The Effect And Mechanism Of Olfactory Mucosa Mesenchymal Stem Cells On The Secretory Pathway Ca²⁺-ATPase Isoform 1 Of Golgi Apparatus After Cerebral Ischemia/Reperfusion Injury

Objective:To investigate the effect and mechanism of olfactory mucosal mesenchymal stem cells（OM-MSCs）on the expression and function of secretory pathway Ca²⁺-ATPase isoform 1（SPCA1）in Golgi apparatus following cerebral ischemia reperfusion injury（IRI）.Methods:1.The morphology of OM-MSCs was observed under light microscope and OM-MSCs were identified using flow cytometry.2.In vitro,the oxygen-glucose deprivation/reoxygenation（OGD/R）model of N2a cells was performed,and OM-MSCs were co-cultured with N2a cells using Transwell plates.3.In vivo,the middle cerebral artery occlusion（MCAO）model of SD rats was established,and 5×10⁶ OM-MSCs were injected via tail vein at 24hours after MCAO model induction.4.In the study that explored the effect of OM-MSCs on SPCA1 after cerebral IRI:（1）N2a cells were divided into three groups:Normal group,OGD/R group,OGD/R+OM-MSC group.The CCK-8,lactate dehydrogenase（LDH）kit and flow cytometry were used to detect cell viability,LDH level,apoptosis and reactive oxygen species（ROS）level,respectively.The m RNA and protein expression level of SPCA1 were analyzed using q RT-PCR and Western Blot.And Ca²⁺detection kit was used to examine the Ca²⁺concentration in the cytoplasm and Golgi apparatus.（2）SD rats were randomly divided into three groups:Sham group,MCAO+Saline group,MCAO+OM-MSC group.The Infarct volumes were evaluated by TTC staining.The modified neurologic severity score（m NSS）and rotarod treadmill were used to evaluate the neurological deficits of rats.Flow cytometry and TUNEL+Neu N double-labeled immunofluorescence staining were performed to assess neuronal apoptosis.Total superoxide dismutase（T-SOD）and lipid peroxide（LPO）kits were used to detect the levels of T-SOD and LPO.The m RNA and protein expression level of SPCA1 were analyzed using q RT-PCR and Western Blot.The flow cytometry was performed to detect the concentration of intracellular Ca²⁺.The ultramicrostructure changes of Golgi apparatus in neurons were observed under a transmission electron microscopy.5.In the study that investigated the effect of pigment epithelium-derived factor（PEDF）secreted by OM-MSCs on SPCA1 after cerebral IRI:（1）N2a cells were divided into five groups:Normal group,OGD/R group,OGD/R+OM-MSC group,OGD/R+OM-MSC^{NC si RNA} group,OGD/R+OM-MSC^{PEDF si RNA} group.The flow cytometry was used to detect apoptosis,ROS level and Ca²⁺concentration.Western Blot was performed to detect the protein expression levels of SPCA1,p-Akt/Akt,and p-m TOR/m TOR.And a transmission electron microscope was used to observe the ultramicrostructure of Golgi apparatus.（2）SD rats were randomly divided into four groups:Sham group,MCAO+Saline group,MCAO+OM-MSC^{NC si RNA} group,MCAO+OM-MSC^{PEDF si RNA} group.The neurological deficits of rats were assessed using m NSS.The pathological damage of cerebral tissue was detected by HE staining.The protein expression level of SPCA1 were analyzed using Western Blot.The level of ROS and the concentration of Ca²⁺were detected by flow cytometry,and the level of LPO was examined using LPO kit.6.In the study that explored the role of the PI3K/Akt/m TOR signaling pathway in the effect of OM-MSCs:N2a cells were divided into four groups:Normal group,OGD/R group,OGD/R+OM-MSC group,OGD/R+OM-MSC+Perifosine group.Western Blot was used to assess the protein expression levels of p-Akt/Akt,p-m TOR/m TOR and cleaved-caspase-3.The protein expression level of SPCA1 was analyzed using Western Blot and immunofluorescence staining.The level of ROS and the concentration of Ca²⁺were detected using flow cytometry.And a transmission electron microscope was used to observe the ultramicrostructure of Golgi apparatus.Results:1.Under light microscope,OM-MSCs adopted a fibroblastic or spindle-shaped morphology;The immunophenotype of OM-MSCs exhibited positive expression of CD73,CD90,and CD105 and negative expression of CD34 and CD45.2.In vitro,OM-MSCs co-culture increased the cell viability of N2a cells after OGD/R,and decreased the level of LDH,apoptosis and ROS in N2a cells after OGD/R（p<0.05）.In vivo,OM-MSCs transplantation reduced the cerebral infarction volume of MCAO rats,improved neurological deficits,and alleviated neuronal apoptosis and oxidative stress levels（p<0.05）.Both in vitro and in vivo,OM-MSCs upregulated the expression level of SPCA1,repressed the increase of Ca²⁺concentration,and alleviated the fragmentation of Golgi apparatus（p<0.05）.3.In vitro,compared to the OGD/R+OM-MSC group,the apoptosis of N2a cells in the OGD/R+OM-MSC^{PEDF si RNA} group was increased（p<0.05）,the expression level of SPCA1 in the OGD/R+OM-MSC^{PEDF si RNA} group was downregulated（p>0.05）,the level of ROS and the concentration of Ca²⁺in the OGD/R+OM-MSC^{PEDF si RNA} group were increased（p<0.05）,and the fragmentation of Golgi apparatus was more remarkable.In vivo,compared to the MCAO+OM-MSC^{NC si RNA} group,the m NSS of rats in the MCAO+OM-MSC^{PEDF si RNA}group was significantly increased,the expression level of SPCA1 in the MCAO+OM-MSC^{PEDF si RNA}group was downregulated,the level of ROS and LPO,and the concentration of Ca²⁺in the MCAO+OM-MSC^{PEDF si RNA} group were increased（p<0.05）.4.OM-MSCs co-culture upregulated the ratio of p-Akt/Akt and p-m TOR/m TOR in N2a cells after OGD/R（p<0.05）.And the ratio of p-Akt/Akt and p-m TOR/m TOR in OGD/R+OM-MSC^{PEDF si RNA} group were lower than that of OGD/R+OM-MSC group（p<0.05）.5.Compared to the OGD/R+OM-MSC group,N2a cells in the OGD/R+OM-MSC+Perifosine group exhibited upregulated expression level of cleaved-caspase-3（p>0.05）,downregulated level of SPCA1（p>0.05）,increased ROS level and Ca²⁺concentration（p<0.05）.Additionally,the fragmentation of Golgi apparatus in OGD/R+OM-MSC+Perifosine group was also more noticeable.Conclusions:1.OM-MSCs exerted neuroprotective effects against cerebral IRI,providing an ideal candidate for the cell therapy of ischemic stroke.2.OM-MSCs promoted the phosphorylation of the PI3K/Akt/m TOR signaling pathway via secretion of PEDF,thereby upregulating the expression level of SPCA1 in Golgi apparatus,reducing the level of oxidative stress and the concentration of Ca²⁺,and alleviating cerebral IRI.