Effects Of RNA Interference Targeting Smad7on Nerve Cells Ischemic Injury Induced In PC12Cells

Ischemic cerebrovascular disease, such as cerebral thrombosis, cerebral infaction,and so on, are the most important causes of morbidity, and the third most commoncause of death in elderly patients in the world. Effective methods of preventing andcontrolling ischemic cerebrovascular disease have been a topic of great interest.Ischemic damage of nerve cells leads to a series of complex signaling pathways thatproduce corresponding biological functions. A better understanding of the role of thesignal transduction mechanisms that underlie brain ischemic injury could identify keytargets for neuroprotective substances.Ischemic stroke occurs when the blood supply to the brain is obstructed.Accumulating evidence suggests that the cell death observed during the first fewhours of cerebellar ischemia is a result of apoptosis as opposed to necrosis, which wasconsidered the predominant form of cerebellar damage generated by ischemia.Moreover, Effective methods of preventing and controlling ischemic cerebrovasculardisease have been a topic of great interest. The ischemic damage of nerve cells leadsto the disruption of a series of complex signaling pathways that produces an effect oncorresponding biological functions and affects the function of the brain. A betterunderstanding of the role of the signal transduction mechanisms that underlie brainischemic injury could identify key targets for neuroprotective substances. Some datasuggest that Smad7can also have proinflammatory effects. Smad7overexpression hasbeen noted in mucosa and mucosal T cells from patients with inflammatory boweldisease, and blockade of Smad7restores TGF-β signaling and reduces proinflammatory cytokine generation in this context. Overexpression of Smad7in mouse Tcells promotes airway hyperreactivity and enhances airway inflammation, whereasinhibition of Smad7suppresses autoimmune encephalitis. Thus, it appears that therole of Smad7in the regulation of inflammation and immunity is complex, is possiblydependent on cell lineage, context, and duration of inflammatory response, and needsto be more completely defined before overexpression of Smad7really becomes viableas a therapeutic goal. For expounding Actin/Smads pathways about ischemic brain injury and signaltransduction mechanism and looking for nerve protective substances that can beapplied to clinical practise and key targets of Actin/Smads pathways. A stable in vitroneuronal ischemia model had been established. Nerve growth factor (NGF) caninduce the differentiation of PC12cells into neuron-like cells. The sympatheticneuron-like cells are characterized by electrical excitability, expression of neuronspecific genes and neurite outgrowth. PC12cells were cultured in different oxygenconditions. The metabolic activity of PC12cells was measured in the final4h prior tocellular characterization using an alamar blue assay following the manufacturer'sinstructions. A neuronal ischemia model was conducted on research the molecularlevel.The experiment small interference targeting Smad7on in vitro neuronal ischemiamodel was taken and observed the changes of cell apoptosis. For study the role ofSmad7on stroke and signal transduction mechanism. And look for nerve protectivesubstances that can be applied for clinic in the furture and key targets in ActA/smadspathways. Protecting and promoting neurological recovery, it will bring the new hopefor the treatment of ischemic cerebrovascular disease.1. NGF combined with OGD induces neural ischemia tolerance in PC12cellsObjective: Ischemic cerebrovascular disease is a major disease in humans. Tobetter study this disease, a good ischemia model of nerve cells is needed.Methods: NGF(100ng/mL)and OGD(5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM) were used to stimulate PC12cells and converted them intoneurons in order to establish an ischemia model. After stimulation with NGF (100ng/mL for6d), PC12cells show a neuron-like function as measured by physiologyand biochemistry. After6d of NGF stimulation, we performed OGD(5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM) treatment for16h to establish anoxygen glucose deprivation model.Results: PC12cells transformed into cells that looked like neurons and thatMAP2was up-regulated in NGF-treated PC12cells. Cell apoptosis was found to beup-regulated after NGF(100ng/mL)stimulation and OGD (5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM for16h). A western blot analysis showed that OGD treatment increased the expression of HIF-1. The apoptosis rate after16hof OGD was19.44%.Conclusions: NGF(100ng/mL)treatment can be combined with OGD(5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM for16h) to establish an in vitromodel of acute ischemic brain damage.2. Effects of RNA interference targeting Smad7on nerve cells ischemic injuryinduced in PC12cellsObjective: In order to further illustrate the role of Smad7genes in ischemiccerebral injury.Methods: Real time-PCR and Western blot were used to determinate the mRNAand protein expression of Smad7by Smad7-siRNA.Results: Smad7-siRNA can be translated into PC12cells by LipofectamineTM2000. The efficiency was higher.Conclusions: LipofectamineTM2000can be used in Smad7-siRNA in PC12cells.Smad7-siRNA can effectively silence Smad7gene expression. The experiment smallinterference targeting Smad7is an effect method in researching signal transductionpathways.3. Effects of RNA interference targeting Smad7on ActA/Smads signallingpathwayObjective: This study was mainly discussed the protein expression levels ofActA and p300in ischemic cerebral injury by targeted silence Smad7.Methods: Real time-PCR was used to detect the mRNA of ActA and p300.Western blot was used to detect the protein expression levels of ActA and p300.Results: The mRNA of ActA and p300were higher by Smad7-siRNA.Theprotein expression of ActA and p300were also higher by Smad7-siRNA in ischemiccerebral injury.Conclusions: The results showed that Smad7-siRNA could sitimute theActA/smads signaling pathways on acute ischemic injury.4. The apoptosis change by Smad7-siRNA on nerve cells ischemic injuryObjective: This experiment is mainly discussed the apoptosis in ischemiccerebral injury by targeted silence Smad7. Methods: FCM and DAPI were used to identify apoptosis rate. Western blot wasused to detect Procaspase-3protein expression.Results: The results showed that OGD16h apoptosis rate is19.54%. OGD16hcombined Smad7-siRNA apoptosis rate is12.34%.Conclusions: The results showed that the apoptosis rate is decreasing inischemic injury by Smad7-siRNA. At the same time, it provide the reference forfurther study ActA/Smads signaling pathways on acute ischemic injury.