| Background:It is widely accepted that ischemia related organ injuries are the primary cause of fatal disease, such as stroke and myocardial infraction attributed to coronary arteriosclerosis. However, the clinic researches found more severe brain dysfunction occured after recovering blood supply during certain period of ischemia, which is called ischemia reperfusion(IR) injury. IR occurred in poor blood circulation like traumatic shock, operation, organ transplantation, empyrosis, frostbite and thrombus. Increasing yearly, stroke became leading cause of mortality and long-term disability in our country. Ischemia stroke is the most common type of stroke, about 70% of strike. The fatality rate of ischemia is 10%, and the disability rate is above 50%. Thus, searching for direct and more effective treatments of brain IR injury has been prioritized.In recent years, a series of studies from our group identified MG53 as an essential component of the cell membrane repair machinery. The effect of repairment is verified in skeletal muscle cells and myocardial cells. MG53 also known as TRIM72, a member of the tripartite motif-containing(TRIM) superfamily which is constituted of proteins that possess a RING finger domain, one or two B-boxes and a coiled-coil(CC) domain. As a muscle specific protein,MG53 mainly expresses on muscle and myocardial, and rarely expresses on other organization. It acts as a sensor of oxidation to oligomerize and recruit intracellular vesicles to site of membrane disruption for the formation of a repair patch. In the presence of external Ca2+,MG53 could move to the cell membrane from cytoplasm, assemble at the breakage of cell membrane and then form a membrane repair patch.The mechanism of cerebral IR are complicated, including free radical formation, intracellular calcium overload, excitability amino acid toxicity, strong leukocyte aggregation, shortage of high energy phosphate compound and et.al. In view of this mechanism, calcium antagonists, gamma-aminobutyric acid receptor agonist, antagonist of AMPA receptor, and free-radical scavenger were confirmed to the therapeutic schedule of IR. However, the therapy is not ideal. Although little MG53 expression in the brain, as a direct repairman of cell membrane,whether there is a protective effect of exogenous administration of MG53 in the cerebral IR should be speculated.In our studies, we used SD rats to created cerebral IR model, and detect the protective effect of MG53 on IR in vitro and in vivo. The research covered four expects:1, The protective effect of MG53 on neuronal cells. 2, The protective effect of rhMG53 on cerebral IR in SD rats. 3, Wether the circulating MG53 could run across the blood brain barrier to play a role in the cerebral IR. 4, The signaling pathway of protective effect of MG53 in the cerebral IR.Methods:1. The expression of MG53 in brain tissue and cellThe brain tissue from MG53(-/-) knockout mice and Sprague-Dawley(SD) rats were used to detect the expression of MG53, the skeletal muscle was extracted as a positive control. Western blot and immunohistochemistry were used to test protein expression and distribution of MG53 in different kinds of brain cells including microgliacyte, astrocyte, neural stem cell,and neurocytoma.2. The protective effect of MG53 on cerebral IR in vivo and in vitroFor IR induced brain injury, SD rats were subjected to 60 min of ischemia to the left carotid artery to establish the IR model. In vitro studies, we established a hypoxia-reoxygenation(HR) protocol that induces membrane injury to cultured HT22 neuron cells. The rh MG53-mediated amelioration of cerebral IR were proved by TTC staining, neurological scores, TUNEL staining and expression of caspase 3.3. Circulating MG53 can cross the blood-brain barrier to protect the mouse brain from IR injuryWe constructed a transgene by adding a tissue plasminogen activator(tPA) leader sequence ahead of the mg53 c DNA(t PA-MG53) to allow for secretion of MG53 into the bloodstream. Western blot to detecte the circulating MG53 in the brain after IR.In the meanwhile,to observe the effect of MG53 on IR in tp A-MG53 mice.Intravenous injection of rhodamin marked rhMG53 and FITC-Annexin V, and then utilized IVIS and laser scanning confocal mnicroscopy to test the expression of rh MG53 could cross the blood-brain barrier and play a role in IR.4. The signaling pathway of rhMG53 in the brain injury protectionThe SD rats were used to establish the IR model. The experiment was divided into four-group:sham, sham+MG53, IR, IR+MG53.Extracting the brain protein after injecting MG53. As RISK and SAFE are predominant signaling pathway in IR,we use western blot to filtrate the key molecules involved in the regulation of protecting effect of MG53 in brain injury.Result:1.The expression of MG53 protein is null in brain.In mouse tissues, Immunoblotting showed no MG53 expression in the mouse brain and immunohistochemical staining of MG53 was negative in the brain tissue. The lack of MG53 expression was further confirmed in cultured neuronal cells, including microglia, astrocytes, neuro-stem cells and neuroblastoma.2. Intravenous delivery of rhMG53 ameliorates IR induced brain injury and protect neuron cells from HR injury2.1 rhMG53 was effective at protecting against IR-induced brain injuries in animal models. It could effectively reduce the area of infarction in the left brain and improved the animal behavior following brain injury.2.2 The incubation of rhMG53 in the culture medium enhanced survival of HT22 cells following HR treatment. Moreover, the application of rhMG53 significantly reduced the release of LDH from HT22 cells into the culture medium after HR treatment, suggesting that rh MG53 can protect neuron cells from IR injury.3. Circulating MG53 can cross the blood-brain barrier to protect the mouse brain from IR injuryThe rhodamine-labeled rh MG53 was intravenously administered to rats that were subjected to IR induced injury to the left brain, whereas the right brain only showed marginal labeling of rhodamine-rhMG53. This result raised the possibility that circulating MG53 can cross the blood-brain barrier(BBB) to target injured brain tissue.4. rh MG53 protects brain injury through activation of the RISK signaling pathway and suppressing Akt-dependent caspase 3 activity.4.1 Cultured neuronal stem cells were infected with Ad-GFP-MG53 in order to investigate the extent of MG53’s participation in the repair of membrane injury. Nucleation of GFP-MG53 at the neuronal membrane injury sites was not observed when dithiothreitol(DTT) was present in the extracellular solution.4.2 We found that IR-induced brain injury led to inactivation of RISK signaling transway and activation of caspase3 activity. The results showed that rhMG53 treatment increased Akt, Gsk3β phosphralation of RISK signaling transway, and could suppress caspase3 activation which could indicated that the reduction of apoptotic cell death contributes to MG53-mediated neuro-protection following IR injury. Whileas STAT3 involved in the SAFE signaling way and ERK1/2is not affected by IR injury to the brain, and rhMG53 did not affect their activation.Conclusion:1. There is no MG53 expression in the brain and different kinds of brain cells.2. Redox-dependent process of MG53-mediated neuron cell membrane repair is observed after HR-induced HT22 cells injury, rh MG53 was effective at protecting against IR-induced brain injuries in animal models, including reducing the infacrtion area, improving the animal behavior following brain injury and suppressing caspase3 activation.3. MG53 in blood circulation is impermeable across the BBB under healthy condition, ischemic injury to the brain increases the permeability of MG53 through the BBB, allowing MG53 to target the injury sites in the brain tissue.4. rhMG53 can activate the RISK survival pathway to elicit neuro-protection. |
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