Baicalin Attenuates Subarachnoid Hemorrhagic Brain Injury by Modulating Blood-Brain Barrier Disruption, Inflammation, and Oxidative Damage in Mice

Abstract

In subarachnoid hemorrhagic brain injury, the early crucial events are edema formation due to inflammatory responses and blood-brain barrier disruption. Baicalin, a flavone glycoside, has antineuroinflammatory and antioxidant properties. We examined the effect of baicalin in subarachnoid hemorrhagic brain injury. Subarachnoid hemorrhage was induced through filament perforation and either baicalin or vehicle was administered 30 min prior to surgery. Brain tissues were collected 24 hours after surgery after evaluation of neurological scores. Brain tissues were processed for water content, real-time PCR, and immunoblot analyses. Baicalin improved neurological score and brain water content. Decreased levels of tight junction proteins (occludin, claudin-5, ZO-1, and collagen IV) required for blood-brain barrier function were restored to normal level by baicalin. Real-time PCR data demonstrated that baicalin attenuated increased proinflammatory cytokine (IL-1β, IL-6, and CXCL-3) production in subarachnoid hemorrhage mice. In addition to that, baicalin attenuated microglial cell secretion of IL-1β and IL-6 induced by lipopolysaccharide (100 ng/ml) dose dependently. Finally, baicalin attenuated induction of NOS-2 and NOX-2 in SAH mice at the mRNA and protein level. Thus, we demonstrated that baicalin inhibited microglial cell activation and reduced inflammation, oxidative damage, and brain edema.

Figure 1

Neurological score and brain edema 24 hours after SAH. (a) SAH grading scores in SAH with either vehicle or baicalin posttreatment groups. (b) Neurological score for each group 24 hours after SAH. Values are mean ± standard deviation. N = 6 in each group, ∗∗∗∗ represents p < 0.0001, and ∗∗ represents p < 0.005. (c) Effect of baicalin on brain water content in the left and right hemispheres 24 hours after SAH. N = 6 in each group, ∗∗∗∗ represents p < 0.0001, and ∗ represents p < 0.05.

Figure 2

Effect of baicalin on BBB permeability and tight junction proteins in SAH mice. (a) Evans blue dye extravasation was observed in each set after SAH. The result demonstrated that BBB permeability was increased and attenuated by baicalin. Values are mean ± standard deviation. N = 6 in each group, ∗∗∗∗ represents p < 0.0001, and ∗ represents p < 0.05. (b) Western blot analyses of occludin, claudin-5, ZO-1, and collagen IV.

Figure 3

Effect of baicalin on proinflammatory cytokine mRNA expression in SAH mice. Real-time PCR analyses of IL-1β, IL-6, and CXCL-3. Values are mean ± standard deviation. N = 6 in each group, ∗∗∗∗ represents p < 0.0001, and ∗∗∗ represents p < 0.0005.

Figure 4

Effect of baicalin on glial cell activation marker IBA-1 expression in SAH mice. (a) Real-time PCR analyses of IBA-1. Values are mean ± standard deviation. N = 6 in each group, ∗∗∗∗ represents p < 0.0001, ∗∗∗ represents p < 0.0005. (b) Immunoblot analyses of IBA-1 along with loading control GAPDH.

Figure 5

Effect of baicalin on glial cell activation in vitro. (a) Baicalin dose dependently inhibited the secretion of proinflammatory cytokines IL-1β and IL-6, which were determined by ELISA. N = 6 in each group, ∗∗∗∗ represents p < 0.0001, and ∗∗∗ represents p < 0.0005. (b) Immunoblot analyses of p65 from nuclear fraction along with loading control histone H3. ∗∗ represents p < 0.005.

Figure 6

Effect of baicalin on NOS-2 and NOX-2 mRNA expression in SAH mice. (a) Real-time PCR analyses were performed on two oxidative stress-generating enzymes NOS-2 and NOX-2. Values are mean ± standard deviation. N = 6 in each group, ∗∗∗∗ represents p < 0.0001, and ∗∗∗ represents p < 0.0005. (b) Immunoblot analyses of NOS-2 and NOX-2 from total brain lysates with loading control beta-actin.

Figure 7

Schematic diagram of baicalin-mediated protection in SAH mice. SAH injury in brain leads to three physiological responses, namely, inflammation, microglial cell activation, and oxidative damage, which lead to cellular injury and cell death. Baicalin modulates all three physiological processes and thus attenuated SAH-mediated brain injury. Baicalin also restores blood-brain barrier function impaired by SAH, where degradation of tight junction proteins was modulated.