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 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 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 investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the synthesis of hyaluronic acid (HA) in human aortic vascular smooth muscle cells (HAVSMC), and explore its molecular mechanism. Methods The T/G HAVSMC were cultured in vitro with different concentration of ox-LDL(0,5, 0,5, 100 mg/L), the CCK-8 method was used to measure the cell proliferation and grouping analysis was carried out. T/G HAVSMC were treated with ox-LDL at concentrations of 25 and 50 mg/L for 48 hours. Natural LDL (N-LDL) group (50 mg/L N-LDL) and control group were set up. HPLC was used to determine the HA content, real-time quantitative PCR was used to detect the mRNA level of hyaluronic acid synthetase 2 (HAS2) and hyaluronic acid synthase 3 (HAS3), Western blot was used to detect the protein level of lectin-like oxidized low density lipoprotein recepter-1 (LOX-1), low-density lipoprotein receptor-related protein-1 (LRP), scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX) and cluster of differentiation 36 (CD36). Results After 48 h intervention, ox-LDL had no significant cytotoxicity on T/G HAVSMC cells. After 25 and 50 mg/L ox-LDL intervention for 48 h, the content of HA were significantly higher than those in the N-LDL group and the control group(P<0.05), the mRNA expression levels of HAS2 and HAS3 were significantly higher than those in the N-LDL group and the control group(P<0.05), and there was no significant difference between the N-LDL group and the control group(P>0.05). The expression of LOX-1 in 25 mg/L ox-LDL group and 50 mg/L ox-LDL group was significantly higher than that in the N-LDL group and the control group(P<0.05), while the expression of LRP-1, SR-PSOX and CD36 showed no significant change(P>0.05). Conclusion ox-LDL could induce the synthesis of HA in human aortic smooth muscle cells, and its mechanism may be related to the combination of LOX-1.

Abstract:Aim To investigate the effect of Xuezhikang (XZK) on apoptosis induced by oxidized low density lipoprotein (ox-LDL) and its possible mechanism. Methods Human umbilical vein endothelial cells (HUVEC) were cultured and divided into three groups, namely control group, ox-LDL group and ox-LDL+XZK(different dosages)groups.Cell counting kit-8 was used to test cell survival, Annexin V-FITC/PI apoptosis detection kit was used to count apoptotic rate of HUVEC among different groups, reactive oxygen species (ROS) were detected by ROS assay kit, expression of cytochrome C (CytC), caspase-3 and PARP-1 were analyzed by Western blot. Results XZK (25 mg/L, 50 mg/L and 100 mg/L) showed an antagonistic effect on apoptosis and ROS production of HUVEC induced by ox-LDL, and 100 mg/L XZK downregulated the protein expression of CytC, caspase-3 and PARP-1. Conclusion XZK reduces ROS production, leading to less expression of CytC, caspase-3 and PARP-1, thus prevents mitochondrial apoptotic signaling from activating, and eventually protects HUVEC from apoptosis.

Abstract:Aim To explore whether the hxeroderma pigmentosum D(XPD) could mediate the apoptosis of human umbilical vein endothelial cells (HUVEC) promoted by oxidized low density lipoprotein (ox-LDL). Methods The model of apoptosis of HUVEC was established by ox-LDL. XPD-siRNA was transfected into HUVEC with liposome,followed by treatment with ox-LDL. This experiment was divided into six groups:blank control group; negative control siRNA group; XPD-siRNA group; ox-LDL group; ox-LDL+negative control siRNA group; ox-LDL+XPD-siRNA group. Cell vitality was detected by MTT; Cell apoptosis rate and cell cycle were assessed with flow cytometry; The expressions of the XPD, Bax and Bcl-2 were detected by RT-PCR and Western blot. Results The optimal concentration of ox-LDL to establish the model of apoptosis of HUVEC was 100 mg/L. Compared with negative control siRNA group, the cell apoptosis rate of XPD-siRNA group was significantly decreased (P<0.05), the survival rate was significantly increased (P<0.01), the cell number in G0/G1 phase decreased (P<0.05), while increased in S phase (P<0.05), the expressions of XPD and Bax were declined (all P<0.05), while the expression of Bcl-2 was elevated (P<0.05); Compared with blank control group, the cell apoptosis rate of ox-LDL group increased significantly (P<0.01), the survival rate was decreased (P<0.05), the cell number in G0/G1 phase increased (P<0.05),while decreased significantly in S phase (P<0.01), the expressions of XPD and Bax were elevated (all P<0.05),while the expression of Bcl-2 was declined (P<0.05). Compared with ox-LDL+negative control siRNA group,the cell apoptosis rate of ox-LDL+XPD-siRNA group was reduced (P<0.05),the survival rate was increased(P<0.01), the cell number in G0/G1 phase decreased (P<0.05),while increased in S phase (P<0.05), the expressions of XPD and Bax were declined (all P<0.05), while the expression of Bcl-2 was elevated (P<0.05). Conclusion XPD can mediate the effect of ox-LDL on promoting the apoptosis of HUVEC.

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.