| Background:Neonatal hypoxic-ischemic Encephalopathy(HIE)is a brain injury caused by perinatal asphyxia,is one of the common causes of severe neurological sequelae in children,and the main cause of neonatal death.Hypoxia accompanied by cerebral hypoperfusion can cause neurological abnormalities,resulting in long-term neurodevelopmental disorders,such as cerebral palsy,epilepsy and autism.Ischemia and hypoxia mainly damage neurons in the cerebral cortex,hippocampus and subventricular region,while the hippocampus and its circuitry,as the cerebral paleo cortex,play an important role in spatial memory,learning ability and emotion regulation.Immature hippocampal neurons are vulnerable to external environmental stimuli during the development of hippocampal structure,and many harmful stimuli such as hypoxia and intrauterine growth restriction can cause fatal damage to hippocampal development.Cellular Repressor of E1A-Stimulated genes(CREG)can inhibit cell apoptosis,inflammatory response and cell migration,which can effectively inhibit the development and progression of cardiac hypertrophy,cardiac fibrosis,atherosclerosis and other diseases.It plays an important role in ischemia reperfusion of multiple organs including liver and heart.These findings suggest that CREG may play a protective role in cerebral ischemia reperfusion,which provides a research basis for evaluating the role of CREG in neonatal HIE.Apoptosis is the main pathophysiological change after acute nerve cell necrosis and reperfusion injury.Reducing and preventing apoptosis of brain cells is an effective means to treat neonatal HIE.Previous studies have shown that CREG is involved in regulating the process of apoptosis.The overexpression of CREG can inhibit the apoptosis of vascular smooth muscle cells,while the silencing of CREG aggravates the degree of apoptosis.CREG treatment can also inhibit renal cell apoptosis and reduce renal fibrosis,thereby improving renal function in hypertensive nephropathy patients induced by a high-salt diet.The above background suggests that CREG may play a role in neuronal apoptosis in neonatal HIE.Objective: Currently,there are no reports on the role of CREG in cerebral ischemia reperfusion process.The purpose of this study is to observe whether CREG is involved in regulating hypoxic-ischemic brain injury in newborn rats and explore its possible mechanism of action,so as to provide an important research basis for HIE treatment.Methods:1.The internationally recognized neonatal HIE rat model was used in this study.SD rats on day 7 of birth were admitted,male and female unlimited.Experimental animals were randomly divided into two groups: sham group and HIE group.There were 42 cases in sham group and 46 cases in HIE group.Brain tissue was collected from rats 3 and 7 days after operation,and neurobehavioral function was evaluated before sampling.Some brain tissues were stained with TTC.Part of brain tissue was immersed in 4%paraformaldehyde,fixed and embedded in paraffin for HE staining and immunofluorescence staining.The remaining hippocampal tissue was frozen at-80℃,and the CREG protein level of hippocampal tissue was measured by Western-blot and quantitative real-time PCR(q-PCR).2.Hippocampal neurons from primary culture to the third generation were randomly divided into normal culture group(Con group)and oxygen glucose deprivation culture group(OGD/R group).The cells prepared for immunofluorescence were rinsed with phosphate buffer saline(PBS)for 3 times and fixed with 4% paraformaldehyde.Cells used for q-PCR were lysed with Trizol total RNA extract and collected in a sterile collection tube,which was stored in a refrigerator at-80℃ for further use.The cells used for Western Blot were washed with PBS for 3 times after the culture medium was abandoned,the adherent cells were digested with 0.25% trypsin,and the supernatant was removed by centrifugation,the cells were collected and precipitated in a sterile collection tube and stored in a refrigerator at-80℃.The levels of CREG protein in hippocampal neurons were detected by Western-blot and q-PCR.3.The primary hippocampal neurons were divided into two groups according to whether they silenced or overexpressed CREG,then oxygen glucose deprivation experiment was performed,and the effects of CREG silencing and overexpression were verified by Western-blot.The activity of hippocampal neurons in each group was detected by MTT kit.TUNEL staining was used to detect the apoptosis of hippocampal neurons.The expressions of apoptosis-related proteins and apoptosis-related pathway proteins in hippocampal neurons were detected by Western-blot.4.Hippocampal neurons in the CREG overexpression group were divided into groups according to whether LY294002,a specific inhibitor of PI3K/Akt signaling pathway,was added or not,and the expression of CREG was detected by Western blot and q-PCR.The activity of hippocampal neurons in each group was detected by MTT kit.TUNEL staining was used to investigate the apoptosis of hippocampal neurons.The expressions of apoptosis-related proteins and apoptosis-related pathway proteins in hippocampal neurons of all groups were detected by Western-blot.Result:1.The experiment time of righting reflex and cliff avoidance in HIE group at 3 and 7days after ischemia and hypoxia was prolonged(P < 0.05).TTC staining showed different degree of infarction in the left cerebral hemisphere of newborn rats in HIE group compared with the sham group(P < 0.05).TUNEL staining showed that the percentage of neuronal apoptosis index in hippocampal CA1 and CA3 areas was significantly increased in HIE group compared with the sham group.Neu N and CREG dual immunofluorescence staining suggested that CREG distribution in hippocampal CA1 and CA3 regions decreased in HIE group.Western-blot analysis showed that CREG expression in the ipsilateral hippocampus of rats with artery ligation in HIE group was lower than that in the sham group(P < 0.05),and q-PCR showed that CREG m RNA expression in the ipsilateral hippocampus of rats with artery ligation in HIE group was lower than that in the sham group(P < 0.05).These results confirmed that CREG protein expression in the hippocampus was decreased after ischemia and hypoxia in neonatal rats.2.24 hours after oxygen glucose deprivation,the result of q-PCR showed that the CREG m RNA expression of hippocampal neurons in OGD/R group decreased compared with the control group(P < 0.05),Western Blot analysis showed that the expression of CREG in hippocampal neurons in OGD/R group decreased compared with the control group(P < 0.05),dual immunofluorescence staining for neuron markers Neu N and CREG showed that the distribution of CREG decreased in hippocampal neurons in OGD/R group.These results confirmed that CREG expression was decreased in hippocampal neurons under oxygen glucose deprivation.3.The expression level and difference of CREG in the cells of control group and LV-sh NC group and LV-shCREG group,control group and LV-ex NC group and LV-ex CREG group were detected by Western-blot technique.The results showed that CREG protein expression level in primary hippocampal neurons of LV-shCREG group was significantly lower than that of control group and LV-sh NC group(P < 0.05),indicating the successful construction of CREG gene silencing.And the expression level of CREG protein in primary hippocampal neurons of LV-ex CREG group is significantly higher than that of control group and LV-ex NC group(P < 0.05),indicating that CREG gene overexpression was successfully constructed.4.The results of MTT assay showed that the activity of hippocampal neurons decreased significantly under oxygen glucose deprivation(P < 0.05).Cell activity decreased further after silencing CREG expression,while neuron activity increased significantly after overexpression of CREG(P < 0.05).These results suggest that inhibition of CREG expression can further reduce cell activity and aggravate cell damage under oxygen glucose deprivation,while overexpression of CREG can significantly improve the cell activity and alleviate cell damage caused by oxygen glucose deprivation.5.TUNEL assay results showed that under oxygen glucose deprivation,apoptosis was further aggravated after CREG expression was silenced,while the degree of apoptosis was alleviated after CREG expression was overexpressed.These results suggest that CREG can improve the apoptosis of hippocampal neurons induced by oxygen glucose deprivation.6.Western Blot results showed that compared with the normal control group,oxygen glucose deprivation could significantly reduce the expression level of Bcl2 in hippocampal neurons,significantly increase the expression level of Bax and Cleaved caspase-3 protein,and decrease the level of P-Akt(P < 0.05).After silencing CREG expression,Bcl2 level was further decreased,Bax and Cleaved caspase-3 protein expression levels were further increased,and p-Akt level was further decreased(P <0.05).These results suggest that under oxygen glucose deprivation,CREG silencing can increase the expression of pro-apoptotic protein and decrease the expression level of anti-apoptotic protein in hippocampal neurons,while CREG silencing can decrease the expression of the apoptotic pathway protein p-Akt.In contrast,CREG overexpression decreased the expression of pro-apoptotic proteins,increased the expression of anti-apoptotic proteins,and increased the expression of the apoptotic pathway protein P-Akt.7.LY294002 had no effect on the expression of CREG protein in primary hippocampal neurons with CREG overexpressed under oxygen glucose deprivation;MTT assay suggested that LY294002 could reduce the activity of hippocampal neurons after CREG overexpression and aggravate cell damage.TUNEL assay indicated that the inhibitory effect of CREG overexpression on apoptosis of hippocampal neurons was reversed after LY294002 was used.Western Blot results showed that the expression level of Bcl2 in hippocampal neurons of OGD/R+LV-ex CREG+LY294002 group was significantly reduced,Bax protein expression level was significantly increased,and p-Akt level was significantly decreased(P < 0.05).These results indicate that P-Akt is a downstream signal molecule of CREG,LY294002 can increase the expression of pro-apoptotic proteins in CREG overexpressed primary hippocampal neurons,decrease the expression level of anti-apoptotic proteins,and decrease the expression of apoptotic pathway protein p-Akt under oxygen glucose deprivation.Conclusion:1.The expression level of CREG in HIE animal model and OGD/R hippocampal neuron model decreased significantly,suggesting that CREG may play an inhibitory role in the occurrence and development of HIE.2.Silencing and overexpression of CREG at the cellular level found that CREG could increase the viability of hippocampal neurons and inhibit the apoptosis of hippocampal neurons under the condition of oxygen glucose deprivation.It further confirmed the inhibitory effect of CREG on the occurrence and development of HIE,and revealed that CREG could achieve its protective effect in cerebral ischemia-reperfusion injury mainly by inhibiting apoptosis and increasing cell viability.3.Through the use of LY294002,a specific inhibitor of PI3K/Akt signaling pathway at the cellular level,it was confirmed that CREG could increase the phosphorylation level of Akt,improve the expression level of Bcl2,and then inhibit the apoptosis of hippocampal neurons by activating the PI3K/Akt signaling pathway.4.The experimental results of this study show that CREG is a negative regulator of cerebral ischemia-reperfusion,and its inhibitory effect mainly depends on the activation of PI3 K / Akt signaling pathway to inhibit the apoptosis of hippocampal neurons.Therefore,CREG is expected to become an important target for the clinical treatment of cerebral ischemia-reperfusion injury. |
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