Effects Of TMP On The Lipopolysaccharide Induced Injury And The Expressions Of MiRNA155 And MiRNA193b In Human Umbilical Vein Endothelial Cells

Protective effect of TMP on lipopolysaccharide induced vascular hyperpermeabilityObjective:To investigate the protective effects and possible mechanisms of pretreatment with TMP on lipopolysaccharide-induced vascular hyperpermeability in guinea pigs.Methods:Eighteen of white male healthy guinea pigs were divided by randomized block design.TMP(3or6mg/kg)or saline was intravenously administered 30 min before LPS injection. LPS(100, 300, and 1,000 ug/0.1 ml/site) and saline(0.1 ml/site) were administered intracutaneously in the dorsum of guinea pigs.Vascular permeability was measured by leakage area and absorbance of 610nm(OD610) of Evans Blue dye after intracutaneous injection of LPS(100-1000 ug/site).Results:Dye leakage in the skin began to increase at 5 min,and increased significantly 2h after the injection of LPS.A significant difference was seen in the dye leakage area(F=5.77,P<0.05) and the OD610(F=7.736, P<0.05)induced by 100, 300 and 1,000 ug/site of LPS administration between the control and TMP groups(3 and 6mg/kg).Conclusion:TMP has protective effect on the hypermeability of endothelial cells induced by lipopolysaccharide, and inhibition of Rho/ROCK signaling pathway is a possible mechanism combined with previous experiments.Effects of TMP on the lipopolysaccharide induced injury in human umbilical vein endothelial cellsObjective:To observe the effects of TMP on the expressions of LIMK1 and cofilin on lipopolysaccharide(LPS)-induced injury in human umbilical vein endothelial cells and the changes of actin cytoskeleton(F-actin) morphology and distribution,with the aim to explore its possible mechanisms.Methods:1. HUVECs were separated from human umbilical vein, digested with collagenase type I and cultured in complete medium ECM. Subculture was obtained with trypsin.cells, morphology was observed under inverted microscope and identified by factor VIII-related antigen immunocytochemistry staining2. HUVECs cultured in vitro were randomly divided into 7 groups, namely the control group(equal volume of medium), LPS group(LPS 0.1ug/ml), fasudil positive control group(fasudil 3.275ug/ml+LPS 0.1ug/ml), TMP group(TMP 1500ug/ml),TMP I group(TMP 500ug/ml+LPS0.1ug/ml), TMPII group(TMP 1000ug/ml+LPS0.1ug/ml), TMPIII group(TMP 1500ug/ml+LPS 0.1ug/ml).3. The changes of Cytoskeleton(F-actin) morphology and distribution were observed under confocal laser scanning microscope.4. Quantitative real-time PCR was applied to detect the expressions of LIM kinase and cofilin m RNA in the endothelial cells.Results:1. Primary HUVECs started to adhere at 4~6h culturation in ECM, completely adhention occurred at about 24 h and cell film forming at about 3~5d.Cells exhibited cobblestone mosaic-like arrangement. Factor VIII-related antigen fluorescent antigen staining were positive, which confirmed the cultured cells were HUVECs.2. Compared with the control group, F-actin morphology changed significantly in LPS group, F-actin filaments within cytoplasmic increased and thickened, densebundles of stress fibers appeared, peripheral dense tape was broken, cells gap increased, while TMP could inhibit the change.3. No significant difference(P>0.05) was observed in the expressions of LIMK1(F=1.095, P=0.412) and cofilin(F=0.729, P=0.634) in the experimental group and the control group.Conclusions:1. The primary HUVECs were successfully isolated and cultured by enzyme perfusion digestion.2. TMP showed protective effect on LPS-induced injury in HUVECs by stabilizing cytoskelet.3. The TMP protective effect may not correlated with the expression leved of LIMK1 and cofilin genes.Effects of TMP on the expression of mi RNA155 and Mi RNA193 b in HUVECs with LPS-induced injuryObjective:To detect the expression changes of mi RNA155 and mi RNA193 in the LPS-induced injury of HUVECs before and after the adding of TMP,to investigate the effect of TMP and the possible mechanism.Methods:1. HUVECs cultured in vitro were randomly divided into four groups, namely: control group: ECM complete culture medium 4ml, without special treatment factors; TMP group: ECM medium 3.7ml and TMP 300ul(1500ug/ml); LPS group: ECM medium4 ml and LPS2ul(0.1ug/ml); TMP+LPS group: ECM medium 3.7ml+TMP 300ul(1500ug/ml) +LPS2ul(0.1ug/ml).2. Quantitative real-time PCR was used to detect expressions of mi RNA155,mi RNA193 b relative gene expression of each group.Results:Compared with the control group, the gene expression levels of mi RNA155(P=0.004)and mi RNA193b(P=0.007) in LPS group were significantly higher(P<0.01), two gene expression in TMP group was not up-regulated or down-regulated(P>0.05);Compared with LPS group, the expression of mi RNA155 decreased significantly in TMP+LPS group(P=0.011), meanwhile no significant expression differences was observed in mi RNA193-b in the two group(P=0.232).Conclusion:1. the expression of mi RNA155 and mi RNA193 b in HUVECs increased significantly after LPS stimulation.2. the protective effects of TMP on HUVECs with LPS-induced injury by down-regulating of mi RNA155 expression,not by regulating of mi R 193 b expression.