Pretreatment with β-Boswellic Acid Improves Blood Stasis Induced Endothelial Dysfunction: Role of eNOS Activation

Abstract

Vascular endothelial cells play an important role in modulating anti-thrombus and maintaining the natural function of vascular by secreting many active substances. β-boswellic acid (β-BA) is an active triterpenoid compound from the extract of boswellia serrate. In this study, it is demonstrated that β-BA ameliorates plasma coagulation parameters, protects endothelium from blood stasis induced injury and prevents blood stasis induced impairment of endothelium-dependent vasodilatation. Moreover, it is found that β-BA significantly increases nitric oxide (NO) and cyclic guanosine 3', 5'-monophosphate (cGMP) levels in carotid aortas of blood stasis rats. To stimulate blood stasis-like conditions in vitro, human umbilical vein endothelial cells (HUVECs) were exposed to transient oxygen and glucose deprivation (OGD). Treatment of β-BA significantly increased intracellular NO level. Western blot and immunofluorescence as well as immunohistochemistry reveal that β-BA increases phosphorylation of enzyme nitric oxide synthase (eNOS) at Ser1177. In addition, β-BA mediated endothelium-dependent vasodilatation can be markedly blocked by eNOS inhibitor L-NAME in blood stasis rats. In OGD treated HUEVCs, the protective effect of β-BA is attenuated by knockdown of eNOS. In conclusion, the above findings provide convincing evidence for the protective effects of β-BA on blood stasis induced endothelial dysfunction by eNOS signaling pathway.

Figure 1. Effects of β-BA on ACh-mediated relaxation of mesenteric artery rings with or without endothelium.

Mesenteric artery rings of blood stasis rats were isolated after oral administration with β-BA (100 mg/kg/d or 200 mg/kg/d) for 7 times. β-BA induced relaxation in aortic rings with (a) or without endothelium (b) was determined. The tension responsible for the vascular relaxation and constriction was tested. The contraction was induced by 0.5 mM phenylephrine. The results are expressed as percentage of relaxation for comparison with the remaining controlled tension (Mean ± SEM, n = 8). Experimental groups: control (■), model (⧫), model + β-BA 200 mg/kg (▲), model + β-BA 100 mg/kg (×). ^##p < 0.01 versus the control group, ^#p < 0.05 versus the control group, **p < 0.01 versus the model group, *p < 0.05 versus the model group.

Figure 2. Representative images of HE staining performed on vascular endothelium in carotid aortas of blood stasis rats.

The microscopic structures of carotid aortas were observed (bar: 100 μm). Arrows: broken endothelium in aorta.

Figure 3. β-BA increased NO/cGMP levels in vascular endothelium of carotid aortas and HUVECs.

After oral administration with β-BA (100 mg/kg/d or 200 mg/kg/d) for 7 times, NO (a) and cGMP (b) production in carotid arotas of rats were examined by Griess reaction and enzyme-linked immunosorbent assay. All data represent the results (Mean ± SD, n = 8). (^#P < 0.05, ^##P < 0.01 versus the control group. *P < 0.05, **P < 0.01 versus the model group). (c,d) HEUVCs were pretreated with β-BA for 24 h before being subjected to 6 h OGD then incubated with β-BA for an additional 24 h. NO production was measured by DAF-FM DA. The amount of NO was evaluated by measuring the fluorescence intensity excited at 495 nm and emitted at 515 nm. Representative images were taken by the confocal microscope (bar: 20 μm). All data represent the results (Mean ± SD) of triplicate independent experiments. ^##p < 0.01 versus the control group, ^#p < 0.05 versus the control group, **p < 0.01 versus OGD group, *p < 0.05 versus OGD group).

Figure 4. Effects of β-BA on the phosphorylation of eNOS at Ser1177 in carotid aortas and HUVECs.

(a) After oral administration with β-BA (100 mg/kg/d or 200 mg/kg/d) for 7 times. Representative immunohistochemistry staining for p-eNOS (Ser1177) was shown on carotid arotas (bar: 100 μm); (b) HEUVCs were pretreated with β-BA for 24 h before being subjected to 6 h OGD then incubated with β-BA for an additional 24 h. Cells were immunostained with anti-phospho eNOS (Ser1177) antibodies, representative immunofluorescent staining for p-eNOS (Ser1177) was shown (bar 20 μm). DAPI: nuclear staining and merged images were also exhibited. Comparison of fluorescence intensity changes in HUVECs are presented by the histogram. (c) Western blot analysis showed the expression of p-eNOS (Ser1177) in HUVECs, and the blots were operated under the same experimental conditions. The data represent the results (Mean ± SD) of triplicate independent experiments.

Figure 5. eNOS activity was essential for β-BA mediated relaxation of mesenteric artery rings and cell viability.

(a) Representative tracings showed the vasorelaxant effects of β-BA (200 mg/kg/d) in phenylephrine pre-contracted aortic rings. β-BA induced relaxation could be significantly attenuated when the endothelium intact specimens were exposed to L-NAME (10⁻⁴ M). The values of results are expressed as Means ± SD (n = 8). Experimental groups: control (■), model (⧫), model + β-BA 200 mg/kg (▲), model + β-BA 200 mg/kg + L-NAME (○). ^##p < 0.01 versus the control group, ^#p < 0.05 versus the control group, **p < 0.01 versus the model group, *p < 0.05 versus the model group. (b) si-con and si-eNOS were transfected to HUEVCs for 12 h, then the cells were pretreated with β-BA for 24 h before being subjected to 6 h OGD then incubated with β-BA for an additional 24 h. Expression of eNOS was determined. Cell viability was measured with MTT assay. The data represent the results of (Mean ± SD) of triplicate independent experiments. ^##p < 0.01 versus the control group, **p < 0.01 versus OGD group, p < 0.01 versus β-BA group.