Baicalin Attenuates Brain Edema in a Rat Model of Intracerebral Hemorrhage

Baicalin was purchased from Sichuan Xieli Pharmaceutical Co. Ltd. (Chinese Drug Approval Number: H20053191, purity was 98.1 %, Chengdu, China). Collagenase VII was purchased from Sigma–Aldrich (St. Louis, MO, USA). Ten percent chloral hydrate was purchased from Qilu Pharmaceutical Co. Ltd. (Jinan, China). MMP-9 and NF-κB mouse monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-IκBα (Ser32/36) and IκBα antibodies were purchased from Cell Signaling Technology (Shanghai, China). Rabbit polyclonal antibody against laminin was purchased from Lab Vision Corporation (USA). Hybond-P PVDF membrane blot was purchased from GE Healthcare Life Sciences (Beijing, China). TRIzol Reagent was purchased from Invitrogen (CA, USA).

The animal experiments were conducted in accordance with the Principles of Laboratory Animal Care (NIH Publication No. 86-23, revised 1985) and approved by the Ethics Committee of The Second Hospital of Shandong University, Jinan, China.

Male Wistar rats, aged 12 weeks and weighing 320–350 g, were provided by the Laboratory Animal Center of Shandong University (Jinan, Shandong, China). Prior to the experiments, the animals were allowed to acclimate to the new environment for 1 week. During the adaptation and the experimental periods, the animals were housed in separate cages under diurnal lightning conditions with free access to food and water. ICH was induced by intracerebral administration of 0.5 U collagenase VII in 1 μL saline into the caudate nucleus of the rats at a rate of 0.2 μL/min as previously described [24]. In brief, under anesthesia with 10 % chloral hydrate (0.36 mg/g body weight, intraperitoneal), the rats were injected unilaterally into the caudate putamen with collagenase VII (0.5 U in 1 μL saline), at the following stereotactic coordinates: 0.2 mm posterior to bregma, 3.0 mm lateral to the midline, and 6.0 mm in depth below the skull. Collagenase was delivered for 5 min, and the needle was left in place for an additional 10 min to prevent any reflux. Rectal temperature was maintained at 37 ± 0.5 °C throughout the experimental and recovery periods. The sham operation rats were administered with an equal volume of saline without collagenase VII. ICH was confirmed by the appearance of hematoma in the caudate nucleus. The rats with ICH were randomly divided into four groups: group B (vehicle-treated), group C (baicalin, 25 mg/kg), group D (baicalin, 50 mg/kg), and group E (baicalin, 100 mg/kg). The rats with sham operation were used as controls (group A). Two hours after the ICH induction, the animals were injected either with saline (groups A and B) or baicalin (groups C, D, and E) through intraperitoneal injection, and the same treatments were conducted once a day thereafter. On day 1, day 3, and day 5, six rats from each group were sacrificed by decapitation under anesthesia with 10 % chloral hydrate. The brain tissues were collected from the perihematoma areas and stored at −70 °C before the assays.

Neurobehavioral function was evaluated by a score system as described previously [25]. The score system consists of three individual tests, each with a score range of 0–4 (0 = best, 4 = worst). The maximum total score value is 12. The tests included: (1) spontaneous ipsilateral circling behavior, (2) contra-lateral forelimb and hindlimb retraction capability, and (3) ability to walk a 70-cm-long 32.4-cm-wide wood beam. The evaluation was conducted by a masked observer at day 1, day 3, and day 5 after the induction of ICH.

RNA concentration was determined by UV absorbance at 260 nm. First-strand cDNA synthesis was performed with 2 μg of total RNA using random hexamers as primers in a final volume of 20 μL (5 μg/μL random hexamers, 1 mm dNTPs, 2 U/L RNasin, and 10 U/L Moloney murine leukemia virus reverse transcriptase). The reaction was carried out at 37 °C for 60-min cDNA encoding β-actin; MMP-9 was amplified from 3 to 5 μL of the cDNA reaction mixture using specific gene primers. Oligonucleotide primers for β-actin and MMP-9 were as follows: β-actin, 5′-TGACGGGGTCACCCACACTGTGCCCATC-TA-3′ (forward primer), 5′-CTAGAAGCATTTGCGGTGGACGATG-3′ (reverse primer); Mmp-9, 5′-AGTTTGGTGTCGCGGAGCAC-3′ (forward primer) and 5′-TACATGAGCGCTTCCGGCAC-3′ (reverse primer).

The amplification conditions were as follows: for MMP-9, 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, extension at 72 °C for 2 min, and then 1 cycle of final extension at 72 °C for 5 min; for β-actin, 25 cycles of denaturation at 94 °C for 45 s, primer annealing at 60 °C for 45 s, extension at 72 °C for 2 min, and then 1 cycle of final extension at 72 °C for 5 min. The expression of β-actin was be used as an internal control for the assay of a constitutively expressed gene.

Western blotting was performed as previously described [26]. Briefly, the brain tissues were homogenized in radio immunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors. The proteins were separated by electrophoresis on a 10 % SDS–PAGE gel and transferred to a Hybond-P PVDF membrane. Subsequently, the membrane was blocked in 5 % nonfat milk and incubated with appropriate primary antibodies (RelA/p65, 1:1,000; MMP-9, 1:1,000) overnight at 4 °C, followed by incubation with the peroxidase-conjugated rabbit anti-goat secondary antibody (1:1,000 dilution) for 1 h at room temperature. After washing with PBS, the bound primary antibody was visualized with the Enhanced Chemiluminescence System from Amersham (Piscataway, NJ, USA) and exposed to film. The same membrane was probed for β-actin for loading control. The relative density of RelA/p65 to β-actin was analyzed with the TotalLab TL120 Image software (TotalLab Life Science Analysis Essentials, Greensboro, NC, USA).

The brain tissues were collected from the perihematoma areas. The samples were weighed. After homogenization, the lysate was mixed with equal volume of RIPA buffer and put on ice for 30 min. After centrifugation (12,000 rotations/min for 20 min), the supernatant was collected for IL-1β and IL-6 measurements using enzyme-linked immunosorbent assay (ELISA) kits (Wuhan Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions. The results were expressed as picograms per milligram of tissues.

Evans blue dye (100 mg/kg, Sigma) was injected into the femoral vein. After 1 h, the rats were perfused with saline through the left ventricle until the perfusion fluid obtained from the right atrium became colorless. After decapitation under anesthesia with ether, the brain tissues were collected from the perihematoma areas. The samples were weighed and soaked in 50 % trichloroacetic acid solution. After homogenization and centrifugation (12,000 rotations/min for 20 min), the supernatant was diluted with ethanol (1:3). The fluorescence intensity was measured at 620 and 680 nm for excitation and emission. The concentration of the Evans blue dye in the brain tissue was calculated according to a linear standard curve obtained from known amounts of the dye. The tissue content of the Evans blue dye was expressed in micrograms per gram of tissues.

All data were presented as means ± SD and analyzed with one-way analysis of variance and Student’s t test. P value <0.05 was considered statistically significant.

In the ICH model group, around 10 % of the rats would die in 24 h following the operation. In the sham operation group, all rats survived. Neurobehavioral deficits were evaluated using a scoring system. After the induction of ICH, all ICH groups showed neurobehavioral deficits as compared with the sham operation group. The neurobehavioral deficits attenuated over time in each group, suggestive of self-recovery ability of the rats. At each time point, baicalin significantly improved the neurobehavioral function in a dose-dependent manner (Fig. 1).

Brain edema was evaluated by measuring the brain water content (Fig. 2). After the induction of ICH, the brain water content in the vehicle-treated ICH group (group B) increased significantly on day 1 compared to the sham operation group (group A) and peaked on day 3. At each time point, the brain water content in group B was significantly higher than that in the control group (P < 0.01, respectively). On day 3 and day 5 after ICH, baicalin significantly reduced the brain water content in a dose-dependent manner. On day 10, the brain water contents in all the ICH groups were still higher than that of the control group, whereas the brain water content of the vehicle-treated ICH group was not significantly different from those of the baicalin-treated groups.

MMP-9 is known to play an essential role in the maintenance of the integrity of blood–brain barrier and has been implicated in brain edema formation. In the present study, we first determined the MMP-9 protein expression in the brain tissues of the rats after ICH by western blotting. As shown in Fig. 3, the MMP-9 expression was significantly increased on day 1 in the vehicle-treated ICH mice (group B), compared to that in the sham operation group (group A), and reached a peak on day 3 after ICH. Baicalin attenuated the MMP-9 expression in a dose-dependent manner at each time point.

The MMP-9 mRNA expression was analyzed by RT-PCR. As shown in Fig. 4, the MMP-9 mRNA level was significantly increased on day 1 in the vehicle-treated ICH mice (group B) compared to that in the sham operation group (group A) and reached a peak on day 3. Baicalin inhibited ICH-induced MMP-9 mRNA expression in a dose-dependent manner.

The effects of baicalin on the NF-κB (RelA/p65) expression and activity were determined by western blotting. As shown in Fig. 5, the RelA/p65 expression was significantly increased on day 1 in the vehicle-treated ICH mice (group B) compared to that in the sham operation group (group A), in which the level decreased over time. Baicalin inhibited the NF-κB expression in a dose-dependent manner at each time point.

The activation of the NF-κB signaling pathway is known to play an essential role in inflammatory processes. In the present study, additional experiments were conducted to observe the effects of baicalin on the production of pro-inflammatory cytokines including IL-1β and IL-6 in the brain tissues at 72 h following ICH. The IL-1β and IL-6 levels were determined by ELISA. As shown in Fig. 6a, b, following ICH, the production of IL-1β and IL-6 was significantly increased in the brain tissues around the perihematoma areas. Baicalin reduced the IL-1β and IL-6 production in a dose-dependent manner.

As MMP-9 is a key regulator of the BBB permeability, next we determined the effect of baicalin on the blood–brain barrier permeability by Evans blue leakage method. As shown in Fig. 7, the BBB permeability significantly increased and peaked at 24 h after ICH, which was attenuated by baicalin dose dependently.