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AIM: To investigate the effect and mechanism of ursolic acid on high glucose and high-fat injury of human aortic endothelial cells. METHODS: MTT method was used to select the appropriate injury concentration of high glucose and sodium palmitate and UA pre incubation concentration. The levels of NO and ROS, the release rate of lactate dehydrogenase and the activities of antioxidant enzymes including total superoxide dismutase, catalase and glutathione peroxidase were detected by kit. The expression of endothelial nitric oxide synthase, intercellular adhesion molecule, vascular cell adhesion molecule caspase-1 and GSDMD were detected by Western blot. The protective effect of UA on HAEC was observed. Hoechst33342 combined with PI fluorescence staining was used to detect the whole state of cell membrane to explore the occurrence of pyroptosis. RESULTS: Pre-incubation with UA (1 and 5 μmol/L) could reduce the damage of HAEC caused by high glucose and high fat (30 mmol/L Glu + 0.1 mmol/L SPA), enhance HAEC activity, increase NO release and eNOS protein expression, alleviate oxidative stress injury, reduce the protein expression of adhesion molecules and reduce the occurrence of pyroptosis. CONCLUSION: UA may reduce the damage of endothelial cells by inhibiting the oxidative stress response and the occurrence of pyroptosis induced by high glucose and high fat.
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AIM: To investigate the effect of curcumin on oxidized low-density lipoprotein (ox-LDL)-induced injury of human aortic endothelial cells (HAECs).METHODS: HAECs were pre-treated with curcumin at different concentrations and then treated with ox-LDL.The cell viability was assessed by MTT assay.The cell proliferation ability was analyzed by EdU assay.ELISA was used to determine the concentrations of interleukin-6 (IL-6), transforming growth factor β1 (TGFβ1), high mobility group box-1 protein (HMGB1) and secretory receptor for advanced glycation end products (sRAGE) in the HAEC culture medium.The binding activity of peroxisome proliferator-activated receptor γ (PPARγ) was evaluated by electrophoretic mobility shift assay.The protein levels of HO-1, HMGB1, RAGE,IL-6,TGFβ1 and phosphorylated PPARγ in the HAECs were determined by Western blot.RESULTS: The viability and the proliferation ability decreased significantly in the HAECs treated with ox-LDL.The PPARγ/HO-1 signaling pathway was inhibited while its down-stream HMGB1/RAGE signaling pathway was activated by ox-LDL.The levels of IL-6, TGFβ1, HMGB1 and sRAGE were increased.Pre-treatment with curcumin activated PPARγ/HO-1 signaling pathway and inhibited HMGB1/RAGE signaling pathway in ox-LDL treated HAECs in a concentration-dependent manner.The levels of IL-6, TGFβ1, HMGB1 and sRAGE were also decreased dramatically by pre-treatment of curcumin in a concentration-dependent manner.CONCLUSION: ox-LDL induces HAEC damage by inhibiting PPARγ/HO-1 to activate HMGB1/RAGE inflammatory signaling.Curcumin exerts protective effect on ox-LDL treated HAECs via activating PPARγ/HO-1 signaling pathway.
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AIM: To investigate the inhibitory effect of L-carnitine on high glucose-induced apoptosis of human aortic endothelial cells (HAECs) and the molecular mechanisms.METHODS: The apoptosis of HAECs was induced by high-glucose incubation.HAECs were treated with L-carnitine at different concentrations (50, 100 and 200 μmol/L).The cell viability was measured by MTT assay.The cell apoptosis was assessed by Hoechst 33258 staining and flow cytometry.Colorimetric method was employed to detect the caspase-3 activity in the HAECs.The protein expression and phosphorylation levels were determined by Western blot.RESULTS: High-glucose incubation dramatically decreased the cell viability and induced apoptosis.The protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6) signaling pathways of endoplasmic reticulum stress were activated to induce cell apoptosis via down-stream caspase-4/3 cascade.However, L-carnitine treatment significantly attenuated the cell apoptosis and increased the cell viability in a concentration-dependent manner.L-carnitine also significantly suppressed endoplasmic reticulum stress and ATF6 signaling in high glucose-incubated HAECs without attenuating PERK and IRE1 signaling.The expression of site-1 protease (S1P) and site-2 protease (S2P) was inhibited by L-carnitine treatment, thus decreasing pro-apoptotic factor ATF6 p50 produced by ATF6 cleavage.CONCLUSION: L-carnitine inhibits high glucose-induced apoptosis of HAECs by inhibiting ATF6 signaling.
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AIM: To clarify whether advanced glycation end products (AGEs) can influence the expression of mitochondrial fusion proteins Mfn1 and Mfn2 in cultured human aortic endothelial cells (HAECs) in vitro.METHODS:AGE-BSA was used as AGEs.Purchased primary human aortic endothelial cell line was multiplied, and transferred to dif-ferent passages for subsequent grouping.For dose-dependent experiment, HAECs were divided into 4 groups, and the con-centrations of AGE-BSA in each group were 0 mg/L (control group), 50 mg/L, 100 mg/L and 200 mg/L, respectively. For time-dependent experiment, HAECs were divided into 5 groups with the same concentration (100 mg/L) of AGE-BSA, but the intervention time was 0 h (control group), 6 h, 12 h, 24 h and 48 h, respectively.The mRNA and protein expres-sion levels of Mfn1 and Mfn2 in the HAECs were detected by real-time PCR and Western blot, respectively.RESULTS:Exposure of the HAECs to AGEs at different concentrations for 24 h all down-regulated the mRNA and protein expression levels of Mfn1 and Mfn2.Except for 6 h intervention group, 100 mg/L AGEs intervention for 12 h, 24 h and 48 h all down-regulated the mRNA and protein expression levels of Mfn1 and Mfn2 in cultured HAECs.CONCLUSION: AGEs down-regulates the expression of mitochondrial fusion proteins Mfn1 and Mfn2 in cultured HAECs, indicating that AGEs may influence mitochondrial dynamics of human aortic endothelial cells.