| ObjectiveIschemic stroke(IS)can induce infarction and neurogenesis around the infarcted area.The endogenous neural progenitor cells in the sub-ventricular zone(SVZ)of the lateral ventricle and the sub-granular zone(SGZ)of the hippocampal dentate gyrus are considered to be an important source of infarction-induced neurogenesis.These cells proliferate,migrate and differentiate into mature neurons which are integrated into the damaged neural network for neural repair function.Endogenous neurogenesis may become a new therapeutic approach for ischemic stroke.Neuroblasts are affected by chemokines and cytokines secreted by activated microglia and astrocytes and migrate to the site of injury.Chemokine receptor 3(CC chemokine re-ceptor3,CCR3)CCR3 is abundantly expressed in the hippocampal neurons and may be involved in the occurrence and development of ischemic stroke.We speculate that CCR3 may be involved in the neurogenesis in the subgranular region of the hippocampal dentate gyrus in adult mammals.In this study,we explored the regulatory effect of CCR3 on neural progenitor cell models under the conditions of ischemic stroke through a variety of cellular and molecular biological methods,so as to provide the basis for further elucidation of the role and mechanism of CCR3 in the neurogenesis of ischemic stroke.MethodsThe SH-SY5 Y cells were transfected with CCR3 overexpression plasmid,and the experimental cells were divided into four groups.One group was the control group,and the other three groups were in the OGD/R condition: the OGD/R model group,the OGD/R CCR3 negative control(NC)group,and the OGD/R CCR3 overexpression group.(1)The intracellular expression of CCR3 was observed by detecting the relative m RNA expression by immunofluorescence and RT-q PCR.(2)cell count kit 8(CCK8)was used to detect the effect of the changes of CCR3 expression under different conditions on the proliferation of SH-SY5 Y cells.(3)The effect of CCR3 expression on the cell cycle of SH-SY5 Y under different conditions was analyzed by flow cytometry.(4)The effect of the change of CCR3 expression on the migration ability of SH-SY5 Y cells was detected by cell scratch test.(5)The protective effect of the change in CCR3 expression on cell damage was detected by lactate dehydrogenase(LDH)kit.(6)The differentially expressed genes of SH-SY5 Y were detected by transcriptomics sequencing technology,and the key genes differentially expressed in pathways were selected by GO gene functional enrichment analysis and KEGG pathway enrichment analysis.RT-q PCR and Western Blotting experiments were performed to verify the expression of the key genes.Key genes included GDF5(growth differentiation factor 5),ID1(DNA binding inhibitor 1),and ID4(DNA binding inhibitor 4).Results1.The CCR3 overexpression vector was transfected into cells and expressed stably.Compared with the OGD/R CCR3 NC group,the m RNA expression level of the CCR3 overexpression group was significantly increased,and the CCR3 overexpression vector was successfully transfected.By immunofluorescence staining,we observed that compared with the CCR3 NC group,the fluorescence signal of the CCR3 overexpression group was significantly increased,indicating that the CCR3 overexpression vector was successfully expressed in SH-SY5 Y cells.In addition,compared with the control group,the m RNA expression level of CCR3 in the OGD/R model group was decreased significantly,indicating that the OGD/R environment affected the expression of CCR3.There was no significant difference in m RNA expression levels between the CCR3 NC group and the OGD/R model group,except that other factors were involved.2.An in vitro OGD/R model was constructed and the effects of increased CCR3 expression on the reduction of SH-SY5 Y cell injury,promotion of cell proliferation,regulation of cell cycle and promotion of cell migration were detected by LDH experiment,CCK8 experiment,flow cytometry detection of cell cycle and cell scratch test.(1)The experimental results of LDH showed that compared with the control group,the release of cell LDH in the OGD/R model group was increased,while compared with the OGD/R model group,the release of cell LDH in the OGD/R CCR3 overexpression group was decreased.(2)The experimental results of CCK8 showed that compared with the control group,the absorbance values of cells cultured for different days in CCK8 experiment were lower than the control group.Compared with the OGD/R model group,the absorbance values of cells cultured for different days in OGD/R CCR3 overexpression group were higher than that of the OGD/R model group.(3)Compared with the control group,the G1 phase block was weakened in the OGD/R model group in cell cycle experiments,but the S phase block was increased.Compared with the OGD/R model group,the overexpression group of OGD/R CCR3 showed no significant difference in the G1 phase of the cell cycle,and the reduction of S-phase block.(4)The cell scratch test showed that the percentage of wound healing in the OGD/R model group was lower than that in the control group by calculating the percentage of wound healing.Compared with the OGD/R model group,the cell scratch test calculated the percentage of wound healing and concluded that the percentage of wound healing in the OGD/R overexpression group was higher than that in the OGD/R model group.(5)Three genes which were enriched in TGF-beta pathway and expressed significantly differently were screened out by transcriptomics sequencing analysis technology: GDF5,ID1 and ID4.As detected by RT-q PCR and Western Blotting experiments,compared with the control group,the m RNA expression and protein expression of key genes ID1 and ID4 enriched in TGF-beta pathway in OGD/R model group were increased.Compared with the OGD/R model group,the ID4 m RNA and protein expressions of key genes ID1 and Id4 enriched in the TGF Beta pathway in the OGD/R CCR3 overexpression group were decreased.Conclusions1.Under OGD/R environment,the increase of CCR3 expression reduces the content of lactate dehydrogenase in cells,weakens cell damage,increases cell vitality and enhances cell proliferation ability.OGD/R condition weakened the proliferation ability of cells,and the increase of CCR3 expression decreased the S phase arrest of cell cycle and promoted the proliferation ability of cells.The results of cell scratch experiment show that the migration ability of cells is increased.2.Transcriptomics showed that CCR3 regulated TGF beta pathway by regulating key genes DGF5,ID1 and ID4,thus affecting DNA synthesis,cell proliferation and cell migration of SH-SY5 Y cells. |
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