Ursolic acid from the Chinese herb Danshen (Salvia miltiorrhiza L Ursolic acid from the Chinese herb Danshen (Salvia miltiorrhiza L.) upregulates eNOS and downregulates Nox4 expression in human endothelial cells  Katja Steinkamp-Fenske, Larissa Bollinger, Natalie Völler, Hui Xu, Ying Yao, Rudolf Bauer, Ulrich Förstermann, Huige Li  Atherosclerosis  Volume 195, Issue 1, Pages e104-e111 (November 2007) DOI: 10.1016/j.atherosclerosis.2007.03.028 Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Total aqueous Danshen extract increases eNOS promoter activity and eNOS mRNA expression in human endothelial cells. Human EA.hy 926 endothelial cells were stably transfected with a 3.5kb human eNOS promoter fragment driving a luciferase reporter gene. These cells as well as normal EA.hy 926 cells were treated for 18h with various dilutions of total aqueous Danshen extract (stock solution corresponding to 5g raw Danshen per ml). In the transfected cells, luciferase activity was analyzed as a determinant of eNOS promoter activity (panel A). In normal EA.hy 926 cells eNOS mRNA expression was analyzed with RNase protection assays. Panel B demonstrates an original gel (performed in triplicate). The gel shows the protected bands for eNOS and for β-actin (used for normalization). Panels C and D demonstrate the concentration- and time-dependency of Danshen's effect on eNOS mRNA expression, respectively. Symbols represent mean±S.E.M., n=12–15 (*p<0.05; **p<0.01; ***p<0.001 compared with control). Atherosclerosis 2007 195, e104-e111DOI: (10.1016/j.atherosclerosis.2007.03.028) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Total aqueous Danshen extract enhances eNOS protein expression and NO production. EA.hy 926 cells were treated for 18h with aqueous Danshen extract (dilution 1:1000, stock solution corresponding to 5g raw Danshen per ml) and eNOS protein expression was analyzed with Western blot using a monoclonal anti-eNOS antibody. Panel A shows a representative blot. Panel B illustrates results of densitometric analyses. Panel C: EA.hy 926 cells were treated for 18h with various dilutions of aqueous Danshen extract. Oxidation products of NO, nitrite and nitrate, in the cell culture supernatant were assayed by NO-ozone chemiluminescence using a NO Analyzer. Columns represent mean±S.E.M., n=12–15 (*p<0.05, **p<0.01, compared with control). Atherosclerosis 2007 195, e104-e111DOI: (10.1016/j.atherosclerosis.2007.03.028) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Effects of different fractions of Danshen on human eNOS promoter activity and eNOS mRNA expression. Human EA.hy 926 endothelial cells were stably transfected with a 3.5kb human eNOS promoter fragment driving a luciferase reporter gene. Cells were treated for 18h with dichloromethane, methanol and water extracts of Danshen (100μg/ml each). Luciferase activity was analyzed as a determinant of eNOS promoter activity. eNOS mRNA expression was analyzed with RNase protection assays. Columns represent mean±S.E.M., n=9–12 (*p<0.05, **p<0.01 vs. untreated cells). Atherosclerosis 2007 195, e104-e111DOI: (10.1016/j.atherosclerosis.2007.03.028) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Ursolic acid enhances eNOS mRNA expression, eNOS protein expression and NO production. Human EA.hy 926 cells (panel A) or HUVEC (panel B) were treated with the alcohol-soluble Danshen constituents baicalin or ursolic acid for 18h and eNOS mRNA expression was analyzed with quantitative Real-Time RT-PCR. Panel C: EA.hy 926 cells were treated for 18h with ursolic acid (10μM) and eNOS protein expression was analyzed with Western blot using a monoclonal anti-eNOS antibody. Panel D: Human EA.hy 926 cells were treated with ursolic acid (10μM) for 18h. Bioactive NO from EA.hy 926 cells was bioassayed using guanylyl cyclase-containing RFL-6 reporter cells. cGMP content in RFL-6 cells was determined with radioimmunoassay. Columns represent mean±S.E.M., n=9–18 (**p<0.01 compared with control). Atherosclerosis 2007 195, e104-e111DOI: (10.1016/j.atherosclerosis.2007.03.028) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Ursolic acid downregulates Nox4 expression and reduces ROS production. Human EA.hy 926 cells were treated with 10μM ursolic acid for 18h and mRNA expression of NADPH oxidase subunits Nox4 and p22phox was analyzed with quantitative Real-Time RT-PCR (panel A). Panel B: EA.hy 926 cells, with or without apocynin pre-treatment (300μM, 48h), were treated with 10μM ursolic acid for 18h. ROS production was measured with L-012-derived chemiluminescence. Columns represent mean±S.E.M., n=9–15 (**p<0.01; n.s., not significant). Atherosclerosis 2007 195, e104-e111DOI: (10.1016/j.atherosclerosis.2007.03.028) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions