Chinese medicine Tongxinluo modulates vascular endothelial function by inducing eNOS expression via the PI-3K/Akt/HIF-dependent signaling pathway.

AIM OF THE STUDY: 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. MATERIALS AND 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. CONCLUSION: 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.

[Effects of Tongxinluo on repeated hypoxic tolerance in mice].

OBJECTIVE: To observe the effects of Tongxinluo (TXL) on repeated hypoxic tolerance in mice and explore the underlying mechanisms. METHODS: Mice were randomly divided into groups of repeated hypoxia (control) and TXL according to body weights. The mice in each group were exposed to acute repeated hypoxia for 0 run (H0), 1 run (H1), 3 runs (H3) and 5 runs (H5). The animal's tolerance time of each hypoxic exposure was recorded. Western blot was used to measure the protein levels of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in cortex tissue. RESULTS: The hypoxic tolerance time in control and TXL groups significantly increased run by run. Both HIF-1alpha and VEGF proteins in two groups increased gradually. Compared with control group, the tolerance time in H1 of TXL group [(18.0 +/- 2.4) minvs (15.6 +/- 2.0) min], H3 [(68.3 +/- 13.2) min vs (41.7 +/- 9.0) min) and H5 (85.9 +/- 7.0) min vs (51.4 +/- 14.4) min] increased (P < 0.05 or P < 0.01); the HIF-1alpha protein expression in H1 of TXL group (0.95 +/- 0.04 vs 0.79 +/- 0.02), H3 (1.01 +/- 0.03 vs 0.85 +/- 0.02), H5 (1.16 +/- 0.02 vs 0.92 +/- 0.03) increased (P < 0.05 or P < 0.01); the VEGF protein expression in H3 of TXL group (1.14 +/- 0.02 vs 0.89 +/- 0.03), H5 (1.34 +/- 0.05 vs 0.99 +/- 0.07) increased (P < 0.05 or P < 0.01). CONCLUSIONS: Under repeated hypoxia, an organism has a strong adaptive ability. The rises of HIF-1alpha and VEGF may be an adaptive mechanism. TXL can increase obviously the adaptive ability of mice to hypoxia.