[Effect of total Panax notoginseng saponins on the activity of human endothelial nitric oxide synthase gene promoter].
OBJECTIVE
To study the mechanism of total Panax notoginseng saponin (tPNS) in regulating the transcription activity of human endothelial nitric oxide synthase (heNOS) gene promoter.


METHODS
With gene recombination technique, we subcloned the heNOS gene promoter sequence (from -1 to -1 600 bp) into the BglII/HindIII sites of the firefly luciferase reporter gene vector, pGL2-Basic (Promega), to yield the recombinant plasmid designated as peNOS-Luc. With lipofectamine- mediated co-transfection technique, peNOS-Luc, pGL2-Basic and pCMV-beta were cotransfected into NIH3T3 cells, which were treated with lipopolysaccharide (LPS), tPNS and transforming growth factor beta1 (TGFbeta1) respectively. The relative activities (Luc/beta-gal) were subsequently determined in the cell lysates to evaluate the effects of these 3 factors on the activity of heNOS gene promoter.


RESULTS
Double restriction enzyme digestion and sequencing both confirmed that the recombinant plasmid, peNOS-Luc, was constructed correctly, which could be effectively expressed in NIH3T3 cells. Upon LPS stimulation, the luciferase activity was obviously decreased, contrary to the results of tPNS and TGFbeta1 treatment, and between the latter two agents, TGFbeta1 produced higher transcription activity.


CONCLUSIONS
A firefly luciferase reporter gene vector containing heNOS gene promoter sequence has been constructed correctly. tPNS can up-regulate the activity of heNOS gene promoter in NIH3T3 cells.
Zhang
Li-hua
LH
Department of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China.
Jia
Yu-hua
YH
Sun
Xue-gang
XG
Xing
Fei-yue
FY
Liu
Zhi-Feng
ZF
Song
Ge
G
Jiang
Yong
Y
D000818:Animals; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000378:metabolism; D053723:Panax notoginseng / Q000737:chemistry*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012503:Saponins / Q000302:isolation & purification / Q000494:pharmacology*; D014158:Transcription, Genetic
D012503:Saponins; D008156:Luciferases; D052250:Nitric Oxide Synthase Type III