Buyang Huanwu Decoction Promotes Angiogenesis after Cerebral Ischemia by Inhibiting the Nox4/ROS Pathway

Abstract

Background: Buyang Huanwu decoction (BYHWD), an important traditional Chinese medicine (TCM), has been used clinically for centuries for the treatment of various diseases. The study aims to explore the BYHWD effects on angiogenesis and neuroprotection after cerebral ischemia/reperfusion (CI/R) injury in rats and to explore the underlying angiogenic roles and mechanisms of BYHWD in hydrogen peroxide (H₂O₂) induced oxidative stress in human umbilical vein endothelial cells (HUVECs) model.

Methods: The effects of BYHWD on neurological function were screened by measuring neurological deficits, spatial memory function, and angiogenesis (by microvascular density (MVD) and cerebral blood flow (CBF)) after CI/R injury in middle cerebral artery occlusion (MCAO) in vivo in rats. In vitro, we examined the angiogenic roles and mechanisms of action of BYHWD in an H₂O₂-induced oxidative stress HUVECs model by measuring cell viability, apoptosis, vascular tube formation, intracellular ROS generation, NADPH oxidase (Nox) activity, and Nox4 protein expression.

Results: BYHWD significantly improved neurological function, including neurological deficits and spatial learning and memory, and significantly increased MVD and CBF in the ischemic penumbra after CI/R injury in rats. BYHWD significantly increased cell viability, inhibited apoptosis, induced vascular tube formation, decreased intracellular ROS generation, and reduced Nox activity and Nox4 protein expression in H₂O₂-treated HUVECs in a dose-dependent manner.

Conclusions: Our study demonstrates that BYHWD promotes neurological function recovery and increases angiogenesis. BYHWD exerts angiogenic effects against cerebral ischemic injury through the downregulation of Nox4, which results in the reduction of ROS generation.

Figure 1

Effects of BYHWD on neurological deficit after focal cerebral ischemia in rats. The neurological deficit for each group was measured at 1, 2, 3, and 4 w after focal cerebral ischemia. ^#P < 0.05 and ^##P < 0.01 versus vehicle group on the same day (n = 10).

Figure 2

Effects of BYHWD in spatial acquisition trials and probe trials of Morris water maze after focal cerebral ischemia in rats. (a) Latency for reaching the hidden platform during 5 consecutive days of spatial acquisition training. (b) Representative search patterns during the probe transfer trial. (c) Time spent in the quadrant where the platform had previously been located during the probe transfer trial. ^#P < 0.05 and ^##P < 0.01 versus vehicle group on the same day (n = 10).

Figure 3

Effects of BYHWD on microvessel density (MVD) after focal cerebral ischemia in rats. Representative microphotographs of factor VIII-positive MVD for each group at 4 w after focal cerebral ischemia (n = 6). Scale bar, 50 μm.

Figure 4

Effect of BYHWD on cerebral blood flow (CBF) after focal cerebral ischemia in rats. CBF for each group before the operation, and 1, 2, 3, and 4 w after focal cerebral ischemia. ^#P < 0.05 and ^##P < 0.01 versus vehicle group on the same day (n = 10).

Figure 5

Effects of BYHWD on H₂O₂-induced cytotoxicity in HUVECs. Cell viability in HUVECs preincubated with BYHWD (10, 20, or 30 mg/ml) for 6 h followed by H₂O₂ for 4 or 6 h ^#P < 0.05 and ^##P < 0.01 versus H₂O₂ group at the same time.

Figure 6

Effects of BYHWD on H₂O₂-induced cell apoptosis in HUVECs. Cells were double-stained with cell membrane-permeable (Hoechst 33342; blue) and impermeable (PI; red) DNA labeling fluorochromes. (a) Vehicle medium (control) for 6 h. (b) Preincubation with BYHWD (30 mg/ml) for 6 h. (c) H₂O₂ (400 μM) for 6 h. (d) Preincubation with BYHWD (10 mg/ml) for 6 h followed by H₂O₂ for 6 h. (e) Preincubation with BYHWD (20 mg/ml) for 6 h followed by H₂O₂ for 6 h. (f) Preincubation with BYHWD (30 mg/ml) for 6 h followed by H₂O₂ for 6 h. (g) The percentage of apoptotic cells for each group and time point. ^#P < 0.05 and ^##P < 0.01 versus the H₂O₂ group at the same time. Scale bars, 50 μm.

Figure 7

Effects of BYHWD on H₂O₂-induced vascular tube formation in HUVECs. (a) Vehicle medium (control) for 10 h. (b) Preincubation with BYHWD (30 mg/ml) for 6 h. (c) H₂O₂ (400 μM) for 10 h. (d) Preincubation with BYHWD (10 mg/ml) for 6 h followed by H₂O₂ for 10 h. (e) Preincubation with BYHWD (20 mg/ml) for 6 h followed by H₂O₂ for 10 h. (f) Preincubation with BYHWD (30 mg/ml) for 6 h followed by H₂O₂ for 10 h. (g) Numbers of vascular tubes in HUVECs cultures for each group and time point. ^##P < 0.01 versus the H₂O₂ group at the same time. Scale bars, 100 μm.

Figure 8

Effects of BYHWD on H₂O₂-induced intracellular ROS in HUVECs. Cells were stained with DCFH-DA. (a) Vehicle medium (control) for 6 h. (b) Preincubation with BYHWD (30 mg/ml) for 6 h. (c) H₂O₂ (400 μM) for 6 h. (d) Preincubation with BYHWD (10 mg/ml) for 6 h followed by H₂O₂ for 6 h. (e) Preincubation with BYHWD (20 mg/ml) for 6 h followed by H₂O₂ for 6 h. (f) Preincubation with BYHWD (30 mg/ml) for 6 h followed by H₂O₂ for 6 h. (g) Preincubation with apocynin (200 μM) for 1 h followed by H₂O₂ for 6 h. (h) Intracellular ROS level in HUVECs for each group. ^∗∗P < 0.01 versus the control group. ^##P < 0.01 versus the H₂O₂ group. Scale bars, 50 μm.

Figure 9

Effects of BYHWD on H₂O₂-induced NADPH oxidase activity in HUVECs. NADPH oxidase activity in HUVECs preincubated with BYHWD (10, 20, or 30 mg/ml) for 6 h or apocynin (200 μM) for 1 h followed by H₂O₂ for 2 h ^∗∗P < 0.01 versus the control group. ^#P < 0.05 and ^##P < 0.01 versus the H₂O₂ group.

Figure 10

Time course of the effects of H₂O₂ on Nox4 protein expression in HUVECs. (a) Western blot of Nox4 protein expression in HUVECs treated with H₂O₂ (400 μmol/L) for various lengths of time (30 min or 1, 2, or 4 h). (b) Quantification of Nox4 protein expression in HUVECs for each group. ^∗∗P < 0.01 versus the control group.

Figure 11

Effects of BYHWD on H₂O₂-induced Nox4 protein expression in HUVECs. (a) Western blot of Nox4 protein expression in HUVECs preincubated with BYHWD (10, 20, or 30 mg/ml) for 6 h followed by H₂O₂ for 2 h. (b) Quantification of Nox4 protein expression in HUVECs for each group. ^∗∗P < 0.01 versus the control group. ^#P < 0.05 and ^##P < 0.01 versus the H₂O₂ group.