The Expression Of Smad2in Oxygen-glucose Deprivation Followed By Reperfusion Model And Its Effect And Underlying Mechanism

Objective:Cerebral ischemia is the most common type of cerebrovascular diseases. However, the pathophysiologic mechanisms involved in ischemia are still far from clear. It has proved that multiple reasons results in neuronal death involving severe failure of metabolic energy support, elevated intracellular Ca+, apoptosis, mitochondrial injury, and so on. Recent studies have shown a neuroprotective activity of TGF-(3against ischemia-induced neuronal death. Smad2is an important cytokine which plays significant role in TGF-β signaling. Little is known, however, the effect and underlying mechanism of Smad2in ischemic stroke. Golgi apparatus (GA) is an important organelle that involves in the metabolism of numerous proteins. Recent evidence highlights the functional importance of the GA in neurological diseases. The functions of the mammalian GA, in addition to the processing and transport of cargo, also include ionic homeostasis. SPCA1can transport Ca2+into the Golgi lumen and therefore is involved in the cytosolic and intra-Golgi Ca2+homeostasis. It has shown that both of the mRNA and protein of SPCAl are highly expressed in brain. In addition, brain is the region with the highest activity of SPCA isoforms, which may be related to the involvement of Ca2+homeostasis in neural functions. Moreover, SPCA1plays the pivotal role of for normal neural development and differentiation. Ischemia is associated with a loss of cytosolic calcium homeostasis. Actually, recently, a small but significant drop in the SPCA1activity was observed in ischemic hippocampus of rats.However, whether the exposed ischemic course is associated with the functional alternation and the changes of expression pattern of SPCA1are still not clear. Disruption of Ca2+homeostasis is a well-established signal for programmed cell death in mammalian cells, and many apoptotic effectors such as calcineurin and calpains show Ca2+-dependent activation. Ischemic stroke is characterized by apoptotic and necrotic cell death leading to neuronal loss. DAPK1is a Ca2+/calmodulin-regulated serine/threonine kinase that acts as a positive mediator of apoptotic pathways, including those involved in neuronal cell death. It has proved that DAPK1was a target of transcriptional activation by Smads in TGF-(3-induced apoptosis in Hep3B hepatoma cells. Whether the same pathway is existed in ischemic is still unknown.The objectives of this study are to clarify the beneficial effects as well as the mechanism of action of Smad2against oxygen-glucose deprivation followed by reperfusion (OGD/R) damage. We are interested in gaining insight into the intricate role of the SPCA pumps and DAPK1to explain the Smad2related functions in mouse N2a cells upon OGD/R.Methods1. We employed a stable OGD/R model to mimic ischemic-like conditions in vitro. The neuronal injury was measured by the lactate dehydrogenase (LDH) release and the MTT assay, as well as the morphologic changes studied under Light Microscopes.2. In order to evaluate the role of Smad2in OGD/R, SB431542was used to inhibit the Smad2phosphorylation by TGF-β type Ⅰ receptor and thus blockade the TGF-β-dependent transcriptional activation. Experimental grouping was designed as follows:control group, common OGD/R group, and OGD/R group with SB431542intervention. The latter two groups were divided into three different subsets for various exposed OGD time (OGD/Rlh, OGD/R4h, OGD6h/R).3. The intracellular free Ca2+concentration and the rate of apoptosis were measured by flow cytometric analysis.4. The protein level of Smad2, phospho-Smad2(pSmad2), SPCA1and DAPK1was determined by western blotting.5. The mRNA levels of Smad2, SPCA1and DAPK1were tested by quantitative Real-Time polymerase chain reaction. 6. The SPCA1-Ca2+-ATPase activity was determined by biochemical method.7. Confocal Immuno-fluorographs of GM130and SPCA1were taken to determine the subcellular location of SPCA1.Result:1. The MTT and the LDH showed the activity of N2a cells decreased upon OGD as well as the changed morphology. When followed by reperfusion, the damage was more severe (P<0.05), indicating that the reperfusion aggravate the OGD injury. The stable OGD/R model was established.2. The alternatin of intracellular free Ca2+concentration:The OGD/R induced severe intracellular Ca2+overloading in a time-dependent manner. The [Ca]i in OGD4h/R group was higher than other groups(P<0.01). The [Ca2+]i in OGD/R groups with SB431542treatment displayed significant increase when compared to simultaneous common OGD/R groups (P<0.05).3. The SPCA1-Ca2+-ATPase activity decreased remarkably in OGD/R groups than in control (P<0.05), in a time-dependent manner. Among the common OGD/R subsets, the minimal level of SPCA1activity was the OGD6h/R group (P<0.05) while among the OGD/R subsets with SB431542treatment, the minimal level of SPCA1activity was the OGD4h/R group (P<0.05). The activity in OGD/R group with SB431542intervention showed significant decrease when compared to simultaneous common OGD/R group (P<0.05). The correlation analysis confirmed that the alternation of [Ca2+]i was inverse correlative to the activity of SPCA1.(r=-0.722, P<0.01).4. Confocal Immuno-fluorographs revealed that SPCA1was colocalized with GM130, a marker molecule for the Golgi apparatus.5. The expression of Smad2was elevated both in protein and mRNA level upon OGD/R damage, in a time-dependent manner. In common OGD/R groups, the highest level of Smad2, as well as the pSmad2 came from the OGD4h/R subset. In the groups with SB431542intervention, the pSmad2was almost negative expressed and the expression of Smad2also decreased remarkably (P<0.05), indicating that the SB431542inhibited the Smad2phosphorylation and thus decreased the total protein level of Smad2. Interesting, the mRNA level of Smad2changed toward SB431542intervention. The expression elevated in OGD1h/R while decreased in OGD4h/R and OGD6h/R than the simultaneous common OGD/R group (P<0.05).6. The expression of SPCAl was down-regulated both in protein and mRNA level upon OGD/R damage, in a time-dependent manner (P<0.05). In the common OGD/R groups, the xpression of SPCA1was clearly suppressed immediately after OGD/R and remained depressed at least up to4h following deprivation and then elevated in the OGD6h/R subset (P<0.05). The tendency in the groups with SB431542intervention was as the same (P<0.01). The expression level of SPCA1mRNA was parallel with the protein. We further studied whether the inhibition of Smad2affect the expression of SPCA1. The result revealed that, only in the OGD6h/R point, the expression of SPCA1elevated both in protein and mRNA level by the SB431542intervention (P<0.05).7. The expression of DAPK1was up-regulated both in protein and mRNA level upon OGD/R damage, also in a time-dependent manner (P<0.05). The expression level in OGD4h/R subset was higher than other subsets both in protein and gene level (P<0.05). The SB431542intervention further elevated the increased expression level of DAPK1(P<0.05).8. The rate of apoptosis significantly increased upon OGD/R damage (P<0.05). The rate in the OGD4h/R subset was maximal (P<0.01). The SB431542intervention further elevated the rate of apoptosis (P<0.05), suggesting that the inhibion of Smad2aggravate the OGD/R injury.Conclusion:1. The expression pattern of Smad2was regulated by the OGD/R. The inhibition of Smad2phosphorylation aggravated the OGD/R injury, involving the mechanism of intracellular Ca2+overloading and apoptosis damage. Smad2displayed neuroprotective effects against OGD/R.2. The OGD/R induced significant depression of SPCA-associated Ca2+-ATPase activity. The impaired activity of SPCA1contributed to the intracellular Ca2+overloading. The OGD/R could also attenuate the protein and gene expression of SPCA1.3. The expression of DAPK1was up-regulated by OGD/R.4. The neuroprotective activity of Smad2against OGD/R may be closed related to maintain the intracellular Ca2+homeostasis. The possible mechanism may involve the regulation of SPCA1and DAPK1...