Torc1, Phospho-CREB And BDNF Dynamic Expression In The Cerebral Ischemia And Tsa's Neuro-protection Against Focal Ischemia In Rat

Objective: Ischemia, with high mortality and serious disability, is the most common type of cerebral vascular disease. A variety of cellular defense mechanisms are required to protect neurons from the resulting damage. Currently, the survival of nerve cells is thought to be associated with some transcription factors, of which CREB family is the most important. Its functions in nervous system dependent ultimately on the precise mechanism of transcriptional regulation to downstream genes in time and space. These genes involved extensively in the many processes of the cell structure, signal transduction, genetic transcription, synaptic transmission and metabolism etc. In some cerebral ischemia and reperfusion model, a variety of stresses induced phosphorylation of CREB Ser133 through intracellular signaling pathway of transduction. Phosphorylation of CREB activated directly or indirectly transcription of related genes, and expressed certain protein molecules and some neurotrophic factors such as c-fos, B cell leukemia/lymphoma-2 (bcl-2), basic fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF) and so on.The gene products regulate the stress-induced neuronal regeneration survival and plerosis.Although CREB can activate its target genes, the effect of activity is lower. Interaction of CREB and its co-activator exert coordinatedly effect. TORCs (transducers of regulated CREB) represent a new family of conserved and powerful CREB coactivators that function as intracellular calcium- and cAMP-sensitive coincidence detectors, controlling the kinetics of CRE-mediated responses and long-term potentiation of synaptic transmission. Thus, whether TORC1, as a subtype of TORCs, promotes the neuroprotective effect of CREB in ischemic cerebrovascular disease should be further studied and explored its potential advantages.TanshinoneⅡA(TSA), a fat soluble active ingredients from Chinese herb Danshen, used commonly in cardiac, liver disease and menopausal syndrome, as well as cerebrovascular disease,by its anti-inflammatory and ant-oxidation effect.It is well known that MCAO is a classical model of cerebral ischemia. This study evaluated the TORC1,phospho-CREB and BDNF expression at protein level in rat MCAO model at different time points, and investigated the role of TORC1 in promoting activation of CREB / CRE pathway during acute ischemia-mediated brain injuries. At 24 h after MCAO, we determined the brain water content, neurological deficit, infarct size and TORC1 phospho-CREB and BDNF expression after Tanshinone IIA (TSA) administration. This research reviewed (1) the relationship between TORC1,phospho-CREB and BDNF expression and TSA'neuroprotection effect, (2) potential mechanism of TSA'neuroprotection in ischemic stroke in vivo, which provides a more favorable theoretical basis for clinical administration.Methods: Male, This study included two parts: Experiment 1was used to evaluate the dynamic expression of TORC1 phospho-CREB and BDNF in the cerebral ischemia, Experiment 2 was used to detect TSA's neuroprotection in cerebral ischemia. Experiment 1, Male, Sprague-Dawley rats were randomly individed sham group and MCAO group, and the latter were subjected to permanent focal cerebral ischemia by right MCA occlusion, neurological deficit was evaluated using a modified six point scale 3 h, 6 h, 12 h, 24 h, 48 h, 72 h after MCAO, 2, 3,4 or 5 scores were brought into this study. Immunohistochemistry, Western blotting were used to analyse the expression of TORC1 phospho-CREB and BDNF. Experiment 2, Male, Sprague-Dawley rats were randomly individed into sham group, vehicle control, TSA low dose group (10mg/kg, TSA-L) and TSA high dose group (20mg/kg, TSA-H). TSA solution (10mg/kg or 20mg/kg) was injected intraperitoneally immediately after MCAO. At 24 h neurological deficit was evaluated; brain water content was measured; infarct size was analyzed with 2, 3, 5-triphenyltetrazolium chloride (TTC). Immunohistochemistry, Western blotting and RT-PCR were used to analyse the expression of TORC1 phospho-CREB and BDNF, and confocal microscope was used to observe the expression and nuclear transduction of TORC1.Experiment 2 was used to detect the neuroprotective role of TSA. Rats were randomly individed into sham group, MCAO group, TSA low dose group (10mg/kg, TSA-L) and TSA high dose group (20mg/kg, TSA-H). TSA was administered by intraperitoneal injection immediately after MCAO. At 24h neurological deficit was evaluated using a modified six point scale; brain water content was measured; infarct size was analysed with 2,3,5- triphenyltetrazolium chloride(TTC); immunohistochemistry, Western blot and RT-PCR were selectionally used to analyse the expression of TORC1, phospho-CREB and BDNF protein and mRNA; and confocal microscope to definite nuclear transduction and expression of TORC1 in neuron.Results:1. Compared with normal control, at the 6 time points after MCAO, expression of phospho-CREB, TORC1 and BDNF were induced at protein level in ischemic brain (P < 0.05), peaking at 3 h and 48 h after MCAO for nuclear transcription and expression of TORC1, and peaking at 3 h and 72 h after MCAO for expression of phospho-CREB and BDNF.2. Neurological deficit evaluation: In Experiment 2, neurological deficit were evaluated with using a modified six point scale. Compared with vehicle-control, TSA-H improved the neurological deficit (P < 0.05), but not in TSA-L group.3. Brain water content measurement: brain water content in sham was 78.50%±0.7%, 84.57%±0.51% in vehicle-control group, compared with them, TSA-H significantly reduced the brain water content (82.16%±0.63%, P < 0.05). But no statistical significance was observed in TSA-L group.4. Infarct size analysis: Compared with vehicle-control (%HLV: 43.54%±3.22%), TSA-H significantly decreased the infarct size (%HLV: 34.49%±1.39%, P < 0.05), but not in TSA-L group compared with vehicle controls.5. TSA's role to TORC1,phospho-CREB and BDNF expression: Compared with vehicle group, the expression of TORC1, phospho-CREB, and BDNF protein and gene level was significantly increased in TSA-H group (P < 0.05). Immunofluorescent intensity with confocal microscope showed a significant increase in TORC1 immunoreactivity in TSA-H group as compared to vehicle control. And enhanced TORC1 are localized in nucleus and cytoplasm of neurons。Conclusion: The expression of TORC1, phospho-CREB and BDNF were induced in the early stage after MCAO. TORC1 may promote activation of CREB /BDNF pathway during 48 h following ischemia. TSA protected the brain from damage caused by MCAO ,TSA-H lessened neurological deficit scores, the brain water content and infarct size, and upregulates the expression of TORC1, phospho-CREB and BDNF. The results suggest that TSA protected rat brain from ischemic damage, which might be associated with the upregulated expression of TORC1, phospho-CREB and BDNF. So TSA should be the better one of the strategic targets for cerebral ischemic therapies.