Abstract
Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is a protective principle in the vasculature. Many cardiovascular diseases are associated with reduced NO bioactivity and eNOS uncoupling due to oxidative stress. Compounds that reverse eNOS uncoupling and increase eNOS expression are of therapeutic interest. Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, an extract of ZSS increased eNOS promoter activity, eNOS mRNA and protein expression, and NO production in a concentration- and time-dependent manner. Major ZSS constituents include saponins, such as jujuboside A and B, and pentacyclic triterpenes, such as betulin and betulinic acid. Jujuboside A, jujuboside B, or betulin had no significant effect on eNOS expression, whereas betulinic acid increased eNOS mRNA and protein expression in HUVEC and EA.hy 926 cells. Interestingly, betulinic acid also attenuated the expression of NADPH oxidase subunits Nox4 and p22phox, thereby reducing oxidative stress and improving eNOS function. Consequently, betulinic acid-treated endothelial cells showed an increased production of bioactive NO (as indicated by a higher efficacy in stimulating cGMP generation in RFL-6 reporter cells). Thus, betulinic acid possesses combined properties of eNOS up-regulation and NADPH oxidase down-regulation. Compounds such as betulinic acid may have a therapeutic potential in cardiovascular disease.
Footnotes
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This work was supported by the Collaborative Research Center SFB 553 (project A1 to H.L. and U.F.) from the Deutsche Forschungsgemeinschaft (Bonn, Germany).
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This article contains parts of the doctoral theses of K.S.-F. and of L.B.: Steinkamp-Fenske K (2007) Identifikation protektiver Naturstoffe aus kardiovaskulär wirksamen chinesischen Heilpflanzen. Doctoral dissertation, Johannes Gutenberg University, Mainz, Germany; and Bollinger L (2005) Regulation der gen-expression der endothelialen NO-synthase durch extrakte chinesischer Heilpflanzen. Doctoral dissertation, Johannes Gutenberg University, Mainz, Germany.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.123356.
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ABBREVIATIONS: NO, nitric oxide; eNOS, endothelial NO synthase; BH4,(6R)-5,6,7,8-tetrahydro-l-biopterin; HUVEC, human umbilical vein endothelial cells; ZSS, Zizyphi Spinosi semen; apoE-KO, apolipoprotein E-knockout; ROS, reactive oxygen species; kb, kilobase; RT, reverse transcription; PCR, polymerase chain reaction; SOD, superoxide dismutase; IBMX, 3-isobutyl-1-methylxanthine; L-012, 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4(2H,3H)dione; PKC, protein kinase C; CAS, Chemical Abstracts Service.
- Received March 24, 2007.
- Accepted May 10, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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