| Intracerebral hemorrhage (ICH) is a devastating neurological disorder with high mortality rate and poor prognosis. ICH-associated brain damage involves numerous mediators, such as thrombin, inflammatory injury, apotosis, matrix metalloproteinase-9, neurotoxicity of excitabilitical amine acid, etc. Thrombin may play an essential role in this process and thrombin-induced extravascular neurotoxicity has been shown to be mediated by protease activated receptors (PARs).To date, no curative therapy is available for the treatment of ICH, and supportive therapy is the major option for most cases. Targeting PAR-1 might offer a potential therapeutic option for patients with ICH.Baicalin (7-glucuronic acid,5,6-dihydroxyflavone) is one of the major flavonoid compounds isolated from the dry roots of Scutellaria baicalensis Georgi. Numerous studies have shown that baicalin has neuroprotective effects on ischemic cerebral injury. However, little is known about the effects of baicalin on hemorrhagic brain injury. Previous studies have shown that complex prescriptions containing Scutellaria baicalensis Georgi were able to inhibit PAR-1 expression in rat brain after ICH. The aim of this study was to evaluate the effects of baicalin on ICH-induced brain injury using an ICH rat model. In addition, the mechanisms underlying were also investigated.Objectives:To observe pathophysiological changes in experimental ICH model of rats. To investigate the effect and mechanisms of baicalin on neurologic impairment, brain edema, neurotic pathomorphology, expression and significance of PAR-1mRNA and PAR-1, Caspase-3 mRNA and Caspase-3, MMP-9 mRNA and MMP-9, NMDA-NR, NF-κB, and number of TUNEL-positive cells in the brain issue of rat with experimental intracerebral hemorrhage, and to explore its possible protective mechanism, to find a effective therapeutic drug.Methods:ICH was induced by intracerebral administration of 0.5 U collagenaseⅦin 1μl saline into the right caudate nucleus of rats, sham operation rats were administered with equal volume of saline without collagenaseⅦ. Rats with ICH were randomly divided into five groups: sham-operated group (Group A), ICH group (Group B) and ICH with baicalin small dose (25 mg/kg, Group C), moderate dose (50 mg/kg, Group D), large dose (100 mg/kg, Group E) treatment group. Rats with sham operation were used as controls. Two hours after the ICH induction, saline was intraperitoneally injected into the animals both in sham-operated group and ICH group, while baicalin into the baicalin treatment Group at the indicated doses. The same treatments were conducted once a day thereafter.On day 1, day 3, day 5 and day 10, rat neurologic defecit scores were observed, six brain tissue samples were collected from the perihematoma areas. The water content of the brain tissue was quantitated with wet/dry weight measurement. Brain tissue pathomorphology was observed in electron microscope by dyeing the brain tissue slice with HE. The perihematomal brain tissue were extracted to determine the differential expression of PAR-1mRNA, Caspase-3 mRNA and MMP-9 mRNA, and protein levels of PAR-1, NMDA-NR, Caspase-3, MMP-9, and NF-κB by RT-PCR, Western Blot and immunohistochemical methods. Cell apoptosis was evaluated by terminal transferase dUTP nick end labeling (TUNEL) staining. All data were presented as means±SD and analyzed with one-way analysis of variance (ANOVA) and Student’s t-test by SPSS 13.0 soft ware package.Results:(1) Neurologic deficit scores were higher in ICH group than those in sham-operated group at each time point, peaking on day 1. Baicalin group got lower neurologic defecit scores than that in ICH group at every time points (P<0.05), there was difference between Group C and Group D, E (P<0.05). (2) After ICH, the brain water content was much higher than that in sham-operated group (P<0.01), it increased on day1, peaked on days 3 and then declined gradually, still higher on day5. The brain water content in baicalin group was obviously lower than that in ICH group at each time point (P<0.01), there was difference between Group C and Group D, E at 3d and 5d post-ICH (P<0.05). (3) After ICH, the expression of brain tissue PAR-1mRNA, Caspase-3 mRNA, MMP-9 mRNA and the protein levels of brain issue PAR-1, Caspase-3, MMP-9, and the number of TUNEL-positive cells were much higher than those in sham-operated group (P<0.01), it was higher on day 1, peaked on days 3 and then declined gradually on day5 and day 10. At each time point, baicalin at all doses significantly reduced PAR-1mRNA, Caspase-3 mRNA and MMP-9 mRNA expression, PAR-1, Caspase-3, MMP-9 protein levels and the number of TUNEL-positive cells in comparison with those of the vehicle treated ICH group (P<0.01, respectively). On day 3 and 5, there was significant difference between Group C and Group D, E. (4) After ICH, the brain tissue NF-κB and NMDA-NR expression was significantly higher than that in sham-operated group (P<0.01), it rose on day1 and then reduced gradually on day3 to day 10. The NF-κB and NMDA-NR expression in baicalin group was obviously lower than that in ICH group at each time point (P<0.01), there was difference between Group C and Group D, E at 1d and 3d post-ICH (P<0.05). (5) Observe the gestalt form of the brain:in ICH groups, the right cerebral hemisphere got swollen, the gyri extended and the sulci shallow; the changes in baicalin treatment groups were slighter; the bilateral hemispheres were approximately symmetric in sham-operated group. The hematoma, which was irregularly spheroid or oval, could been viewed at the area of basal nucleus in the coronal slice. Observe the HE-stained tissue with a light-microscope:in ICH groups, the red cells were of integrity, the brain edema and neural injury surrounding the hematoma were observed, inflammatory cells transmigrated, the tissues were loose, nerve cells and cotloidal cells swollen, and vacuoles of different sizes were formed in cell space and the cell space increased, the cell ballooning degeneration and necrosis were observed, that peaked on days 3 and then reduced gradually; in baicalin treatment groups, the brain tissues were swollen and loose around the injected area, inflammatory cells transmigrated, the changes in baicalin treatment groups were slighter. There were not obviously pathological changes in sham-operated groups.Conclusions:(1) The results demonstrated that the upregulation expression of PAR-1mRNA, Caspase-3 mRNA, MMP-9 mRNA, PAR-1, Caspase-3, MMP-9, NF-κB, NMDA-NR and the increased number of TUNEL-positive cells after ICH were in a time-dependent manner, and the neurologic deficit and brain edema formation were correlated with brain tissue PAR-1, MMP-9, NMDA-NR, NF-κB expression and cell apoptosis in rats. (2) Baicalin effectively reduced the brain edema, inhibited cell apoptosis and inflammatory injury and improved the neurologic deficit following ICH in a dose- and time-dependent manner, with concomitant suppression of brain tissue PAR-1, Caspase-3 and MMP-9, NF-κB, NMDA-NR expression at both mRNA and (or) protein levels, which may be one of the mechanisms of neuroprotective effects of baicalin on ICH-induced brain injury. (3) The study raised the possibility that baicalin may be a potential agent for ICH therapy. |
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