Abstract:Aim To observe the effects of receptor interacting protein 2 (RIP2) on macrophage inflammatory activation and polarity transformation, and to explore the mechanism of RIP2 in macrophage phagocytosis of oxidized low density lipoprotein (ox-LDL). Methods THP-1 derived macrophages were treated with different doses (0,5 and 50 mg/L) of ox-LDL for 24 hours, and treated with 50 mg/L ox-LDL for 8,6 and 24 hours. Real-time quantitative PCR and Western blot were used to detect the expression of RIP2 mRNA and protein in THP-1 derived macrophages, and ELISA was used to detect the secretion of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). Three pairs of RIP2 siRNA were designed, transfecting them into cells using hiperfict transfection reagent, real-time quantitative PCR and Western blot were used to detect the expression of RIP2 mRNA and protein in THP-1 derived macrophages after transfection, in order to screen for the optimal siRNA transfection concentration and the most effective pair of siRNA.After transfection with the most effective RIP2 siRNA, cells were treated with 50 mg/L ox-LDL for 24 hours, ELISA was used to detect the secretion of TNF-α, MCP-1, interleukin-10 (IL-10) and interleukin-12 (IL-12), real-time quantitative PCR was used to detect the expression of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), flow cytometry was used to detect the expression of cell surface antigens CD86, CD80 and CD163. Results Ox-LDL induced the expression of RIP2 in macrophages in a dose-dependent and time-dependent manner. With the increase of ox-LDL treatment dose and time, the expression of RIP2 mRNA and protein increased. Among them, the expression of RIP2 protein in the 50 mg/L group was 7.6 times of the control group, and the expression of RIP2 protein in the 24 h group was 17.9 times of the control group (P＜0.001). The ELISA results showed that with the increase of ox-LDL treatment dose and time, the secretion of TNF-α and MCP-1 increased (P＜0.05). After transfection of RIP2 siRNA into cells, ELISA results showed that the secretion of TNF-α, MCP-1 and IL-10 in the ox-LDL group was 2.4 times, 2.9 times and 1.8 times of the control group (P＜0.01), and the secretion of IL-12 decreased by 34.2% compared to the control group (P＜0.01); the secretion of TNF-α, MCP-1 and IL-10 in the siRNA+ox-LDL group decreased by 37.4%, 45.3% and 27.4%, respectively, compared to the ox-LDL group (P＜0.01), while the secretion of IL-12 increased by 31.4% (P＜0.05). The results of flow cytometry and real-time quantitative PCR showed that the expression of CD86, CD80 and iNOS mRNA in the ox-LDL group was 14.2 times, 33.8 times and 4.5 times of those of the control group, respectively, while the expression of CD163 and Arg-1 mRNA decreased by 33.4% and 43.0%, respectively, compared with the control group (P＜0.05); the expression of CD86, CD80 and iNOS mRNA in the siRNA+ox-LDL group decreased by 27.6%, 29.3% and 34.3%, respectively, compared to the ox-LDL group, while the expression of CD163 and Arg-1 mRNA increased by 30.3% and 38.6%, respectively (P＜0.05). Conclusion RIP2 expression in macrophages can be induced by ox-LDL in a dose-dependent and time-dependent manner. RIP2 gene silencing can inhibit ox-LDL induced M1 macrophage transformation.

Abstract:Aim To investigate the influences of ginkgetin on the ferroptosis of vascular endothelial cells induced by oxidized low density lipoprotein (ox-LDL) by regulating the nuclear factor erythroid-2 related factor 2 (Nrf2)/solute carrier protein 7 family member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling pathway. Methods Human umbilical vein cell fusion cells EA.hy926 were grouped into normal control group (normal culture), ox-LDL group (50 mg/L ox-LDL), and ginkgetin low dose group (50 mg/L ox-LDL+10 μmol/L ginkgetin), middle dose group (50 mg/L ox-LDL+20 μmol/L ginkgetin), high dose group (50 mg/L ox-LDL+40 μmol/L ginkgetin), ML385 group (50 mg/L ox-LDL+40 μmol/L ginkgetin+1 μmol/L Nrf2 inhibitor ML385), Erastin group (50 mg/L ox-LDL+40 μmol/L ginkgetin+5 μmol/L SLC7A11 inhibitor Erastin), RSL3 group (50 mg/L ox-LDL+40 μmol/L ginkgetin+0.5 μmol/L GPX4 inhibitor RSL3). Cell viability was detected by tetramethylazolium salt (MTT) method. The kits were applied to detect the levels of cellular superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH). The intracellular iron content was detected by specific fluorescent probe method. The levels of intracellular reactive oxygen species (ROS) and lipid ROS were detected by 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe method and boron dipyrrole (BODIPYTM) method; Western blot was applied to detect the protein expressions of cellular Nrf2, SLC7A11, GPX4,4-hydroxynonenoic acid (4-HNE), cyclooxygenase 2 (COX2), and p53. Results Compared with the normal control group, the cell viability, SOD content, GSH content, expressions of Nrf2, SLC7A11 and GPX4 were obviously decreased in the ox-LDL group (P＜0.05); the MDA content, Fe2＋ content, ROS, lipid ROS, expressions of 4-HNE, COX2, p53 were obviously increased (P＜0.05). Compared with the ox-LDL group, the cell viability, SOD content, GSH content, expressions of Nrf2, SLC7A11 and GPX4 were obviously increased in low, middle and high dose groups of ginkgetin (P＜0.05); the MDA content, Fe2＋ content, ROS, lipid ROS, expressions of 4-HNE, COX2, p53 were obviously decreased (P＜0.05). Compared with the high dose ginkgetin group, the ML385 group, Erastin group and RSL3 group attenuated the inhibitory effect of ginkgetin on ox-LDL-induced vascular endothelial cell ferroptosis. Conclusion Ginkgetin inhibits ox-LDL-induced vascular endothelial cell ferroptosis by activating the Nrf2/SLC7A11/GPX4 pathway.

Abstract:Aim To explore the clinical value of transcutaneous oxygen pressure (TcPO2) combined with serum oxidized low density lipoprotein (ox-LDL) in predicting restenosis after interventional therapy for lower arteriosclerosis obliterans (LASO). Methods 113 patients with LASO underwent interventional therapy in Yueyang Integrated Traditional Chinese and Western Medicine Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from January 2020 to June 2021 were enrolled, and classified into non-stenosis group (n＝79) and restenosis group (n＝34) according to the occurrence of restenosis 1 year after interventional treatment. Serum ox-LDL levels were detected by ELISA and corresponding assay kits, and TcPO2 was measured by laser Doppler flowmeter. Then comparison was conducted on general data, TcPO2 and ox-LDL. Multivariate Logistic regression model was used to screen the influencing factors of restenosis after intervention therapy for LASO. Spearman test was used for correlation analysis, ROC curve was used to evaluate the efficacy of TcPO2 and ox-LDL in predicting restenosis in LASO patients after intervention therapy. Results There was no significant difference between the two groups in age, body mass index (BMI), sex, alcohol consumption, history of cerebrovascular disease and coronary heart disease, number of diseased vessels, fasting blood glucose, and homocysteine (P＞0.05). Compared with the non-stenosis group, smoking, irregular drug use, the number of implanted stents, ox-LDL, and the level of blood uric acid in the restenosis group were significantly higher (P＜0.05), and TcPO2 was significantly lower (P＜0.05). Logistic regression model analysis showed that smoking, irregular drug use after LASO intervention, large number of implanted stents, decreased TcPO2, ox-LDL, and increased blood uric acid were the risk factors for restenosis after LASO intervention (P＜0.05). Spearman test showed that ox-LDL was positively correlated with restenosis after LASO intervention (r＝0.513, P＜0.001), and TcPO2 was negatively correlated with it (r＝－0.524, P＜0.001). The ROC curve showed that the predictive efficacy of TcPO2＋ox-LDL in predicting restenosis after LASO interventional therapy (AUC＝0.802) was higher than that of each indicator alone. The predictive sensitivity and specificity were 67.60% and 94.90% respectively, and the critical point was 37.23 mmHg and 5.31 mmol/L. Conclusion TcPO2 is decreased and ox-LDL is increased in patients with restenosis after interventional therapy for LASO, both indicators have certain predictive value for restenosis, and combined detection of the two can reflect restenosis condition more completely.

Abstract:Aim To investigate the correlation between small dense low density lipoprotein (sdLDL), high sensitivity C-reactive protein (hs-CRP), mean platelet volume/platelet count (MPV/PLT) and the severity of coronary artery lesions in patients with coronary heart disease. Methods From August 2020 to August 1,0 patients with coronary heart disease who were hospitalized in the Department of Cardiology of Shenzhen Third Peoples Hospital and underwent coronary angiography were selected. According to the results of coronary angiography, they were divided into single-vessel lesion group (n＝50), double-vessel lesion group (n＝50), and multi-vessel lesion group (n＝50). According to clinical classification, they were divided into stable angina pectoris group (SAP group, n＝26), unstable angina pectoris group (UAP group, n＝48), and acute myocardial infarction group (AMI group, n＝76). Serum levels of sdLDL, hs-CRP, and MPV/PLT were measured among various subgroups, and their correlation with the number and clinical classification of coronary artery disease was analyzed. The ROC curve was used to analyze their effectiveness in predicting the severity of coronary artery disease. Results ①The levels of serum sdLDL, hs-CRP and MPV/PLT in the multi-vessel lesion group were 1.2,1.96 and 1.16 times of those in the double-vessel lesion group (all P＜0.05), and 2.8,3.32 and 1.50 times of those in the single-vessel lesion group (all P＜0.05). Serum sdLDL, hs-CRP and MPV/PLT levels in the double-vessel lesion group were 1.7,1.69 and 1.29 times higher than those in the single-vessel lesion group (all P＜0.05). Serum sdLDL and MPV/PLT levels in AMI group were 1.39 and 1.29 times higher than those in UAP group (all P＜0.05), and 1.37 and 1.38 times higher than those in SAP group (all P＜0.05). There were no significant differences in serum sdLDL and MPV/PLT levels between UAP group and SAP group (P＞0.05). The serum hs-CRP level in AMI group and UAP group was 2.59 and 1.85 times of that in SAP group (both P＜0.05), but there was no statistical significance in hs-CRP level between AMI group and UAP group (P＞0.05). ②Ordered Logistic regression analysis showed that increased levels of serum sdLDL, hs-CRP and MPV/PLT were independent risk factors for predicting the severity of coronary heart disease. ③Spearman correlation analysis showed that serum sdLDL, hs-CRP and MPV/PLT levels were not only positively correlated with the number of coronary lesions (correlation coefficients were 0.5,0.569 and 0.495, respectively, P＜0.05), but also positively correlated with clinical classification of coronary heart disease (correlation coefficients were 0.2,0.40 and 0.414, P＜0.05). ④The ROC curve showed that the combined detection of serum sdLDL, hs-CRP and MPV/PLT was more effective in predicting the severity of coronary heart disease than that of a single indicator detection. Conclusion Increased levels of serum sdLDL, hs-CRP and MPV/PLT are independent risk factors for predicting the severity of coronary heart disease, and are positively correlated with the number of coronary artery lesions and clinical classification of coronary heart disease.

Abstract:Aim To explore the effects of rapamycin liposomes (RL) on the migration of human aortic vascular smooth muscle cells (HA-VSMC) induced by oxidized low density lipoprotein (ox-LDL) and its mechanism related to S100 calcium binding protein A4 (S100A4). Methods Vascular smooth muscle cells were transfected with lentivirus knockdown S100A4 gene, and then added puromycin to screen stable strain of S100A4 gene knockdown. Vascular smooth muscle cells were treated with 50 mg/L ox-LDL, and different doses of RL (3,6 and 12 mg/L) were added to observe the effect on cell migration before and after treatment. Cell migration was detected by cell scratch method and Transwell, and the expression of S100A4, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), typeⅠcollagen protein (COLⅠ), and vimentin were detected by Western blot. Results After 48 h treatment with ox-LDL, compared with the blank control group, the expression of S100A4, p-PI3K, p-Akt, p-mTOR, COLⅠ and vimentin was significantly increased (P＜0.05), and the speed of cell migration was significantly accelerated (P＜0.05). Compared with ox-LDL group, different doses of RL significantly inhibited the expression of S100A4, p-PI3K, p-Akt, p-mTOR, COLⅠ and vimentin and significantly inhibited cell migration after 48 h treatment (P＜0.05), of which 6 mg/L and 12 mg/L of RL had more significant inhibitory effects (P＜0.05). After S100A4 gene knockdown, the cell migration rate was significantly reduced (P＜0.05). Conclusion RL can significantly inhibit the migration of HA-VSMC induced by ox-LDL, which may be related to the down-regulation of S100A4, p-PI3K, p-Akt, p-mTOR, COLⅠ and vimentin expression by RL.

Abstract:Aim To explore the role of circular RNA spindle and kinetochore-associated protein 3 (circ-SKA3) in regulating Toll-like receptor 4 (TLR4) axis in atherosclerosis (As) through miR-1303. Methods Carotid artery plaque, diseased vascular tissue, normal tissue adjacent to plaque and venous blood were collected from 30 patients with As treated by carotid endarterectomy from April 2019 to April 2022. Another 30 normal venous blood samples were collected. Microarray analysis, quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization (FISH) were used to detect the expression and localization of circ-SKA3 in plaque tissue of As patients, control samples, plasma exosome, human umbilical vein endothelial cell (HUVEC) and aorta of As model mice. The relationship among circ-SKA3, miR-1303 and TLR4 was verified by bioinformatics, double luciferase reporter gene detection and RNA immunoprecipitation. The proliferation, migration and angiogenesis of HUVEC were detected by CCK-8, scratch, Transwell and angiogenesis experiments. TLR4 axis-related protein expression was detected by Western blotting. Pathological changes of aorta in As model mice was observed by Oil red O staining, HE staining and Masson staining. TLR4 expression in aorta of As model mice was detected by immunohistochemistry and immunofluorescence. Results The expression levels of circ-SKA3 and TLR4 in plaque tissue, plasma exosome, oxidized low density lipoprotein (ox-LDL) treated HUVEC and circ-SKA3, TLR4 in aortic of As model mice were up-regulated (P＜0.05), while the expression level of miR-1303 was down-regulated (P＜0.05). Functional analysis showed that circ-SKA3 promoted HUVEC damage in vitro and As progress in vivo. Mechanism analysis showed that circ-SKA3 could promote TLR4 expression by adsorbing miR-1303 by sponge. Inhibition of circ-SKA3/miR-1303/TLR4 axis can inhibit the formation of As lesions. Conclusions circ-SKA3 is overexpressed in carotid plaque, plasma exosome, ox-LDL-treated HUVEC and As model mouse aorta in As patients. circ-SKA3/miR-1303/TLR4 axis can promote the development of As model in vivo and in vitro.

Abstract:Aim To investigate the relationship between small and dense low density lipoprotein cholesterol (sdLDLC) and atherogenic index of plasma (AIP) in patients with coronary heart disease (CHD). Methods A total of 525 patients admitted to our hospital due to chest pain and chest tightness from September 2019 to June 2020 and suspected CHD underwent coronary angiography were retrospectively collected as the research subjects. According to coronary angiography results, they were divided into CHD group (n＝422) and non-CHD group (n＝103). At the same time, they were divided into three groups according to AIP tertiles:AIP≤－0.056 group (n＝176), －0.056＜AIP＜0.208 group (n＝175), AIP≥0.208 group (n＝174). Each lipid index was measured and AIP was calculated. sdLDLC and AIP levels were compared between CHD group and non-CHD group. Influencing factors for AIP and risk factors for CHD were analyzed. Results The sdLDLC, AIP, low density lipoprotein (LDL) subtype LDL3, LDL4 in CHD group were significantly higher than those in non-CHD group, and the LDL1, high density lipoprotein cholesterol in CHD group were significantly lower than those in non-CHD group (P＜0.05). With the increase of AIP, sdLDLC and LDL4 increased significantly, while LDL1 and LDL2 decreased significantly (P＜0.001). Multiple linear regression analysis showed that sdLDLC and history of hypertension were independent influencing factors of AIP (P＜0.05). Multivariate Logistic regression analysis showed that sdLDLC and AIP were independent risk factors for CHD (P＜0.05). Conclusion There is a significant positive correlation between sdLDLC and AIP in CHD patients, and sdLDLC and AIP are independent risk factors for CHD.

Abstract:Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serine protease, which is mainly expressed in hepatocytes. PCSK9 binds to low density lipoprotein receptor (LDLR) forming PCSK9-LDLR complex, and leads to LDLR degradation in lysosomes, which finally results in the reduction of cell membrane LDLR protein level and the elevation of plasma low density lipoprotein cholesterol (LDLC) level. PCSK9 inhibitors have become a hot spot in the research of hypercholesteremia and atherosclerotic cardiovascular disease (ASCVD) drugs discovery. There are three globally marketed PCSK9 inhibitors including evolocumab, alirocumab and PCSK9 siRNA drug inclisirian (Leqvio). This article reviewed the structure, functions and inhibitors of PCSK9.

Abstract:Aim To evaluate the diagnostic value of high resolution nuclear magnetic resonance imaging (HR-MRI), oxidized low density lipoprotein (ox-LDL) and lipoprotein associated phospholipase A2 (Lp-PLA2) in serum for the prognosis of patients with middle cerebral atherosclerotic stenosis (MCAS). Methods From August 2015 to August 7,6 patients (stenosis group) who were diagnosed as cerebral infarction by neurology department, and diagnosed as MCAS by transcranial Doppler (TCD), magnetic resonance imaging (MRI), magnetic resonance angiography (MRA) and clinical manifestations were selected, and another 90 patients with normal physical examination were selected as control group. The parameters of plaque size and plaque load were obtained by HR-MRI vascular wall imaging technology. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of ox-LDL and Lp-PLA2 in serum. After 3 months of treatment, the prognosis was assessed by modified Rankin Scale (mRS scores ＞2 was poor prognosis), and the patients were divided into 85 cases of poor prognosis group and 21 cases of good prognosis group. Pearson method was used to analyze the correlation between plaque size, plaque load, levels of ox-LDL and Lp-PLA2 in serum and mRS scores. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic effect of HR-MRI combined with serum ox-LDL and Lp-PLA2 on the prognosis of patients with MCAS. Results Compared with the control group, the plaque size, plaque load, serum ox-LDL and Lp-PLA2 levels in the stenosis group were significantly increased (P＜0.05). Compared with the good prognosis group, the plaque size, plaque load, serum ox-LDL and Lp-PLA2 levels in the poor prognosis group were significantly increased (P＜0.05). Plaque size, plaque load, serum ox-LDL and Lp-PLA2 levels were all positively correlated with mRS scores (r＝0.5,0.4,0.7,0.679, P＜0.05), and have high diagnostic efficiency, the combined detection was more effective in predicting the prognosis of patients with MCAS. Conclusion Plaque size, plaque load, serum ox-LDL and Lp-PLA2 levels have high diagnostic efficacy for the prognosis of MCAS patients, and the diagnostic efficacy of combined detection is higher, HR-MRI vascular wall imaging combined with serum ox-LDL and Lp-PLA2 levels has certain reference value for clinical diagnosis of MCAS patients.

Abstract:Aim To explore the expression of proprotein convertase subtilisin kexin 9 (PCSK9) in polycystic ovary syndrome (PCOS) ovarian granulosa cells, and the relationship between PCSK9 expression and the lipid status of PCOS ovarian tissue and testosterone-treated KGN cells. Methods A rat model of PCOS was constructed by letrozole combined with high-fat diet. Oil red O staining was used to observe the lipid distribution in the ovarian tissue of PCOS rats. Transcriptome data from PCOS ovarian tissue and normal ovarian tissue was analyzed by bioinformatics, and the differentially expressed genes involved in lipid metabolism pathway were screened. Western blot and immunohistochemical techniques were used to detect the protein expression of PCSK9 in PCOS rat tissues. Immunofluorescence, qRT-PCR and Western blot techniques were used to detect the expression differences of PCSK9 and low density lipoprotein receptor (LDLR) in KGN cells treated with different concentrations of testosterone. Immunofluorescence microscope was used to observe the ability of KGN cells uptake Dil-LDL. Results The results of Oil Red O staining showed that the distribution of lipid droplets in granular cells of PCOS ovarian tissue was significantly less than that of normal ovarian tissue. The results of bioinformatics analysis showed that the up-regulated expression of PCSK9 in the ovarian tissue of PCOS rats was mainly involved in the cholesterol metabolism pathway. The results of immunohistochemistry and Western blot showed that the PCSK9 protein was mainly localized in granulosa cells and its expression increased in the granulosa cells of PCOS rats.The results of immunofluorescence, qRT-PCR and Western blot showed that PCSK9 expression was up-regulated with the increased concentration of testosterone, and LDLR expression was decreased with the increased concentration of testosterone in KGN cells. Testosterone (100 nmol/L) could reduce the uptake of Dil-LDL by KGN cells. Conclusion PCSK9 was highly expressed in PCOS ovarian granulosa cells, which could reduce the expression of LDLR protein and lead to insufficient lipid uptake of granulosa cells.