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 lipopolysaccharide on the expression of tribbles homolog 3 in acute lung injury / 安徽医科大学学报

Objective To examine the expression of tribbles homologous 3 (TRB3) on lipopolysaccharide (LPS) induced acute lung injury (ALI) and its relationship with p38-mitogen-activated protein kinase (MAPK) pathway.Methods Rats received a intravenous injection by LPS (5 ml/kg) as models of ALI and a intravenous injection by NS (5 ml/kg) as the control.In rat lung tissue the expression of TRB3 protein was examined using immunohistochemical staining, the expression of TRB3 mRNA was determined by reverse transcript polymerase chain reaction (RT-PCR).Cultured rat pulmonary microvascular cells (PMVEC) were randomly divided into dose-dependent, time-dependent and intervention groups in vitro.In dose-dependent group, PMVEC were stimulated by various concentrations of LPS (0, 2, 4, 10 μg/ml) for 4 h, and in time-dependent group PMVEC were challenged by 10 μg/ml LPS for different time (0, 4, 8, 12 h).In intervention group, PMVEC grown in normal medium or medium with 10 μg/ml LPS for 4 h were pretreated using p38-MAPK inhibitor (10 μmol/L SB203580) for 2 h.Western blot was used to examine expression of TRB3, p-p38 and p38-MAPK.Results Immunohistochemical staining showed that TRB3 protein distributed in rat alveolar walls and glandular epithelium.Increased TRB3 mRNA expression using RT-PCR were found in lung tissue of rats injected by LPS when compared to those in NS group (t=15.524, P<0.01). Increased TRB3 mRNA expression using RT-PCR had also been found in PMVEC stimulated by LPS when compared to those in NS group (t=7.549, P<0.01). In PMVEC, LPS significantly increased the expression of TRB3 protein in a dose-dependent manner (0, 2, 4, 10 μg/ml) after stimulation for 4 h (F=12.619, P<0.001).At indicated time-points after PMVEC were challenged by 10 μg/ml LPS, the expression of TRB3 protein raised at 4 h , then decreased gradually at 8 h , but still was higher than 0 h group , there were significant difference (F=11.273, P<0.001). LPS significantly increased the expression of p-p38 protein after stimulation for 4 h when compared to the control group, LPS also increased the expression of TRB3 protein after stimulation for 4 h when compared to the control group (t=49.121,15.113,P<0.001). SB203580 decreased the protein levels of p-p38 in response to LPS, SB203580 also decreased the protein levels of TRB3 in response to LPS(t=7.040,11.900,P<0.05,0.001).SB203580 alone had no effect on the expression of p-p38 and TRB3,when compared to the control group,there was not statistically significant.Conclusion Levels of TRB3 protein increase in LPS-induced acute lung injury, and is regulated by p38-MAPK pathway.

RhoA/mDia1 pathway involved in the expression of p-ERM in the pulmonary micro-vascular endothelial cell induced by lipopolysaccharide / 中华急诊医学杂志

Objective To investigate the possibility of the involvement of RhoA/mDia1 pathway to cause the expression of phosphorylate ezrin-radixin-moesin (p-ERM) in rat pulmonary micro-vascular endothelial cell (PMVEC) after the stimulation of lipopolysaccharide (LPS).Methods The specimens of lung tissue were taken from healthy male SPF grade SD rat with 100-120 g body weight which was purchased from the laboratory animal center of Anhui province.After culture,the PMVECs were randomly divided into dose-dependent groups (0,0.1,1,10 μg/mL LPS added in PMVECs and cultured for30 min,n =8 in each),time-dependent groups (10 μg/mL LPS added to PMVECs cultured for 0,15,30,60,120 min,n =8 in each) and intervention group (n =8).In the intervention group,PMVECs were cultured with 1 μg/mL C3 transferase in serum free media for 240 min,followed by treatment with 10 μg/mL LPS for 30 min.Meanwhile,two control groups in serum-free DMEM medium were made by adding 10.μg/mL LPS to PMVECs and 1 μg/mL C3 transferase to PMVECs respectively cultured for 30 min (n =8 in each).Western blot was used to detect the level of p-ERM,ERM and mDia1.Data were analyzed with SPSS 16.0 software,while one way analysis of variance (ANOVA) was used to compare multiple sets of variables,the intergroup comparisons were analyzed by the least-significant-difference (LSD) tests,with P ＜0.05 for the statistically significant difference.Results ERM,p-ERM and mDia1 were presented in rat PMVEC.Stimulation with LPS up-regulated p-ERM,mDia1 in a dose-dependent manner:LPS [0 μg/mL LPS group:(0.520±0.101),0.1 μg/mL LPS group:(0.657 ±0.092),1 μg/mL LPS group:(0.891 ±0.167),10 μg/mL LPS group:(1.227 ±0.106);0 μg/mL group vs.0.1 μg/mL group,P ＞0.05;the rest P ＜0.01];and mDia1 [0 μg/mL LPS group:(0.200 ±0.102),0.1 μg/mL LPS group:(0.430 ±0.121),1 μg/mL LPS group:(0.603 ±0.154),10 μg/mL LPS group:(0.887 ±0.204);0.1 μg/mL group vs.1 μg/mL group,P ＞ 0.05;the rest P ＜ 0.05].In time-dependent group,the level of p-ERM protein increased at 15 min (0.670 ±0.149),peaked at 30 min (1.175 ±0.103),then decreased,at 60 min (0.959 ±0.189),90 min (0.842 ±0.129),but kept at higher level at 120 min (0.767 ±0.097) than that in control group (0.471 ±0.157,15 min group vs.120 min group,60 min group vs.90 min group and 90 min group vs.120 min group,P ＞ 0.05;the rest P ＜ 0.05);and the level of mDia1 increased at 15 min (0.779 ±0.035),peaked at 30 min (0.889 ±0.036) then decreased at 60 min (0.648 ±0.019),90 min (0.582 ±0.068),but kept at higher level at 120 min (0.526 ±0.059) than that in control group (0.284±0.118,all P ＜ 0.01).C3 transferase caused a marked attenuation of LPS induced p-ERM expression [control group:(0.339 ± 0.069);C3 + LPS group:(0.438 ± 0.07);C3 control group:(0.352 ± 0.071);LPS control group:(0.634 ± 0.191),C3 + LPS group vs.LPS control group,P =0.01],as the same in mDia1 [control group:(0.449 ±0.122);C3 + LPS group:(0.380 ±0.148);C3 control group:(0.404 ±0.164);LPS control group:(0.622 ±0.174),C3 + LPS group vs.LPS control group,P ＜ 0.01].Conclusions LPS could up-regulated the expression of p-ERM protein,and inhibition of RhoA/mDia1 signal pathway by C3 transferase could down-regulated the p-ERM levels.

Study of the level of ERM proteins in pulmonary microvascular endothelial cells induced by tumor necrosis factor-α / 中华急诊医学杂志

Objective To investigate the effect of tumor necrosis factor-α (TNF-α) on the levels of ezrin-radixin-moesin (ERM) proteins and the phosphorylated ERM proteins (p-ERM) in rat pulmonary microvascular endothelial cells (PMVEC),and to explore Rho kinase (ROCK) influencing on modulation of the ERM proteins phosphorylation.Methods Cultured rat pulmonary microvascular endothelial cells were randomly divided into dose-dependent and time-dependent groups.In dose-dependent group,cells were cultured with different doses of TNF-α (0,0.1,1,10 μg/LTNF-α) for 60 min.In time-dependent group,cells were cultured with TNF-α (10 μg/L) for 0,15,30,60,90,120,180 min.In ROCK inhibitor (Y27632) intervention group,cells were cultured with TNF-α (10 μg/L) or Y27632 (30 μmol/L) + TNF-α (10 μg/L) for 60 min respectively.The levels of ERM proteins and p-ERM were determined by western blot.One way analysis of variance (ANOVA) was employed for statistical analysis by using SPSS version 16.0 software to compare values among all groups.A significant difference was presumed as a P value ＜ 0.05.Results Western blot revealed that ERM and p -ERM proteins were present in rat PMVEC.Stimulation withTNF-α gradually up-regulated the level of pERM proteins in a dose-dependent manner [0 μg/LTNF-α group:(0.648 ± 0.102),0.1 μg/LTNF-αgroup:(0.728-±0.082),1 μg/LTNF-α group:(0.926±0.121),10 μg/LTNF-α group:(1.245 ±0.134),all P =0.000].In time-dependent group,the level of p-ERM proteins rose at 15 min (0.777 ±0.151),peaked at 90 min (1.295 ±0.176),then decreased gradually at 120 min (0.802 ±0.139),but stayed higher level at 180 min (0.669 ±0.128) than that in un-stimulated 0 min group (0.631 ±0.123,P=0.004,0.000,0.001,0.016,respectively).When PMVEC pre-incubated with ROCK inhibitor and TNF-t,the level of p-ERM proteins caused a marked attenuation of TNF-αstimulation [(0.634 ± 0.112) vs.(0.875 ± 0.164),P =0.002],however,there are no significant differences compared to ROCK inhibitor alone group (0.661 ± 0.108) and no intervention group (0.654 ± 0.125),P =0.973,P =0.900,respectively).Conclusions TNF-α could induce up-regulation of the level of the phosphorylated ERM proteins in rat PMVEC,and ROCK signal molecules might involve in modulation of the ERM proteins phosphorylation.

Role of CREB in LPS-induced injury of RPMVEC / 中国药理学通报

Aim To investigate the role of cAMP re-sponse element binding protein (CREB)in the injury of rat pulmonary microvascular endothelial cell (RPM-VEC)induced by LPS.Methods RPMVECs were i-solated and cultured in vitro,Western-blot was used to assay phosphorylation levels of CREB.Endothelial per-meability was determined by measuring the influx of Evans blue-labeled albumin across endothelial mono-layer.Results LPS increased CREB phosphorylation at Ser 1 3 3 in RPMVEC in a time-dependent manner , peaked at 30 min,but still higher at 120 min compared with basal control group.Pretreatment of cells with PKA inhibitor V5681 nearly suppressed the CREB phosphorylation stimulated in the presence of LPS,and the monolayer permeability of PMVEC was significantly increased. Conclusions LPS rapidly induces the phosphorylation of CREB in RPMVEC,and PKA me-diates the process.During the process of LPS-stimula-ted injury of RPMVEC,phosphorylation of CREB may play a protective role.

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.