ABSTRACT
AIM: To investigate the effects of norepinephrine(NE)on vascular endothelial cell damage in-duced by lipopolysaccharides(LPS).METHODS: Human umbilical vein endothelial cells(HUVEC-12)were cultured with LPS at 100 mg/L to establish the cell damage model.Real-time PCR and Western blot were used to determine the ex-pressions of VE-cadherin at mRNA and protein levels.The levels of TNF-α,IL-1β,IL-2 and IL-10 in culture supernatant were measured by ELISA.The reactive oxygen species(ROS)production in the endothelial cells was detected by ROS as-say kit.RESULTS: LPS decreased both mRNA and protein levels of VE-cadherin accompanied by increased levels of TNF-α,IL-1β,IL-2 and intracellular ROS,and decreased level of IL-10 in the endothelial cells.NE reversed the expres-sion of VE-cadherin at mRNA and protein levels under the condition of LPS treatment in a dose -dependent manner,and al-so alleviated the intracellular oxidative stress.CONCLUSION: NE reverses the endothelial damage induced by LPS, which may be related to the up-regulation of VE-cadherin level and the decreases in oxidative stress and inflamatory media-tors.
ABSTRACT
<p><b>OBJECTIVE</b>This study examined the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), on eukaryotic initiation factor (eIF- 4E) expression in rat myocardial fibroblasts infected by Coxsackievirus B3 (CVB3) in order to identify the drug target for treatment of viral myocarditis.</p><p><b>METHODS</b>Primary cultured rat myocardial fibroblasts were treated with CVB3 with multiplicity of infection (MOI=0.5 PFU/cell). The experiment consisted of four groups in which the cultured rat fibroblasts cells were treated with CVB3, rapamycin (10 nM) and CVB3 + rapamycin or placebo (control). Experimental model of CVB3-infected myocardial fibroblasts was confirmed by detection of CVB3 mRNA expression with RT-PCR and observation of morphological changes of the infected cells with microscopy. eIF-4E expression was determined by both RT-PCR and Western Blot methods.</p><p><b>RESULTS</b>Morphological changes were found in the fibroblasts treated with MOI 0.5 PFU/cell of CVB3 by transmission electron microscope and the viral particles were found in the cytoplasm. CVB3 mRNA was expressed in CVB3-infected fibroblasts after 1, 2, and 3 days after infection and 2 days after passage. The gray scale values of the eIF- 4E /beta -actin in the control, the CVB3, the rapamycin and the CVB3+rapamycin groups were 0.73 +/- 0.07, 0.87 +/- 0.03, 0.32 +/- 0.03 and 0.56 +/- 0.04 respectively detected by RT-PCR, and were 0.79 +/- 0.09, 1.35 +/- 0.12, 0.55 +/- 0.04, and 0.62 +/- 0.07 respectively detected by Western blot. EIF- 4E expression in the CVB3 group was higher than that in the control group. Both the rapamycin and the CVB3+rapamycin groups had lower eIF- 4E expression than the control and the CVB3 groups.</p><p><b>CONCLUSIONS</b>CVB3 can infect myocardial fibroblasts and up-regulate the eIF- 4E expression in rat myocardial fibroblasts. Rapamycin can inhibit eIF- 4E expression and may be a potential medicine for treatment of viral myocarditis. It was suspected that mTOR/eIF- 4E signal pathway in rat myocardial fibroblasts might play an important role in the pathogenesis of viral myocarditis.</p>