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Weifang Medical University, No.7166, Baotong West Street, Weifang, PR ChinaDepartment of Cardiology, Zibo Central Hospital Affiliated to Binzhou Medical College, NO.10, South Shanghai Road, Zibo, PR China
Sirt4 deficiency aggravates inflammation and promotes the development of atherosclerosis.
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Sirt4 deficiency activates the phosphorylation of NF-κB.
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Sirt4 exhibits a protective effect in atherosclerosis.
Abstract
Background and aims
As a member of mitochondrial sirtuins, Sirt4 plays a vital role in cellular metabolism and intracellular signal transduction; however, its effect on atherosclerosis is unclear. This study aimed to explore the effect of Sirt4 on atherosclerosis and its underlying mechanism.
Methods
In vivo, Apoe−/− and Apoe−/−/Sirt4−/− mice were fed a high-fat diet to induce atherosclerosis. In vitro, peritoneal macrophages from two mouse types were extracted and treated with oxidized low-density lipoprotein to establish a cell model, THP-1 cells were used to observe the effect of Sirt4 on the adhesion ability of monocytes. The growth and composition of aortic plaques in two mouse types were analyzed by H&E staining, Oil Red O staining, Dil oxidized low-density lipoprotein, immunohistochemistry, real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Transcriptome analysis and Western blotting were performed to explore the specific mechanism.
Results
Sirt4 deficiency aggravated atherosclerosis in mice. In vivo, aortic plaque size, lipid content, and expression of related inflammatory factors in Apoe−/−/Sirt4−/− mice were higher than those in the control group, whereas the content of collagen Ⅰ and smooth muscle actin-α was significantly lower. Sirt4-deficient macrophages exhibited stronger lipid phagocytosis in vitro, and the adhesion ability of monocytes increased when Sirt4 expression decreased. Transcriptome analysis showed that the expression of CXCL2 and CXCL3 in Sirt4-deficient peritoneal macrophages increased significantly, which may play a role by activating the NF-κB pathway. In further analysis, the results in vitro and in vivo showed that the expression of VCAM-1 and pro-inflammatory factors, such as IL-6, TNF-α and IL-1β, increased, whereas the expression of anti-inflammatory factor IL-37 decreased in Sirt4-deficient peritoneal macrophages and tissues. After blocking the effect with NK-κB inhibitor BAY11-7082, the inflammatory reaction in sirt4 deficient macrophages was also significantly decreased.
Conclusions
This study demonstrates that Sirt4 deficiency promotes the development of atherosclerosis by activating the NF-κB/IκB/CXCL2/3 pathway, suggesting that Sirt4 may exhibit a protective effect in atherosclerosis, which provides a new strategy for clinical prevention and treatment of atherosclerosis.
Atherosclerosis (AS) is one of the most common cardiovascular diseases and a leading cause of death and disability worldwide. Although the clinical diagnosis and treatment approach of the condition are constantly improving, its morbidity and mortality continue to rise every year [
]. As a chronic inflammatory disease of the large arteries, the pathogenesis of AS involves the focal accumulation of low-density lipoprotein (LDL) cholesterol, and its oxidized products in the tunica intima, stimulating endothelial cell activation and monocytes recruitment, and triggering a series of inflammatory responses. During disease progression, monocytes differentiate into macrophages, engulf LDL to form foam cells, which gradually develop into atherosclerotic plaques [
Nuclear factor κB (NF-κB) is a key regulator of vascular inflammatory reactions and drives the expression of various atherogenic molecules, including TNF-α and IL-1β. Therefore, abnormal activation of the NF-κB pathway is closely associated with AS development [
RIPK1 expression associates with inflammation in early atherosclerosis in humans and can Be therapeutically silenced to reduce NF-kappaB activation and atherogenesis in mice.
]. In the inflammatory microenvironment, pro-inflammatory factors can futher stimulate NF-κB to respond to inflammation, which leads to or enhances the expression of a series of inflammatory factors such as IL-6 [
]. During the development of AS, some inflammatory chemokines, such as CXCL2 and CXCL3, have been found to be involved in mediating recruitment, chemotaxis, and proliferation of monocytes and neutrophils in the lesion [
Adventitial CXCL1/G-CSF expression in response to acute aortic dissection triggers local neutrophil recruitment and activation leading to aortic rupture.
], further accelerating the development of AS. This series of inflammatory reactions continues to develop until the formation of atherosclerotic plaques in the arteries [
Sirtuins is a NAD + -dependent class III histone deacetylase and mono-ADP-ribosyltransferase that play a crucial role in various cellular processes, including cell metabolism, oxidative stress, and cell senescence [
]. Seven different sirtuins, Sirt1 to Sirt7, have been identified in mammals. Among them, Sirt4, Sirt3, and Sirt5 exist in the mitochondria of cells and are highly expressed in the heart, brain, liver, and islet β cells [
]. Sirt4 has been shown to play an essential role in cardiovascular diease. Liu et al. found that expression of Sirt4 in H9c2 cells under hypoxia decreased significantly. When Sirt4 was overexpressed, the activity of H9c2 cells under hypoxia was significantly increased, while the activity of caspase-3/7 was significantly decreased [
]. Luo et al. found that Sirt4 played a positive regulatory role in Angiotensin II-induced cardiac hypertrophy. Mice overexpressing heart-specific Sirt4 showed a more significant myocardial hypertrophy and higher ROS levels under Angiotensin II stimulation [
]. However, the effect of Sirt4 on AS and its underlying mechanisms have not been clearly confirmed. This study aimed to explore the effect of Sirt4 gene dificiency on AS and its possible mechanism. At the same time, the effect of NF-κB was specifically blocked by BAY11-7082, and futher reverse verification was carried out in vitro. This study provides a feasible new strategy for clinical diagnosis and treatment of AS.
2. Materials and methods
2.1 Acquisition of human arterial specimens
Human artery specimens used in this study were all from blood vessels isolated during the operation of patients with arteriosclerosis occlusion of the lower extremities. Prior to the start of the study, each patient was informed of the study and written consent was obtained. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and the ethics committee of Zibo Central Hospital approved the acquisition of human arterial specimens.
2.2 Establishment of animal model and organization preparation
Male C57BL/6, Apoe−/−, and Apoe−/−/Sirt4−/− mice were purchased from Beijing Weishanglide Biotechnology Co., Ltd., and raised in the animal room of Zibo Central Hospital. The temperature was 25 ± 2 °C, humidity was 50%, and the light-dark cycle was 12 h. All mice were free to eat and drink. After 1 week of adaptive feeding, mice in all three groups were fed a high-fat diet with 40% fat content for 12 weeks. Afterward, blood samples were drawn from the three groups of mice after fasting for 8 h for follow-up experiments. After blood collection, all mice were euthanized. Five mice from each group were randomly selected and perfused with 0.9% normal saline. Thereafter, heart and aortic tissues were collected, frozen with liquid nitrogen, and stored at −80 °C for real-time quantitative polymerase chain reaction (RT-qPCR). The remaining mice in each group were perfused with 0.9% saline and 4% paraformaldehyde for histological and morphological analyses. After perfusion, the heart and aorta were carefully separated and the tissue was immersed in 10% paraformaldehyde overnight. During the section, the aorta were embedded in the compound of optimal cutting temperature (OCT) for freezing, and 5 μm-thick continuous frozen sections of the aortic root, aortic arch and abdominal aorta were obtained. The human arterial sections were cut into 5 μm-thick paraffin sections, and all sections were stored at-20 °C for follow-up studies.
2.3 Extraction and treatment of peritoneal macrophages
A 6% soluble starch (Solarbio, Beijing, China) solution was prepared with pure sterile water, and 2 ml solution was injected into the abdominal cavity of Apoe−/− and Apoe−/−/Sirt4−/− mice. After 72 h, mice in the two groups were euthanized and injected with 10 ml sterilized phosphate-buffered saline (PBS) solution into the abdominal cavity. After gently massaging the abdomen, the abdominal fluid was collected and centrifuged at 800 rpm for 5 min at 25 °C. The cells were collected and washed with PBS. The cells were re-suspended in Dulbecco modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) and cultured at 37 °C with 5% CO2 in the incubator. After 2 h, non-adherent cells were removed by mild washing, fresh DMEM was added, and peritoneal macrophages were used for the follow-up experiment. In this study, cells were treated with 50 μg/ml oxidized low-density lipoprotein (ox-LDL, Yiyuan Biotechnologies, Guangzhou, China), 50 μg/ml Dil ox-LDL (Yiyuan Biotechnologies, Guangzhou, China) or 10 μM of BAY11-7082 (MedChemExpress, New Jersey, USA).
2.4 Cell lines and virus infection
The human monocyte THP-1 cell line from Punosai Biotechnology Co., Ltd. (Wuhan, China) was preserved in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10%FBS. Mouse aortic endothelial cells (MAECs) were obtained from Beina Chuanglian Biotechnology Research Center (Beijing, China) and preserved in DMEM containing 10%FBS. Both cells were cultured in a humidified incubator at 37 °C with 5% CO2. Sirt4-interfered lentivirus was synthesized by Genechem Co. Ltd. (Shanghai, China). The cells were infected with lentivirus at different multiplicity of infection and were detected by RT-qPCR, and optimal experimental conditions were selected. The infected cells were selected by puromycin of 3 μg/ml.
2.5 Immunofluorescence
Peritoneal macrophages and tissues sections obtained using the method described above were fixed for 20 min with precooled 4% formaldehyde. Afterward, cells and tissues were rinsed with PBS, permeabilized for 20 min with 0.25% Triton X-100 solution, and blocked with 5% BSA for 1 h after PBS cleaning. After blocking, the cells and tissues were incubated overnight with anti-Sirt4 (1:100, ABclonal, Wuhan, China), anti-F4/80 (1:500, ServiceBio, Wuhan, China), anti-CD68 (1:500, Servicebio, Wuhan, China), and anti-NF-κB (1:100, ZEN-BIOSCIENCE, Chengdu, China) antibodies at 4 °C. The next day, immunoglobulin G conjugated with Alexa Fluor 488 or Cy3 (1:500, Abcam, Cambridge, UK) was added in the dark at room temperature for 2 h. Afterward, nuclei were stained with DAPI (Beyotime, Beijing, China), and tablets were sealed with anti-fluorescence quenching tablets after cleaning.
2.6 Detection of lipids in cells and tissues
The lipid content of aortic tissue sections and peritoneal macrophages was detected with the Oil Red O staining kit (Solarbio, Beijing, China). Before staining, peritoneal macrophages were treated with 50 μg/ml ox-LDL for 24 h. Peritoneal macrophages and sections were fixed with Oil Red O fixed solution for 30 min, washed with distilled water, washed with 60% isopropanol for 5 min, then dyed with newly-prepared Oil Red O staining solution for 30 min, hematoxylin dye solution was added for 3 min after washing, Oil Red O buffer was added for 1 min, rinsed with distilled water, and cells were observed and photographed under the microscope. Cells treated with Dil ox-LDL were observed and photographed at 1, 6, 12 and 24 h by fluorescence microscopy. For Oil Red O staining of tissue sections, the method was the same as for cell staining, and pictures were taken after dehydration and sealing. Oil Red O staining was used to observe adipose tissue around the aorta. The adipose tissue around the aorta, separated by the method highlighted above, was removed as much as possible with tweezers and washed twice with PBS. The blood vessels were carefully cut longitudinally along the vessel wall using anatomical scissors and washed slightly with tap water for 5 s. The blood vessels were first immersed in 60% isopropanol 3 s and then immersed in 37 °C Oil Red O dye solution for 60 min staining. Thereafter, the blood vessels were removed with tweezers and immersed in 60% isopropanol, with the adipose plaques in the lumen appearing orange or bright red, and other parts nearly colorless. Afterward, the blood vessels were washed with distilled water to terminate differentiation. After removing the blood vessels, filter paper was used to absorb the excess moisture. A slide was placed on a black or white background plate with a scale, the blood vessels were spread on the slide, a pace with good lighting was selected, the focal length and exposure of the camera were adjusted, and pictures were taken. The lipid composition of the cells and aorta was calculated by ImageProPlus6.0 software image analysis system.
2.7 Monocyte adhesion assay
THP-1 cells were treated with ox-LDL for 24 h, and labelled with 5 μg/ml Calcein AM (Beyotime, Beijing, China) and co-incubated with MAECs at 37 °C for 30 min. Non-adherent cells were removed by repeated washing with PBS. Adherent monocytes were fixed with 4% paraformaldehyde, and counted under the microscope.
2.8 Enzyme-linked immunosorbent assay (ELISA)
The blood of the two groups of mice was centrifuged, and the serum was collected. Concentrations of IL-6 and TNF-α were determined using ELISA (Neobioscience, Shenzhen, China), according to the manufacturer's instructions.
2.9 RT-qPCR
Total RNA was extracted from the cells and tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). PrimeScripRT kit (Accurate Biology, Hunan, China) was used for reverse transcription at 37 °C. Thereafter, according to the manufacturer's instructions, the corresponding genes were amplified using SYBR Pre MixEx Taq (Accurate Biology, Hunan, China) amplification and ABI StepOne Plus detection system. The expression of related genes was calculated using the 2−ΔΔCt method. Each measurement was repeated at least three times at each temperature and the primers (Sangon Biotech, Shanghai, China) of the related genes were as follows:
The frozen slices were taken out of the environment at −20 °C, reheated at room temperature for 30 min, fixed with 80% ethanol for 30 min, and then washed with distilled water. Afterward, H&E staining kit (Solarbio, Beijing, China) was used for staining. The procedure was as follows: the slices were first stained with hematoxylin for 3 min, rinse with distilled water for 3 min, and then rinsed with tap water for 2 min each time. After that, they were stained with hematoxylin for 40 s, rinsed slightly with distilled water, and then quickly dehydrated and sealed. The percentage of plaque area was calculated by ImageProPlus6.0 software image analysis system.
2.11 Immunohistochemistry
Paraffin slices were baked in an oven at 65 °C for 2 h, and dewaxed according to the standard procedure. After incubation with 3% hydrogen peroxide to reduce the activity of endogenous peroxidase, antigen repair was performed by EDTA method, was blocking with 5% goat serum at room temperature. Next, the slices were incubated with anti-Sirt4 (1:100) aitibody at 4 °C overnight. The next day, the sectinos were incubated for 60 min with the secondary antibody at room temperature, stained with a 3.3-diaminobenzidine (DAB) kit (ZSBIO, Beijing, China), re-stained with hematoxylin, and finally dehydrated and sealed. The negative control group was incubated with PBS instead of antibody. When the frozen sections were used, the dewaxing step was omitted, and the remaining steps were as mentioned above. The antibodies used were: anti-Sirt4 (1:100), anti-p-NF-κB (1:100), anti-IL-1β (1:200), anti-IL-6 (1:200), anti-TNF-α (1:200), anti-CXCL2/3 (1:100, Boster Biological, California, USA), anti-VCAM-1 (1:100, ABclonal, Wuhan, China), anti-α-SMA and anti-collagen I (1:200, Abcam, Cambridge, UK). The percentage of positive area was calculated by the ImageProPlus6.0 software image analysis system.
2.12 Western bloting
The extracted proteins were separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Afterward, the proteins were transferred to methanol-activated polyvinylidene fluoride membrane, sealed with TBST containing 5% skim milk, and incubated overnight with anti-GAPDH (Santa Cruz, Texas, USA), anti-CXCL2/3, anti-NF-κB, anti-p-NF-κB, anti-IκB, anti-p-IκB, anti-IL-1β, anti-IL-6, anti-TNF-α, and anti-IL-37 (ZEN-BIOSCIENCE, Chengdu, China) antibodies at 4 °C. Incubation with the second antibodies (1:5000, Abcam, Cambridge, UK) was done at room temperature the next day, and the antigen-antibody complex was visualized with enhanced electrochemiluminescence system after washing.
2.13 Statistical analysis
The Shapiro-Wilk normality test was used to test the normality of the measurement data. GraphPad Prism 6 and SPSS16.0 were used for data analysis. The normally distributed data are presented as mean ± SD, and statistical differences were analyzed with unpaired 2-tailed Student t-test for 2-group comparisons. A value of p < 0.05 was considered statistically significant.
3. Results
3.1 Differences of Sirt4 expression between atherosclerotic and non-atherosclerotic tissues and extraction and identification of peritoneal macrophages
Previous studies have found that serum SIRT4 levels are closely related to the incidence of AS [
Circulating levels of sirtuin 4, a potential marker of oxidative metabolism, related to coronary artery disease in obese patients suffering from NAFLD, with normal or slightly increased liver enzymes.
]. Initially, we detected the expression of SIRT4 in human arterial tissues with AS, and found that SIRT4 was expressed in the intima, media and adventitia of diseased vessels (Fig. 1A), which confirmed that SIRT4 was involved in the process of AS. Immunohistochemistry and RT-qPCR were used to detect Sirt4 expression in the aorta of mice with AS and without AS. Our results were consistent with the previous results, that is, Sirt4 expression was significantly decreased in the tissues with AS (Fig. 1B and C, n = 10, ***p < 0.001, ****p < 0.0001). The results of immunofluorescence showed that Sirt4 and macrophages were widely co-expressed in human and mouse arterial plaques (Fig. 1D and E). Therefore, in the follow-up study, we extracted peritoneal macrophages from Apoe−/− mice and Apoe−/−/Sirt4−/− mice for the in vitro study and identified the macrophages with F4/80 and CD68 double labeling (Fig. 1F). RT-qPCR results confirmed the difference in Sirt4 expression between the two groups (Fig. 1G, n = 10, ****p < 0.0001).
Fig. 1Expression of SIRT4 and macrophages in human and mouse arterial plaques and identification of peritoneal macrophages.
(A) Expression of SIRT4 in intima, media and adventitia in human arterial plaques. Bar = 200 μm or 50 μm. (B) Representative Sirt4 staining and quantitative analysis of Sirt4 in the aorta of mice without AS and with AS. N = 10, ****p < 0.0001. Bar = 50 μm. (C) mRNA expression of Sirt4 in the aorta of mice without AS and with AS was detected by RT-qPCR. N = 10, ***p < 0.001. (D) SIRT4 co-expression with macrophages in human arterial plaques. Bar = 75 μm. (E) Sirt4 co-expression with macrophages in mice arterial plaques. Bar = 50 μm. (F) Identification of peritoneal macrophages. Macrophages were identified by F4/80 and CD68 co-labeling. Red represents F4/80, green represents CD68, and blue (DAPI) represents the nucleus. Bar = 150 μm. (G) mRNA expression of Sirt4 in peritoneal macrophages of Apoe−/− mice and Apoe−/−/sirt4−/− mice was detected by RT-qPCR. N = 10, ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.2 Sirt4 deficiency aggravates lipid accumulation and plaque instability during AS
After treatment with 50 μg/ml ox-LDL for 24 h, cells’ ability to phagocytize lipids was observed by Oil Red O staining. The results showed that the lipid phagocytosis ability of Sirt4-deficient macrophages was significantly higher than that of the control group (each group n = 6, *p < 0.05, **p < 0.01, Fig. 2A). At the same time, the lipid phagocytosis progression of the two groups of cells was observed using 50 μg/ml Dil ox-LDL. Fluorescence microscopy was used to observe lipid phagocytosis at different time points (1 h, 6 h, 12 h and 24 h), the result was consistent with that of Oil Red O staining (Fig. 2B, n = 6, **p < 0.01, ***p < 0.001, ****p < 0.0001). In vivo, H&E staining and Oil Red O staining of tissues of two groups of mice also suggested that the degree of AS in Apoe−/−/Sirt4−/− mice was more severe and lipid composition in plaques was higher (Fig. 2C–G, n = 6 or 10, ***p < 0.001, ****p < 0.0001). The histological quantification of the plaques size in sections of the aortic arches and abdominal aorta in Apoe−/−/Sirt4−/− and Apoe−/− mice is shown in Supplementary Fig. 6). Immunohistochemistry showed that the expression of collagen Ⅰ and α-SMA in plaque of Apoe−/−/Sirt4−/− mice was significantly lower than in the control group, suggesting that plaques of Apoe−/−/Sirt4−/− mice were more unstable (Fig. 2H, n = 10, ***p < 0.001, ****p < 0.0001).
Fig. 2Sirt4 deficiency promotes the ability of macrophages to phagocytose lipids and contributes to AS and plaque instability.
(A) After peritoneal macrophages of Apoe−/− mice and Apoe−/−/Sirt4−/− mice were incubated with ox-LDL (50 μg/ml) for 24 h, lipid phagocytosis of macrophages was observed by Oil Red O staining, and quantitative analysis in the two groups. N = 6, *p < 0.05, **p < 0.01, bar = 50 μm. (B) Dil ox-LDL (50 μg/ml) was used to observe lipid phagocytosis of peritoneal macrophages in the two groups at different time points and quantitative analysis. N = 6, **p < 0.01, ***p < 0.001, ****p < 0.0001, bar = 150 μm. (C) The percentage of total lesion area of aorta in the two groups of mice was evaluated by Oil Red O staining and quantitative analysis. N = 6, ***p < 0.001. (D and E) Representative images of cross-sectional aortic root lesions by H&E staining (n = 10) and Oil Red O staining (n = 10), and (F and G) quantitative analysis in the two groups. ***p < 0.001,****p < 0.0001, bar = 300 μm. (H) Representative collagene I and α-SMA staining and quantitative analysis in the two groups of mice. N = 10, ***p < 0.001, ****p < 0.0001, bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3 Transcriptome screen design, verification, and identification of enriched genes
To further explore the promoting effect of Sirt4 deficiency on AS and its mechanism, peritoneal macrophages of Apoe−/− and Apoe−/−/Sirt4−/− mice were treated with 50 μg/ml ox-LDL for 24 h, thereafter, the two groups of cells were analyzed by transcriptome analysis (each group n = 3). A total of 55 differentially expressed genes were identified, of which 11 were upregulated and 44 were downregulated. A Volcanic map (Fig. 3A) was used to elucidate the overall distribution of differentially expressed genes. The result of a cluster analysis of different groups is shown in Fig. 3B. The significance of KEGG pathway was analyzed (Fig. 3C). The transcriptome results showed that the expression of inflammatory chemokine CXCL2 and CXCL3 in the Sirt4-deficient group was significantly increased. This was verified by Western blotting and immunohistochemistry, and the results were consistent with the transcriptome analysis results (Fig. 3D and E, n = 3 or 6, ***p < 0.001, ****p < 0.0001) since chemokines are involved in monocyte chemotaxis and inflammation in the process of AS [
]. In vitro, we used Sirt4-interfered lentivirus to infect THP-1 cells and observed their adhesion ability, and the results showed that the adhesion ability of THP-1 cells infected by Sirt4-interfered lentivirus was stronger than that of the control group (Fig. 3F, n = 6, ****p < 0.0001. The results of the letivirus transfection experiments are shown in Supplementary Fig. 7). In addition, we also detected the expression of VCAM-1 and some inflammatory factors in the plaques of two groups of mice, which are closely related to AS [
]. The results showed that the expression of VCAM-1 and inflammatory factors in sirt4-deficient arterial plaques was significantly higher than in the control group (Fig. 3G, n = 6, **p < 0.001, ***p < 0.0001). The serum levels of IL-6 and TNF-α in the two groups of mice was detected by Elisa, and the results are shown in Supplementary Fig. 8.
Fig. 3Transcriptome analysis and further verification based on it.
Peritoneal macrophages of Apoe−/− (n = 3) and Apoe−/−/Sirt4−/− mice (n = 3) were treated with ox-LDL for 24 h and then transcriptome analysis was performed. (A) Volcano plot of differentially expressed genes. Grey represents non-significant difference genes, while red and green represent significant difference genes. (B) Cluster analysis of differential gene expression level. Red indicates relatively high expression protein coding gene, blue indicates relatively low expression protein coding gene.(C) KEGG pathway significance analysis.(D) Protein expression of CXCL2 and CXCL3 in the two groups of peritoneal macrophages by Western blotting and quantitative analysis. N = 3, ****p < 0.0001. (E) Representative CXCL2 and CXCL3 staining and quantitative analysis in the two groups of mice. N = 6, ***p < 0.001, bar = 50 μm. (F) THP-1 transfected with lentivirus were treated with ox-LDL for 24 h, and then were labbeled with 5 μg/ml Calcein AM fluorescent dye (green) and incubated on top of a monolayer of MAECs for 30 min at 37 °C. Cells were washed extensively before visualization by microscopy and quantification by plate reader. N = 6, ****p < 0.0001, bar = 150 μm. (G) Representative VCAM-1, IL-1β, IL-6 and TNF-α staining and quantitative analysis in the two groups of mice. N = 6, **p < 0.01,***p < 0.001, bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.4 Sirt4 deficiency promotes the development of AS by activating NF-κB/IκB pathway
Transcriptome analysis results suggested that Sirt4 deficiency accelerated the development of AS and may play a role by activating the NF-κB/IκB pathway; therefore, we verified this pathway. Two groups of cells were treated with 50 μg/ml ox-LDL for 24 h, and the expression of NF-κB/p-NF-κB and IκB/p-IκB was detected by Western blotting and immunohistochemistry. The results showed that the expression of p-NF-κB and p-IκB in Sirt4-deficient cells and tissues increased significantly, whereas the expression of IκB decreased significantly (Fig. 4A and B, n = 3 or 10, **p < 0.01,***p < 0.001, ****p < 0.0001). To further verify this, the two groups of cells were pretreated with NF-κB inhibitor BAY 11–7082 (BAY, MedChemExpress, New Jersey, USA) at the concentration of 10 μM for 1 h and thereafter treated with 50 μg/ml ox-LDL for 24 h. The expression was detected by Western blotting, and we found that expression of NF-κB/p-NF-κB and IκB/p-IκB in the two groups of cells pretreated with inhibitors showed the opposite trend to the previous results (Fig. 4A, n = 3, ***p < 0.001, ****p < 0.0001). In addition, we observed nuclear translocation of NF-κB in the treated cells via immunofluorescence. The results showed significant nuclear translocation of NF-κB in Sirt4-deficient cells treated with ox-LDL, but not in the control group (Fig. 4C). However, when the cells of the two groups were pretreated with BAY before ox-LDL treatment, there was no difference in nuclear translocation of NF-κB between the two groups (Fig. 4D). In addition, we compared lipid phagocytosis in the two groups of cells pretreated with BAY. Previous results have shown that Sirt4-deficient cells have a stronger ability to phagocytize lipids after ox-LDL treatment. The cells in the control group and the experimental group were treated with ox-LDL or pretreated with BAY and afterward treated with ox-LDL. The results showed that lipid phagocytosis of Sirt4 deficient cells was significantly decreased after BAY pretreatment; that is, after blocking NF-κB, lipid accumulation caused by Sirt4 deficiency was significantly decreased (Fig. 4E, n = 6, **p < 0.01, ***p < 0.001). These results indicated that Sirt4 deficiency activated the NF-κB/IκB pathway and promoted the development of AS.
Fig. 4Sirt4 deficiency promotes the development of AS by activating the NF-κB pathway.
(A) Protein expression of NF-κB, p-NF-κB, IκB and p-IκB in four groups of peritoneal macrophages by Western blotting and quantitative analysis. The concentrations of ox-LDL and BAY were 50 μg/ml and 10 μM, respectively. **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Representative p-NF-κB staining and quantitative analysis in the two groups of mice. N = 10, ***p < 0.001, bar = 50 μm. (C) Nuclear translocation of NF-κB in the two groups of peritoneal macrophages treated with ox-LDL (50 μg/ml) for 24 h. Green stands for NF-κB, and the nucleus was stained with DAPI (blue). Bar = 10 μm. (D) NF-κB nuclear translocation in the two groups of peritoneal macrophages pretreated with BAY 11–7082(10 μM) for 1 h before ox-LDL (50 μg/ml) treatment for 24 h. Green stands for NF-κB, and the nucleus is stained with DAPI (blue). Bar = 10 μm. (E) Oil red O staining was used to observe the lipid phagocytosis ability of peritoneal macrophages of four groups under different treatment conditions and quantitative analysis was performed. N = 6, **p < 0.01, ***p < 0.001, bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.5 Sirt4 deficiency promotes the development of AS through the NF-κB/κB/CXCL2/CXCL3 pathway
According to the transcriptome analysis results, Western blotting and RT-qPCR were used to detect the expression of inflammatory chemokines CXCL2 and CXCL3 before and after NF-κB inhibition. When only treated with ox-LDL, CXCL2 and CXCL3 expression in Sirt4-deficient foam cells was significantly higher than in the control group, whereas their expression in both groups was completely reversed after NF-κB inhibition, and the expression of CXCL2 and CXCL3 in Sirt4-deficient macrophages was lower than in the control group (Fig. 5A and B, n = 3 or 10, **p < 0.01, ***p < 0.001, ****p < 0.0001). To further confirm our conclusion, we detected the expression of pro-inflammatory factors (IL-1 β, IL-6 and TNF-α) and anti-inflammatory factor (IL-37), which are closely related to AS (Fig. 5C and D). The results were consistent with our expectations: when only treated with ox-LDL, the expression of pro-inflammatory factors in Sirt4-deficient macrophages was higher and the anti-inflammatory factor was lower. When the effect of NF-κB was inhibited, the expression of these inflammatory factors in the two groups was reversed (Fig. 5C and D, n = 3 or 10, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). In summary, our study demonstrates that Sirt4 deficiency promotes the development of AS by activating the NFκB/IκB/CXCL2/3 pathway.
Fig. 5Sirt4 deficiency promotes the development of AS through the NF-κB/κB/CXCL2/CXCL3 pathway.
The concentrations of ox-LDL and BAY were 50 μg/ml and 10 μM, respectively. (A and B) Protein (n = 3) and mRNA (n = 10) expression of CXCL2 and CXCL3 in four groups of peritoneal macrophages assessed by Western blotting and RT-qPCR, and quantitative analysis. **p < 0.01, ***p < 0.001, ****p < 0.0001. (C and D) Protein (n = 3) and mRNA (n = 10) expression of IL-1β, IL-6, TNF-α and IL-37 in four groups of peritoneal macrophages assessed by Western blotting and RT-qPCR, and quantitative analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
As a member of sirtuins, Sirt4 has received much attention for its role in cardiovascular disease. A previous study has found that serum SIRT4 levels in obese people are significantly lower than those in healthy people, and there is no significant correlation with the degree of obesity, but is closely related to some indicators considered to be risk factors for coronary heart disease, such as low serum levels of high-density lipoprotein [
Circulating levels of sirtuin 4, a potential marker of oxidative metabolism, related to coronary artery disease in obese patients suffering from NAFLD, with normal or slightly increased liver enzymes.
]. Another study showed that the expression of SIRT4 in human umbilical vein endothelial cells (HUVECs) treated with LPS was significantly decreased. Sirt4 silencing could increase the expression of many cytokines such as IL-1β, IL-6, and adhesion molecule ICAM-1, whereas overexpression of SIRT4 could inhibit inflammation of HUVECs induced by LPS [
]. In our study, we found that Sirt4 expression in the aorta of mice with AS was significantly lower than that in the aorta of mice without AS, which was consistent with the results of previous studies. In vivo, we knocked out the Sirt4 gene of Apoe−/− mice, and the results showed that the AS lesions of Apoe−/−/Sirt4−/− mice were more severe. Since the extensive co-expression of Sirt4 and macrophages was found in the arterial plaques of mice with AS, we chose the classical model of peritoneal macrophages for in vitro studies [
]. Oil Red O staining results in vivo and in vitro showed that lipid accumulation in aortic plaques and peritoneal macrophages with Sirt4-deficient was more severe. Further studies showed that Sirt4 deficiency may lead to the aggravation of AS by promoting inflammation, suggesting that Sirt4 may exhibit a protective effect in AS.
It is well known that inflammation promotes the progression of AS [
]. Many inflammatory chemokines control the infiltration of monocytes and the accumulation of macrophages as a result of their recruitment of leukocytes, and play an important role in the development of AS lesions [
]. Copin et al. found that chemokine was significantly expressed in Apoe−/− mice plaque through a study of carotid atherosclerotic Apoe−/− mice. After treatment with evasin-3, a chemokine inhibitor, inflammation of carotid plaque in mice improved [
]. Boro et al. found that chemokine CXCL2 was involved in the activation of NLRP3 inflammatory bodies in macrophages, and the blockage of chemokines such as CXCL2 could significantly reduce the production of bioactive IL-1β in vivo [
]. In this study, transcriptome analysis results showed that the expression of CXCL2 and CXCL3 in Sirt4-deficient foam cells was significantly higher than that in the control group. Based on this result, we performed a series of validations. In vivo, we found that expression of VCAM-1 and inflammatory factors IL-1β, IL-6, and TNF-α was significantly higher in aortic plaques of Sirt4-deficient mice than in controls. In vitro, infection of THP-1 cells using Sirt4-interfering lentivirus also revealed increased adhesion of THP-1 when Sirt4 expression was decreased. These results all suggested that sirt4 deficiency promotes AS development by increasing chemokines expression, enhancing monocytes adhesion, and exacerbating the inflammatory response.
NF-κB is an important inflammatory transcription factor, which is a dimer composed of polypeptide chain p-65 and p-50 protein subunits [
Danlou tablet inhibits the inflammatory reaction of high-fat diet-induced atherosclerosis in ApoE knockout mice with myocardial ischemia via the NF-kappaB signaling pathway.
]. In resting cells, the NF-kB dimer exists in the cytoplasm in an inactive form and bind to the inhibitory protein IκB. When stimulated by inflammation, IκB is phosphorylated and degraded, which activates NF-κB and its translocation to the nucleus, and binds to the promoters or enhancers of specific genes to initiate transcription and regulate inflammatory response [
RIPK1 expression associates with inflammation in early atherosclerosis in humans and can Be therapeutically silenced to reduce NF-κB activation and atherogenesis in mice.
Low doses of lipopolysaccharide and minimally oxidized low-density lipoprotein cooperatively activate macrophages via nuclear factor kappa B and activator protein-1: possible mechanism for acceleration of atherosclerosis by subclinical endotoxemia.
Cholesterol crystals promote endothelial cell and monocyte interactions via H(2)O(2)-mediated PP2A inhibition, NFkappaB activation and ICAM1 and VCAM1 expression.
]. In our study, the transcriptome analysis results suggested that the atherosclerotic phenomenon caused by Sirt4 deficiency may play a role by activating the NF-κB/IκB pathway. Therefore, we verified this pathway. Western blotting results showed that, compared with the control group, IκB was significantly activated and degraded in Sirt4 deficient foam cells, and the expression of p-IκB was significantly increased, which further led to the activation of NF-κB and a significant increase in the expression of p-NF-κB. Immunohistochemistry also showed significantly higher p-NF-κB expression in aortic plaques of Apoe−/−/Sirt4−/− mice than in controls. This phenomenon reasonably explained the changes of lipid accumulation and chemokines and inflammatory factors mentioned above. Afterward, the effect of NF-κB was blocked by its inhibitor BAY 11–7082, and the result of Western bloting was completely in contrast to the previous result. In addition, we observed nuclear translocation of NF-κB. Compared with the control group, Sirt4 deficient cells had obvious NF-κB nuclear translocation only after ox-LDL treatment; however, no obvious NF-κB nuclear translocation was observed in the two groups after BAY pretreatment. The same results were obtained by Oil Red O staining; that is, Sirt4-deficient macrophages had stronger lipid phagocytosis ability than the control group, however, when we blocked the effect of NF-κB, the lipid phagocytosis ability of Sirt4-deficient macrophages decreased significantly and was much lower than that of the control group. Concurrently, we detected whether the expression of CXCL2/3 and related inflammatory cytokines changed after BAY pretreatment. As expected, when the effect of NF-κB was inhibited, the expression of chemokines and inflammatory factors was reversed. Our series of studies have shown that Sirt4 deficiency activates the NF-κB/IκB pathway, which promotes the development of AS by aggravating inflammatory response, suggesting that Sirt4 exhibits a protective effect in AS.
Author contributions
Bo Li and Yunshan Wang designed the study; Shuting Chang and Guanzhao Zhang performed the experiments and wrote the original manuscript; Lanlan Li revised the manuscript; and Haiying Li analyzed the data and drew the figures; Bo Li and Xiaodong Jin provided financial support for this study.
Declaration of competing interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (Grant No. 81700321) and Natural Science Foundation of Shandong Province (Grant No. ZR2020MH025).
Appendix A. Supplementary data
The following is the Supplementary data to this article:
RIPK1 expression associates with inflammation in early atherosclerosis in humans and can Be therapeutically silenced to reduce NF-kappaB activation and atherogenesis in mice.
Adventitial CXCL1/G-CSF expression in response to acute aortic dissection triggers local neutrophil recruitment and activation leading to aortic rupture.
Circulating levels of sirtuin 4, a potential marker of oxidative metabolism, related to coronary artery disease in obese patients suffering from NAFLD, with normal or slightly increased liver enzymes.
Danlou tablet inhibits the inflammatory reaction of high-fat diet-induced atherosclerosis in ApoE knockout mice with myocardial ischemia via the NF-kappaB signaling pathway.
RIPK1 expression associates with inflammation in early atherosclerosis in humans and can Be therapeutically silenced to reduce NF-κB activation and atherogenesis in mice.
Low doses of lipopolysaccharide and minimally oxidized low-density lipoprotein cooperatively activate macrophages via nuclear factor kappa B and activator protein-1: possible mechanism for acceleration of atherosclerosis by subclinical endotoxemia.
Cholesterol crystals promote endothelial cell and monocyte interactions via H(2)O(2)-mediated PP2A inhibition, NFkappaB activation and ICAM1 and VCAM1 expression.
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