Chinese medicine Tongxinluo modulates vascular endothelial function by inducing eNOS expression via the PI-3K/Akt/HIF-dependent signaling pathway.
OBJECTIVE
To investigate the molecular mechanisms whereby the Chinese medicinal compound Tongxinluo improves vascular endothelial function through studying the induction of endothelial nitric oxide synthase (eNOS) and its upstream signaling pathway.


METHODS
Hyperhomocysteinemia was induced in Wistar rats by a methionine-rich diet followed by Tongxinluo treatment. The aorta ring was isolated for measuring vascular dilation of aorta and eNOS expression. Human umbilical vein endothelial cells (HUVECs) were transfected with AP-1, NF-κB, HRE or eNOS reporter plasmid followed by Tongxinluo exposure. Expression of the reporter genes was measured by luciferase assay. The level of eNOS was studied by western blot and the nitric oxide content was measured using the nitrate reductase method. HUVECs were also transiently transfected with the dominant negative mutant of HIF-1, PI-3K or Akt to explore the role of HIF and PI-3K/Akt pathway in eNOS induction by Tongxinluo.


RESULTS
Tongxinluo could significantly up-regulate the expression of eNOS in the aortic tissue and improve the endothelium-dependent vasodilation of the aorta ring. Additionally, Tongxinluo at various doses could significantly enhance the expression of HRE and eNOS reporter gene as well as up-regulate the protein level of eNOS. Meanwhile, Tongxinluo caused a dose-dependent increase in the NO content in the supernatant of HUVECs. Suppression of HIF-1 activation by DN-HIF or inhibition of PI-3K/Akt pathway by ΔP85 or DN-Akt both attenuated HRE reporter gene activation and eNOS induction by Tongxinluo.


CONCLUSIONS
Tongxinluo, a compound Chinese traditional medicine, up-regulates the expression of eNOS via the PI-3K/Akt/HIF-dependent signaling pathway, thus improving the endothelium-dependent vasodilation.
Liang
Jun Qing
JQ
The International Cooperation Laboratory on Signal Transduction of Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China.
Wu
Kun
K
Jia
Zhen Hua
ZH
Liu
Chang
C
Ding
Jin
J
Huang
Shan Na
SN
Yin
Pei Pei
PP
Wu
Xiang Chun
XC
Wei
Cong
C
Wu
Yi Ling
YL
Wang
Hong Yang
HY
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002681:China; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000502:physiology*; D020834:Ethnopharmacology; D017930:Genes, Reporter; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008516:Medicine, Chinese Traditional; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology
D004365:Drugs, Chinese Herbal; D051793:Hypoxia-Inducible Factor 1; C513941:tongxinluo; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt