Effect of Xiaozhongzhitong mixture on vascular regeneration of random flap in rats

AIM To observe the effect of Xiaozhongzhitong （detumescence and relieving pain） mixture on vascular regeneration and vascular endothelial growth factor （VEGF）-Dll4/Notch signaling pathway of random flap in rats. METHODS A total of 240 SD rats were randomly divided into 6 groups： blank group, sham group, model group, Xiaozhongzhitong mixture group （detumescence group）, Xiaozhongzhitong mixture+Notch blocker MK-0752 group （detumescence+MK group） and Xiaozhongzhitong mixture+VEGF receptor inhibitor axitinib group （detumescence+AXI group）. The capillary filling time, the number of new capillaries, the microvascular density, the microvascular diameter and the vascular survival area were observed by HE staining. The serum level of VEGF was measured by ELISA, and the mRNA expression of VEGFA, Notch and Dll4 in rat flap tissues was detected by RT-qPCR. RESULTS On the 10th day, compared with model group, the capillary filling time in detumescence group was decreased （P<0.05）. The capillary filling time in detumescence+AXI group was increased compared with detumescence group （P<0.05）. The flap tissue cells inmodel group were disordered and the nucleus was sparse, which showed obvious edema. Compared with model group, the number, diameter and area of microvessels, the serum content of VEGF, and the mRNA expression of VEGFA, Notch and Dll4 were significantly increased in detumescence group （P<0.05）. Compared with detumescence group, the number, density and area of microvessels, the serum content of VEGF, and the mRNA expression of VEGFA, notch and Dll4 was significantly decreased in detumescence+AXI group （P<0.05）. After the intervention with Notch blocker, the mRNA expression of VEGFA was increased, and the mRNA expression of Notch and Dll4 was inhibited （P<0.05）. CONCLUSION Xiaozhongzhitong mixture promotes the regeneration of blood vessels and enhances the survival rate of random flap, which may be related to the regulation of VEGF-Dll4/Notch signaling pathways.     

Notch1 regulates activation of pancreatic stellate cells by non-classical Notch signaling pathway

AIM: To investigate the effect of Notch1 on the activation of pancreatic stellate cells (PSCs). METHODS: The expression of Notch1 in pancreatic duct adenocarcinoma (PDAC) tissues was detected by the immunohistochemical and immunofluorescence double staining. The PSCs were isolated, cultured, and identified by oil red O staining, Western blot and RT-qPCR. The expression of Notch1 and HES1 was detected by Western blot and RT-qPCR. After transfection of Notch1 siRNA to PSCs, Western blot was used to detect the protein expression of α-smooth muscle actin (α-SMA), fibronectin and collagen type Ⅰ (ColⅠ) in activated PSCs. The expression of Notch1 and HES1 was also detected by Western blot. The effects of Notch1 siRNA on migration ability and viability of PSCs were determine by scratch test and CCK-8 assay. RESULTS: The results of immunohistochemical and immunofluorescence double staining showed that Notch1 expressed in α-SMA positive cells in PDAC stroma. The mouse PSCs were successfully cultured, and the expression of α-SMA, fibronectin, ColⅠ, Notch1 and HES1 in activated PSCs were significantly increased compared with unactivated PSCs (P<0.01). After transfection of Notch1 siRNA to mouse PSCs, the expression of α-SMA and ColⅠ was significantly reduced compared with negative groups, but the expression of fibronectin and HES1 did not change significantly. After knock-down of Notch1 expression in activated PSCs, the migration ability and viability of PSCs were significantly reduced compared with negative group. CONCLUSION: Notch1 is involved in regulating the activation of PSCs. Knock-down of Notch1 expression inhibits the expression of the markers of activated PSCs, α-SMA and ColⅠ, reduces the activation of PSCs, and attenuates the migration capacity and viability of PSCs. Notch1 regulates the activation of PSCs without relying on the classic Notch signaling pathway.     

Role of PI3K/Akt/eNOS signaling pathway in inhibitory effects of puerarin on ox-LDL-induced TF expression in vascular endothelial cells

AIM:To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS:The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS:Compared with control group,incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein,and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels,increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation,and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells,and reduced Akt protein phosphorylation and NO production.CONCLUSION:Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.     

Shear stress regulates NO production in vascular endothelial cells through Pim1/eNOS signaling pathway

AIM: To determine whether laminar shear stress regulates nitric oxide (NO) production in vascular endothelial cells through Pim1/endothelial nitric oxide synthase (eNOS) signaling pathway. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress using a parallel-plate flow system. NO production is evaluated by NO assay kit. Pim1 protein expression and eNOS phosphorylation were determined by Western blot. A specific small interfering RNA was used to knock down Pim1 gene expression, and then the changes of above indicators were detected. RESULTS: After 15-min exposure of HUVECs to laminar shear stress (15 dyn/cm²), rapid increases in Pim1 protein expression and NO production were observed (P < 0.05). Shear stress also caused time-dependent stimulation of eNOS phosphorylation (P < 0.05). The shear-induced Pim1 expression and NO production were abrogated in the HUVECs transfected with siPim1 (P < 0.05). Pim1 silencing also prevented shear-induced rise of eNOS-Ser1177 phosphorylation (P < 0.05). CONCLUSION: Pim1 may account for shear-induced NO production in endothelial cells due to phosphorylation activation of eNOS.     

Hydrogen sulfide attenuates human umbilical vein endothelial cell senescence via modulation of Sirt1/eNOS pathway

AIM: To explore the role of Sirt1/eNOS signalling pathway in the protective effect of hydrogen sulphide (H₂S) against endothelial cell senescence induced by high glucose.METHODS: High glucose (33 mmol/L) was applied to induce senescence in primary human umbilical vein endothelial cells (HUVECs). The cell viability, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. Mean while, Sirt1 siRNA was used to examine the effect of Sirt1 on eNOS expression and the senescence-related parameters.RESULTS: Treatment of HUVECs with high glucose decreased the cell viability slowly with a larger proportion of the cells stained with SA-β-Gal, and the protein expression of PAI-1 was dramatically increased. The increased cell viability, reduced SA-β-Gal positive cells and decreased protein expression of PAI-1 were detected after sodium hydrosulfide (NaHS, 100 μmol/L) treatment. Furthermore, NaHS treatment upregulated the protein expression of Sirt1 and eNOS, and eventually increased the production of nitric oxide (NO).CONCLUSION: Exogenous H₂S modulates Sirt1/eNOS/NO pathway to prevent HUVECs against high glucose-induced senescence.     

NBP promotes angiogenesis of HUVECs by activating VEGF/VEGFR2-Notch1/Dll4 signal pathway

AIM: To investigate the effects of DL-3-n-butylphthalidle (NBP) on angiogenesis of human umbilical vein endothelial cells (HUVECs) and the role of vascular endothelial growth factor (VEGF)/VEGF receptor 2(VEGFR2)-Notch1/Delta-like ligand 4 (Dll4) signaling pathway in this process. METHODS: The serum-free medium and anoxic tank were used to simulate the conditions of hypoxia and ischemia (H/I). HUVECs were divided into control group, H/I group, H/I+NBP_high group and H/I+NBP_low group. The HUVECs in control group were conventionally cultured, and those in H/I group were cultured under H/I intervention. The HUVECs in H/I+NBP_high group were treated with NBP at 20 μmol/L under H/I intervention. The HUVECs in H/I+NBP_low group were treated with NBP at 5 μmol/L under H/I intervention. The cell viability of each group was measured by CCK-8 assay. The migration ability of the HUVECs in each group was detected by cell scratch test. The vessel formation ability of the HUVECs was examined by in vitro angiogenesis assay. The expression of VEGFR2, Notch1 and Dll4 at mRNA and protein levels was determined by qPCR and Western blot, and the expression of VEGF was determined by qPCR and ELISA. RESULTS: NBP increased the viability of HUVECs, and promoted the migration ability and the formation of blood vessels in vitro under H/I intervention. These effects of NBP at high dose were more significant than those at low dose. NBP increased the expression of VEGF, VEGFR2, Notch1 and Dll4 at mRNA and protein levels (P<0.05). CONCLUSION: NBP promotes HUVECs to form blood vessels under H/I intervention. The mechanism may be related to the activation of VEGF/VEGFR2-Notch1/Dll4 signaling pathway.     

Effect of shikonin on high glucose-induced apoptosis and oxidative stress in vascular endothelial cells

AIM:To investigate the effect of shikonin on the apoptosis and oxidative stress induced by high concentration of glucose in vascular endothelial cells. METHODS:Rat thoracic aortic endothelial cells were randomly divided into 5 groups:normal control group (with glucose at concentration of 5.5 mmol/L in cell culture medium), high glucose group (with glucose at concentration of 33 mmol/L in cell culture medium), high glucose+low shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 0.1 μmol/L in cell culture medium), high glucose+medium shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 1 μmol/L in cell culture medium), and high glucose+high shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 10 μmol/L in cell culture medium). After treatments, the cell viability was measured by CCK-8 assay and cell apoptotic rate was analyzed by flow cytometry. In addition, the status of oxidative stress was evaluated by determining the levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activation of Nrf2/HO-1 signaling pathway was determined by Western blot. RESULTS:Compared with high glucose group, shikonin reversed high glucose-induced decrease in cell viability and increase in apoptosis in a concentration-dependent manner. High concentration of glucose induced high levels of MDA and ROS, while decreased the levels of SOD and GSH-Px. However, after treatment with shikonin, the contents of MDA and ROS were decreased, while the activities of SOD and GSH-Px were increased as compared with high glucose group. Furthermore, the high concentration of glucose up-regulated the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear). Compared with high glucose group, the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear) were partly decreased after treatment with shikonin. CONCLUSION:Shikonin alleviates high glucose-induced vascular endothelial cell apoptosis. Its mechanism may be related to activation of Nrf2/HO-1 signaling pathway and down-regulation of oxidative stress in vascular endothelial cells.     

Inhibitory effect of recombinant human endostatin on angiogenesis in atherosclerotic plaque of rats by regulating Dll4/Notch pathway

AIM: To observe the inhibitory effect of recombinant human endostatin (rhES) on plaque angiogenesis, and to explore the regulatory mechanism of Dll4/Notch pathway in the anti-angiogenic effect of rhES. METHODS: Male Wistar rats were randomized into 3 groups:normal control group (N group), atherosclerotic model group (AS group), and rhES treated group (AS+rhES group). The rats in N group were fed a normal diet, while the remaining 2 groups were established to atherosclerotic rat model via high-cholesterol diet, intraperitoneal injection of vitamin D₃ and aortic balloon injury. The rats in AS+rhES group received intraperitoneal injection of rhES. The blood total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), interleukin-1 (IL-1) and troponin I (TnI) were measured. The atherosclerotic abdominal aortas were taken for pathological observation. Immunohistochemical staining was used to measure the density of neovessels in the plaques, which were marked by CD31. The protein levels of Dll4 and Notch1 in the aortas were analyzed by Western blot. RESULTS: The levels of blood TC, TG, LDL-C, CRP and IL-1 in AS group and AS+rhES group were much higher than those in N group (P<0.05), and no statistical difference between AS group and AS+rhES group was observed. The expression of CD31 in AS group was the highest among all groups. Compared with AS group, the density of neovessels in the plaques of AS+rhES group decreased significantly (P<0.05). The protein expression of Dll4 and Notch1 in AS group was lower than that in N group (P<0.05). Compared with AS group, the protein expression of Dll4 and Notch1 increased significantly (P<0.05). CONCLUSION: rhES has the ability to inhibit plaque angiogenesis in rats. The activation of Dll4/Notch pathway may be the mechanism of rhES in inhibiting plaque angiogenesis.     

Tongxinluo improves anti-hypoxia ability of vascular endothelial cells via PI-3K/Akt/HIF pathway

AIM: To investigate the effects of PI-3K/Akt/HIF pathway on anti-hypoxia ability of vascular endothelial cells influenced by Tongxinluo under hypoxic condition. METHODS: Human umbilical vein endothelial cells (HUVECs) were divided into the following groups: control group, Tongxinluo (100 mg/L) group, hypoxia group and hypoxia+Tongxinluo (100 mg/L) group. The CCK-8 assay were used to detect the viability and proliferation rate of the cells in each group. The protein levels of HIF-1α, Bcl-2, Mcl-1, Bax and phosphorylated Akt were studied by immunoblotting analysis. The HUVECs were transiently transfected with the dominant negative mutant of HIF-1α (DN-HIF). The apoptotic rates were analyzed by flow cytometry (FCM). The HUVECs were transiently transfected with the dominant negative mutant of PI-3K (Δp85) or Akt (DN-Akt) to investigate the role of PI-3K/Akt signal pathway in the anti-hypoxia ability of Tongxinluo on endothelial cells. RESULTS: Under hypoxic condition, although the proliferation rate increased significantly in Tongxinluo group compared with hypoxia group, the degree was notably weak compared with control group. The protein levels of HIF-1α, Bcl-2, Mcl-1 and phosphorylated Akt were up-regulated by Tongxinluo. Meanwhile, the expression of Bax was down-regulated. Inhibition of HIF-1α activation by DN-HIF and inhibition of the PI-3K/Akt pathway by Δp85 or DN-Akt attenuated the increase in HIF-1α expression and HUVEC viability induced by Tongxinluo. The percentage of apoptotic HUVECs was down-regulated to a certain extent by Tongxinluo. CONCLUSION: Tongxinluo improves the anti-hypoxia ability of vascular endothelial cells by up-regulating the protein level of HIF-1α, promoting the expression of anti-apoptotic factors, improving the cell viability and eventually reducing the apoptotic rate.These effects of Tongxinluo depend on PI-3K/Akt signal pathway.     

DL-3-n-butylphthalide attenuates mitochondrial damage in vascular endothelial cells under the condition of oxygen-glucose deprivation

AIM:To investigate the mechanism in which DL-3-n-butylphthalide (NBP) protects the mitochondria from the damage of oxygen-glucose deprivation (OGD). METHODS:Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Mitochondrial morphology and mitochondrial reactive oxygen species (ROS) were examined using MitoTracker Green and MitoSOX Red, respectively. The activity of superoxide dismutase (SOD) was evaluated by SOD assay kit. RESULTS:NBP significantly attenuated OGD-induced mitochondrial fragmentation, reduced the content of mitochondrial ROS and increased the activity of SOD. CONCLUSION:NBP alleviates OGD-induced damage in the mitochondria. Reduction of mitochondrial ROS and enhancement of SOD activity may be the mechanism in which NBP protects mitochondria.     

Effect of salidroside on activity of endothelial progenitor cells and phosphoinositide 3-kinase/Akt signaling pathway

AIM：To investigate whether salidroside has influence on the activities of endothelial progenitor cells (EPCs) and its mechanism. METHODS：Mononuclear cells from normal human peripheral blood were cultured in fibronectin coated flasks in endothelial progenitor medium. After 7 d, EPCs were characterized as adherent cells with acLDL-DiI uptaking and lectin binding by direct fluorescent staining. The proliferation and migration of EPCs were analyzed by MTT assay and Transwell chamber assay, respectively. The EPCs adhesion assay was performed by re-plating the cells on fibronectin-coated dishes, and then adherent cells were counted. NO and Akt protein were also detected. RESULTS：Salidroside promoted EPCs proliferative, migratory and adhesive capacities in a concentration dependent manner. Salidroside also increased NO secretion, and the level of phosphorylated Akt protein. However, the effects of salidroside on EPCs were inhibited by phosphoinositide 3-kinase inhibitor LY294002. CONCLUSION：Salidroside regulates the activity of EPCs by phosphoinositide 3-kinase/Akt signaling pathway.     

Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Asiaticoside attenuates hypoxic pulmonary hypertension in mice by inhibiting NF-κB/p38 signaling pathway

AIM: To investigate whether asiaticoside attenuates hypoxic pulmonary hypertension by inhibiting p38/NF-κB signaling pathway. METHODS: BALB/c mice (n=30) were randomly divided into normoxia (N) group, hypoxia (H) group, and hypoxia+asiaticoside group. Right ventricular systolic pressure (RVSP), mean carotid artery pressure (mCAP), the weight ratio of right ventricle/(left ventricle+ventricular septum)[RV/(LV+S)], the ratio of right ventricle/body weight (RV/BW), vessel wall area/vessel total area (WA/TA) and vessel wall diameter/vessel wall total diameter (WT/TT) were determined after the model was established. The protein levels of p38, p-p38, NF-κB and p-NF-κB in the lung tissues were detected by Western blot. The fluorescence intensity of p-p38 and p-NF-κB were measured by immunofluorescence method. The serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. RESULTS: Compared with N group, the levels of RVSP, RV/(LV+S), RV/BW, WA/TA and WT/TT were significantly increased in H group, while administration of asiaticoside decreased the levels of RVSP, RV/(LV+S), RV/BW, WA/TA and WT/TT (P<0.05). Compared with N group, the relative protein levels of p-p38 and p-NF-κB in H group were significantly increased (P<0.05), and the concentrations of IL-6 and TNF-α were significantly increased, which were apparently attenuated by asiaticoside injection. CONCLUSION: Inhibition of p38/NF-κB signaling pathway and reduction of inflammatory responses may be the important mechanisms of asiaticoside in the prevention and treatment of hypoxic pulmonary hypertension.     

Doxorubicin induces stemness of mouse TNBC 4T1 cells through Stat3-Oct-4/CD44 signaling pathway

AIM:To investigate the stemness of mouse triple-negative breast cancer (TNBC) 4T1 cells induced by doxorubicin (DOX) and the underlying mechanism. METHODS:The 4T1 cells and MDA-MB-468 cells were treated with DOX at different concentrations (0, 0.05, 0.1 and 0.5 μmol/L) for 24 h, and the shape and viability of the cells were observed. The concentration of DOX at 0.1 μmol/L was chosen as the optimal concentration for the following experiments. The 4T1 cells and MDA-MB-468 cells resistant to DOX were established by continuous stimulation with DOX for 4 weeks, and named as 4T1-DOX and MDA-MB-468-DOX. Sphere formation assay was used to detect the stemness of 4T1 cells and MDA-MB-468 cells. The expression of CD133 was observed by immunofluorescence staining. The expression of CD44 was analyzed by flow cytometry. The protein levels of Stat3, phosphorylated Stat3 (p-Stat3) and Oct-4 were determined by Western blot. RESULTS:The sphere formation ability of the 4T1-DOX cells was stronger than that of the 4T1 control cells. The 4T1-DOX cells expressed high levels of the stemness markers CD133 and CD44 as compared with the 4T1 cells (P<0.05). Furthermore, the 4T1-DOX cells exhibited enhanced activation of Stat3 (p-Stat3) and increased expression of Oct-4 (P<0.05), while the expression of total Stat3 had no obvious variation. In addition, when activation of Stat3 was inhibited by WP1066, the protein levels of p-Stat3, Oct-4 and CD44 were down-regulated (P<0.05). Furthermore, inhibition of Stat3 phosphorylation reduced the sphere formation ability of the 4T1-DOX cells (P<0.05). CONCLUSION:DOX induces the stemness of mouse TNBC 4T1 cells through Stat3-Oct-4 signaling pathway.     

Salvianolate attenuates oxidative stress injury of human endothelial EA.hy926 cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups:control group, damage group, and anti-damage groups (salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide (NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor (VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased (P<0.05). The apoptotic rate was significantly decreased (P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased (P<0.05), and the intracellular ROS level was significantly decreased (P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.     

Notch signaling in bone marrow mesenchymal stem cells induced by lipopolysaccharide

AIM: To investigate the roles of Notch signaling in lipopolysaccharide (LPS)-induced proliferation and secretion of interleukin-6 (IL-6) and chemokine CXCL1 in bone marrow mesenchymal stem cells (BMSCs).METHODS: BMSCs were isolated by whole bone marrow culture. The expression levels of Notch signaling pathway receptors and ligands in the BMSCs treated with LPS were measured by qPCR and Western blot. The proliferation of BMSCs was analyzed by MTT assay and viable cell counting. The secretion levels of IL-6 and CXCL1 induced by LPS were measured by ELISA.RESULTS: Treatment with LPS at 1 mg/L effectively induced the proliferation of BMSCs and the secretion of IL-6. Obvious expression of Notch receptors and ligands in the BMSCs was observed, and LPS had little effect on the mRNA and protein levels of Notch receptors and ligands, but LPS increased the protein levels of Hes1 and Hey1, the target genes of Notch signaling. LPS at 1 mg/L increased the proliferation of BMSCs, whereas DAPT (Notch signal inhibitor) reduced the basal and LPS-induced proliferation of BMSCs (P<0.01). LPS treatment robustly increased the secretion of IL-6 and CXCL1 as assessed by ELISA. However, inhibition of Notch signaling almost completely abolished LPS-induced secretion of IL-6 and CXCL1 (P<0.05).CONCLUSION: Inhibition of Notch signaling reduced not only the proliferation of BMSCs but also IL-6 and CXCL1 secretion induced by LPS.     

Role of SIRT1/eNOS/NO signaling pathway in protective effect of ginsenoside Rb1 against replicative senescence of endothelial cells

AIM: To explore the effect of ginsenoside Rb1 on replicative senescence of endothelial cells and the role of SIRT1/eNOS/NO signaling pathway in this process. METHODS: The replicative senescence model of primary human umbilical vein endothelial cells (HUVECs) was established. The morphological change of the cells, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. The expression of eNOS and PAI-1 at mRNA and protein levels in the aging cells was determined by real-time PCR and Western blot before and after silencing of SIRT1 was performed. The NO concentration in the cell culture supernatant was measured by nitrate reductase assay. RESULTS: HUVECs with cumulative population-doubling level (CPDL) at 16 were chosen as the replicative senescence model in this research. Ginsenoside Rb1 at 80 μmol/L significantly reduced the expression of PAI-1 at mRNA and protein levels. Furthermore, ginsenoside Rb1 increased the expression of SIRT1 and eNOS at mRNA and protein levels, and increased the NO content. SIRT1 silencing inhibited the expression of eNOS at mRNA and protein levels and reduced NO generation, leading to an increase in the expression of PAI-1 at mRNA and protein levels. Upon intervention of ginsenoside Rb1, the eNOS and PAI-1 expression and the level of NO were not reversed. CONCLUSION: Ginsenoside Rb1 modulates SIRT1/eNOS/NO signaling pathway to prevent the replicative senescence of HUVECs.