterial vascular injury.

In the present invention, the vascular injury refers to vascular injury caused by atherosclerosis, or vascular injury caused when atherosclerosis is treated, such as vascular injury caused by balloon expansion or stent placement, or vascular injury caused by post-transplantation arterial disease or pulmonary hypertension.

In the present invention, the atherosclerosis includes both the stage of vascular stenosis at the earlier stage of atherosclerosis and the stage of vascular infarction when atherosclerosis is severe.

In the invention, the artery stent is a tubular device for supporting a blood vessel which is narrowed or occluded due to pathological changes in a human body and recovering blood circulation, is processed and made of metal or high polymer materials, and can be remained in the blood vessel of the human body for a long time or temporarily. On the basis of the expansion and the formation of the saccule of the lumen, the stent is placed in the lesion section to achieve the purposes of supporting the blood vessel of the stenotic occlusion section, reducing the elastic retraction and the reshaping of the blood vessel and keeping the blood flow of the lumen unobstructed, and the stent comprises a peripheral artery stent and a coronary artery stent.

In the present invention, the restenosis refers to a general biological response that leads to restenosis of the vascular lumen when a local vascular injury occurs. This refers to restenosis caused by iatrogenic injury, which consists mainly of arterial remodeling and endothelial hyperplasia.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention discovers a new function of the Tollip gene, namely the Tollip gene has the functions of inhibiting the hyperplasia of intima of blood vessels and inhibiting the occurrence of restenosis after the blood vessels are damaged.

(2) Based on the role of tollid in inhibiting restenosis following vascular injury, it can be used for the preparation of a medicament for the prevention, alleviation and/or treatment of restenosis following vascular injury.

(3) Tollid can be used for the preparation of an arterial stent for the prevention, alleviation and/or treatment of restenosis following vascular injury.

Drawings

FIG. 1 is a histogram of the EVG staining and intima area results 14, 28 days post-operatively in WT and Tollip-KO mice;

wherein, A is EVG staining graph, B is inner membrane area statistical histogram (p <0.05vs WT VI group).

FIG. 2 is an immunofluorescence staining and statistical histogram of results for WT and Tollip-KO mice expressing the vascular wall cell proliferation level markers PCNA, CyclinD1 at day 14 and day 28 after surgery;

wherein, A is immunofluorescence staining, B is a result statistical histogram (p <0.05vs WT VI group).

FIG. 3 is an immunofluorescence staining and statistical bar graph of results for WT and Tollip-KO mice expressing the smooth muscle cell phenotype transformation markers SMA, SM22 alpha at day 14, 28 post-surgery;

immunofluorescent staining and B statistical histogram of results (p <0.05vs WT VI group).

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

Experimental animal and breeding

Experimental animals: male C57BL/6 mice (WT mice, purchased from Beijing Huafukang Biotechnology Co., Ltd.) and Tollip knockout mice (Tollip-KO, purchased from European EMMA, Cat.: 01970) which are 8-10 weeks old and 24-27g in weight were selected.

A breeding environment: all experimental mice were housed in the SPF-class laboratory animal center, university of Wuhan. SPF mouse feed is purchased from Beijing Huafukang Biotech limited, and the feeding conditions are as follows: the room temperature is 22-24 ℃, the humidity is 40-70%, the illumination time is 12h with alternating light and shade, and the drinking water can be freely taken.

Example 1 mouse vascular injury model (VI) acquisition

1. Grouping experimental animals: WT and Tollip-KO mice, 8-10 weeks old and 24-27g in weight, were used, and divided into 2 groups in total: WT vascular injury groups, Tollip-KO vascular injury groups, 20 mice each. Mice were sacrificed at 14 and 28 days post-surgery, respectively, and injured segment vessels were taken for analysis.

2. Mouse vascular injury model operation procedure:

1) accurately weighing the weight (g) of the mouse by using an electronic balance in a dynamic mode, preparing a 3% sodium pentobarbital solution by using double distilled water, slightly shaking to fully dissolve the solution, calculating the volume of the required sodium pentobarbital solution by adopting 80mg/kg weight dose, accurately extracting the corresponding volume of the solution by using a 1mL syringe, carrying out intraperitoneal injection to anaesthetize the mouse, and carrying out 8% sodium sulfide neck depilation after the mouse is fully drunk and fell (about 3 min).

2) The internal and external carotid arteries were isolated.

3) The external carotid artery was tied with 8-0 thread at the bifurcation of the internal and external carotid arteries, while the blood supply to the internal and common carotid arteries was temporarily blocked with a vascular clamp (WPI, 501784-G).

4) A small incision was cut transversely above the external carotid artery ligature using microshear (WPI, 501839). A0.015 inch diameter guidewire (No. C-SF-15-15, Cook, Bloomington, Indiana) was inserted through this vascular incision and rotated 5-6 times.

5) Ligating external carotid artery at the heart-entering end of the incision, loosening vascular clamps arranged in the neck and common carotid artery, cutting off thread ends, cleaning operative field, and suturing the neck incision.

Example 2 vascular injury model (VI) determination of neointimal neogenesis in mice

1. Mouse selection

1) Mice were anesthetized and the hearts were cut open for exsanguination.

2) Cutting the carotid artery from the position near the bifurcation of the carotid artery, taking the carotid artery to be 0.5-0.6cm long, and keeping the external carotid artery knot.

3) The carotid artery was placed in PBS and the residual blood in the organ cavity was gently drained with micro-forceps.

4) The vessels were fixed in 1.5mL EP tubes containing 1mL of 4% paraformaldehyde.

2. Pathology detection

2.1 preparation of Paraffin specimen sections

Prepare paraffin sample section by laboratory professional pathology staff, main operating procedure includes: after 4% paraformaldehyde overnight fixation, the blood vessels are carefully wrapped with filter paper and placed in an embedding frame → washing with running water → dehydrating → transparency → wax-dipping → embedding → slicing (3 μm) → spreading → drying in the air or baking for later use.

2.2EVG staining

The method mainly comprises the following steps: baking at 55 ℃ for 30min → xylene for 5min, 3 times → 100% alcohol for 3min, 2 times → 95% alcohol for 3min, 1 time → 70% alcohol for 3min, 1 time → double distilled water for 1min → potassium permanganate solution for 5min (zhhaibeisuo, BA-4083B) → water washing for 1min → oxalic acid solution for 5min (zhhaibeisuo, BA-4083B) → water washing for 1min → 95% alcohol differentiation for 2-3 sec → Elastin dye solution (zhhaibeiso, BA-4083B) for 8-24 h → 95% alcohol differentiation for 1s → flowing water washing for 10min → double distilled water for 1min → Van Gieson dye solution (zhhaibeiso, BA-4083B) for 1min → 95% alcohol differentiation for 2-3s → 100% alcohol 2min, 2 times → xylene for 2min, 3 times → xylene is not dried and is immediately dried and dried in a microscope.

The blood vessel inner elastic fiber and the outer elastic fiber are used as boundaries, the inner part of the inner elastic plate is a blood vessel intima, the outer part of the outer elastic plate is a blood vessel adventitia, and the blood vessel intima is arranged between the inner elastic plate and the outer elastic plate. The lumen area of each vessel was encircled with Image-Pro Plus 6.0 software.

The calculation of the area size of the inner membrane is as follows:

the area of the neogenetic inner membrane is equal to the area of the inner elastic plate-the area of the tube cavity;

the area of the middle membrane is equal to the area of the outer elastic plate-the area of the inner elastic plate.

The results of neointimal neogenesis after EVG staining in mice are shown in fig. 1. The normal vascular wall has complete structure and regular arrangement, the vascular intima is a single-layer endothelial cell, the structure is complete, and the mesodermal smooth muscle cells are arranged in order. The HE staining shows that the vascular wall structure of the vascular injury group (VI group) is incomplete, vascular endothelial cells are deleted, neointimal hyperplasia is obvious, and a large amount of inflammatory cells infiltrate; the Tollip-KO group showed a significantly larger neointimal area at 14 and 28 days post-surgery than WT mice. Similarly, the ratio of intima/media area was higher in the VI post-operative Tollip-KO group than in the WT group. This suggests that deletion of the tollid gene can promote neointimal neogenesis following vascular injury.

Example 3 detection of the proliferation level of vascular wall cells

Immunofluorescent staining detects the expression of Cell Proliferating Nuclear Antigen (PCNA) and cyclin (CyclinD 1). The required primary antibody information: PCNA (# 2586; 1: 100; mouse; CellSignaling Technology), cycle D1(# 2978; 1: 25; rabbit; Cell Signaling Technology); required secondary antibody information: alexa Fluor 568-conjugated coat antai-rabbitIgG (A11011; Invitrogen, Carlsbad, CA), Alexa Fluor 568-conjugated coat antai-mouse IgG (A11004; Invitrogen, Carlsbad,150d, CA).

The method mainly comprises the following steps:

1) baking slices: the paraffin sections were placed in an oven at 55 ℃ for more than 60 min.

2) Dewaxing: xylene 8min × 3.

3) Hydration: 100% ethanol 5min × 2; 95% ethanol for 5 min; 70% ethanol for 5 min; ddH2O was rinsed for 5min × 2.

4) Citrate tissue antigen repair (hyperbaric repair): taking a certain amount of pH6.0 citrate antigen repairing working solution (purchased from New Biotechnology Co., Ltd., Fuzhou, product number MVS-0100) into a repairing box, wherein the amount of the repairing working solution is enough to immerse a whole slice, putting the repairing box into a pressure cooker added with a proper amount of tap water, heating to boil with strong fire, putting the dewaxed and hydrated tissue slice on a high-temperature-resistant dyeing rack, slowly putting the dyeing rack into the repairing box, covering a pot cover, fastening a pressure valve, continuously heating to jet air, starting timing for 5min, disconnecting a power supply of the pressure cooker, removing the valve, opening the cover, and taking out the repairing box; and standing at room temperature for 20min, naturally cooling, and taking out the slices.

5)ddH₂O rinsing 5min × 2 times, PBS rinsing 5min × 2 times.

6) Grouping stroke circle, dropping 10% donkey serum (GTX27481, GeneTex) for sealing, and sealing in a wet box at 37 deg.C for 60 min.

7) Removing the blocking solution, adding dropwise primary antibody diluted in a proper proportion, incubating overnight at 4 deg.C, rewarming at 37 deg.C for 30min, removing the primary antibody, and washing with PBS for 8min × 4 times.

8) Add the secondary antibody drop by drop, incubate in a wet box at 37 ℃ for 60min, discard the secondary antibody, and wash with PBS 5min × 4 times.

9) SlowFade Gold anti reagent with DAPI (S36939, Invitrogen) coverslips.

10) And (5) observing under a fluoroscope, and taking a picture. If necessary, the cells were stored in a dark and wet box at 4 ℃.

The fluorescence statistical method comprises the following steps: counting PCNA immunofluorescence staining by using IPP software, wherein the percentage of PCNA positive cells is 100 percent of the total DAPI number of the PCNA positive cells/(intima + media); statistics of immunofluorescent staining for cyclinD1 Positive absorbance was measured directly using IPP software.

The expression changes of the smooth muscle cell proliferation markers PCNA and CyclinD1 after WT and Tollip-KO mouse vascular injury were observed by immunofluorescence, and the results are shown in FIG. 2. PCNA and CyclinD1 are expressed in vascular tissues, the number of PCNA positive cells and the fluorescence intensity of CyclinD1 of a Tollip-KO mouse are increased to WT mice in the same group 14 and 28 days after operation, and the Tollip-gene knockout can promote the expression of PCNA and CyclinD1 and the proliferation of smooth muscle cells and the neogenesis of blood vessels intima.

Example 4 detection of smooth muscle cell phenotype

Immunofluorescence staining detection of smooth muscle cell differentiation markers: expression of Smooth Muscle cell differentiation specific antigen (smoothenin), Smooth Muscle Actin (smoothen Muscle Actin, SMA), Smooth Muscle22 α (smoothen Muscle22 alpha, SM22 α). The required primary antibody information: SMA (ab 5694; 1: 100; rabbitt; Abcam) and SM22 α (ab 14106; 1: 100; rabbitt; Abcam); required secondary antibody information: alexa Fluor 488-conjugated Goatanti-rabbitIgG (A11008; Invitrogen, Carlsbad, Calif.).

The main steps refer to example 3.

The fluorescence statistical method comprises the following steps: positive absorbance was measured directly using IPP software.

Under normal physiological state, vascular smooth muscle cells are in a static state and mainly show a contraction type; after the blood vessel is damaged, the smooth muscle cells of the blood vessel migrate from the tunica media to the tunica intima, the proliferation and the apoptosis of the smooth muscle cells are out of balance, the phenotype is changed from a contraction type to a synthesis type, and the blood vessel wall is discomfortably remodeled, thereby causing the tunica intima hyperplasia. The expression change of SMA and SM22 alpha after WT and XX-KO mouse vascular injury was observed by immunofluorescence, and the result is shown in FIG. 3. The fluorescence intensity of smoothening, SMA and SM22 alpha in vascular tissues is lower than that of XX-KO mice in smoothening, SMA and SM22 alpha at 14 and 28 days after operation than that of WT mice in the same group, which shows that the Tollip gene knockout can inhibit the expression of smoothening, SMA and SM22 alpha and promote the phenotype conversion of smooth muscle cells from contraction type to synthesis type, thereby promoting intimal hyperplasia.

As shown in the above example results, both wild-type mice and Tollip-KO mice developed restenosis after vascular injury induced by vascular injury model (VI). The tollid knockout mice all had significant intimal neogenesis, cell proliferation levels, and smooth muscle cell phenotype switching over that of wild-type mice. These results indicate that tollid can improve vascular injury-induced smooth muscle cell proliferation, phenotypic transformation, and neointimal formation of blood vessels.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

The application of the Tollip gene as a drug target in screening drugs for preventing and treating restenosis after vascular injury is characterized in that: the screened medicine for preventing and treating restenosis after vascular injury is a medicine for promoting Tollip gene expression; said use is non-diagnostic and non-therapeutic.
2. Use according to claim 1, characterized in that: the vascular injury is caused by atherosclerosis, atherosclerosis interventional therapy, arterial disease after transplantation or pulmonary hypertension.
3. An arterial stent for preventing and treating restenosis after vascular injury, which is characterized in that the stent is coated with Tollip.