Abstract:Atherosclerosis is an important factor in cardiovascular events. Relevant epidemiological surveys show that the burden of atherosclerotic cardiovascular disease in China has increased rapidly and significantly in recent years, which has brought major challenges to Chinas public health problems. Myocardin-related transcription factor-A (MRTF-A) plays an important role in the development of atherosclerosis, including participating in inflammatory responses, promoting lipid accumulation, and phenotypic transformation of vascular smooth muscle cells (VSMC) in the process of atherosclerosis, and exploring the correlation between MRTF-A and atherosclerosis is of great significance for finding new therapeutic targets. This article reviews the role of MRTF-A in the process of atherosclerosis lesions, which can provide a reference for targeted therapy of coronary atherosclerosis.

Abstract:Aim To investigate the mechanism of perilipin 2 (PLIN2) increasing lipid accumulation in macrophages. Methods The experiments were divided into oxidized low density lipoprotein (ox-LDL) group, different PLIN2 expression groups, and different activity Rab18 groups, the levels of Rab18 and acyl-CoA long chain synthetase 3 (ACSL3) protein in high expression and silent expression of PLIN2 macrophages were measured, and the levels of PLIN2, Rab18, and ACSL3 protein expression in high expression PLIN2 macrophages with different activity Rab18 were measured.Western blot was used to detect protein expression levels, immunofluorescence was used to observe the localization of intracellular related proteins, and oil red O staining was used to observe intracellular lipid accumulation. Results The expression levels of Rab18 and ACSL3 in cells with high expression of PLIN2 were increased significantly (P＜0.05), and there was an intracellular phenomenon of co-localization of PLIN2 with Rab18 and ACSL3. The expression level of ACSL3 in high expression macrophages of PLIN2 after transfection with the Rab18 dominant mutant Q67L plasmid (Rab18 activity increased) was increased significantly (P＜0.05), and the number of intracellular lipid droplets was also increased significantly (P＜0.05). Conclusion PLIN2 promotes macrophage lipid accumulation through Rab18 by up-regulating ACSL3.

Abstract:Aim To investigate the effects of astragalus polysaccharide and hirudin combined intervention on lipid accumulation, mitochondrial membrane potential and apoptosis-related proteins in macrophages induced by oxidized low density lipoprotein (ox-LDL). Methods RAW264.7 cells were incubated with 100 mg/L ox-LDL for 24 h to establish a foam-cell model, which were treated by astragalus polysaccharide and hirudin after dose optimization. The control group, model group, astragalus polysaccharide group, hirudin group and the combined intervention of the two drugs group were established. Oil red O staining and oxidase method were used to detect the cholesterol contents in macrophages induced by ox-LDL, flow cytometry was used to detect the early apoptosis rate, late apoptosis rate and total apoptosis rate of macrophages, the changes of mitochondrial membrane potential were detected by laser confocal microscopy, Western blot was used to detect the expression levels of anti-apoptotic protein Bcl-2, proapoptotic protein Caspase-3 and Bax. Results The contents of cholesterol in macrophages of the astragalus polysaccharide and hirudin combined intervention group were decreased than those in the hirudin group or astragalus polysaccharide group significantly (P＜0.05). Compared with the model group, the astragalus polysaccharide and hirudin combined intervention could reduce the early apoptosis rate and total apoptosis rate of macrophages induced by ox-LDL (P＜0.01), increase the mitochondrial membrane potential of macrophages (P＜0.01), reduce the expressions of Caspase-3 and Bax, and increase the expression of Bcl-2 protein (P＜0.05) significantly. Conclusions The astragalus polysaccharide and hirudin combined intervention in reducing lipid accumulation in macrophages is superior to drug treatment alone. The combined treatment of the two drugs can reduce the apoptosis rate of macrophages induced by ox-LDL. Its mechanism may be related to regulate mitochondrial membrane potential and regulate the expressions of proapoptotic protein Caspase-3, Bax and anti-apoptotic protein Bcl-2.

Abstract:Aim To investigate the relationship between visceral adipose index (VAI) and lipid accumulation index(LAP) and carotid atherosclerosis (CAS) in high risk stroke patients. Methods The source of the case was 9 215 cases of high risk stroke population screened by 8 community hospitals in Fengtai District, Beijing. Collect complete demographic information and TCM syndrome scale through face-to-face questionnaire survey. Test items included physical examination, blood test and carotid ultrasound examination. The two indexes of VAI and LAP can be calculated by the formula. Statistical methods was used to explore the correlation between various indicators and carotid atherosclerosis, and stratified research was carried out. Results 9 215 subjects (mean age 60±9 years, 61.4% of women) were analyzed. The prevalence of carotid atherosclerosis was 74.7%. The levels of waist circumference, waist to height ratio (WHtR), female VAI, and female LAP was significantly higher in CAS group than those in control group (P＜0.001). Female visceral obesity replacement indexes were significantly correlated with carotid atherosclerosis (P＜0.001). Multivariate Logistic regression showed that age, gender (female), previous stroke history, hypertension, diabetes, smoking, and lack of physical exercise were independent risk factors for CAS in high risk stroke populations. Conclusion High high density lipoprotein cholesterol (HDLC) levels, eating vegetables, and fruits have a protective effect on carotid atherosclerosis. Female visceral obesity is significantly positively correlated with carotid atherosclerosis in stroke populations. The main risk factors affecting the formation of CAS are VAI, age, gender (female), history of previous stroke, hypertension, diabetes, smoking, lack of physical exercise.

Abstract:Aim To investigate the effect and mechanism of metformin on lipid accumulation in Huh7 hepatocytes. Methods Huh7 cells were cultured in vitro and divided into:blank control group, 5 mmol/L, 10 mmol/L and 15 mmol/L metformin treatment group. After determing the optimal concentration, LDL was used to load fat, and then divided into:blank control group, LDL group, LDL+15 mmol/L metformin treatment group. Cell proliferation and toxicity test kits were used to detect cell survival rate, oil red O and BODIPY lipid probes were used to detect intracellular lipid content, Dil-LDL was used to detect cell lipid uptake capacity, and Western blot analysis was performed on sterol regulatory element binding protein-2 (SREBP-2), sterol regulatory element binding protein-1c (SREBP-1c), low density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin 9 (PCSK9) protein expression. Results Metformin can inhibit LDL-induced lipid accumulation in Huh7 hepatocytes and reduce protein expression of SREBP-2, SREBP-1c, LDLR, and PCSK9. Conclusion Metformin reduces intracellular lipid uptake by reducing LDLR expression and inhibiting the expression of the transcription factors SREBP-2 and SREBP-1c.

Abstract:Aim To investigate the mechanism of fibroblast growth factor 21 (FGF21) in promoting cholesterol efflux from foam cells. Methods THP-1 derived foam cells were treated with FGF21 at different concentrations (0,0, 0,0, 400 μg/L) for 24 hours and 200 μg/L FGF21 at different time (0,6, 2,4, 48 h), Western blot, laser confocal were used to detect LC3, and MDC staining for autophagosomes analysis, HPLC, oil red O staining for intracellular cholesterol accumulation measurement, and liquid scintillation counting for cholesterol efflux detection. Results After 200 μg/L, 400 μg/L FGF21 and 200 μg/L FGF21 were applied for 24 h and 48 h, the levels of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) in foam cells were significantly reduced, while their cholesterol efflux was significantly enhanced. Mechanism studies found that FGF21 induced the formation of autophagosomes in foam cells, and the conversion rate of microtubule-associated protein Ⅰ light chain3 (LC3-Ⅰ) to microtubule-associated protein Ⅱ light chain3 (LC3-Ⅱ) was significantly increased. However, after autophagy was inhibited with autophagy-related gene 5(ATG5) siRNA or 3-methyladenine (3-MA) or Bafilomyein A1 (BafA1), effect of FGF21 on decreasing TC, FC, CE and lipid accumulation, and increasing cholesterol efflux were reduced. Conclusion FGF21 promotes cholesterol efflux in foam cells by up-regulating autophagy.

Abstract:Aim To observe the targeting regulation of miR-497-5p on nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP1) and its effect on cholesterol efflux. Methods Bioinformatics and luciferase reporter gene assay were used to verify the targeted binding of miR-497-5p and NLRP1. Human THP-1 monocytes were induced into foam cells after treatment with phorbol ester and oxidized low density lipoprotein. miR-497-5p mimic and miR-497-5p inhibitor were used to treat cells. Real-time fluorescence quantitative PCR and Western blot were used to detect the expressions of NLRP1, cysteinyl aspartate specific proteinase 1 (Caspase-1) and apoptosis-associated speck-like protein containing a Caspase-recruitment domain (ASC). The contents of interleukin-1β (IL-1β) and IL-18 in cell culture medium were determined by enzyme-linked immunosorbent assay. Cholesterol efflux detection was performed by liquid scintillator. Lipid content in foam cells was detected by high performance liquid chromatography. Results The luciferase activity of wild type NLRP1 3′UTR reporter gene was significantly reduced by miR-497-5p mimic. Compared with the control group, the mRNA and protein expression levels of NLRP1, ASC and Caspase-1 in miR-497-5p mimic group were significantly down regulated, and the secretions of IL-1β and IL-18 was significantly reduced. Compared with the control group, miR-497-5p mimic significantly promoted the cellular cholesterol efflux and reduced the contents of total cholesterol, free cholesterol and cholesterol ester in cells. Conclusion miR-497-5p may inhibit the inflammatory response of macrophage-derived foam cells and promote cholesterol efflux by targeting regulation of NLRP1.

Abstract:Aim To explore the effect of sorting receptor Sortilin on lipid metabolism in THP-1 macrophages. Methods THP-1 macrophages were incubated with oxidized low density lipoprotein (ox-LDL). Change of Sortilin expression in lipid-loaded THP-1 macrophages were detected by Western blot. Under the overexpression and silence of Sortilin in THP-1 macrophages, intracellular cholesterol efflux was measured by liquid scintillation counting apparatus, intracellular lipid content was detected by high performance liquid chromatography, and the situation of intracellular lipid droplets was observed by oil red O staining. Results The expression of Sortilin in THP-1 macrophages was increased in the manners of dose- and time-dependence of ox-LDL. The expression level of Sortilin in THP-1 macrophages reached the peak after 24 hours treatment with 50 mg/L ox-LDL. Compared with the control group, after Sortilin overexpression, the cholesterol efflux of macrophages was decreased, the intracellular lipid content was increased, and the lipid droplets was increased significantly, while Sortilin was silent, it just happened to be the opposite. Conclusion Sortilin inhibits the cholesterol efflux of macrophages and promotes the accumulation of intracellular lipid.

Abstract:Aim The previous work showed that momordicin(MD28) can up-regulate the expression of ATP-binding cassette transporter A1 (ABCA1) in THP-1-derived foam cells and reduce the intracellular lipid accumulation, but its mechanism is unclear. This study is to analyze the mechanism of action from the post-transcriptional level. Methods MiRNA chip was used to analyze the microRNA (miRNA) that were down-regulated by 1.5-fold in the miRNA of THP-1-derived foam cells treated with 50 mg/L ox-LDL for 48 h after the intervention of MD28 and compared with the genecards miRNA which regulates the ABCA1 gene expression. Of common miRNA, target gene relationship between this miRNA and ABCA1 was verified by the luciferase reporter gene system. qRT-PCR was used to detect ABCA1 mRNA and miR-23b-3p expression levels, protein was detected by Western blot, oil red O staining was used to detect intracellular lipid accumulation. Results miR-23b-3p was the intersection of miRNA chip and genecards. The target gene validation experiment confirmed that ABCA1 was the target gene of miR-23b-3p. MD28 dose-dependently (0 g/L, 0.4 g/L, 1.2 g/L, 3.6 g/L, 5 g/L) and time-dependently (0 h, 6 h, 12 h, 24 h, 48 h) up-regulated ABCA1 expression, the highest expression level of ABCA1 was at 1.2 g/L for 12 h of MD28. ox-LDL up-regulated the expression of miR-23b-3p and was inhibited by MD28, while MD28 decreased intracellular lipid accumulation, and the inhibitor of miR-23b-3p antagonized the effect of MD28 on ABCA1 expression and intracellular lipid accumulation. Conclusion MD28 can up-regulate the expression of ABCA1 and decrease the intracellular lipid accumulation in THP-1 macrophage-derived foam cells by decreasing the expression of miR-23b-3p.

Abstract:Aim To explore whether nuclear factor kappa B (NF-κB) manipulates Sortilin expression to regulate lipid metabolisms and foam cell formation of lipid-laden THP-1 macrophage. Methods The levels of Sortilin mRNA and protein were detected by quantitative real-time PCR (qRT-PCR) and Western blot in lipid-laden THP-1 macrophage incubated with NF-κB activator phorbol-12-myristate-13-acetate (PMA) or its specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC). Under the lipid-laden THP-1 macrophage treated with Sortilin shRNA and together with PMA, cholesterol efflux from macrophage was measured by liquid scintillation counting apparatus, intracellular lipid contents were detected by high performance liquid chromatography, and the intracellular lipid droplets were stained with oil red O. Results PMA treatment increased the levels of Sortilin mRNA and protein in lipid-laden THP-1 macrophage, whereas PDTC incubation decreased macrophage Sortilin expression. When lipid-laden THP-1 macrophage was treated with PMA alone, the cholesterol efflux of macrophages was reduced, the intracellular lipid accumulation was increased, and foam cell formation was increased. Reversely, the cholesterol efflux of lipid-laden macrophage was increased, the intracellular lipid accumulation was decreased, and foam cell formation was reduced under the treatment with Sortilin shRNA and supplemented with PMA. Conclusion NF-κB promotes Sortilin expression to inhibit the cholesterol efflux from lipid-laden macrophage and accelerates the accumulation of intracellular lipid and the formation of foam cell.