The effect of nuclear factor erythroid 2 related factor 2-induced inhibition of ferroptosis on hyperoxia lung injury / 中华实用儿科临床杂志

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.

MicroRNA-128b mediates lipopolysaccharide-induced apoptosis via reactive oxygen species in human pulmonary microvascular endothelial cells

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.

Study of mesenchymal stem cells transfering mitochondria to repair pulmonary microvascular endothelial cells / 医学研究生学报

Objective To study and confirm that mouse bone marrow-derived mesenchymal stem cells (MSCs) could promote the repair of mouse pulmonary microvascular endothelial cells (PMVECs) by transferring mitochondria, providing a new idea for the treatment of ARDS. Methods This study was divided into four groups: control group, injuryed group, group of MSC and MSC-oli (loss-of-function of mitochondrial). PMVECs induced by lipopolysaccharide (LPS) were co-cultured with MSC and MSC-oli in transwell chamber. The transfer of mitochondria was observed under confocal microscopy. The expression of mitochondria-associated proteins(CYP1A1,CYP1A2), synthesis-related proteins (eNOS, iNOS) and connexin protein (VE-cadherin) were detected by Western Blot. The permeability of endothelial cell was evaluated by fluorescein isothiocyanate-dextran method. And the apoptosis rate of endothelial cells was evaluated by flow cytometry and JC-1 membrane potential. Results It showed that both MSC and MSC-oli groups could deliver mitochondria from MSC to PMVEC under confocal fluorescence microscopy. The results of Western blot showed the expression of CYP1A1, CYP1A2 and eNOS in endothelial cells of MSC-oli group was significantly lower than that in MSC group (P<0.05). The expression of iNOS was significantly higher than that in MSC group (P<0.05). But there was no significant difference in the expression of VE-cadherin (P>0.05). The dextran method showed that the permeability was significant reduced in MSC-oli group compared with the MSC group (P<0.05). Flow cytometry and JC-1 membrane potential showed that the rate of apoptosis was lower in MSC-oli group compared to the MSC-oli group (P<0.05). Conclusion MSCs could alleviate the damage of PMVECs by transmitting mitochondria.

Effects of curcumin on pro-inflammatory factors in pulmonary microvascular endothelial cell thrombus model stimulated by thrombus / 中国中西医结合急救杂志

Objective To explore the effects of curcumin on pro-inflammatory factors in the lung microvascular endothelial cells (LMVEC) model stimulated by thrombus. Methods The LMVECs were divided into six groups according to the random number table method. No treatment was given to the blank control group ; the model group was cultured for 7 hours in normal medium; the curcumin group was treated with 40 μmol/L curcumin for 72 hours ; the shRNA group was infected with shRNA adenovirus for 72 hours; the irregular chemokines (CX3CL1) overexpression group was infected with CX3CL1 overexpressing adenovirus for 72 hours; the shRNA+curcumin group infected with shRNA adenovirus and treated with 40 μmol/L curcumin together for 72 hours; CX3CL1 overexpression +curcumin group infected with CX3CL1 overexpressing adenovirus and treated with 40 μmol/L curcumin together for 72 hours. After each group was given the corresponding pretreatment, the thrombus natural precipitation was added each group for 12 hours. The contents of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the mRNA expression levels of CX3CL1, CX3CL1 receptor (CX3CR1), IL-6, TNF-α and the protein expression levels of CX3CL1/CX3CR1, CX3CR1/NF-κB in various groups were observed, repeat 3 times in each group. Results The contents and mRNA expression of IL-6, TNF-αand protein expression of CX3CR1, NF-κB in the LMVEC group were significantly higher than those in blank control group [IL-6 (ng/L): 207.90±16.69 vs. 85.93±20.32, TNF-α (ng/L): 239.60±15.27 vs. 101.23±11.92; IL-6 mRNA: 0.66±0.05 vs. 0.11±0.02, TNF-α mRNA: 1.06±0.04 vs. 0.02±0.01; CX3CR1 protein:3.94±0.58 vs. 1.00±0.31, NF-κB protein: 1.20±0.07 vs. 1.00±0.10; all P < 0.05]; the contents of IL-6 in shRNA group, CX3CL1 overexpression group, shRNA + curcumin group, CX3CL1 overexpression + curcumin group were all obviously lower than those in LMVEC group (ng/L: 183.60±11.52, 159.27±15.02, 117.03±7.91, 119.97±11.43 vs. 207.90±16.69, all P < 0.01); the content of TNF-α was markedly increased in shRNA group compared with that of LMVEC group (ng/L: 282.00±5.63 vs. 239.6±15.27), while the contents of TNF-α in CX3CL1 overexpression group, shRNA+ curcumin group, CX3CL1 overexpression + curcumin group were all lower than those in LMVEC group (ng/L: 216.97±9.20, 203.97±19.03, 191.97±17.50 vs. 239.6±15.27, all P < 0.05). The mRNA expression levels in CX3CL1 overexpression group and CX3CL1 overexpression + curcumin group were significantly higher than those in the blank control group and the LMVEC group (CX3CL1 mRNA: 55 210.3±1 209.2, 165 296.3±8 082.4 vs. 3.3±0.6, 2.0±0.0, all P < 0.01). The mRNA expression level of IL-6 in shRNA group was higher than that in LMVEC group (0.82±0.17 vs. 0.66±0.05), the mRNA expression level of IL-6 in CX3CL1 overexpression was lower than that in LMVEC group (0.29±0.03 vs. 0.66±0.05), the changes after pretreatment with curcumin were more significant (1.06±0.03 vs. 0.66±0.05 and 0.15±0.01 vs. 0.66±0.05); the mRNA expressions of TNF-α in shRNA group, CX3CL1 overexpression group, shRNA+ curcumin group were significantly lower than those in LMVEC group (0.41±0.04, 0.88±0.07, 1.01±0.02 vs. 1.06±0.04), the mRNA expression level of TNF-α in CX3CL1 overexpression + curcumin group was significantly higher than that in LMVEC group (1.36±0.01 vs. 1.06±0.04). The protein expression of CX3CL1, CX3CR1, NF-κB in shRNA group, CX3CL1 overexpression group, shRNA + curcumin group, CX3CL1 overexpressing + curcumin group were significantly higher than those in the LMVEC group (CX3CL1 protein: 0.41±0.07, 0.59±0.09, 0.69±0.61, 1.02±0.23 vs. 1.33±0.33, CX3CR1 protein: 0.85±0.18, 1.10±0.16, 1.32±0.18, 1.54±0.08 vs. 3.94±0.58, NF-κB protein: 0.33±0.07, 0.41±0.08, 0.41±0.07, 0.63±0.08 vs. 1.20±0.07). Conclusion Curcumin can inhibit the secretion of IL-6, TNF-α, CX3CR1 and NF-κB in thrombus-stimulated LMVEC model.

Brain-Derived Glia Maturation Factor β Participates in Lung Injury Induced by Acute Cerebral Ischemia by Increasing ROS in Endothelial Cells / 神经科学通报·英文版

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia (ACI), we established a middle cerebral artery occlusion (MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor β (GMFB) based on quantitative analysis of the global rat serum proteome. Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was over-expressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation (OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium (CM) after OGD. We then used the CM to culture pulmonary microvascular endothelial cells (PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover, ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells. In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.

p38 siRNA reduced lung ischemia-reperfusion injury of pulmonary microvascular endothelium after lung transplantation through NF-κB pathway inhibition / 中华器官移植杂志

Objective Using small interfering RNA (siRNA) against p38 and simulated lung transplantation model,we discussed the effect of p38 siRNA on hypoxia/reoxygenation injury of pulmonary microvascular endothelial cells (PMVECs) after lung transplantation.Methods We transfected the PMVECs with p38 siRNA or non-targeting (NT) siRNA.After 48 h,these cells were exposed to simulated ischemia-reperfusion.At 2 h and 4 h of reperfusion,we detected lactate dehydrofenase (LDH) leakage rate,malondialdehyde (MDA) levels,superoxide dismutase (SOD) activity,cell apoptosis,and the serum levels of interleukin (IL)-1,IL-6 and tumor necrosis factor (TNF)-α.Protein levels of p38,NF-κB and AP-1 were detected.Untreated PMVECs served as the negative control.Results As compared with NT siRNA,p38 siRNA reduced LDH leakage rate (22.3 ± 5.7 vs.45.1 ± 6.2 and 46.3 ± 7.3 vs.75.6 ± 12.4),decreased MDA levels (4.1 ± 2.2 vs.7.1 ± 2.1 and 3.9 ± 0.5 vs.6.1 ± 1.2),increased SOD activity (12.8 ± 3.2 vs.9.4 ± 1.1 and 10.8 ± 1.2 vs.7.0 ± 1.1),and inhibited apoptosis (2.8 ± 0.6 vs.4.1 ± 1.4 and 3.1 ± 1.1 vs.5.8 ± 1.3).p38 siRNA reduced the levels of IL-1 (288 ± 89 vs.592 ± 95 and 380 ± 94 vs.775 ± 175) and IL-6 (38 ± 5 vs.70 ± 12 and 80 ± 20 vs.118-± 17),however,had no influence on TNF-α level.Silencing p38 gene decreased phosphorylation of p65 and inhibitor of nuclear factor kappa-B kinase β,and increased inhibitor of nuclear factor kappa-B expression.However,p38 siRNA had no effect on the phosphorylation of c-Jun and c-Fos.Conclusion Through inhibiting the NF-κB classic activation pathway,p38 siRNA reduced oxidative stress,inflammation and apoptosis of rat PMVECs,protected membrane integrity,and reduced hypoxia/reoxygenation injury.

The Sonic hedgehog signaling pathway involved in the expression of RACK1 in the pulmonary microvascular endothelial cells induced by lipopolysaccharide / 中华急诊医学杂志

Objective To explore the effect of expression of protein kinase C receptor 1(RACK1) induced by lipopolysaccharide (LPS) on Sonic hedgehog(SHH) signaling pathway in rat puhnonary microvascular endothelial cells (RPMVEC).Methods The healthy male SPF grade SD rat with 100-120 g body weight were gotten from the laboratory animal center of Anhui province.Using immunocytochemistry method,the expression of RACK1 protein in RPMVECs was detected,cultured RPMVECs were randomly divided into different groups as LPS dose-dependent group,SAG(smoothened Agonist,a SHH signaling pathway specific agonist) dose-dependent group,LPS time-dependent group,SAG time-dependent group and LPS+SAG group.In LPS dose-dependent groups,RPMVECs were cultured with 0.1,1,10 mg/L LPS for 8 h.In LPS time-dependent groups,RPMVECs were cultured with 10 mg/L LPS for 0,2,4,8,12,24 h.In SAG dose-dependent groups,RPMVECs were cultured with 0.1,1,10 μ mol/L for 8 h.In SAG time-dependent groups,RPMVECs were cultured with 1 μ mol/L SAG for 0,2,4,8,12,24 h.In LPS+SAG group,RPMVECs were cultured with 1 μ mol/L SAG 8 h after 10 mg/L LPS treatment for 1 h.In addition,blank group,LPS group and SAG group were set for control.Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the expression of GLI-1 mRNA after intervention.Results Immunocytochemistry revealed that RACK1 were present in RPMVEC.1.In LPS dose-dependent groups (0,0.1,1,10 mg/L),the level of RACK1 elevated as LPS dose increased correspondingly with inter-group difference (P＜0.05);the relative expression levels of GLI-1 mRNA were (1.109 + 0.063),(1.039 + 0.135),(0.813 ± 0.066),(0.770 + 0.105),(1 mg/L vs.10 mg/L,P＞0.05;the rest P＜0.05).In LPS time-dependent groups,the relative expression level of RACK1 at 2 h (0.370 + 0.010) was higher than that at 0 h (0.329 ± 0.008),peaked at 12 h (1.296 ± 0.048),and compared with 0 h,there was significant differences (F=1 272.204,P＜0.05).The relative expression level of GLI-1 mRNA was decreased at 2 h (0.929 ±-0.007),and compared with 0 h(1.089 ± 0.042),there was significant differences (F=306.609,P＜0.05).2.In SAG dose-dependent groups,there was no significant difference in level of RACK1 between groups(all P＞0.05).The relative expression levels ofGLI-1 mRNA were (1.109 ± 0.063),(1.169 ± 0.052),(3.468 ± 0.128),(3.434 ± 0.054),(0 μ mol/L vs.0.1 μ mol/L and 1 μ mol/L vs.10 μ mol/L,P＞0.05,the rest P＜0.05).Among SAG time-dependent groups,there was no significant difference in levels of RACK1 protein(P＞0.05).The relative expression level of GLI-1 mRNA increased at 2 h (3.027 ± 0.065),and compared with 0 h (2.651 + 0.123),there was significant differences (F=132.841,P＜0.05).3.In LPS+SAG intervention groups,the expression of RACKI was lower than that in LPS group (0.831 ±0.040 vs.1.189 ± 0.149,P＜0.05),and the expression of GLI-1 mRNA was higher than that in LPS group (2.720 + 0.130 vs.0.796-4-0.082,P＜0.05).Conclusions The LPS up-regulates the expression of RACK1 in RPMVECs,and the activated SHH signaling pathway can down-regulate the expression of RACK1 induced by LPS in RPMVECs.

Effects of MAPKs signaling on heat stress-induced apoptosis of pulmonary microvascular endothelial cells and its mechanism / 解放军医学杂志

Objective To investigate the effect of mitogen-activated protein kinases (MAPKs) activation on the heat stressinduced apoptosis of pulmonary microvascular endothelial cells (PMVECs).Methods A mouse model of severe heat stroke was made and TUNEL and immunohistochemistry were employed to detect lung tissue damage.MACS separation was used for isolation of neonatal PMVECs,and TUNEL was utilized to detect the apoptosis of PMVECs.Western blotting was used for determining the MAPKs activation during heat stress recovery (0,2,6h).The monolayer permeability of endothelial cells was detected in terms of transmembrane resistance (TEER) and horseradish peroxidase (HRP).Cells were pretreated with MAPKs activation inhibitors to examine the effect of heat stress on the monolayer cell permeability and apoptosis.Results In mice with severe heat stroke,extensive apoptosis of PMVECs was found in their pulmonary tissues.TUNEL revealed that the number of apoptotic cells increased over time during heat stress recovery period and heat stress could activate MAPKs in PMVECs.Compared with heat stress group,in the cells pretreated with p38 or ERK activation inhibitor PD98059 and SB203580,the monolayer permeability and apoptosis increased while in cells pretreated withJNK inhibitor SP600125,the cellular permeability and apoptosis decreased.Conclusion In mice with severe heat stoke,PMVECs might experience apoptosis and p38 and ERK could inhibit apoptosis while JNK could promote apoptosis.

Xijiao Dihuang decoction combined with Yinqiao powder inhibits TNF-α-induced permeability increase in PMVEC via PKC-SSeCKS pathway / 中国病理生理杂志

AIM:To investigate the molecular mechanism of Xijiao Dihuang decoction combined with Yinqiao powder (XDY) in treating viral pneumonia, and the effects of XDY on TNF-α-induced permeability in pulmonary microvascular endothelial cells (PMVEC) and the role of PKC-SSeCKS pathway involved.METHODS:The electric conductivity method was used to detect transendothelial electrical resistance (TER) of primarily cultured PMVEC on Transwell chamber at different time points to determine the permeability of PMVEC.After pretreatment for 24 h, the activity of PKC, TER, and the expression of SSeCKS at mRNA and protein levels were detected.Laser scanning confocal microscopy was used to observe the location of SSeCKS and construction of F-actin in PMVEC.RESULTS:The permeability of PMVECs induced by TNF-α reached the peak at 24 h.Compared with control group, the TER in TNF-α group was decreased, and the activity of PKC was increased.Compared with TNF-α group, the activity of PKC in TNF-α with PKC inhibitor group and TNF-α with XDY group was decreased, while the TER was increased, without difference from control group.Compared with control group, the mRNA expression of SSeCKS and phospho-SSeCKS was increased in PMVEC of TNF-α group, but decreased in TNF-α with XDY group compared with TNF-α group.In control group, F-actin was mainly located around the nucleus and at cytoplasmic borders of PMVEC, forming the dense peripheral bundle, and SSeCKS was evenly scattered in the cell.In TNF-α group, the dense peripheral bundle of F-actin surrounding the cells almost disappeared, and SSeCKS was concentrated around the nucleus.Compared with TNF-α group, the distribution and the structure of F-actin and SSeCKS nearly returned to normal in TNF-α with XDY group.CONCLUSION:XDY inhibits the activation of PKC signaling pathway in PMVEC caused by TNF-α to reduce the mRNA expression of SSeCKS and the phosphorylation of SSeCKS, thus preventing the deformation of endothelial cells and reducing the permeability of PMVEC.

Effects of Alprostadil on the Expression of VEGF and eNOS in Human Pulmonary Microvascular Endothe-lial Cells with Hypoxia-induced Injury / 中国药房

OBJECTIVE:To study the effects of alprostadil on the expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in human pulmonary microvascular endothelial cells (HPMECs) with hypoxia-in-duced injury. METHODS:HPMECs were divided into group A(normal cultural environment),group B(3%O2),group C(3%O2+15 μg/L alprostadil)and group D(3%O2+45 μg/L alprostadil)for 24 h culture. Cell morphology was observed;MTT method was conducted to detect cell activity(recorded by absorbance);flow cytometry was adopted to detect apoptosis rate;Western blot was used to detect relative expressions of VEGF and eNOS in cells. RESULTS:Cell density in group A and D was relatively high, that in group B and C was low. Compared with group A,cell activities decreased and apoptosis rates increased in group B,C and D,expressions of VEGF and eNOS enhanced,eNOS/VEGF decreased (P<0.05). Compared with group B,cell activities in-creased in group C and D,VEGF expression decreased (P<0.05);apoptosis rate in group D decreased,eNOS expression de-creased and eNOS/VEGF increased. Compared with group C,cell apoptosis rate was decreased in group D,VEGF expression de-creased and eNOS/VEGF increased(P<0.05). CONCLUSIONS:Alprostadil maybe play effect on HPMECs with hypoxia-induced injury by up-regulating the expression level of eNOS/VEGF.

Impact of perfluoroisobutylene exposure on pulmonary microvascular endothelial cells of rats / 中国药理学与毒理学杂志

OBJECTIVE To investigate the change of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),intercellular adhesion molecules(ICAM-1),matrix metalloproteinases 2 (MMP-2) and MMP-9 contents in cultured pulmonary microvascular endothelial cells (PMVECs) in rats after perfluoroisobutylene (PFIB) exposure. METHODS PMVECs were separated and purified using a modified method of implantation of pulmonary tissues. After identification,PMVECs were divided into the normal control group and the PFIB-exposed groups(n=3). The PFIB-exposed groups inhaled PFIB at the concentration of 200 mg · m-3 for 5 min in a flow-past header,while the normal control group were PMVECs in quiescent condition. The supernatants and lysates of PMVECs were harvested at 0.5,1,2,4 and 8 h,respec?tively, after execution. The contents of TNF-α,IL-1β,ICAM-1,MMP-2 and MMP-9 were measured by ELISA,and the activity of MMP-2 and MMP-9 was measured by gelatin zymography. RESULTS① According to the morphologic characteristics of cell growth and the expression of specificity antigens and the bind experiment of phytohemagglutinin,the cells separated and purified by modified method shared the characteristics of PMVECs.②TNF-αwas rapidly expressed by PMVECs at 0.5 h post PFIB stimulation and the maximum value was achieved at 2 h post PFIB stimulation(P<0.05). The newly synthesized TNF-α was slowly released out of the cells. The maximum TNF-α in the supernatant was achieved at 4 h post stimulation.③Within 2 h of stimulation,PMVECs synthesized a large amount of IL-1β and peaks at 2 h. However,IL-1βwas never released to the extracellular milieu.④The amount of ICAM-1 was rapidly synthesized by PMVECs after PFIB stimulation,but at a low level.⑤After stimulation with PFIB,MMP-2 in the supernatant of PMVECs culture was gradually increased,peaked at 2 h and then decreased subsequently. The biological activity of MMP-2 in the supernatant was also enhanced after PFIB stimulation. PFIB did not stimulate synthesis or secretion of MMP-9,indicating that PMVECs were not the main source of MMP-9 during PFIB inhalation-induced acute lung injury. CONCLUSION PFIB stimulates the surviving PMVECs to synthesize a large amount of TNF-α,IL-1β, MMP-2 and conjunctive ICAM-1.

The changes and effects of Apelin/APJ system in LPS-induced injury of rat pulmonary microvascular endothelial cells / 中国药理学通报

Aim To explore the changes of Apelin/APJ system in LPS-induced injury of rat pulmonary mi-crovascular endothelial cells( PMVECs) , and the effect and mechanism of Apelin. Methods PMVECs were cultured with the explant technique, and the identifica-tion of rat PMVECs was carried out by immunocyto-chemical staining of factorⅧrelated antigen. MTT as-say was used to evaluate the viability of PMVECs. The mRNA expression of Apelin and APJ was detected by RT-PCR. The protein expression of PCNA and the phosphorylation of Akt was analyzed by Western blot. Results The mRNA expression of Apelin and APJ showed a compensatory increase after LPS treatment for a short period of time ( P<0. 01 ) , but with the exten-sion of time, which was significantly inhibited, even lower than the control group ( P<0. 05 or P<0. 01 ) , suggesting that Apelin/ APJ system might be involved in LPS-induced PMVECs injury. MTT results showed that 10 -6 ~10 -9 mol · L-1 Apelin obviously promoted the proliferation of rat PMVECs ( P <0. 05 or P <0. 01 ) , and with certain concentration and time de-pendence. Moreover, Apelin also improved the LPS-induced PMVECs injury in different degrees ( P<0. 05 or P < 0. 01 ) . In addition, Western blot analysis showed that Apelin significantly reversed the decrease of the protein expression of PCNA and the Akt phos-phorylation level induced by LPS ( P <0. 05 or P <0. 01 ) . Conclusions The Apelin/APJ system is in-volved in LPS-induced PMVECs injury. Apelin plays an important role in protecting the pulmonary microvas-cular endothelial function and reversing the LPS-in-duced PMVECs injury, which might be related to the activation of Akt phosphorylation pathway.

Autophagy attenuates injury of human pulmonary microvascular endothe-lial cells under mimic ischemia/reperfusion microenvironment / 中国病理生理杂志

[ ABSTRACT ] AIM: To detect the autophagic changes of human pulmonary microvascular endothelial cells ( HPMVECs) under ischemia/reperfusion ( I/R) microenvironment, and to clarify the effects of autophagy on the HPM-VECs survival and endothelial barrier integrity under I/R condition.METHODS:Rapamycin ( RAP) was applied to pro-mote autophagy of HPMVECs.These cells were then incubated under the condition of oxygen-glucose deprivation/oxygen-glucose restoration ( OGD) .After exposure to OGD, the changes of autophagy, cellular death and permeability of the cells were determined by transmission electron microscopy, flow cytometry and transwell assay, respectively.RESULTS:Com-pared with the control cells, OGD-challenged cells had a much higher level of autophagy.The apoptotic rate was much higher and endothelial permeability was more serious in OGD group than those in control group.Preconditioning with RAP effectively improved OGD induced autophagy, it did not affect the cell survival and endothelial permeability under normal living condition, but obviously decreased the cells apoptotic rate, and remarkably lowered OGD-induced high permeability of the cells.CONCLUSION:Autophagy protects HPMVECs against I/R-induced injury.Promotion of autophagy is help-ful for attenuating I/R-induced cell death and sustaining the endothelial barrier integrity.

Effect of penehyclidine hydrochloride on -arrestin-1 expression in lipopolysaccharide-induced human pulmonary microvascular endothelial cells

β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M3 receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury.

Regulation of src-suppressed C kinase substrate on the expression of TNF-α in endothelial cells / 中华急诊医学杂志

Objective To study the role of src-suppressed C kinase substrate (SSeCKS) in the secretion of tumor necrosis factor (TNF-α) in rat pulmonary micro-vascular endothelial cells (PMVEC) induced by lipopolysaccharide (LPS).Methods Wistar rat PMVEC cultured in vitro were randomly (random number) divided into several groups (n =4) as per exposure to given dosage of LPS for different lengths of time and to different dosages of LPS for given length of time.After PMVEC exposed to 10 mg/L LPS for 1 hour (h),3 h,6 h,12 h and 24 h or 0.1 mg/L,1 mg/L and 10 mg/L LPS for 24 h,the levels of TNF-αin the supernatant of culture medium were examined by the method of enzyme linked immunosorbent assay (ELISA).Another PMVEC was pre-treated by protein kinase C (PKC) inhibitor bis-indolylmaleimide (BIM) for 0.5 h or had the transfection of SSeCKS-specific small interfering RNA (siRNA) for 48 h before 10 mg/L LPS challenge for 24 h,and subsequently the supernatant was also examined by ELISA.One-way analysis of variance (ANOVA) was employed for statistical analysis by SPSS version 10.0 to compare values among all groups.A significant difference was presumed as a probability value ＜ 0.05.Results After PMVEC incubated with 0.1 mg/L,1 mg/L and 10 mg/L LPS for 24 hours,the levels of TNF-αsecreted were (253.70 ± 23.55),(327.88 ± 37.25),(403.20 ± 36.22),respectively,which were higher than that in un-stimulated PMVEC (82.28 ± 22.56,all P =0.000).After 10 mg/L LPS challenge for one hour,the level of TNF-αin the supernatant of PMVEC raised substantially (170.11 ±49.22),peaked at the time of 6 h (404.82 ± 13.78),then persisted at a higher level until 24 h (395.67 ± 36.23) than that in un-stimulated PMVEC (84.60 ± 23.61,P =0.001,0.000,0.000,respectively).After PMVEC pre-incubated with BIM,the level of LPS-induced TNF-αdecreased obviously (200.44 ± 27.39 vs.402.28 ± 31.07,P =0.000).Compared with LPS challenged PMVEC (407.28 ± 32.64),depletion of endogenous SSeCKS in PMVEC after inhibited by SSeCKS-siRNA significantly attenuated increase in the level of LPS-induced TNF-α (195.20 ± 13.28,P =0.000).Conclusions Down-activation of SSeCKS and PKC can inhibit the secretion of TNF-αin PMVEC induced by LPS,relieving the inflammatory response of PMVEC.

Improvement of rat pulmonary microvascular endothelial cell primary culture methods / 军事医学科学院院刊

Objective:To improve the primary culture method of rat pulmonary microvascular endothelial cells(PMVECs) and obtain purified PMVECs.Methods:The modified tissue block pasted culture method was used to isolate and culture Wistar rat PMVECs. The morphous of cultured cells were observed by microscopy. The cultured cells were identified by detecting factor Ⅷ related antigen and binding isolectin B4. Results and Conclusion:The morphous of cultured primary PMVECs in vitro showed short fusiform shape or polygon, and the monolayer of cultured cells displayed the shape of pavingstone. But the morphous changed followed the transfer of culture and the change of culture condition. The cultured cells had characterization of binding isolectin B4 and negative immunocytochemical staining for factor Ⅷ related antigen. The cultured PMVECs have good growth state and purity,and can be subcultringed stably.The observation of cell morphous integrating with immunocytochemical staining is a reasonable identification method for PMVECs.

Detection on mRNA exepression and activity of phosphodiesterase in rat pulmonary microvascular endothelial cells / 解剖学报

Objective To study the main subtypes messenger ribonucleic acid(mRNA) and the basal enzyme activity of phosphodiesterase (PDE) in rat pulmonary microvascular endothelial cells (PMVECs) through the examination mRNA expression and activity of PDE in vitro. The data were offered to reveal the relationship between PDE distributions, activity change and to accumulate data for the possibility of drug regulation of its functional alteration. Methods The cells were cultured with tissue-sticking method;the gene expression of PDEs was detected by reverse transcript polymerase chain reaction (RT-PCR), and the activity of PDEs was calculated by cyclic nucleotides content change examined with high performance liquid chromatogram (HPLC) before and after the PDE reaction( n ＝3). Results The PMVECs identified by cell immunofluorescence with polyclonal antibody of CD31 were dissociated and cultured, mRNAs of PDE1A, 1C, 2A,3A, 3B, 4A, 4D, 5A, 7A, 7B, 8A, 8B, 9A, 10A,11A were expressed in PMVECs, but there was no mRNA of PDE1B expressed in PMVECs. cAMP/cGMP-PDE in the extent of 5-20μl had a good linear correlation with its activity. Conclusion There are 17 kinds of PDE gene expression existing in PMVECs which contain of the basic enzyme with a higher activity.

Comprehensive characterization of rat pulmonary microvascular endothelial cells cultured by tissue-sticking method / 第三军医大学学报

Objective To establish a comprehensive characterization protocol of rat pulmonary microvascular endothelial cells(RPMVECs) cultured by tissue-sticking method.Methods RPMVECss were cultured with peripheral lung tissue-sticking method.Histological sections from peripheral lung tissue pieces used for cell culture were examined.With rat pulmonary artery smooth muscle cells and human umbilical vein endothelial cells as controls,the cultured cells were identified by immunocytochemical staining using CD34 antibody,lectin from Bandeiraea simplicifolia and factor Ⅷ related antigen.The cell morphology and ultrastructure were observed with inverted light microscope and transmission electron microscope respectively.Results Histological sections showed that tissue pieces were scissored from peripheral lung lobes accurately.The cultured cells had characterization of binding lectin from Bandeiraea simplicifolia and positive immunocytochemical staining with CD34 antibody,but negative for factor Ⅷ related antigen.Weibel-Palade bodies were not found in cells.Conclusion Factor Ⅷ related antigen and Weibel-Palade body are not ideal indexes for RPMVECs identification.The combination of peripheral lung tissue section,CD34 immunocytochemical staining and lectin from Bandeiraea simplicifolia binding assay provides a simple and reasonable comprehensive characterization protocol for RPMVECs.