Application of JunB expression inhibitor in preparation of medicine for treating atherosclerosis

Abstract

The invention belongs to the field of biotechnology and medical technology, and particularly relates to application of a JunB expression inhibitor in preparation of a medicine for treating atherosclerosis. According to the application disclosed by the invention, homocysteine is found to induce atherosclerosis by promoting JunB expression and reducing the autophagy level of macrophages; after the JunB plasmid is transfected and knocked down to the macrophages, the number of the autophagosomes and the number of the autophagosomes and the number of the autophagosomes are increased; and after the overexpressed JunB plasmid is transfected to the macrophage, the number of the autophagosome and the number of the autolysosome are reduced, which indicates that the expression inhibitor of the JunB can be effectively used for preparing the medicine for treating the atherosclerosis, and provides possibility for developing a new medicine for treating the atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medical technology, specifically relating to the application of JunB expression inhibitors in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] Atherosclerosis (As) is a chronic degenerative disease characterized by the formation of atherosclerotic plaques. Unstable plaques (vulnerable plaques) are at risk of hemorrhage, rupture, detachment, and thrombosis, which are the initiating factors for acute cardiovascular and cerebrovascular events, seriously threatening human life and health. Studies have shown that hyperhomocysteinemia (Hcy) is an independent risk factor for the formation of vulnerable atherosclerotic plaques, and its harm is no less than that of hyperlipidemia, earning it the nickname "21st-century cholesterol." An increase of 5 μM in serum homocysteine ​​levels per liter increases vascular risk by 60% in men and 80% in women, with a relative risk of 1.6 for men and 1.8 for women, equivalent to the increased risk of a 20 mg / dL increase in total cholesterol. Although adequate folic acid and B vitamin supplementation is currently the main measure for controlling homocysteine ​​levels, hyperhomocysteinemia is one of the important markers of arterial plaque progression. Due to the many factors that cause hyperhomocysteinemia, the currently developed therapeutic drugs are not very effective. Therefore, in-depth exploration of prevention and treatment strategies for homocysteine-induced atherosclerotic vulnerable plaques has become an important issue that urgently needs to be addressed.

[0003] JunB, a key member of the AP-1 complex, promotes fetal angiogenesis and cardiovascular development. As a novel factor in angiogenesis, JunB regulates the "tip cell" properties of endothelial cells in the neurovascular interactions of mouse embryonic skin and retina. Increasing evidence suggests that JunB plays a crucial role in determining the fate and regulating the function of immune cells, particularly CD4, by modulating the expression of target genes. + During T cell differentiation, the transcription of key downstream genes such as IL-17A and IL-23R is initiated, thereby participating in autoimmune diseases and immune system disorders. Therefore, abnormal expression of JunB may cause various pathological states in organisms. However, whether JunB can regulate atherosclerosis remains to be studied. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides the application of JunB expression inhibitors in the preparation of drugs for treating atherosclerosis. This invention reveals for the first time that inhibiting JunB expression can effectively increase the number of autophagosomes and autolysosomes, thereby providing a possibility for the development of new drugs for treating atherosclerosis.

[0005] To achieve the above object, the specific technical solutions of the present application are as follows: The present application provides, in a first aspect, an application of an expression inhibitor of JunB in the preparation of a drug for treating atherosclerosis, wherein the expression inhibitor of JunB is a small interfering RNA si-JunB targeting a JunB gene.

[0006] Further, the si-JunB has a sense strand and an antisense strand, wherein the sequence of the sense strand is a modified or unmodified sequence as shown in SEQ ID NO. 1, and the sequence of the antisense strand is a modified or unmodified sequence as shown in SEQ ID NO. 2.

[0007] Further, the modification is a 3' overhang of deoxyribonucleotides dTdT on the sense strand and / or the antisense strand.

[0008] Further, the atherosclerosis is induced by homocysteine.

[0009] Further, the si-JunB slows down the development of atherosclerotic plaques by increasing the autophagy level of macrophages.

[0010] Further, the autophagy level is the number of autophagosomes or the number of autolysosomes.

[0011] The present application provides, in a second aspect, a drug for treating atherosclerosis, wherein the drug comprises the above-mentioned si-JunB as the only effective component.

[0012] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0013] Further, the excipient comprises any one or more of a filler, a stabilizer, a diluent, and an adjuvant.

[0014] Further, the atherosclerosis is induced by homocysteine.

[0015] Compared with the prior art, the present application has the following beneficial effects: The application discloses an application of an expression inhibitor of JunB in preparation of a medicine for treating atherosclerosis, and the expression inhibitor of JunB is small interfering RNA si-JunB targeting a JunB gene. JunB The expression of JunB is promoted by Hcy, and the autophagy level of macrophages is reduced to induce atherosclerosis. JunB The expression of JunB is promoted by Hcy, and the autophagy level of macrophages is reduced to induce atherosclerosis. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0017] Figure 1 The influence of Hcy on the autophagy of macrophages. Figure 1 A in the figure is the co-localization expression of CD68 (green, a macrophage marker) and LC3 (red, an autophagy marker) in a normal feed group (CD) and a 1.7% high methionine feed group (HMD) observed by an immunofluorescence experiment, wherein the upper row of pictures is the CD group, the lower row of pictures is the HMD group, and from left to right, they are CD68 staining images, LC3 staining images, DAPI staining images and superimposed images of CD68, LC3 and DAPI staining, and the scale is 50um. Figure 1 B in the figure is a statistical chart of the co-localization expression of CD68 and LC3 in the CD group and the HMD group. Figure 1C in FIG. 1 is the co-localization expression of CD68 (green, macrophage marker) and p62 (red, autophagy marker) in the normal feed group (CD) and the 1.7% high methionine feed group (HMD) observed by immunofluorescence experiment, wherein the upper row of pictures is the CD group, and the lower row of pictures is the HMD group, from left to right are CD68 staining images, p62 staining images, DAPI staining images, and overlay images of CD68, p62 and DAPI staining, respectively, and the scale bar is 50 μm. Figure 1 D in FIG. 1 is a statistical chart of the co-localization expression of CD68 and p62 in the CD group and the HMD group. Figure 1 E in FIG. 1 is the expression level of LC3II and p62 protein in BMDM cells in the CD group and the HMD group detected by Western blot experiment. Figure 1 F in FIG. 1 is Figure 1 E in FIG. 1 is a statistical chart of protein expression. Figure 1 G in FIG. 1 is the expression level of LC3II and p62 protein in RAW264.7 cells in the Control group and the Hcy group detected by Western blot experiment. Figure 1 H in FIG. 1 is Figure 1 G in FIG. 1 is a statistical chart of protein expression. Figure 1 I in FIG. 1 is the change in the number of autophagosomes and autolysosomes in BMDM cells observed by immunofluorescence, wherein the upper row is the CD group, and the lower row is the HMD group, from left to right are GFP (green fluorescent protein) channel images, mRFP (red fluorescent protein) channel images, and overlay images, respectively, and the scale bar is 10 μm. Figure 1 J in FIG. 1 is a statistical chart of quantitative analysis of the number of autophagosomes in the CD group and the HMD group. Figure 1 K in FIG. 1 is a statistical chart of quantitative analysis of the number of autolysosomes in the CD group and the HMD group. Figure 1 L in FIG. 1 is the change in the number of autophagosomes and autolysosomes in RAW264.7 cells observed by immunofluorescence, wherein the upper row is the Control group, and the lower row is the Hcy group, from left to right are GFP (green fluorescent protein) channel images, mRFP (red fluorescent protein) channel images, and overlay images, respectively, and the scale bar is 10 μm. Figure 1 M in FIG. 1 is a statistical chart of quantitative analysis of the number of autophagosomes in the Control group and the Hcy group. Figure 1 N in FIG. 1 is a statistical chart of quantitative analysis of the number of autolysosomes in the Control group and the Hcy group. P <0.05, ** represents P <0.01.

[0018] Figure 2 FIG. 1 is a chart of the experimental results of screening for differentially expressed proteins in the Control group and the Hcy group. Figure 2A in FIG. 1 is a two-dimensional principal component analysis diagram of the Control group and the Hcy group samples. Figure 2 B in FIG. 1 is a MA diagram of the differential proteins in the Control group and the Hcy group, showing the logarithmic fold change of protein expression and the average expression. Figure 2 C in FIG. 1 is a peptide length distribution diagram of the differential proteins in the Control group and the Hcy group. Figure 2 D in FIG. 1 is a heat map of the differential expression proteins in the Control group and the Hcy group, showing the high expression proteins; Figure 2 E in FIG. 1 is a Western blot verification of three types of proteins with higher score in the differential proteins between the two groups, namely Cyp4V2, JunB and Uqcrc2, wherein (a) is a Cyp4V2 protein expression diagram, (b) is a JunB protein expression diagram, and (c) is a Uqcrc2 protein expression diagram. Figure 2 F in FIG. 1 is a statistical diagram of the relative expression amounts of Cyp4V2, JunB and Uqcrc2 in the Control group and the Hcy group. Figure 2 G in FIG. 1 is the co-localization expression of CD68 (macrophage marker) and JunB in the plaques of the CD group and the HMD group, wherein the upper row of pictures is the CD group, the lower row of pictures is the HMD group, from left to right are CD68 staining images, JunB staining images, DAPI staining images and superimposed images of CD68, JunB and DAPI staining, scale bar: 50 μm. Figure 2 H in FIG. 1 is a statistical diagram of the co-localization expression levels of CD68 and JunB in the CD group and the HMD group. * represents P <0.05, *** represents P <0.001.

[0019] Figure 3 FIG. 2 is a result of detecting the effect of JunB on macrophage autophagy. Figure 3 A in FIG. 2 is a Western blot detection of the expression levels of autophagy proteins after the knockdown JunB of JunB. Figure 3 B in FIG. 2 is a statistical diagram of the expression levels of autophagy proteins after the knockdown JunB of JunB. Figure 3C is the immunofluorescence analysis of the effect of JunB knockdown on the number of autophagosomes and autolysosomes, wherein the upper row of images from left to right are the distribution of GFP (autophagosome) under the conditions of si-NC, si-JunB, si-NC+Hcy and si-JunB+Hcy, the middle row of images from left to right are the distribution of mRFP (autolysosome) under the conditions of si-NC, si-JunB, si-NC+Hcy and si-JunB+Hcy, and the lower row of images is the merged image of the upper row and the middle row, scale bar: 10 μm. Figure 3 D is the quantitative analysis of the number of autophagosomes and autolysosomes, (a) is the quantitative analysis of the number of autophagosomes, and (b) is the quantitative analysis of the number of autolysosomes. Figure 3 E is the Western blot analysis of the effect of overexpression of JunB on autophagy protein expression. Figure 3 F shows the relative quantification of LC3II, LC3I and p62 protein expression. Figure 3 G is the immunofluorescence result of the effect of overexpression of JunB on the number of autophagosomes and autolysosomes, wherein the upper row of images from left to right are the distribution of GFP (autophagosome) under the conditions of si-NC, si-JunB, OE-NC and OE-JunB, the middle row of images from left to right are the distribution of mRFP (autolysosome) under the conditions of si-NC, si-JunB, OE-NC and OE-JunB, and the lower row of images is the merged image of the upper row and the middle row, scale bar: 10 μm. Figure 3 H is the quantitative analysis of the number of autophagosomes and autolysosomes after overexpression of JunB, (a) is the quantitative analysis of the number of autophagosomes, and (b) is the quantitative analysis of the number of autolysosomes. * indicates that the si-NC group is significantly different from the si-JunB group P <0.05; # indicates that the si-NC+Hcy group is significantly different from the si-JunB+Hcy group P <0.05, or OE-NC group and OE-JunB group P <0.05; ## indicates that the si-NC+Hcy group is significantly different from the si-JunB+Hcy group P <0.01, or OE-NC group and OE-JunB group P <0.01.

[0020] Figure 4 is an experimental analysis of the interaction between JunB and Trim21. Figure 4 A is a scatter plot of the coverage and the number of unique peptides of the mass spectrometry analysis result. Figure 4 B is a list of proteins identified in the mass spectrometry analysis. Figure 4C in FIG. 6 is a network analysis diagram of the binding ability of JunB to Trim21. Figure 4 D in FIG. 6 is an IP experiment to verify the interaction between JunB and Trim21; wherein, (a) is an IP experiment of JunB, showing the binding of Trim21 to JunB; (b) is an IP experiment of Trim21, showing the binding of JunB to Trim21. Figure 4 E in FIG. 6 is an immunofluorescence experiment to observe the co-localization expression of JunB and Trim21 in macrophages, wherein (a) shows the co-expression localization of JunB (green) and Trim21 (red) in macrophages, the upper row of pictures from left to right are the distribution of JunB protein in macrophages, the distribution of Trim21 protein in macrophages, the lower row of pictures from left to right are the staining of the nucleus, the co-localization of JunB and Trim21 with the nucleus in macrophages; (b) shows the merged image of JunB and Trim21, yellow indicates the co-localization area; (c) shows the intensity distribution diagram of the co-localization of JunB and Trim21. Scale bar: 50 μm.

[0021] Figure 5 F in FIG. 6 is the effect of JunB and Trim21 on the regulation of autophagy in macrophages. Figure 5 A in FIG. 6 is an IP experiment showing that Hcy promotes the binding of Trim21 to JunB. Figure 5 B in FIG. 6 is the effect of Hcy on Trim21 mRNA the expression level. Figure 5 C in FIG. 6 is the effect of Hcy on the expression of Trim21 protein. Figure 5 D in FIG. 6 is a quantitative analysis of the relative protein expression of Trim21 under Hcy treatment. Figure 5 E in FIG. 6 is the effect of JunB knockdown on Trim21 mRNA the expression. Figure 5 F in FIG. 6 is the effect of JunB knockdown on the expression of Trim21 protein. Figure 5 G in FIG. 6 is a quantitative analysis of the relative protein expression of Trim21 under the condition of JunB knockdown. Figure 5 H in FIG. 6 is a Western blot analysis showing the effect of JunB knockdown and Trim21 knockdown on the expression of autophagy proteins LC3II, LC3I and p62. Figure 5 I in FIG. 6 is a quantitative analysis of the relative protein expression of autophagy proteins LC3II and p62 under the condition of JunB knockdown and Trim21 knockdown. Figure 5J in FIG. 6 is the influence of knockdown of JunB and Trim21 on the number of autophagosomes and autolysosomes by immunofluorescence analysis, wherein the upper row of pictures from left to right are the distribution of GFP (autophagosome) under si-NC, si-JunB, si-Trim21 and si-JunB+si-Trim21 conditions, the middle row of pictures from left to right are the distribution of mRFP (autolysosome) under si-NC, si-JunB, si-Trim21 and si-JunB+si-Trim21 conditions, and the lower row of pictures is the combined image of the upper row and the middle row of pictures, the scale: 10 μm. Figure 5 K in FIG. 7 is the quantitative analysis of the number of autophagosomes. Figure 5 L in FIG. 8 is the quantitative analysis of the number of autolysosomes. P <0.05, ** represents P <0.01. DETAILED DESCRIPTION

[0022] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0023] Atherosclerosis is a chronic degenerative disease, and the bleeding and rupture of vulnerable plaques are the starting links of acute cardiovascular and cerebrovascular events. Studies have shown that hyperhomocysteinemia is an independent risk factor for the formation of vulnerable plaques in atherosclerosis. For every 5 μM increase in serum homocysteine content, the risk of blood vessels increases by 60% in men and 80% in women, and the relative risk is 1.6 for men and 1.8 for women. Although supplementation of folic acid and B vitamins can control Hcy levels, hyperhomocysteinemia is still an important marker of atherosclerotic plaque progression, and the current therapeutic drugs have poor effects, so it is essential to further explore the prevention and treatment strategies of Hcy-induced vulnerable plaques in atherosclerosis. JunB, as an important member of the AP-1 complex, plays a key role in fetal angiogenesis and cardiovascular development, and can affect immune cell function by regulating target gene expression, and is involved in autoimmune diseases and immune system disorders. However, the regulatory role of JunB in atherosclerosis remains to be further studied.

[0024] The application discloses an application of an expression inhibitor of JunB in the preparation of a drug for treating atherosclerosis. ApoE - / -The mouse atherosclerosis model first revealed the key role of JunB in atherosclerosis. At the animal level, immunofluorescence experiments showed that compared with the CD group (control group), the co-localization expression of CD68 (macrophage marker) and LC3 (autophagy marker) in the plaque of the HMD group (1.7% high methionine diet group) was reduced, and the co-localization expression of p62 (autophagy marker) was increased. Western blot detected the expression of LC3II and p62 in BMDM (bone marrow-derived primary macrophages) in the CD group and the HMD group, and RAW264.7 (mouse macrophages) in the Control group and the Hcy intervention group. The results showed that compared with the CD group and the Control group, the expression of LC3II was reduced and the expression of p62 was increased in the HMD group and the Hcy group; at the same time, the above groups were transfected with autophagy double-label adenovirus (mRFP-GFP-LC3), and the results showed that under the condition of Hcy, the number of autophagosomes and autolysosomes in macrophages was significantly reduced compared with the CD group and the Control group. This indicates that Hcy can inhibit the autophagy level of macrophages. (2) In order to explore how Hcy regulates macrophage autophagy, protein expression of differential proteins in the Control group and the Hcy group was analyzed by proteomics, and high-throughput sequencing screened out the protein of JunB which was significantly increased. Immunofluorescence experiment results showed that in the plaque of the HMD group, the expression of JunB and CD68 was significantly increased compared with the CD group. Hcy promotes the expression of JunB. At the cellular level, by constructing JunB knockdown and overexpression plasmids and transfecting them into macrophages, Western blot results showed that overexpression of JunB inhibited LC3II protein expression, promoted p62 protein expression, and reduced the number of autophagosomes and autolysosomes in macrophages; knockdown of JunB promoted LC3II protein expression and inhibited p62 protein expression, and increased the number of autophagosomes and autolysosomes in macrophages, and after Hcy stimulation, the above indicators decreased and the autophagy level decreased. (3) In order to further explore how JunB affects macrophage autophagy, mass spectrometry results showed that Trim21, as an autophagy regulator, is a downstream protein of JunB. At the cellular level, IP (immunoprecipitation) experiments confirmed that JunB could bind to Trim21. Immunofluorescence confirmed that in macrophages, Trim21 and JunB co-localized expression was significantly increased, and the expression of Trim21 decreased when Hcy was intervened. Therefore, Hcy promotes the binding of JunB and Trim21, inhibits the expression of Trim21, and thus inhibits the autophagy level of macrophages. IP results confirmed that when Hcy was intervened, the binding of JunB and Trim21 in macrophages increased, and the expression of Trim21 decreased.By constructing a Trim21 knockdown plasmid, a JunB knockdown plasmid is simultaneously transfected into macrophages, and Western blot results show that, compared with the single JunB knockdown group, in the double transfection JunB knockdown and Trim21 knockdown group, the expression of LC3II is reduced, the expression of p62 is increased, and the number of autophagosomes and autolysosomes is reduced; compared with the single Trim21 knockdown group, the expression of LC3II in the double transfection group is increased, the expression of p62 is reduced, and the number of autophagosomes and autolysosomes is increased. The above results all show that Hcy promotes the combination of JunB and Trim21, inhibits the expression of Trim21, and inhibits the autophagy level of macrophages. Hcy inhibits the expression of macrophage autophagy level by combining JunB with Trim21, and the expression inhibitor of JunB can be used for preparing a drug for treating atherosclerosis.

[0025] Example 1: Application of the expression inhibitor of JunB in the preparation of a drug for treating atherosclerosis I. Experimental method The expression inhibitor of JunB used in the present application ApoE - / - The mice were purchased from Beijing Weishanglide Biotechnology Co., Ltd. (Animal License No.: SCXK (Jing) 2021-0010), and the animal experiment was approved by the Ethics Committee of Ningxia Medical University (Ningxia Medical University Ethics No. 2021-G076). The mice were all raised in the SPF level environment of the Experimental Animal Center of Ningxia Medical University, and 12 8-week-old C57BL / 6J mice were randomly selected ApoE - / - The mice were divided into two groups: 6 ApoE - / - Standard rodent feed group (CD); 6 ApoE - / - The mice were fed with 1.7% high methionine feed (HMD) for 22 weeks.

[0026] Specimen collection: The heart aortic arch of the mice was sampled within 10 minutes after the mice were sacrificed. The residual blood was removed by flushing with physiological saline for 3 times, the surface moisture was absorbed with filter paper, and the tissue was immediately immersed in 4% paraformaldehyde. The remaining tissue was divided into cryogenic tubes, frozen in liquid nitrogen, and then transferred to a-80℃ ultra-low temperature refrigerator for storage.

[0027] Extraction of bone marrow-derived macrophages (BMDM): The bone marrow of each group of mice was stripped from the tibia and femur in a clean bench, soaked in 75% alcohol for 30 min, washed with sterile PBS for 2 times, and immersed in DMEM medium. The bone marrow cavity was repeatedly washed with a 1 mL syringe until the bone marrow cavity turned white. The DMEM suspension was collected in a 15 mL centrifuge tube, centrifuged at 1,200 rpm for 10 min. The supernatant was removed. 10 mL of red blood cell lysis solution was added to the centrifuge tube until the liquid turned white. Centrifugation at 1,000 rpm for 5 min, remove the supernatant. Add the specific mouse bone marrow macrophage culture medium, adjust the cell concentration to 2.5 x 10 6 cells / mL, and place the cells in a 25 cm 2 culture bottle in a 37°C, 5% CO2 incubator until the cells adhere.

[0028] The mouse bone marrow macrophage RAW264.7 cell line was purchased from Shanghai Mingke Biological Technology Co., Ltd. The cells were stored in liquid nitrogen for standby, and the freezing solution was prepared according to the volume ratio of DMSO and serum being 1:9.

[0029] 1. Immunofluorescence staining of the aortic root of mice in the conventional feed group (CD) and the 1.7% high methionine diet group (HMD) By immunofluorescence staining, the expression changes of macrophages and autophagy-related proteins in the aortic root of mice in the NC group and the HMD group were observed.

[0030] Immunofluorescence experiment was used to detect the expression of macrophage CD68, autophagy-related LC3 and p62, and JunB.

[0031] 1) 4% paraformaldehyde fixation for 30 min; 2) Prepare PBS solution according to Table 1, wash with PBS for 5 min, 3 times; Table 1 Preparation of PBS solution 3) Blocking: Add endogenous peroxidase blocking agent to the section tissue area, cover all section tissue areas, incubate at room temperature for 30 min, wash the section with PBS for 3 times, 5 min / time; 4) Immunolabeling: Use absorbent paper to gently absorb the water around the tissue, and use a special oil pen to draw a circle around the tissue. Add 80 L of goat serum blocking solution, and incubate at room temperature for 60 min. After discarding the blocking solution, incubate the prepared primary antibody at 4°C overnight. The primary antibody is diluted at a ratio of 1:200. After washing with PBS the next day, incubate the fluorescent secondary antibody at room temperature for 2 h;

[0032] 5) Nucleus restaining: After washing with PBS for 3 times, add hematoxylin restaining for 1 min, and wash with PBS for 3 times again; 6) Covering: After adding neutral gum, observe under microscope.

[0033] 2、Cell experiment grouping and culture The effects of Hcy on macrophage autophagy were studied: Control group, 100 μM Hcy intervention group, knockdown JunB group (si-JunB), 100 μM Hcy intervention knockdown JunB group (Hcy+si-JunB), overexpression JunB group (OE-JunB), knockdown Trim21 group (si-Trim21), and co-knockdown JunB and Trim21 group (si-JunB+si-Trim21); The constructed JunB and Trim21 small interfering viruses and JunB overexpression virus were synthesized by Shanghai GenePharma Company, denoted as si-JunB and si-Trim21. The sense strand and antisense strand sequences of si-JunB are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the sense strand and antisense strand sequences of si-Trim21 are shown in SEQ ID NO. 3 and SEQ ID NO. 4. The sequence 3' end of SEQ ID NO. 1 to SEQ ID NO. 4 is overhanging deoxyribonucleotide (dT) (dT), which is introduced into the mouse monocyte macrophage leukemia cell line RAW264.7, thereby realizing the expression of the target gene.

[0034] SEQ ID NO. 1: 5'-GGAACAGCCUUUCUAUCAC-3'; SEQ ID NO. 2: 5'-GUGAUAGAAAGGCUGUUCC-3'; SEQ ID NO. 3: 5'-GGAGCCUAUGAGUAUCGAA-3'; SEQ ID NO. 4: 5'-UUCGAUACUCAUAGGCUCC-3'.

[0035] 2.1、Cell culture (1) The cell culture medium is complete medium, specifically: high-sugar DMEM medium containing 10% (volume fraction) serum and 1% (volume fraction) penicillin-streptomycin; (2) Control group: RAW264.7 was cultured in 7% FBS high-sugar DMEM medium and placed in a cell incubator at 37°C and 5% CO2; (3) Hcy group: 100 moL Hcy+7% FBS high-sugar DMEM was used to culture RAW264.7, which was placed in a cell incubator at 37°C and 5% CO2; (4) si-JunB group: RAW264.7 was cultured in 7% FBS high glucose DMEM medium, and transfected with JunB knockdown virus; (5) Hcy+si-JunB group: RAW264.7 was cultured in 100 moL Hcy+7% FBS high glucose DMEM, and transfected with JunB knockdown virus; (6) OE-JunB group: RAW264.7 was cultured in 7% FBS high glucose DMEM medium, and transfected with JunB overexpression virus; (7) si-Trim21 group: RAW264.7 was cultured in 7% FBS high glucose DMEM medium, and transfected with Trim21 knockdown virus; (8) si-JunB+si-Trim21 group: RAW264.7 was cultured in 7% FBS high glucose DMEM medium, and transfected with JunB knockdown and Trim21 knockdown viruses; (9) The above groups of cells were transfected with plasmids for 24 hours, and then collected after Hcy intervention for 24 hours for subsequent experiments.

[0036] 2.2, Cell freezing Freezing solution was prepared according to DMSO: serum = 1:9, the cells were taken out of the incubator, and the cells in the logarithmic growth phase were selected, then the adherent cells were blown off and mixed evenly on the clean bench, and then transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the freezing solution was added, the cells were mixed by blowing, and then divided into 1.5 mL-1.8 mL freezing tubes, and the cell name, freezing time and operator were noted, and the gradient freezing sequence of 4°C for 30 min, -20°C for 2 h, -80°C overnight was followed, and then transferred to a liquid nitrogen tank for storage.

[0037] 2.3, Small interfering RNA transfection The logarithmic growth phase cells were gently blown and shaken to prepare a suspension, and 500 μL was inoculated in a 24-well plate overnight; the next day, the cells were taken out and observed under a microscope when the cells grew to 60%-70%, one tube of high glucose DMEM medium was diluted to a concentration of 4 μM with siRNA targeting JunB. In another tube of high glucose DMEM medium, Lipofectamine 2000 was diluted according to the volume ratio of siRNA: Lipofectamine 2000 = 1:1. The diluted siRNA and Lipofectamine 2000 were gently mixed, and the complex was formed at room temperature for 5 minutes. The complex was added dropwise to the cell culture dish, and mixed gently. After transfection for 4-6 hours, the complete medium containing antibiotics was replaced, and the culture was continued for 24-48 hours.

[0038] Then use real-time fluorescent quantitative polymerase chain reaction to detect transfection efficiency. After qRT-PCR, suitable knockdown JunB and knockdown Trim21 are obtained, and stable knockdown JunB and stable knockdown Trim21 cell models are successfully constructed.

[0039] 2.4, qRT-PCR 1) Total RNA extraction in macrophages (1) Preparation: Place the required items for extracting RNA in the ultraviolet irradiated clean bench according to the instructions; (2) Sample lysis: After plasmid transfection of the cell culture medium, incubate at 37°C, 5% CO2 for 72 hours. After adding the prepared lysis solution, lyse at 37°C for 5 min;

[0040] (3) Put into the pre-cooled centrifuge at 12,000 rpm / min for 5 min, take out and move to the workbench, transfer the supernatant to the pre-prepared new enzyme-free EP tube; (4) Add 200 μL of chloroform to the sample, vortex vigorously for 30 s, and stand still in the workbench for 3 min. After centrifugation at 4°C for 10 min, the sample is divided into three layers: organic layer, middle layer and aqueous phase. RNA mainly accumulates in the aqueous phase. 450 μL of colorless aqueous phase layer is removed and moved to a new EP tube; (5) Add 225 μL of anhydrous ethanol to the EP tube and mix well by inverting, then move the mixed liquid to the labeled adsorption column and centrifuge at 4°C 12000 rpm / min for 30 s; (6) Discard the liquid in the collection tube and add 500 μL of deproteinization eluent RD to the adsorption column and centrifuge at 4°C 12000 rpm for 30 s; (7) Discard the liquid in the collection tube and add 500 μL of rinse solution RW to the adsorption column. Place in the workbench for 2 min, then centrifuge at 4°C 12,000 rpm / min for 30 s. Repeat this step twice; (8) After discarding the waste liquid, centrifuge at 4°C 12,000 rpm / min for 2 min. After centrifugation, aspirate the residual liquid; (9) In the clean bench, let the adsorption column stand for 10 min and dry completely. Finally, move to the labeled RNase-free EP tube, add 40 μL of RNase-free ddH2O, stand at room temperature for 2 min, and centrifuge at 4°C 12,000 rpm / min for 30 s to collect the RNA. Use a spectrophotometer to detect the RNA concentration and store it in a -80°C refrigerator for long-term preservation.

[0041] 2) Reverse transcription (1) After the RNA is prepared, if the RNA stored at -80℃ is extracted in advance, it is placed on ice, and after melting, the RNA concentration is measured again, and the reverse transcription system of each sample is configured, as shown in Table 2.

[0042] Table 2 Reverse transcription system (2) The prepared reverse transcription system is oscillated and mixed, and bubbles are removed. Reverse transcription is performed according to the conditions in Table 3: Table 3 Reverse transcription conditions (3) After the reaction of reverse transcription of RNA into cDNA is completed, the sample is collected and stored in a refrigerator at -20℃.

[0043] 3) PCR reaction (1) The reagents required in the PCR reaction are prepared according to the following table, and the eight continuous rows are placed on the pre-cooled constant temperature module to prepare the reaction solution, as shown in Table 4: Table 4 Reaction system (2) The reaction solution is added to each eight continuous row tube, and after slight oscillation and mixing without bubbles, PCR amplification is performed, and the program is as shown in Table 5: Table 5 Amplification program (3) The relative expression amount of the target gene is calculated by 2-Ct method.

[0044] (4) According to the reverse transcription steps described above, the RNA is reverse transcribed into cDNA; (5) The expression of the target gene is detected by qRT-PCR experiment. JunB

[0045] The nucleotide sequence of the target gene is shown as SEQ ID NO. 5, JunB The nucleotide sequence of the target gene is shown as SEQ ID NO. 6. Trim21 SEQ ID NO. 5:

[0046] ​

[0047] SEQ ID NO. 6:

[0048] The present application uses specific primers to amplify JunB, Trim21 and GAPDH genes.

[0049] Amplification JunB The primer set sequence is as follows: The forward primer sequence is 5'-TCACGACGACTCTTACGCAG-3' (SEQ ID NO. 7), 21 bases in length, and the melting temperature (Tm) is 62.6°C; the reverse primer sequence is 5'-CCTTGAGACCCCGATAGGGA-3' (SEQ ID NO. 8), 19 bases in length, and the melting temperature (Tm) is 62.6°C. The primer pair in the PrimerBank database has an ID of 126012538c1, and the fragment size of the amplification product is 95 base pairs.

[0050] Amplification Trim21 The primer set sequence is as follows: The forward primer sequence is 5'-TGGTGGAGCCTATGAGTATCG-3' (SEQ ID NO. 9), 21 bases in length, and the melting temperature (Tm) is 60.5°C; the reverse primer sequence is 5'-GGCACTCGGGACATGAACTG-3' (SEQ ID NO. 10), 20 bases in length, and the melting temperature (Tm) is 62.8°C. The primer pair in the PrimerBank database has an ID of 127139139c1, and the fragment size of the amplification product is 96 base pairs.

[0051] Amplification GAPDH The primer set sequence is as follows: The forward primer sequence is 5'-AGGTCGGTGTGAACGGATTTG-3' (SEQ ID NO. 11), 21 bases in length, and the melting temperature (Tm) is 62.6°C; the reverse primer sequence is 5'-GGGGTCGTTGATGGCAACA-3' (SEQ ID NO. 12), 19 bases in length, and the melting temperature (Tm) is 62.6°C. The primer pair in the PrimerBank database has an ID of 126012538c1, and the fragment size of the amplification product is 95 base pairs.

[0052] 2.5, Co-IP experiment (1) Collect the Control group, Hcy group macrophages, discard the original culture medium, and wash with pre-cooled sterile PBS for 3 times, and completely discard the PBS at the last time; (2) Add 500 mL NP40 (NP40:PMSF=100:1) into each bottle of cells, and place on a 4°C shaker to allow the cells to be fully lysed; (3) Collect the cell lysate, centrifuge at 12,000 rpm for 10 min, and collect the supernatant. Divide the supernatant into IP, IgG, and Input groups. Add 4 μL of JunB and Trim21 antibodies to the IP group, add 4 μL of IgG antibodies to the IgG group, and the Input group is the total protein of the cells;

[0053] (4) Place the cell antibody mixture in a 360° rotator and rotate for 2 h; (5) Add 30 μL of magnetic beads to each of the IP and IgG groups of cells, and rotate at 4°C and 360° overnight; (6) The next day, use a magnetic stand to clean the protein-antibody-magnetic bead complex 3 times for 5 min each time; finally, retain the complex (7) Use the protein lysate to prepare 2x loading buffer, suspend the protein-antibody-magnetic bead complex, and place in a 99°C metal bath for 10 min; (8) Discard the magnetic beads, and follow the subsequent steps of Western blot.

[0054] 2.6, Western blot experiment to detect autophagy-related indicators LC3I / LC3II, p62, and JunB, Trim21 (1) Extract macrophage proteins: After the cells are taken out from the cell interstice, place on ice to prevent protein degradation, wash with PBS, scrape the adherent cells into PBS with a cell scraper, then use a pipette to transfer to a 1.5 mL enzyme-free EP tube, centrifuge at 5,000 rpm for 5 min at 4°C, and discard the supernatant; (2) Prepare the protein lysate, as shown in Table 6.

[0055] Table 6 Preparation of protein lysate (3) Protein extraction: Add 0.5 mL of lysate to each EP tube, vortex vigorously for 30 sec, stand for 4 min, and repeat 5 times. After the last operation, centrifuge at 12,000 rpm for 5 min at 4°C, transfer the supernatant to a new centrifuge tube, detect the protein concentration, add loading buffer, protein solution:loading buffer=4:1, and place in a 99°C metal bath for 5 min. After cooling to room temperature, transfer to a -20°C refrigerator for storage;

[0056] (4) SDS-polyacrylamide gel electrophoresis: prepare 10% gel in advance, mark the experimental groups and add samples, set the voltage to 80 V; soak the PVDF membrane in 100% anhydrous ethanol for 5 min 5 min before the end of electrophoresis. After electrophoresis, cut the gel and transfer the mold, avoid bubbles between the gel and the PVDF membrane during transfer, keep the gel / membrane in a wet state, clamp it with a clamp and put it in a membrane transfer tank filled with electrotransfer liquid, set 0.3 A, 90 min;

[0057] (5) Blocking: prepare the blocking solution in advance and mix well on the shaker, after the transfer is completed, put the membrane into the blocking solution, incubate on the shaker at room temperature for 2 h; (6) Primary antibody incubation: after blocking is completed, wash with PBST for 3 times, 10 min each time, prepare the primary antibody diluent, put the membrane into the primary antibody incubation box, incubate at 4°C overnight; (7) Secondary antibody incubation: the next day, wash the membrane with PBST for 3 times, 10 min each time, add the secondary antibody, incubate at room temperature for 2 h; (8) Exposure: soak the membrane incubated with the secondary antibody in the luminescent solution, take out the membrane and absorb the luminescent solution with a water-absorbing paper after a short time, put it into the gel imager, adjust the parameters for exposure, collect the data for further analysis.

[0058] 2.7, Detection of autophagy flow changes in macrophages (1) One day in advance, transfer the cells in each group into a confocal dish; (2) After the cells adhere to the wall the next day, the cell density is about 30%, and there is no aggregation. Discard the original culture medium, and add 1 mL of pure culture medium to the confocal microscope. Add the culture medium according to the ratio of virus transfection agent: autophagy virus = 1:1. After 6 h of transfection, replace it with normal culture medium and culture for 24 h;

[0059] (3) After 24 h, take out the cells. Discard the original culture medium and rinse with sterile PBS for 3 times;

[0060] (4) Discard the PBS, add an appropriate amount of anti-fluorescence quencher, and immediately observe the changes of autophagy flow under the confocal microscope.

[0061] 2.8, Statistical method Prism 8.0 and SPSS 23.0 software were used to process the data. The measurement data was represented by mean ± standard deviation, the independent sample t test was used for comparison between two groups, one-way analysis of variance was used for comparison among multiple groups, and the Student-Newman-Keuls test was used for comparison between two groups. The count data was represented by the number or percentage, and the chi-square (χ 2 ) test was used for comparison between two groups. P P < 0.05 was considered statistically significant.

[0062] II. Experimental results 1. Effect of Hcy on macrophage autophagy As shown in the immunofluorescence staining, the co-localization expression of CD68 and LC3 was lower and the co-localization expression of p62 and CD68 was higher in the HMD group than in the CD group. The expression of LC3II was lower and the expression of p62 was higher in the BMDM of the Hcy group than in the Control group. The number of autophagosomes and autolysosomes in the macrophages was reduced in the presence of Hcy. Figure 1 The above results suggest that Hcy can inhibit the level of macrophage autophagy.

[0063] 2. Hcy promotes the expression of JunB protein

[0064] As shown in the immunofluorescence staining, the co-localization expression of CD68 and LC3 was lower and the co-localization expression of p62 and CD68 was higher in the HMD group than in the CD group. The expression of LC3II was lower and the expression of p62 was higher in the BMDM of the Hcy group than in the Control group. The number of autophagosomes and autolysosomes in the macrophages was reduced in the presence of Hcy. Figure 2 3. Effect of JunB on autophagy As shown in the Western blot, the expression of LC3II was higher and the expression of p62 was lower when the macrophages were transfected with the JunB knockdown plasmid. The expression of LC3II was lower and the expression of p62 was higher when Hcy was added. The immunofluorescence experiment also confirmed that the number of autophagosomes and autolysosomes increased when the macrophages were transfected with the JunB knockdown plasmid, and the number of autophagosomes and autolysosomes decreased when Hcy was added. The Western blot showed that the expression of LC3II was lower and the expression of p62 was higher when the macrophages were transfected with the JunB overexpression plasmid compared with the JunB knockdown group. The number of autophagosomes and autolysosomes was reduced in the JunB overexpression group. The above results all showed that Hcy inhibited the level of macrophage autophagy by promoting the expression of JunB.

[0065] 4. JunB and Trim21 binding regulate macrophage autophagy Figure 3 The results showed that Trim21, as an autophagy regulator, is a downstream protein of JunB, and JunB can bind to Trim21.

[0066] 5. JunB and Trim21 binding regulate macrophage autophagy Figure 4 The results showed that Trim21, as an autophagy regulator, is a downstream protein of JunB, and JunB can bind to Trim21.

[0067] ​5. Hcy inhibits Trim21 by promoting the binding of JunB to Trim21, and inhibits macrophage autophagy Figure 5 To explore how JunB and Trim21 regulate macrophage autophagy. IP results show that when Hcy interferes with macrophages, it promotes the binding of Trim21 to JunB; qPCR and Western blot verify the effect of Hcy on Trim21. The results show that when Hcy is added for stimulation, the expression of Trim21 in macrophages is reduced; qPCR and Western blot verify that when the JunB plasmid is transfected in macrophages, the expression of Trim21 in macrophages is increased. Western blot results and immunofluorescence results show that when the JunB and Trim21 are transfected in macrophages, compared with single transfection of JunB, the expression of LC3II is reduced, the expression of p62 is increased, and the number of autophagosomes and autolysosomes is reduced; compared with single transfection of Trim21, the expression of LC3II is increased, the expression of p62 is reduced, and the number of autophagosomes and autolysosomes is increased. The above results show that Hcy inhibits the expression of Trim21 by promoting the binding of JunB to Trim21, and inhibits the level of macrophage autophagy.

[0068] It should be noted that when the present application involves a numerical range, it should be understood that each numerical range has two endpoints and any number between the two endpoints can be selected. Since the same steps and examples are used, the preferred embodiments are described in the present application to prevent repetition. Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0069] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. Use of an expression inhibitor of JunB for the manufacture of a medicament for the treatment of atherosclerosis, characterized in that, The expression inhibitor of JunB is a small interfering RNA si-JunB targeting JunB gene.

2. Use according to claim 1, characterized in that, The si-JunB has a sense strand and an antisense strand, the sequence of the sense strand is a modified or unmodified sequence as shown in SEQ ID NO. 1, and the sequence of the antisense strand is a modified or unmodified sequence as shown in SEQ ID NO.

2.

3. Use according to claim 2, characterized in that, The modification is overhanging deoxyribonucleotide dTdT at the 3' end of the sense strand and / or the antisense strand.

4. Use according to claim 1, characterized in that, The atherosclerosis is induced by homocysteine.

5. Use according to claim 4, characterized in that, The si-JunB slows down the development of atherosclerotic plaques by increasing the autophagy level of macrophages.

6. Use according to claim 5, characterized in that, The autophagy level is the number of autophagosomes or the number of autolysosomes.

7. An agent for treating atherosclerosis, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The medicine takes the si-JunB as claimed in claim 1 as the only effective component.

8. The medicament according to claim 7, characterized in that, The medicine further comprises a pharmaceutically acceptable excipient.

9. The medicament according to claim 8, characterized in that, The excipient comprises any one or more of a filler, a stabilizer, a diluent, an adjuvant.

10. The medicament according to claim 7, characterized in that, The atherosclerosis is induced by homocysteine.

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