The Anti-inflammatory Neuroprotection And Anti-apoptosis Mechanisms Of DAHP Involved In Inhibiting NOS Expression And TP Via Up-regulating PI3K/Akt Cell Signal In Ischemia Stroke Models

Part1The neuroprotective anti-apoptosis mechanisms of DAHP involved in alleviating NOS up-regulation in rats MCAO modelsAims:BH4,as the co-factor of nitric oxide synthetase (NOS), mediates the synthesis of nitric oxide (NO), and GTPCH-I is an endogenous rate-limiting enzyme for the de nove synthesis of BH4. In the study, we plan to administrate DAHP, the inhibitor of GTPCH-I, to reduce the product of BH4and sequent expression of NOS to investigate its neuroprotective mechanisms.Methods:DAHP was injected12hours before the operation of MCAO on rats. The rats sacrificed24hours after the surgery, and their brain was harvested for the tests of TTC stain, MRI scan, TUNEL assay, RT-PCR, Western Blot and immunohistochemistry.Results:As the inhibitor of GTPCH-I, the rate-limiting enzyme of BH4de nove synthesis, DAHP treatment effectively alleviate the up-regulation of GTPCH-I, iNOS and nNOS after MCAO/R. However, it is not involved in the early generation of eNOS. Compared with the MCAO/R injury group, DAHP treatment can decrease the infarction and edema ranges based on the TTC stain and MRI tests; the data of TUNEL assays and expressions of key proteins related to apoptosis shows that DAHP plays an neuroprotective role in anti-apoptosis; In addition, the anti-inflammation of DAHP may work via the down-regulation of NF-kB/COX-2/iNOS signal pathway.Conclusion:Our study shows that the administration of DAHP makes contribution to anti-apoptosis by alleviating the up-regulation of iNOS and nNOS expression and its neuroprotection may involve in down-regulation the NF-kb signaling pathway after MCAO injury.Part2The anti-inflammative neuroprotection and anti-apoptosis mechanism of TP via up-regulating PI3K/Akt cell signal in ischemic stroke modelsAims:In this experiment, we’d like to explore insights to the anti-inflammation and anti-apoptosis mechanisms of TP in both MCAO/R and OGD/R models.Methods:In vitro,30SD rats were divided into three groups:Sham Group, MCAO Group and TP Treatment Group. TP was administrated12hours before the operation, and rats sacrificed after1.5hours’cerebral ischemia and24hours’reperfusion and, The brains were harvested for tests (Like TTC Stain, MRI Scan, Western Blot, RT-PCR, Immunohistochemistry, TUNEL assay) before the ethologic evaluation of neurologic symptoms. In vivo, we use MTT assay to screen an optimal dose of TP that can be administrated into experimental treatment for PC12cells in OGD/R models, and also investigate the effectiveness in anti-inflammation and anti-apoptosis.Results:In vitro, compared with MCAO Injury Group we found that:in TTC stain test, TP treatment reduced the infarction volume; the ethnology scores show that TP can release the injury after MCAO/R; the data from TUNEL assay suggests that TP has the neuroprotection in anti-apoptosis since the number of apoptotic cells dramatically dropped in TP Treatment Group; in the meanwhile, it also work in anti-inflammation according to the immunohistochemistry, Western Blot and RT-PCR data; By testing the key proteins related to apoptosis, TP can up-regulate the pAkt/Akt, which suggested it may play an anti-apoptotic role via PI3K/Akt cell signal pathway. In vivo, TP can significant alleviate the up-regulation of inflammatory reaction and prevent PC12cells from apoptosis in the treatment groups with different doses. Moreover, the optimal dose is0.4ng/ml for TP treatment in PC12cells OGD/R model.Conclusion:TP treatment contributes to anti-inflammation and anti-apoptosis via PI3K/Akt cell signal pathway in rats MCAO/R model. In the meanwhile, it also plays an anti-apoptotic role in PC12cells OGD/R model, and the optimal dose is0.4ng/ml.