| Activin A(Act A), as a member of transforming growth factor-β(TGF-β)superfamily,expresses in central nervous system. It plays a neuroprotective role through Act A/Smads signaling in multiple neurological diseases, such as hypoxic-ischemic brain injury, neuropsychiatric diseases and neurodegenerative disorder. During these pathological processes, the up-regulation of Act A gene induced by external damage shows the signal activation of Act A/Smads as a linear pathway.Previously, we found that Act A/Smads signal activation in response to Oxygen–Glucose Deprivation(OGD) led to the increased extracellular Act A accumulation. These results suggested a feedback loop in Act A signaling. But the specific behaviors of Act A signal loop and its effect on ischemic injury were still not fully understood. To clarify these issues, we established the OGD model to PC12 cells,which simulates the pathological process in ischemic brain injury. Then the expression of Act A was stably up-regulated through recombinant plasmid transfection and G418 screen. That simulates the increased expression of Act A in injury and avoids the cellular damage and expression changes accompanied with injury. Through monitoring extracellular Act A accumulation and examining the activity of Act A signaling, we elaborated the characteristics of Act A signal loop in PC12 cells. Flow cytometry, MTT assay and Hoechst staining were used to explore the effect of Act A loop on ischemic injury. Further more, to explore the regulation mechanism of SARA on Act A signal loop, the RNA interference technology was used to suppress the expression of SARA in Act A over-expressional PC12 cells. These practical discoveries will bring new insight on the functional outcome, interference site and time window of Act A signaling in ischemic injury by the further understanding: loop signaling.1. Establishment of ischemic injury model in vitroObjective: To establish Oxygen-Glucose Deprivation(OGD) model to PC12 cells and simulate the ischemic injury in vitro.Methods: Glucose free DMEM and Na S2O4 were introduced to PC12 cells to establish OGD model in vitro. Then MTT assay, immunofluorescence staining of Hoechst33342 and flow cytometry were used to exam the survival rates, apoptosis unclei and the number of apoptosis cells after OGD 0, 3, 6 and 12 h.Results: Establish the OGD model to PC12 cells. With the extension of OGD,the survival rates of cells decreased gradually. Hoechst33342 staining showed the increased number of dense blue unclei after OGD. And apoptosis was the predominant methods of cell death according to the flow cytometry.Conclusions:Establish the ischemic injury model in vitro successfully.2. Establishment of Act A over-expressioned PC12 cellsObjective: To establish Act A over-expressioned PC12 cells and simulate the increased endogenous Act A expression in response to injury.Methods: To increase the expression of Act A gene, an Activin βA plasmid was recombined with the use of clonig technology. The recombinant and blank plasmids were transfected into PC12 cells using liposomes transfection reagent. After G418 screen, stably transfected PC12 cells were observed. Then Real time RT-PCR,Western blot and ELISA were used to explore the expression of Act A in Positive(P),Negative(N) and Control(C) groups.Results: Establish the eukaryotic expression vector of Act A(Act A-p IRES2-EGFP). The total expression of Act A m RNA dramatically increased in Positive group compared to the other two groups. Besides, in Positive group the level of Activin βA protein doubled compared to that in Negative and Control groups.ELISA showed that Act A accumulation in Negative group did not change over time,but it gradually increased in Positive group.Conclusions:Establish the Act A over-expressioned PC12 cells successfully. The intracellular levels of Act A increased in PC12 cells, so did the extracellular Act A protein. That could simulate the injury induced increased expression of Act A.3. Behaviors of Act A signal loop and its effects on ischemic injuryObjective: To explore the general characteristics of Act A signal loop and its effect on ischemic injury.Methods: Positive and Negative groups of cells were treated with fresh cellular media(1, 3, 6, 12 and 24 h). To clarify the characteristic of this loop signaling, we monitored the expression Act A by Real-Time RT-PCR and Western blot, andexamined the activity of Act A signaling, which was quantified by the expression of phosphorylated Smad3 and the fluorescence intensity of Smad4 in nuclei through Western blot and Immunofluorescence staining. To explore the biological outcome of Act A signal loop on ischemic injury, flow cytometry, MTT assay and Hoechst staining were carried out to detect the proliferation of PC12 cells and sensitivity of cells to OGD injury.Results: Western blot showed that the expression and phoshporylation of Smad3 in Negative group were not changed with time, while with the accumulation of excellular Act A, they significantly increased in P-3 h. Then they dramatically decreased in P-6 h, and then slightly enhaced in P-24 h which was higher than that in N-24 h. The distributions of Smad4 were observed by a confocal laser scanning microscope. In Negative group, the fluorescence intensity of Smad4 was weak and did not change following media exchange. The nuclear distribution of Smad4 dramatically increased in Positive group after media exchange 3 h and then in P-6 h it decreased before slightly increased in P-12 h and P-24 h. In all the five time points of Positive group, the fluorescence intensity of Smad4 in nuclei was stronger than that in the corresponding Negative group. To investigate the amplifying mechanism underlying Act A/Smads signaling, Act A m RNA and Activin βA protein were measured. The endogenous expression of Act A m RNA did not increase until 3 h after media exchange, when Act A/Smads signaling was activated by increased extra Act A accumulation. And it was more than 7 times compared to that in P-24 h. The protein level of Act A increased by 51 % in P-3 h. However, even if the self-amplifying effect of Act A persisted until at least 12 h after treatment, the up-regulation level of Act A dramatically decreased at 6 and 12 h compared to that at 3 h. Flow cytometry showed that in Positive group the percentage of cells in G1 stage decreased by 43.7 %compared to that in Negative group, and the percentage in G2 stage significantly increased by more than 300 %. MTT assays showed that the survival rates of cells in Positive group were higher than that of the other two groups in all the three time points of OGD injury. Hoechst33342 staining showed that the number of dense blue apoptosis nuclei in Positive group was slightly less than that in Negative group.Conclusions: Act A/Smads positive signal loop existed in the non-pathological state of PC12 cells. It was initiated by up-regulation of Act A gene and activated in extra Act A concentration dependent manner. The activation stage of Act A signalingwas short and self-limiting, which restricted the protective effect of Act A signal loop on ischemic injury in vitro. Increased Act A signal loop promoted PC12 cell proliferation.4. Regulation of Act A/Smads signal loop through SARA expressionObjective: To explore the relationship and mechanism between SARA and the self-limiting characteristic of Act A/Smads signal loop.Methods: The dynamic changes of SARA protein in Act A signaling were explored after media exchange for 1, 3, 6,12 and 24 h at first. Then sh RNA plasmid was synthesized targeting to SARA gene with the use of RNA interference technology.After transfected into Act A over-expressioned PC12 cells, the effect of SARA suppression on Smad3 expression and phosphorylation was detected by Western blot.Results: Western blot showed that the expression of SARA in P-24 h subgroup decreased by 50 % compared to that in N-24 h. After 1 h of media exchange the level of SARA protein increased by 40.2 % compared to that in P-24 h. Then in P-3 h subgroup it reached to the peak, and gradually decreased after 6 h of media exchange.In Negative group, it did not alter with time. After 48 h of sh RNA plasmid transfection, the transfection efficacies was observed under a fluorescence microscope,which was more than 70 %. The delivery of p Genesil-3-SARA-sh RNA(SARA-sh RNA group) resulted in an approximately 74.9 % knockdown of SARA protein. In NC-sh RNA group, the expression of phosphorylated Smad3 dramantically increased after 3 h of media exchange, then decreased at 6 h. In SARA-sh RNA group,the expression of SARA remained at a low level. Thus SARA suppression reduced the expression and phosphorylation of Smad3 in response to Act A signal activation after media exchange. However, after 6 h of media exchange, the expression and phosphorylation of Smad3 in SARA-sh RNA transfection group did not dramatically decrease as that in NC-sh RNA group. Instead, they were almost the same as that in SARA-sh RNA group 3 h.Conclusions: SARA, as a regulator of Act A/Smads signaling, took part in the self-limiting behavior of Act A signal loop. The strength of Act A signaling could be regulated through the level of SARA. |
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