| Ischemic preconditioning(IPC) represents an important adaptation mechanism of CNS, which results in ischemic tolerance(IT) to lethal cerebral damage. The mechanism investigation of this protective phenomenon is worth for clinical use to ischemic injured defence and long term prognosis. Activin A(Act A), as a member of transforming growth factor-β(TGF-β) superfamily, was found over-expressed during cerebral ischemia in our previous work and represented a neuroprotective role through Act A/Smads signaling pathway. Such protection was limited in changing the destiny of neural demise followed by ischemic insult if without blood resupply. IPC was processing quite a similar way as lethal ischemic stimuli essentially but with less injurious damage. The hypothesis that Act A signaling pathway plays a crucial role in IPC induced IT seems reasonable. We intensively focused the sight on the protective mechanisms of Act A signaling involved in IPC and tryed to identify new potential targets for innovative therapeutic strategies to keep brain more resistant.1. Culture and identification of neuronal PC12 cells in vitro.Objective: To culture and identify neuronal PC12 cells in vitro.Methods: PC12 cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 20% fetal bovine serum(FBS) containing100ng/ml nerve growth factor(NGF) and transformed into neurons. Cells were identified by immunofluorescence of MAP2.Results: Initial recovered PC12 cells looked round. A majority of adherent cells started to gather into a group after 1d cultured with 20% fetal bovine serum DMEM medium. After culturing 3 ~ 6 d, the gathered PC12 cell proliferated significantly. NGF induced culture 6 d, PC12 cells were significantly grown with longer neurites, interleaving, showed typical neuronal morphology. Immunofluorescence staining of MAP2 was strongly positive.Conclusions:Differentiated PC12 cells showed typical neural morphology and behaviors after NGF stimuli.2. Establishment of IS and IPC neuronal PC12 cell model in vitro.Objective: To establish IS and IPC neuronal PC12 cell model in vitro.Methods: Glucose free DMEM and Na2S2O4 were introduced to neuronal PC12 cells to establish ischemia(IS) and ischemic preconditiong(IPC) model in vitro. Test microenvironment conditions surrounded cells such as oxygen glucose concentration, p H, major ions concentration and other indicators by blood gas analyzer.Then MTT assay was used to exam cell survival situation.Results: IPC model was established by 6 m M Na2S2O4 treatment for 30 min. Microenvironment conditions didn ’ t change significantly. Cell activity was maitaining stable within 10~40 min treatment in comparison with 10 min.(P>0.05). Cell activity dropped significantly after 40 min treatment(P<0.05). Na2S2O4 treatment at a 18 m M concentration for 3-24 h created an acidic condition for PC12 cells. Cell activity decreased significantly with prolongation of exposure time.(P<0.05).Conclusions:Successfully established IS and IPC model by neuronal PC12 cells in vitro.3. Mechanisms of self-targeting protective effect induced by IPC.Objective: To investigate the process and mechanisms of self-targeting protection induced by IPC.Methods: Hoechst33342 immunofluorescence staining and Annexin V-FITC/PI flowcytometry were performed to test apoptosis situation of neuronal PC12 cells.Results: Blue fluorescence stained nucleus were found in each group detected by Hoechst 33342 immunofluorescence. Compared with control, no severe morphological changes of apoptosis was found in IPC 40 min group. Apoptotic cells with stronger blue fluorescence or chromatin condensation were found in IS 6h group. Apoptotic cells found decreased in IPC40min+IS6h group in comparison with IS 6h group. Apoptotic rate of each group was determined by flowcytometry analysis with Annexin V/PI double staining assay. Statistic analysis showed no significant changes between IPC 40 min group and control(1.8±0.2% VS 1.7±0.2%,P>0.05). Apoptotic rate of IS 6h group increased significantly compared to control(37.4±2.1% VS 1.7±0.2%,P<0.05)and dropped deeply after IPC treatment.(18.2±1.7% VS 37.4±2.1%,P<0.05)(Figure 5.3). The results suggested that IPC began to induce protective effect after an one hour regain treatment(21.3±1.7%),and became strongest at 3h(16.3±1.0%),then slightly declined after 3h.(Figure 5.4, 5.5)Conclusions:IPC activated a self-targeting protection procedure to defend ischemic injury. Protective effect may be influenced by IPC post-regaining time.4. Role of Act A signaling involved in IPC induced IT.Objective: To investigate the expression level and biological role of Act A, Act RIIA and Smad3, as the key sites in Act A signaling pathway involved in IPC procedure.Methods: The m RNA and protein expression of Act A, Act RIIA and Smad3 were measured by Realtime-PCR,Western-blot and ELISA. Act RIIA inhibitor was used to block Act A signaling pathway. Annexin V-FITC/PI flowcytometry was performed to test apoptosis rate in all groups.Results: The m RNA expression of Act A, Act RIIA and Smad3 were all up-regulated significantly after IPC treatment(p<0.05). Protein expression of Act A and Act RIIA trended the same way with m RNA(p<0.05), meanwhile p-Smads showed no significant changes(p>0.05). The apoptosis rate was increased significantly caused by the inhibition of Act RIIA in IPC procedure(p<0.05).Conclusions:IPC activated Act A/Act RIIA signaling pathway in PC12 cells. Over-expressed Act RIIA mediated the signal transduction in later phase of IS injury. Multiple regulative mechanisms may involved in intracellular Act A/Smad3 signaling pathway during IPC.5. Downstream regulators of Act A receptor signaling pathway modulate IPC induced IT process.Objective: To locate the downstream regulators of Act A/Smads signaling pathway mediating IPC-triggered neuroprotection.Methods: Protein expression of JNK1 and p-JNK1 after IPC stimuli were detected by Western-blot. JNK1-inhibitor SP100625 was used to block JNK1 signaling. Western-blot were performed to exam the expression and phosphorylation level of Smad3. Nuclear migration rate of Smad4 was tested by immunofluorescence staining. Hoechst33324 staining and flowcytometry were performed to measure apotosis rate after IS injury when JNK1 signaling were blocked.Results: The expression of JNK1 and p-JNK1 were all up-regulated significantly in IPC group in comparison with control(p<0.05). Protein expression of Smad3 and p-Smad3, the nuclear migration rate of Smad4 were all increased in IPC when JNK1 signaling were blocked. Blockade of JNK1 enhanced Smad3 expression, phosphorylated effect but promoted cell apotosis during IS injury(p<0.05).Conclusions:Crosstalk between JNK1 and Act A/Smads signaling pathway was found in IPC induced IT. JNK1 activation stimulated by IPC suppressed the phosphorylation of Smad3 and the combination with Smad4, inhibited the Smads complex tanslocating into nuclei, suggessed one of the underling mechanism. JNK1 inhibitor was presumed to be a potential target of drug preconditioning since it induced ischemic tolorance of neuronal PC 12 cells in vitro. |
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