| Background:Clinic researches showed more severe brain dysfunction occured after recovering blood flow during certain period of ischemia,accompanied with worsening of tissue injury and inflammatory response,which is called ischemia reperfusion(I/R)injury.However,ischemia/ reperfusion injury has been as a major cause of disability and death in today' s world.The reticulon family proteins have been identified to induce apoptosis,inhibit axon regeneration and regulate protein trafficking.Notably,the reticulon family gene 1(RTN1)was also called neuroendocrine specific protein,expressed abundantly in the brain.Yet,the mechanisms by which RTN1 exerts these effects in the context of cerebral ischemia/reperfusion injury have not been illuminated.Here,we report that the expression of RTN1-C was significantly increased in the middle cerebral artery occlusion stroke(MCAO)model of rats and the model of oxygen glucose deprivation followed by reoxygenation(OGD/R).And we demonstrated that RTN1-C induced neuronal apoptosis through ER stress and mitochondria associated pathways.Moreover,knockdown of RTN1-C in vivo attenuated neural apoptosis in MCAO rats and reduced the extent of cerebral ischemia-reperfusion injury,as assessed by infarct volume and neurological score.Therefore,we confirmed that RTN1-C is involved in cerebral ischemia reperfusion injury,and its mechanism is related to the ER stress and mitochondria associated pathways.Objective:To explore the effect of RTN1-C on neuronal apoptosis during cerebral ischemia reperfusion,which provide a new clue for therapies against cerebral ischemia reperfusion injury.Methods:Western blot and immunofluorescence were performed to detect the expression of RTN1-C.Immunofluorescence staining,cck-8,and flow cytometry suggested RTN1-C overexpression induced apoptosis and increased the cell vulnerability to ischemic injury,conversely,RTN1-C knockdown reversed ischemia-induced apoptosis and attenuated the vulnerability of OGD/R-treated neural cells.WB showed that RTN1-C can induce apoptosis during OGD/R through endoplasmic reticulum stress and mitochondrial related pathways.Immunoprecipitation experiments demonstrated that RTN1-C interacted with Bcl-x L and increased its localization in ER,thereby decreasing the anti-apoptotic activity of Bcl-x L.The neural apoptosis was attenuated in MCAO rats after knockdown of RTN1-C expression in vivo,as assessed by immunofluorescence staining,ttc staining,and neurological score experiments.Results:1.RTN1-C is upregulated during cerebral ischemia/reperfusion.(1)Western blotting: The expression of RTN1-C increased gradually at 12 and 24 hours after cerebral ischemia-reperfusion,but not RTN1-A or RTN1-B.During cerebral ischemia/reperfusion,the expression of RTN1-C was upregulated,while decreasing in ischemic post-conditioning and remifentanil post-conditioning models.(2)Immunofluorescence staining: Following the reperfusion time prolonging,the brain tissues were fixed and stained with RTN1-C(green)and DAPI(blue)at 0h,12 h and 24 h after reperfusion,respectively,RTN1-C expression gradually increased.2.RTN1-C promotes apoptosis during cerebral ischemia/reperfusion.(1)Immunofluorescence staining : The brain tissues were fixed and stained with RTN1-C(green),activated Caspase3(red),and DAPI(blue)after cerebral ischemia/reperfusion for 24 h,the merged images showed that activated caspase3 positive cells increased significantly comparing with the normal group.(2)Cell viability assay: The cck-8 assay showed that the viability of nerve cells decreased after the overexpression of RTN1-C and the activity of nerve cells enhanced after the knockdown of RTN1-C in OGD/R-treated N2 a cell.(3)Flow cytometry analysis: Overexpression of RTN1-C enhanced the levels of apoptosis,while RTN1-C knockdown reduced it in the OGD/R treated cells.3.RTN1-C induces ER stress-associated apoptosis during OGD/R(1)Western blotting detected that the levels of endoplasmic reticulum stress-related protein Grp78,activated Caspase-12,CHOP,activated Caspase-3 significantly increased in the case of RTN1-C overexpression in OGD/R-treated N2 a cells.(2)In order to further confirm the involvement of RTN1-C in endoplasmic reticulum stress-related apoptosis,N2 a cells were overexpressed RTN1-C-GFP plasmid and treated with endoplasmic reticulum stress inhibitor 4-PBA for 6 hours.Then OGD / R treatment was performed.Western blotting was used to detect the protein levels of Grp78,activated Caspase-12,CHOP and activated Caspase-3.Results showed that pretreatment with 4-PBA suppressed the expressions of Grp78,activated Caspase-12,CHOP,activated Caspase-3.4.RTN1-C induces mitochondrial-associated apoptosis during OGD/R(1)To ascertain RTN1-C contributes to ischemia/reperfusion injury via mitochondria-dependent apoptosis,western blotting analyzed the levels of associated apoptotic markers cytochrome C and cleaved Caspase-3.The results showed that after OGD / R treatment,cytoplasm cytochrome C and cleaved Caspase-3 protein levels were greatly increased.RTN1-C over-expression resulted in a dramatic increase of these proteins,conversely,RTN1-C knockdown reduced these protein levels.(2)To explore the interaction between RTN1-C and Bcl-x L in OGD / R model and whether RTN1-C alters the subcellular location of Bcl-x L,we preformed the immunoprecipitation assay and found that the interaction between RTN1-C and Bcl-x L enhanced and cytosolic Bcl-x L was increased after OGD / R treatment,we also confirmed that overexpression of RTN1-C significantly increased the expression of cytosolic Bcl-x L in the OGD/R-treated cells.5.RTN1-C knockdown reduced apoptosis during cerebral ischemia/reperfusion.(1)Immunofluorescence staining: To further explore whether RTN1-C knockdown protects neurons against apoptosis in I/R rats.Tissue immunofluorescence results showed that after RTN1-C downregulation,activated Caspase3 positive cells were significantly decreased compared to LV-sh NC in the MCAO groups.The data showed that RTN1-C knockdown reduces the apoptosis of nerve cells.(2)TTC staining: To further explore the therapeutic effect of knockdown of RTN1-C on cerebral ischemia-reperfusion injury,ttc staining of rat brain tissue indicated that there was no infarct area in the sham operation group,but obvious pale infarct area was found in reperfusion 24 hours group.Compared with the control group,knockdown of RTN1-C brain infarction area was significantly reduced.(3)Neurological function score: We used the Bederson score standards to evaluate neurological function and study the effects of RTN1-C in MCAO rats.The results showed that LV-sh RTN1-C treated rats exhibited significant improvement in neurological functions compared with LV-sh NC groups.Therefore,we confirmed that RTN1-C downregulation could protect the brain from ischemic damage and restore neurologic function impaired by ischemia.Conclusions:Our research shows RTN1-C in cerebral ischemia-reperfusion through endoplasmic reticulum stress and mitochondria-related apoptosis and promote apoptosis of nerve cells,which may provide a new way for treatment of cerebral ischemia-reperfusion injury. |
PDF Full Text Request