Neuroprotective Capabilities Of Tanshinone Iia Against Cerebral Ischemia/reperfusion Injury In Rats And Its Mechanism

Objective:Danshen, a very important traditional Chinese medicinal compoundderived from the dried root or rhizome of Salviae miltiorrhizae Bge, hasTanshinone IIA (TSA) as one of its active ingredients. Studies have shownthat TSA has multiple pharmacological actions such as anti-tumor,anti-bacterial, anti-inflammatory, antioxidant and anti-apoptotic. Recentstudies have shown that TSA could significantly improve the neurologicalfunction and reduce infarct volume in rats, which induced by cerebralischemia and reperfusion injury. However,there is no unified understandingof the optimal dosing concentration and therapeutic time window of it, andthe mechanism of its neuroprotection is not clear.Methods:Transient cerebral ischemia was induced by Middle cerebral arteryocclusion (MCAO) in rats, in which cerebral ischemia had been induced2hearlier, followed by reperfusion for24h. Rats were randomly divided intodifferent groups, sham group, vehicle group and different concentration of TSA groups. TSA was injected intraperitoneally in TSA groups,1mL/kgPBS including1%DMSO was given in control groups. The neuroprotectiveeffects of TSA and its possible mechanisms were studied.(1) Theneurological score was studied2h after cerebral ischemia. The infarctvolume and brain water content were measured to investigate itsneuroprotective effects, the optimal dosing concentration and therapeutictime window24h after reperfusion.(2)Nissl-staining and HE-staining wereused to observe neuron survival and cell morphology. Apoptotic cells werecounted by Terminal Deoxyribonucleotidyl Transferase-MediatedDeoxyuridine Triphosphate Biotin Nick-End Labeling (TUNEL) Staining.The expression of cleaved caspase-3and B-cell lymphoma2(bcl-2) proteinsin the ischemic cortex were detected by Western blot.(3) The accumulationof neutrophils in the brain tissue was assessed by measuring MPO activity.Levels of MIF, tumor necrosis factor-α (TNF-α), and interleukin-6(IL-6)were determined using commercially available ELISA kits. Western blotanalysis and immunofluorescence staining were used to detect theexpression of MIF. Western blot analysis and EMSA were used to detect theexpression and activity of NF-κB.Results:(1)Neurological scores, cerebral infarct volumes and brain watercontent were significantly decreased after treatment with different doses ofTSA such as20mg/kg,25mg/kg,30mg/kg,35mg/kg and40mg/kg, of which 25mg/kg TSA was most significant. Treatment with25mg/kg TSA wasprotective at four different points in time compared to the vehicle group, ofwhich10min after MCAO was most significant. We chose to administer25mg/kg TSA10min after MCAO in subsequent experiments.(2) Treatment with25mg/kg TSA significantly improved neuronalmorphology, increased the number of survival neurons, and reduced thenumber of TUNEL-positive cells in the damaged brain. The expression ofcleaved caspase-3was increased in the vehicle group but significantlydecreased in the25mg/kg TSA group relative to the vehicle group. Theexpression level of bcl-2was significantly increased in the25mg/kg TSAtreated group relative to the vehicle group.(3) The neutrophil infiltration was significantly higher in the vehiclegroup than in the TSA group. ELISA demonstrated that TSA could inhibitMIF expression and the release of TNF-α and IL-6induced by I/R injury.Western blot analysis and immunofluorescence staining showed that MIFexpression was significantly lower in the TSA group than in the vehiclegroup. MIF was found almost all located in neurons and hardly any locatedin astrocytes in the cerebral cortex. Western blot analysis and EMSAdemonstrated that NF-κB expression and activity were significantlyincreased in the vehicle group. However, these changes were attenuated byTSA.Conclusion: (1) Treatment with TSA is beneficial to protect brain againstischemia/reperfusion injury.(2) The neuroprotective effects of TSA against focal cerebralischemic/reperfusion injury are likely to be related to the attenuation ofapoptosis.(3) Our results suggest that TSA helps alleviate the proinflammatoryresponses associated with I/R-induced injury and that this neuroprotectiveeffect may occur through downregulation of MIF expression in neurons.