Traditional Chinese medicine xin-mai-jia recouples endothelial nitric oxide synthase to prevent atherosclerosis in vivo

Ya-Ling Yin, Mo-Li Zhu, Jia Wan, Chong Zhang, Guo-Pin Pan, Jun-Xiu Lu, Song Ping, Yuan Chen, Fan-Rong Zhao, Hai-Ya Yu, Tao Guo, Xu Jian, Li-Ying Liu, Jia-Ning Zhang, Guang-Rui Wan, Shuang-Xi Wang, Peng Li, Ya-Ling Yin, Mo-Li Zhu, Jia Wan, Chong Zhang, Guo-Pin Pan, Jun-Xiu Lu, Song Ping, Yuan Chen, Fan-Rong Zhao, Hai-Ya Yu, Tao Guo, Xu Jian, Li-Ying Liu, Jia-Ning Zhang, Guang-Rui Wan, Shuang-Xi Wang, Peng Li

Abstract

Endothelial dysfunction, which is caused by endothelial nitric oxide synthase (eNOS) uncoupling, is an initial step in atherosclerosis. This study was designed to explore whether Chinese medicine xin-mai-jia (XMJ) recouples eNOS to exert anti-atherosclerotic effects. Pretreatment of XMJ (25, 50, 100 μg/ml) for 30 minutes concentration-dependently activated eNOS, improved cell viabilities, increased NO generations, and reduced ROS productions in human umbilical vein endothelial cells incubated with H2O2 for 2 hours, accompanied with restoration of BH4. Importantly, these protective effects produced by XMJ were abolished by eNOS inhibitor L-NAME or specific eNOS siRNA in H2O2-treated cells. In ex vivo experiments, exposure of isolated aortic rings from rats to H2O2 for 6 hours dramatically impaired acetylcholine-induced vasorelaxation, reduced NO levels and increased ROS productions, which were ablated by XMJ in concentration-dependent manner. In vivo analysis indicated that administration of XMJ (0.6, 2.0, 6.0 g/kg/d) for 12 weeks remarkably recoupled eNOS and reduced the size of carotid atherosclerotic plaque in rats feeding with high fat diet plus balloon injury. In conclusion, XMJ recouples eNOS to prevent the growth of atherosclerosis in rats. Clinically, XMJ is potentially considered as a medicine to treat patients with atherosclerosis.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. XMJ increases eNOS phosphorylation and…
Figure 1. XMJ increases eNOS phosphorylation and activity, enhances NO generation, and improves cell viabilities in HUVECs.
HUVECs were pretreated with XMJ (25, 50, 100 μg/ml) for 30 minutes and then incubated with H2O2 (0.2 mM) for 2 hours. (A) Differentially expressed genes were analyzed by mRNA array in cells treated with vehicle and XMJ (50 μg/ml). Signaling pathway analysis was performed by mapping genes to KEGG pathways. The p-value denotes the significance of the Pathway correlated to the conditions. (B) The eNOS phosphorylation was assayed by western blot in total cell lysates. The representative blots were cropped and the gels were run under the same experimental conditions. (C) The eNOS activity was determine by H3-L-arginine-based method. (D) Intracellular NO levels were detected by DAF fluorescence. (E) Cell viability was measured by MTT. All data were expressed as mean ± SEM. N is 3 in each group. *P < 0.05 VS Control, #P < 0.05 VS H2O2 alone.
Figure 2. XMJ reduces oxidative stress in…
Figure 2. XMJ reduces oxidative stress in HUVECs treated with H2O2.
HUVECs were pretreated with XMJ (25, 50, 100 μg/ml) for 30 minutes and then incubated with H2O2 (0.2 mM) for 2 hours. (A) Distributions of eNOS dimer and monomer were determined by cold western blot analysis. (B) ROS productions were detected by DHE fluorescence. (C) MDA levels in total cell lysates. (D) SOD activity in total cell lysates. All data were expressed as mean ± SEM. N is 3 in each group. *P < 0.05 VS Control, #P < 0.05 VS H2O2 alone. NS indicates no significance.
Figure 3. XMJ increases BH4 levels in…
Figure 3. XMJ increases BH4 levels in H2O2-treated HUVECs.
HUVECs were pretreated with XMJ (25, 50, 100 μg/ml) for 30 minutes and then incubated with H2O2 (0.2 mM) for 2 hours. (A) GTPCH1 protein level was measured by western blot in total cell lysates. The representative blots were cropped and the gels were run under the same experimental conditions. (B and C) The levels of total biopterins in (B) and BH4 in (C) were determined by HPLC. (D) BH2/BH4 ratio was calculated. All data were expressed as mean ± SEM. N is 3 in each group. *P < 0.05 VS Control, #P < 0.05 VS H2O2 alone.
Figure 4. Inhibition of eNOS abolished the…
Figure 4. Inhibition of eNOS abolished the beneficial effects of XMJ in HUVECs.
(A–C) HUVECs were pretreated with XMJ (50 μg/ml) plus L-NAME (1 mM) for 30 minutes and then incubated with H2O2 (0.2 mM) for 2 hours. eNOS activity was determined by H3-L-arginine-based method in (A). Intracellular NO levels were detected by DAF fluorescence in (B). ROS productions were detected by DHE fluorescence in (C). All data were expressed as mean ± SEM. N is 3 in each group. *P < 0.05 VS Vehicle or Control, #P < 0.05 VS H2O2 alone. NS indicates no significance. (D–F) HUVECs were transfected with control or eNOS siRNA for 48 hours followed by treated with XMJ (50 μg/ml) in presence or absence of H2O2 (0.2 mM) for 2 hours. The levels of eNOS protein in (D), intracellular NO in (E), and ROS productions in (F) were determined, respectively. All data were expressed as mean ± SEM. N is 3 in each group. *P < 0.05 VS Control siRNA alone, #P < 0.05 VS control siRNA plus H2O2. NS indicates no significance.
Figure 5. XMJ prevents endothelial dysfunction in…
Figure 5. XMJ prevents endothelial dysfunction in aortic arteries isolated from rats ex vivo.
The isolated rat aortic rings were incubated with XMJ (25, 50, 100 μg/ml) for 30 minutes followed by exposure to H2O2 (0.2 mM) for 6 hours. (A) NO levels and (B) MDA levels in aortic tissues were assessed, respectively. (C) The endothelium-dependent relaxation induced by acetylcholine (Ach) and (D) endothelium-independent relaxation elicited by sodium nitroprusside (SNP) were assayed in organ chamber. All data were expressed as mean ± SEM. N is 5 in each group. *P < 0.05 VS Control, #P < 0.05 VS H2O2 alone.
Figure 6. XMJ reduces the formation of…
Figure 6. XMJ reduces the formation of carotid atherosclerotic plaque in rats.
(A) The protocol of animal experiment. (B and C) At the end of experiment, carotid artery was subjected to perform morphological analysis by Oil Red staining in (B) and quantitative analysis of atherosclerotic plaque size in (C). (D and E) Histological analysis by HE staining in (D) and quantitative analysis of atherosclerotic plaque size in (E) were performed. a, Sham; b, Sham + XMJ-6.0; c, Atherosclerosis (AS); d, AS + XMJ-0.6; e, AS + XMJ-2.0; f, AS + XMJ-6.0; g, Lovastatin; h, ZBT. All data were expressed as mean ± SEM. 10–15 rats per group. *P < 0.05 VS Control, #P < 0.05 VS AS alone.
Figure 7. XMJ recouples eNOS and inhibits…
Figure 7. XMJ recouples eNOS and inhibits oxidative stress in atherosclerotic rats.
The protocol of animal experiment was described in Fig. 6A. Carotid artery was subjected to assess the levels of (A) BH4 by HPLC and (B) p-eNOS by IHC. Blood was collected to assay (C) serum NO level by Griess method. (D) Distributions of eNOS dimer and monomer were determined by cold western blot analysis. a, Sham; b, Sham + XMJ-6.0; c, Atherosclerosis (AS); d, AS + XMJ-0.6; e, AS + XMJ-2.0; f, AS + XMJ-6.0; g, Lovastatin; h, ZBT. All data were expressed as mean ± SEM. 10–15 rats per group. *P < 0.05 VS Control, #P < 0.05 VS AS alone. (E) Proposed mechanism of XMJ against atherosclerosis.

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Source: PubMed

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