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Objective:To observe the expression changes of nuclear factor erythroid 2 related factor 2 (Nrf2) and glutathione peroxidase (GPX4) in human pulmonary microvascular endothelial cells (HPMEC) under different experimental conditions, and to explore the role of Nrf2 in inhibiting ferroptosis in the process of alleviating hyperoxic lung injury(HLI).Methods:Hyperoxic model was established by hyperoxia exposure.HPMEC were treated with blank control (control group), oxygen exposure at the concentration of 950 mL/L (hyperoxia group), oxygen exposure at the concentration of 950 mL/L+ 10 μmol/L Ferrostatin (ferroptosis inhibitor group) and oxygen exposure at the concentration of 950 mL/L + 10 μmol/L ML385 (Nrf2 inhibitor group). Cell viability at 24 h and 48 h was tested by the Cell Counting Kit-8 assay, and reactive oxygen species (ROS) levels were detected by a commercial ROS kit.The mRNA and protein levels of Nrf2 and GPX4 were detected by real-time quantitative polymerase chain reaction and Western blot, respectively.Differences were analyzed using the Student′s t-test for a two-group comparison or one-way ANOVA test among groups. Results:(1)Compared with the control group, significantly decreased viability and increased ROS levels were detected in hyperoxia group.Meanwhile, the mRNA (24 h: 0.750±0.010 vs.1.010±0.160, 48 h: 0.690±0.050 vs.1.000±0.070) and protein levels of GPX4 (24 h: 0.160±0.010 vs.0.290±0.010, 48 h: 0.190±0.010 vs.0.250±0.010) at 24 h and 48 h were significantly downregulated, while the mRNA (24 h: 1.740±0.050 vs.1.000±0.050, 48 h: 2.130±0.020 vs.1.000±0.030) and protein levels of Nrf2 (24 h: 0.840±0.010 vs.0.480±0.010, 48 h: 0.840±0.010 vs.0.550±0.030) at 24 h and 48 h were significantly upregulated in hyperoxia group than those of control group (all P<0.05). (2)Compared with the hyperoxia group, significantly increased viability and decreased ROS levels were detected in ferroptosis inhibitor group.Meanwhile, the mRNA (24 h: 1.520±0.110, 48 h: 1.880±0.050) and protein levels of GPX4 (24 h: 0.290±0.010, 48 h: 0.250±0.004) at 24 h and 48 h were significantly upregulated, while the mRNA (24 h: 0.780±0.040, 48 h: 0.760±0.030) and protein levels of Nrf2 (24 h: 0.480±0.010, 48 h: 0.540±0.020) at 24 h and 48 h were significantly downregulated in ferroptosis inhibitor group than those of hyperoxia group (all P<0.05). (3)Compared with the hyperoxia group, significantly decreased viability and increased ROS levels were detected in Nrf2 inhibitor group.Meanwhile, the mRNA (24 h: 0.600±0.030, 48 h: 0.590±0.003) and protein levels of GPX4 (24 h: 0.150±0.001, 48 h: 0.180±0.001) at 24 h and 48 h were significantly downregulated, while the mRNA level of Nrf2 was significantly upregulated at 24 h (3.360±0.130), but downregulated at 48 h (1.430±0.130) (all P<0.05). No significant difference was detected in the protein level of Nrf2 at 24 h and 48 h between hyperoxia group and Nrf2 inhibitor group ( P>0.05). Conclusions:Ferroptosis is involved in the development of HLI, and Nrf2 is able to alleviate hyperoxic lung injury by inhibiting ferroptosis.Therefore, inhibition of ferroptosis by Nrf2 may provide a new therapeutic target for HLI.
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OBJECTIVE@#To investigate the effects of Naoluo Xintong Decoction (NLXTD) on pyroptosis and angiogenesis of brain microvascular endothelial cells (BMECs) and explore the possible mechanisms in rats with oxygen-glucose deprivation/ reperfusion (OGD/R).@*METHODS@#Rat BMECs with or without caspase-1 siRNA transfection were cultured in the presence of 10% medicated serum from NLXTD-treated rats (or blank serum) and exposed to OGD/R. CCK-8 assay, Transwell chamber assay, and tube formation assay were used to assess proliferation, migration, and tube-forming abilities of the cells. The activity of lactate dehydrogenase (LDH) in the culture supernatant was determined using a commercial assay kit, and the levels of inflammatory factors IL-1β and IL-18 were detected with ELISA. The cellular expressions of pro-caspase-1, caspase-1, NLRP3, Gasdermin D, and angiogenesis-related proteins VEGF and VEGFR2 were detected using Western blotting.@*RESULTS@#The BMECs showed obvious injuries after OGD/R exposure. Compared with the blank serum, the medicated serum significantly improved the cell viability, migration ability, and lumen-forming ability (P < 0.01) and lowered the levels of IL-1β and IL-18 and the LDH release (P < 0.01) of the cells with OGD/R exposure. Western blotting showed that in the BMECs exposed to OGD/R, the medicated serum strongly upregulated the expression of VEGF and VEGFR2 proteins (P < 0.01) and reduced the protein expressions of pro-caspase-1, caspase-1, NLRP3, and Gasdermin D (P < 0.01), and transfection of the cells with caspase-1 siRNA further promoted the expressions of VEGFR2 protein in the cells (P < 0.01).@*CONCLUSION@#NLXTD can improve the proliferation, migration, and tube- forming ability and promote angiogenesis of BMECs with OGD/R injury probably by inhibiting the caspase-1/Gasdermin D pathway in pyroptosis, alleviating cell injury, and upregulating the expressions of VEGF and VEGFR2.
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Animals , Rats , Endothelial Cells , Caspase 1 , Gasdermins , Interleukin-18 , NLR Family, Pyrin Domain-Containing 3 Protein , Vascular Endothelial Growth Factor A , Reperfusion Injury , Brain , Angiogenic Proteins , GlucoseABSTRACT
Aim To study the ability of tetramethylpyrazine (TMP) on promoting neurogenesis in neural stem cell microenvironment after oxygen-glucose deprivation (OGD) injury in vitro. Methods Neural stem cells (NSCs), astrocytes (ACs) and cerebral microvascular endothelial cells (BMECs) were respectively extracted and separated to establish a co-culture system. The OGD modeling conditions were optimized by NSCs activity, and the concentration of TMP was optimized by Nissl staining. Then CCK-8 and Nestin
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Aim To investigate the effect of XNST and its monomeric components on the barrier structure and tight junction protein expression of brain microvascular endothelial cells damaged by oxygen glucose deprivation/reoxygenation (OGD/R) and the possible mechanism. Methods The mouse brain microvascular endothelial cell line bEnd. 3 was inoculated in the upper layer of the Transwell chamber to establish an OGD/R damage model, and the effect of the drug on the integrity of the endothelial cell barrier was investigated by the transmembrane resistance value and fluorescein-so-dium transmittance. Claudin-5 immunofluorescence staining was used to observe the changes of tight junction structure between endothelial cells. RT-PCR and Western blot were employed to detect mRNA and protein expression levels of tightly linked proteins Claudin-5 , Occludin, ZO-1. Western blot was applied to detect the expression levels of MAPKs (JNK, p38, ERK) , I kappa B a, I kappa B kinase phosphorylated protein expression, and Western blot and immunofluorescence were utilized to detect NF-K.B/p65 nucleation expression. Results XNST and its three monomers could significantly increase endothelial cell resistance and de- crease fluorescein-sodium transmittance. Claudin-5 fluorescence staining showed that the tight junction between cells in the model group was significantly damaged , while XNST and its monomer components could significantly improve its tight structure. RT-PCR and Western blot results showed that it could significantly upregulate the expression of mRNA and protein of Claudin-5, Occludin and ZO-1, and further study on the mechanism showed that XNST and its monomer components could significantly inhibit the phosphoryla-tion of JNK, p38 and ERK, inhibit the phosphorylation of I kappa B a and I kappa B kinases, and significantly inhibit the nuclear translocation of NF-KB/p65. Conclusion Both XNST and its monomeric components can exert cerebroprotective effects by increasing the tight junction structure between cells to promote barrier integrity, and the mechanism may be related to inhibition of NF-kB and MAPKs signaling pathway activation.
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OBJECTIVE To study the improvement effect and mechanism of Gastrodia elata active ingredient 3，4- dihydroxybenzaldehyde （3，4-DD） on oxygen-glucose deprivation/reoxygenation（OGD/R） injury in rat primary brain microvascular endothelial cells （BMECs）-rat adrenal chromaffin cells PC12 co-culture system. METHODS The co-culture model of BMECs and PC12 cells was replicated in the Transwell chamber， and divided into control group， model group， butylphthalide group （positive control group， 0.1 mmol/L） and 3，4-DD group （0.1 μmol/L）. OGD/R injury model of the co-culture system was induced in those groups except for the control group. After preventively intervention in BMECs with relevant medicine or culture medium for 24 h， cell transendothelial electronic resistance （TEER） value， lactate dehydrogenase （LDH） activity， brain-derived neurotrophic factor （BDNF） level and mRNA expressions of TrkB， Plc-γ， Map-2， GAP-43 in PC12 cells was detected. RESULTS Compared with the control group， TEER of the co-culture model， LDH activity and BDNF level of PC12 cells were decreased significantly in the model group （P＜0.01）， while mRNA expressions of TrkB， Plc-γ， Map-2 and GAP-43 in PC12 cells were increased significantly （P＜0.01）. Compared with the model group， TEER of the co-culture model， LDH activity， BDNF level， and the mRNA expressions of TrkB， Plc-γ， Map-2 and GAP-43 in PC12 cells were increased significantly in the 3，4-DD group and butylphthalide group （P＜0.05 or P＜0.01）. CONCLUSIONS 3，4-DD can relieve the damage of neuronal OGD/R by acting on BMECs， the mechanism of which may be associated with activating the BDNF/TrkB signaling pathway.
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Objective:To isolate and identify the phenotype and biological characteristics of human placental microvascular endothelial cells(HPMEC).Methods:The HPMEC were isolated from term placenta and cultured in ECM medium. The morphology of the cells was observed under microscope. The functions of HPMEC were detected by uptake of Dil-Ac-LDL, tube formation and cell proliferation. Western Blot was used to detect the expression of endothelial nitric oxide synthase (eNOS), PROKR1 and PROKR2.Results:The HPMEC isolated from the placenta could expressed eNOS, PROKR1 and PROKR2, and were capable of the functions of Dil-Ac-LDL uptaking, tube formation, cell proliferation.Conclusions:HPMEC can be isolated from the placenta, proliferate effectively in vitro and maintain the characteristics of endothelial cells.
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Objective:To explore the effect of myricitrin on the injury of human retinal microvascular endothelial cells (HRMECs) induced by high glucose and its regulation mechanism.Methods:HRMECs were divided into normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml myricitrin groups.HRMECs transfected with pcDNA and pcDNA-circZNF292, respectively and then cultured in high-glucose medium containing 25 mmol/L D-glucose for 24 hours were assigned as pcDNA group and pcDNA-circZNF292 group.HRMECs transfected with siR-NC and siR-circZNF292, respectively and then cultured in medium containing 50.0 μg/ml myricitrin and 25 mmol/L D-glucose for 24 hours were assigned as myricitrin+ siR-NC group and myricitrin+ siR-circZNF292 group.The cell apoptosis rate was detected by flow cytometry.The concentration of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in cells were detected by enzyme-linked immunosorbent assay (ELISA) kits.The expression levels of circZNF292 and miR-23b-3p were detected by real-time fluorescence quantitative PCR.The targeting relationship between circZNF292 and miR-23b-3p was detected by dual-luciferase reporter assay.The relative expression levels of B-cell lymphoma-2 (bcl-2) and bcl-2-related X protein (bax) were assayed by Western blot.Results:Significant differences were found in the relative expressions of bax and bcl-2 proteins, cell apoptosis rate, MDA concentration, SOD activity, circZNF292 and miR-23b-3p among normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml, 50.0 μg/ml myricitrin groups ( F=105.707, 111.835, 74.515, 109.651, 135.020, 219.919, 116.304; all at P<0.001).With the increase of myricitrin concentration, the relative expression levels of bax protein, cell apoptosis rate, MDA concentration and miR-23b-3p in cells gradually decreased, while the relative expression levels of bcl-2 protein, SOD activity and circZNF292 increased, with statistically significant differences among groups with different concentrations of myricitrin (all at P<0.05).In the co-transfected wild-type (WT)-circZNF292 cells, the relative luciferase activity in miR-23b-3p group was 0.35±0.03, which was lower than 0.96±0.09 in microRNA-negative control group, and the difference was statistically significant ( t=11.137, P<0.001).Compared with pcDNA group, the relative expression levels of bcl-2 protein, circZNF292 and MDA concentration in cells of pcDNA-circZNF292 group were significantly increased, and the relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and SOD activity were significantly decreased (all at P<0.05).The relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and MDA concentration were reduced and relative expression levels of bcl-2 protein, circZNF292 and SOD activity were enhanced in myricitrin group and myricitrin+ siR-NC group in comparison with high glucose group and myricitrin+ siR-circZNF292 group, showing statistically significant differences (all at P<0.05). Conclusions:Myricitrin can inhibit cell apoptosis and oxidative stress by regulating the expression of circZNF292/miR-23b-3p, thereby reducing the damage of HRMECs induced by high glucose.
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Objective:To investigate the effect of interleukin-33 (IL-33) on lipopolysaccharide (LPS)-induced permeability of rat cardiac microvascular endothelial cells (RCMECs).Methods:RCMECs were cultured in vitro to be divided into control group, LPS group, IL-33 group and LPS+IL-33 group. The effect of IL-33 on the proliferation of RCMECs was detected by cell counting reagent (CCK8). Fluorescein isothiocyanate (FITC)-dextran assay was used to evaluate the permeability of RCMECs. The expression of vascular endothelial calmodulin, ras homologous gene family (Rho) member A (RhoA) and phosphorylated Rho-associated coiled-coil-containing protein kinase (p-ROCK2) proteins were tested by western blot. High-throughput sequencing and gene ontology (GO) were performed for gene expression in LPS and LPS+IL-33 groups.Results:No significant effect of IL-33 at 10-50 ng/ml on the proliferation of RCMECs was observed ( P>0.05). Compared with the control group, the permeability of RCMECs (permeability coefficient ratio 1.404±0.029 vs. 1.000±0.200, P<0.05) was significantly increased in LPS group and the expression of vascular endothelial calmodulin (relative gray value 0.429 5±0.012 9 vs. 0.594 9±0.014 2, P<0.05) was down-regulated, while the permeability of monolayers (permeability coefficient ratio, 0.948±0.013, P<0.01) was decreased in LPS+IL-33 group and the expression of vascular endothelial calmodulin (relative grayscale value 0.549 1±0.012 0, P<0.005) was up-regulated compared with the LPS group. High-throughput sequencing data revealed that the differential genes downregulated in the LPS and LPS+IL-33 groups were associated with cytoskeleton and Rho signaling pathway. Compared with the control group, RhoA (relative gray value 0.211 4±0.009 9 vs. 0.135 0±0.007 6, P<0.000 1) and p-ROCK (relative gray value 0.656 3±0.013 2 vs. 0.503 6±0.036 2, P<0.000 1) protein expression was upregulated in the LPS group. When compared with LPS group, RhoA (relative gray value 0.157 7±0.010 7, P=0.000 2), p-ROCK (relative gray value 0.427 7±0.003 8, P<0.000 1) protein expression was decreased in LPS+IL-33 group. Conclusion:IL-33 may improve LPS-induced hyperpermeability of RCMECs by inhibiting RhoA and p-ROCK protein expression in Rho/Rho-associated coiled-coil-containing protein kinase signaling pathway.
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Objective:To investigate the effect of N-acetylcysteine (NAC) on endoplasmic reticulum stress (ERS) and oxidative stress(OS) induced by tunicamycin (Tm) and its mechanism.Methods:Mouse derived brain microvascular endothelial cells cultured in vitro were divided into control group (normal cell culture), TM group (cells were intervened with 5 μg/mL Tm for 24 h), NAC + TM group (cells were pretreated with 1 mmol/L NAC for 1 h, then were intervened with 5 μg/mL Tm for 24 h) and NAC group (cells were intervened with 1 mmol/L NAC for 24 h) according to different intervention methods.CCK-8 and FITC-Annexin V/PI were used to detect the survival rate and apoptosis rate of cells.Western blot was used to detect the expression of GRP78、CHOP、p-eNOS and caspase-12 protein. Laser confocal microcopy was used to detect the expression of ROS, and colorimetry was used to detect the activity of MDA and SOD.Results:There were significant differences in apoptosis rate and survival rate among the four groups ( F=62.57, 35.00, both P<0.05). The apoptosis rate of TM group ((25.49±1.55)%) was higher than that of Control group ((13.76±1.48)%)( P<0.01), while the apoptosis rate of NAC+ TM group ((17.65±1.00)%) was lower than that of TM group ( P<0.01). The survival rate of TM group ((66.33±5.69)%) was lower than that of Control group ((100.00±2.12)%)( P<0.01), while the survival rate of NAC+ TM group ((85.67±4.04)%) was higher than that of TM group ( P<0.01). Western blot showed that there were significant differences in the expression levels of GRP78、CHOP and p-eNOS among the four groups ( F=32.39, 68.66, 13.12, all P<0.01). The expression levels of GRP78 and CHOP protein in TM group were higher than those of Control group (both P<0.05), while the expression level of p-eNOS was lower than that of Control group ( P<0.01). The expression levels of GRP78 and CHOP protein in NAC+ TM group were lower than those of TM group (both P<0.05), while the expression level of p-eNOS was higher than that of TM group ( P<0.01). There was no significant difference in the expression level of caspase-12 protein among the four groups ( F=0.33, P>0.05). Laser confocal showed that there was significant difference in the average fluorescence intensity of ROS among the four groups ( F=77.66, P<0.01). The average fluorescence intensity of ROS in TM group (32.67±1.53) was higher than that in Control group (12.67±2.08) and NAC+ TM group (18.33±1.53) (both P<0.01). Colorimetry showed that there were significant differences in the activity of SOD and the concentration of MDA among the four groups ( F=40.53, 34.99, both P<0.01). The results of colorimetry showed that the activity of SOD in TM group((41.60±1.53)U/mg) was lower than that in Control group((65.39±4.60)U/mg) and NAC+ TM group((58.72±1.64)U/mg)(both P<0.01). The concentration of MDA in TM((2.27±0.11)μmol/mg) group was higher than that in Control group((1.39±0.13)μmol/mg) and NAC+ TM group((1.44±0.11)μmol/mg) (both P<0.01). Conclusion:NAC can reduce Tm-induced apoptosis of cerebral micro-vascular endothelial cells, which may be related to its inhibition of ERS/ OS-related pathways.
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ObjectiveThis study was designed to observe the effect of Didang Xianxiong decoction on the cardiac myocardial microvascular endothelial cells （CMECs） injury， and to explore its related mechanism based on the CMECs model induced by high glucose. MethodRat primary myocardial cells were cultured in vitro and 33 mmol·L-1 glucose was added for modeling. After modeling， the rats were randomly divided into model group （final glucose concentration： 33 mmol·L-1）， normal group， Didang Xianxiong decoction low dose group （glucose + 5% Didang Xianxiong decoction containing serum）， Didang Xianxiong decoction medium dose group （glucose+10% Didang Xianxiong decoction containing serum）， Didang Xianxiong decoction high dose group （glucose+20% Didang Xianxiong decoction containing serum） and alagebrium chloride （ALT-711） group （glucose+10% ALT-711 containing serum）. The influence of drug-containing serum on the proliferation of CMECs was detected by MTT tetrazolium salt colorimetric assay. The relative mRNA expression of c-Jun was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression of phosphorylated Janus kinase 1 （p-JAK1）， phosphorylated signal transducer and activator of transcription 1 （p-STAT1） and transforming growth factor-β1 （TGF-β1） was determined by Western blot. ResultCompared with the conditions in normal group， the mRNA expression of c-Jun and protein expression of p-JAK1， p-STAT1 and TGF-β1 were up-regulated in model group （P<0.01）. Compared with model group， all treatment groups had decreased mRNA expression of c-Jun （P<0.01）. Didang Xianxiong decoction medium and high dose groups and ALT-711 group showed reduced protein expression of p-JAK1 and p-STAT1 （P<0.05， P<0.01）， while there was no significant change in Didang Xianxiong decoction low dose group. TGF-β1 protein expression was lowered in all treatment groups （P<0.05， P<0.01）， and the decrease was more significant in Didang Xianxiong decoction medium and high dose groups than Didang Xianxiong decoction low dose group. ConclusionDidang Xianxiong decoction can protect CMECs with high glucose-induced injury， and the mechanism may be related to reducing the activity of JAK/STAT signaling pathway in cells.
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ABSTRACT Objectives: This study aimed to explore the effects of miR-128b in the regulation of Lipopolysaccharide (LPS) induced apoptosis. Methods: Human Pulmonary Microvascular Endothelial Cells (HPMECs) were transfected with an miR-128b inhibitor and stimulated with LPS for 24 h. FCM was performed to detect apoptosis and Reactive Oxygen Species (ROS) production. In addition, miRNA and caspase-3 expression levels were determined using real-time quantitative polymerase chain reaction and western blotting. Results: LPS significantly induced apoptosis and ROS production and upregulated miR-128b and caspase-3 expressions in HPMECs. However, LPS-induced effects were suppressed when an miR-128b inhibitor was used. Preincu-bation with NAC decreased the LPS-induced apoptosis of HPMECs. Conclusions: These effects were mediated by miR-128b via the caspase-3 pathway.
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Objective:To observe the effects of four prostaglandin E2 (PGE2) receptors (EP 1-4R) on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells (hRMEC) in a high glucose environment. Methods:The hRMEC were divided into normal group and high glucose group, and they were cultured in Dulbecco modified Eagle medium containing 5.5 and 30.0 mmol/L glucose, respectively. Flow cytometry was used to observe the apoptosis rate of the high glucose group and the normal group; enzyme chain immunosorbent assay (ELISA) was used to detect the level of PGE2 in the culture supernatant of hRMEC cells. Western blot was used to detect the protein expression of cyclooxyganese (COX2) and EP 1-4R in hRMEC. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of EP 1-4R mRNA in hRMEC. After 72 h of culture, the cells in the high glucose group were divided into control group, PGE2 group, EP 1-4R agonist group, PGE2+EP 1-4R inhibitor group, and dimethylsulfoxide group. According to the group, each group was given the corresponding agonist or inhibitor to continue the culture for 24 h. QRT-PCR was used to detect the expression of nucleotide-binding oligomerization structure-like receptor protein (NLRP3) and pro-interleukin (IL)-1β mRNA in each group of cells. ELISA was used to detect the content of IL-1β and lactic dehydrogenase (LDH) in the cell culture supernatant. Western blot was used to detect the expression of cleaved Caspase-1 in each group of cells. At the same time, hRMEC in a high glucose environment was given IL-1β stimulation for 24 h, and the activity of LDH in the supernatant of the cell culture medium was detected. Results:The apoptotic rate, COX2 protein expression, and PGE2 protein content in hRMEC in the high glucose group were significantly higher than those in the normal group, and they were time-dependent. Compared with the normal group, the expression levels of EP 1R, EP 2R, EP 4R protein and mRNA in hRMEC in the high glucose group were higher than those in the normal group ( P<0.05). Compared with the control group, PGE2 group ( t=4.627, P<0.01), EP 1-4R agonist group ( t=3.889, 3.583, 2.445, 3.216; P<0.05) hRMEC NLRP3 mRNA expression level was significantly increased; the expression level of pro-IL-1β mRNA increased, however the difference was not statistically significant (PGE2 group: t=1.807, P>0.05; EP 1-4R agonist group: t=1.807, 1.477, 0.302, 1.926, P>0.05). Compared with the PGE2 group, the expression of NLRP3 mRNA in hRMEC in the PGE2+EP 2R inhibitor group was significantly reduced ( t=2.812, P<0.05); the expression of pro-IL-1β mRNA in hRMEC in the PGE2+EP 3R inhibitor group was significantly increased ( t=4.113, P<0.01). The protein content of IL-1β in the cell culture supernatant of the PGE2 group, EP 1R agonist group and EP 2R agonist group was significantly higher than that of the control group ( t=5.155, 4.136, 4.817; P<0.01). Compared with PGE2 group, the protein content of IL-1β in the cell culture supernatant of the PGE2+EP 2R inhibitor group and the PGE2+EP 4R inhibitor group were significantly lower than that of the PGE2 group ( t=1.964, 4.765; P<0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2 group and EP 2R agonist group was significantly higher than that in the control group ( t=5.332, 4.889; P<0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2+EP 2R inhibitor group was significantly lower than that of the PGE2 group ( t=6.699, P<0.01). The LDH activity in the cell culture supernatant of the PGE2 group and the EP 2R agonist group was significantly higher than that of the control group ( t=4.908, 4.225; P<0.05). The activity of LDH in the cell culture supernatant of the PGE2+EP 2R inhibitor group was significantly lower than that of the PGE2 group ( t=5.301, P<0.01). Compared with the control group, the LDH activity in the culture supernatant of hRMEC cells in the high glucose environment was significantly increased ( t=3.499, P<0.05). Conclusions:The four receptors of PGE2 can activate NLRP3 and its effector molecules to varying degrees. EP 2R mainly mediates hRMEC damage under high glucose environment.
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Objective:To investigate the inhibitory effects of rosmarinic acid (RA) on high glucose-induced angiogenesis of human retinal microvascular endothelial cells (HRMEC) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome pathway-related proteins.Methods:The HRMEC were divided into control group, high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group, and high glucose+ high concentration RA group, and were cultured in vitro with conventional medium, 30 mmol/L D-glucose medium, 30 mmol/L D-glucose+ 25 μmol/L RA medium, 30 mmol/L D-glucose+ 50 μmol/L RA medium and 30 mmol/L D-glucose+ 100 μmol/L RA medium accordingly.The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to detect the cell proliferation.Transwell assay was performed to detect the cell migration.Matrigel assay was employed to determine the tube formation ability of cells.Western blot was utilized to detect the expression levels of NLRP3, apoptosis-associated speck like protein (ASC) and cysteinyl aspartate-specific protease-1 (Caspase-1). Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the concentrations of interleukin (IL)-1β and IL-18 in supernatant of cell culture. Results:The cell proliferation rate, the number of migrated cells and the number of formed tubes were (100.00±0.92)%, 37.67±9.02 and 45.00±4.58 in the control group, (163.56±1.46)%, 117.33±7.23 and 95.00±9.54 in the high glucose group, (152.29±2.90)%, 78.67±4.04 and 84.67±1.53 in the high glucose+ low concentration RA group, (147.72±2.22)%, 65.33±4.16 and 71.00±3.61 in the high glucose+ medium concentration RA group, (132.47±0.74)%, 52.67±6.81 and 60.00±1.00 in the high glucose+ high concentration RA group, respectively.There were statistically significant differences in cell proliferation rate, the number of migrated cells and formed tubes among all groups ( F=537.07, 64.63, 45.58; all at P<0.001). Compared with the control group, the cell proliferation rate, the number of migrated cells and formed tubes were significantly increased in the high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group and high glucose+ high concentration RA group, showing statistical significances (all at P<0.05). Compared with the high glucose group, the cell proliferation rate, the number of migrated cells and formed tubes were significantly decreased in the different concentrations RA groups (all at P<0.05). With the increase of RA concentration, the cell proliferation rate, the number of migrated cells and formed tubes were decreased, and there were statistical differences among high glucose+ low/medium/high concentrations RA groups (all at P<0.05). There were significantly differences in the relative expression levels of NLRP3, ASC and Caspase-1 proteins in cells and the concentrations of IL-1β and IL-18 in cell culture supernatant among all the five groups ( F=145.12, 422.82, 463.79, 2 019.96, 33 406.97; all at P<0.001). Compared with the control group, the relative expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 in cell culture supernatant were significantly increased in the high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group and high glucose+ high concentration RA group (all at P<0.05). Compared with the high glucose group, the expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 were decreased in the different concentrations RA groups, and the differences were statistically significant (all at P<0.05). With the increase of RA concentration, the expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 were decreased, and there were statistically significant differences among high glucose+ low/medium/high concentrations RA groups (all at P<0.05). Conclusions:RA can inhibit proliferation, migration and tube formation of HRMEC induced by high glucose, and inhibit high glucose-induced activation of NLRP3 inflammasome signaling pathway.
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Blood-brain barrier (BBB) damage after ischemia significantly influences stroke outcome. Compound LFHP-1c was previously discovered with neuroprotective role in stroke model, but its mechanism of action on protection of BBB disruption after stroke remains unknown. Here, we show that LFHP-1c, as a direct PGAM5 inhibitor, prevented BBB disruption after transient middle cerebral artery occlusion (tMCAO) in rats. Mechanistically, LFHP-1c binding with endothelial PGAM5 not only inhibited the PGAM5 phosphatase activity, but also reduced the interaction of PGAM5 with NRF2, which facilitated nuclear translocation of NRF2 to prevent BBB disruption from ischemia. Furthermore, LFHP-1c administration by targeting PGAM5 shows a trend toward reduced infarct volume, brain edema and neurological deficits in nonhuman primate
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OBJECTIVE@#To investigate the synergistic effect of Naoxintong Capsule (NXTC, ) and Guhong Injection (GHI, ) on cerebral ischemia-reperfusion (I/R) injury.@*METHODS@#Forty-eight Sprague-Dawley rats were divided into 6 groups: control group, oxygen and glucose deprivation (OGD) group, nimodipine group (9.375 mg/kg), NXTC group (0.5 g/kg), GHI group (5 mL/kg) and NXTC+GHI group (0.5 g/kg NXTC+5 mL/kg GHI), after the onset of reperfusion and once per day for the following 7 days. Blood was collected 1 h after final administration, and the sera were collected. Cultured primary rat brain microvascular endothelial cells (rBMECs) were subjected to OGD to establish a cell injury model. Untreated rBMECs were used as blank control. The cell counting kit-8 assay was used to assess cell viability using the sera. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were assessed using an enzyme-linked immunosorbent assay. Apoptosis was evaluated after Hoechst33342 staining using fluorescence microscopy and flow cytometry. JC-1 staining was performed to assess changes in mitochondrial membrane potential.@*RESULTS@#Statistical analysis indicated that more than 95% of the cells were rBMECs. Compared with the OGD group, the cellular morphology of the all drug delivery groups improved. In particular, the combined drug group had the most significant effect. Compared with the OGD group, all drug intervention groups induced a decrease in the apoptotic rate of rBMECs, increased the SOD levels, and decreased the MDA levels (all P<0.01). Compared with the mono-therapy groups, the NXTC+GHI group exhibited a significant improvement in the number of apoptotic rBMECs (P<0.01). All drug intervention groups showed different degrees of increase in membrane potential, and the NXTC+GHI group was higher than the NXTC or GHI group (P<0.01).@*CONCLUSION@#The combinationa application of NXTC and GHI on cerebral I/R injury clearly resulted in protective benefits.
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Animals , Rats , Apoptosis , Brain , Brain Ischemia/drug therapy , Drugs, Chinese Herbal , Endothelial Cells , Glutamine/analogs & derivatives , Plant Extracts , Rats, Sprague-Dawley , Reperfusion Injury/drug therapyABSTRACT
Objective:To investigate the protective effect of cerebrospinal fluid containing Tongqiao Huoxuetang (TQHXT) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cells (BMECs), in order to explore the underlying mechanisms. Method:Primary BMECs were extracted by enzymatic digestion, and the cells were randomly divided into six groups: the normal control group, the OGD/R group, the TQHXT group（20%）, the nimodipine（NMDP） group (10 μmol·L-1), the cabozanix group (1 μmol·L-1) and the combination group. Except for the normal control group, the cells in the other groups were rapidly reoxygenated for 24 h after 2 h of oxygen-glucose deprivation, the OGD/R modeling was performed, and the rats were administered with drugs by groups. BMECs were identified by cell immunofluorescence staining, morphological and ultrastructural changes of OGD/R-induced BMECs were observed, and changes in cell transmembrane resistance (TEER) were detected. The levels of nitric oxide (NO), the activity of lactate dehydrogenase (LDH), the fluorescence intensity of reactive oxygen species (ROS) and the content of tissue-type plasminogen activator (tPA) were measured with kits. Intracellular Ca2+ concentration and cell apoptosis were detected by flow cytometry, and the expression of CD34 was observed. The protein expressions of zonula occluden-1 (ZO-1), vascular endothelial growth factor (VEGF), adhesion kinase (FAK), and Paxillin were detected by Western blot. Result:Compared with the normal control group, the cells in the OGD/R group were shrinking and rounded, TEER value and ZO-1 protein expression in cells were significantly decreased, the contents of NO, LDH and ROS in cells were significantly increased, the content of tPA was significantly decreased, the concentration of Ca2+ and the apoptosis in the cells were significantly increased, CD34 was expressed in cells, and the protein expressions of VEGF, FAK and Paxillin were significantly increased (P<0.01). Compared with the OGD/R group, cell damage in the TQHXT group was significantly improved, the TEER value and ZO-1 protein expression in cells were significantly increased, the contents of NO, LDH and ROS in cells were significantly reduced, the content of tPA was significantly increased, the concentration of Ca2+ and the apoptosis in the cells were significantly reduced, CD34 expression increased in cells, and the protein expressions of VEGF, FAK and Paxillin were significantly increased (P<0.05，P<0.01). Conclusion:CSF containing TQHXT protects BMECs from OGD/R injury possibly by promoting angiogenesis through the VEGF-VEGFR2/FAK/Paxillin signaling pathway.
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Objective To improve the method about the primary culture of rat brain microvascular endothelial cells in vitro. Methods The SD rats aged from 4 to 6 weeks were chosen as research object. After craniotomy, washing and cutting, sieving, density gradient concentration of BSA, digestion of type II collagenase and collagenase dispersive enzyme twice, the primary culture was carried out. The target cells were indentified by morphological abservation and immunocytochemical staining of facter VI. Results Cultured for 12 to 24 hours,the cells in vitro migrated outward from the microvascular section. The cells appeared polygonal-shaped, and proliferated in a clustered monolayer, the cell growth density reached 70% - 80% of the bottle bottom after 3 days, and arranged like cobbles. The correlation antigen of VI factor was positive, they reached confluence with over purity 99%. Conclusion The method is available that can successfully separate and cultivate microvascular endothelial cells of rat brains in vitro.
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@#AIM:To investigate the effect of silencing glial fibrillary acidic protein gene(GFAP)on proliferation and apoptosis of high glucose-induced human retinal microvascular endothelial cells(hRMECs)and its mechanism.<p>METHODS: Expression of GFAP in hRMECs treated with high sugar(30mmol/mL)and low sugar(5mmol/mL)was detected by qRT-PCR. The high glucose-induced hRMECs cells of silencing GFAP gene was established by lentiviral-mediated method. High glucose-induced hRMECs cells were treated with SRI-011381(TGF-β signaling pathway activator)and dimethyl sulfoxide(DMSO); Expression of GFAP, transforming growth factor-1(TGF-β1), activating transcription factor2(Smad2), Smad3 proteins were measured by Western blot, and cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively.<p>RESULTS: Expression of GFAP was significantly increased in high glucose treated hRMECs. The high glucose induced hRMECs cell model of GFAP gene silencing was successfully constructed by lentivirus mediation, and the cell proliferation ability was significantly improved, the apoptosis rate is significantly inhibited, and expression of TGF-1, Smad2 and Smad3 proteins in the TGF-β signaling pathway was significantly inhibited after silencing GFAP, while activation of TGF-β signaling pathway could reverse the inhibitory effect of silencing GFAP on the proliferation and apoptosis in high glucose hRMECs.<p>CONCLUSION: Silencing GFAP gene can promote the proliferation of high glucoseinducedhuman retinal microvascular endothelial cells and inhibit cell apoptosis,the mechanism may be related to the inactivation of TGF-β signaling pathway.
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OBJECTIVE@#To explore the effect of heparanase (HPSE) on apoptosis of microvascular endothelial cells (MVECs) and trans-endothelial migration of hepatocellular carcinoma (HCC) cells.@*METHODS@#A HCC cell line with high HPSE expression was selected by real-time quantitative PCR (qRT-PCR) and Western blotting and transefected with a lentiviral vector containing an interfering RNA sequence of HPSE. Transwell migration assay was performed to detect the trans-endothelial migration (TEM) rate of the transfected HCC cells across human umbilical vein endothelial cells (HUVECs). In a Transwell indirect co-culture system, the effect of HPSE silencing in the HCC cells was determined on apoptosis of HUVECs . A nude mouse model of HCC was used to verify the effect of HPSE on apoptosis of MVECs and liver metastasis of the tumor.@*RESULTS@#HCCLM3 cell line highly expressing HPSE was selected for the experiment. Transfection of the HCC cells with the lentiviral vector for HPSE interference the HCC cells resulted in significantly lowered TEM rate as compared with the cells transfected with the control vector ( < 0.01). In the indirect co-culture system, the survival rate of HUVECs co-cultured with HCCLM3 cells with HPSE interference was significantly higher and their apoptotic index was significantly lower than those in the control group ( < 0.05). Ultrastructural observation showed no obvious apoptosis of HUVECs co-cultured with HCCLM3 cells with HPSE interference but revealed obvious apoptotic changes in the control group. In the animal experiment, the tumor formation rate in the liver was 100% (6/6) in the control group, significantly higher than that in RNAi group (33.3%, 2/6) ( < 0.05). Under optical microscope, necrosis and apoptosis of the MVECs was detected in the liver of the control mice, while the endothelial cells remained almost intact in RNAi group.@*CONCLUSIONS@#HPSE promotes the metastasis of HCC cells by inducing apoptosis of MVECs.
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Animals , Humans , Mice , Apoptosis , Carcinoma, Hepatocellular , Cell Line, Tumor , Cell Movement , Cell Proliferation , Endothelial Cells , Gene Expression Regulation, Neoplastic , Glucuronidase , Liver NeoplasmsABSTRACT
To investigate the effect of α-asarone on the function and expression of P-glycoprotein(P-gp)in rat brain microvascular endothelial cells(rBMECs). rBMECs were exposed to L-glutamate(100 μmol/L) for 30 mins to induce the overexpression of P-gp/multidrug resistance gene 1a(Mdr1a)on the cell membranes,which mimicked the overexpression of P-gp/Mdr1a in blood brain barrier(BBB) when drug-resistant epilepsy attacked.MTT assay was used to detect the safe range of α-asarone concentration.The model cells were intervened with different concentrations of α-asarone at 12.5,25.0,and 50.0 μg/μl for 24 hours.After the treatment of α-asarone,the expression and the function of P-gp/Mdr1 were measured by Western blotting,real-time PCR,and intracellular rhodamine 123 accumulation assays. The rBMECs,stimulated by glutamine,showed a high expression of P-gp(=1.924,=0.020)/Mdr1a(=1.788,=0.019) compared to the normal rBMECs.The treatment with 25.0(=1.924,=0.025;=1.788,=0.017) and 50.0 μg/μl(=1.924,=0.035;=1.788,=0.026) α-asarone significantly depressed the expression of P-gp/Mdr1a.The treatment with 25.0 and 50.0 μg/μl α-asarone significantly increased intracellular accumulation of Rhodamine 123 by 40% and 60% respectively. α-asarone down-regulates the high expressions of P-gp and Mdr1a mRNA in rBMECs induced by L-glutamate.Moreover and increases intracellular accumulation of rhodamine-123.Thus,α-asarone may reverse drug resistance in P-gp-mediated drug-resistant epilepsy.