| ObjectiveTo observe the effects of folic acid deficiency(FD)on microglia/astrocyte activation and the expression of inflammatory cytokines after rat cerebral ischemia-reperfusion model.The combination of related inhibitors and folic acid deficiency was used to explore the mechanism of microglia/astrocyte activation after cerebral ischemia-reperfusion.This study provided new therapeutic methods and experimental basis for the prevention and treatment of the cerebral ischemic diseases associated with folate deficiency.Methods Part ⅠThirty male Sprague-Dawley rats weighing 120-150 g were randomly divided into sham operation group(SHAM),middle cerebral artery ischemia-reperfusion group(MCAO),and folic acid deficiency+middle cerebral artery ischemia-reperfusion group(FD+MCAO),and there were 10 rats per group.The focal cerebral ischemia-reperfusion model was performed after normal/folic acid deficiency feed for 28 d.Paraffin sections were made from brain tissue after modeling.Hematoxylin-eosin staining(HE,n=4)and Fluoro-jade B(FJ-B,n=4)method were used to detect the morphological changes of neural cells in the hippocampus of rat brain.The expression levels of Iba-1,TNF-α,IL-1β,IL-6 and Notch1 in hippocampal subregion was detected by immunofluorescence assay(n=4).Western blot was used to detect the expression of Iba-1,Notch1 and NF-κB p65 in hippocampal(n=4).The BV-2 cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM)/folic acid deficient DMEM for 7 d in vitro.The OGD/R model was established after intervention with DAPT.After the BV-2 cells was homogenized in RIPA buffer and extracted,the protein expression of TNF-α,IL-1β,IL-6,Notch1 and NF-κB p65 were detected by western blot(n=4).Part Ⅱ90 male Sprague-Dawley rats weighing 120-150 g were randomly divided intosham operation group(SHAM),middle cerebral artery ischemia-reperfusion 3 h group(MCAO 3 h),MCAO 6 h group,MCAO 12 h group,MCAO 18 h group,MCAO 24 h group,MCAO 72 h group,folic acid deficiency+MCAO 12 h group(FD+MCAO 12 h)and FD+MCAO 24 h group,and there were 10 rats per group.MCAO model was performed after normal/folic acid deficiency feed for 28 d.Paraffin sections were made from brain tissue after modeling.The expression of TNF-α,IL-1β,IL-6,GFAP,pSTAT3 and caspase3 in brain tissue was detected by immunofluorescence(n=4).Western blot was used to detect the protein expression of GFAP,caspase3 and pSTAT3 in cortex(n=4).The primary astrocytes were cultured in Dulbecco’s modified Eagle’s medium(DMEM)/folic acid deficient DMEM for 7 d in vitro,LMT-28,Filgotinib,C188-9,AG490 are inhibitors of IL-6,JAK-1,STAT3 and JAK-2 respectively,and were used to inhibit the expression of corresponding proteins.The cells were performed to establish OGD/R model after intervention.MTS was used to detect cell viability,and the protein expression of TNF-α,IL-1β,IL-6,GFAP,caspase3,pSTAT3 and pJAK-1was detected by Western blot(n=4).Results Part ⅠCompared with the MCAO group,the results of HE staining and FJ-B staining in MCAO+FD group showed that the neurons were severely damaged in hippocampal DG,CA1 and CA3 regions,and the degree of nerve damage in the CA1 region was significantly higher than that in the CA3 and DG regions.Western blot analysis showed that the expression of Iba-1,Notch1 and NF-κB p65 in hippocampus of cerebral ischemia-reperfusion was significantly increased,and the results of immunofluorescence showed that microglia cells were significantly activated and the expression of inflammatory factors and Notch1 was significantly augmented in the hippocampal CA1,CA3 and DG regions.The inflammatory factors expression of microglia in CA1 region was significantly higher than that in the CA3 and DG regions.The Western blot analysis showed that compared with the OGD/R group,the folic acid deficiency resulted in the significantly increased expression of inflammatory factors,Notch1 and NF-κB p65 protein in BV-2 cell after OGD/R.The expression of Notch1,NF-κB p65 and inflammatory factor protein was significantly inhibited after DAPT intervention.The difference between the above two groups was statistically significant(P<0.05).Part ⅡCompared with the SHAM group,the expression of IL-6 in astrocytes of MCAO12 h group was significantly increased(P<0.05),while the expression of TNF-α and IL-1β was not significantly changed(P>0.05),the astrocytes in MCAO 12 h group and MCAO 24 h group were greatly activated.Compared with the MCAO group,the expression of GFAP did not significantly change after folic acid deficiency intervention,while the expression of IL-6,pSTAT3 and caspase3 increased significantly(P<0.05).The results of MTS showed that the cell viability began to decrease after OGD/R.After 3 h of reoxygenation,the cell viability decreased to the lowest level and returned to normal level after 24 h of reoxygenation.The expression of IL-6 and pSTAT3 protein in primary astrocytes reached the peak after 3 h of OGD/R.However,the activation time of IL-6 was earlier than pSTAT3.Compared with the OGD/R group,the protein expression of IL-6,pSTAT3 and caspase3 was further increased after folic acid deficiency intervention(P<0.05).After the cells were treated with AG490,Filgotinib,LMT-28 and C188-9 in combination with folic acid deficiency,the results of Western blot showed that compared with the FD+OGD/R group,the proteins of IL-6 and pSTAT3 in FD+OGD/R+Filgotinib group were significantly suppressed,while the protein of IL-6 and pSTAT3 in FD+OGD/R+AG490 group did not change significantly(P>0.05).The protein of IL-6 and pSTAT3 in FD+OGD/R+LMT-28 group was significantly inhibited.Meanwhile,the proteins of IL-6 and pSTAT3 were also significantly reduced in the FD+OGD/R+C188-9 group.ConclusionsThe model of cerebral ischemia-reperfusion in rats was successfully established.Folic acid deficiency aggravated the pathological damage of neurons in the hippocampus after cerebral infarction.In particular,the CA1 region was more severely affected.Meanwhile,the microglia were activated and the inflammatory responses related to activated microglia and the portein expression of Notch1 and NF-κB p65 in the hippocampus was increased.The expression of inflammatory factors,Notch1 and NF-κB p65 in the BV-2 cell model was also significantly up-regulated.Blocking of Notch1 with DAPT partly attenuated the protein expression of neuroinflammatory cytokines and NF-κB p65 in FD-treated hypoxic BV-2microglia.The results suggested that the microglia immune response mediated by the Notch1/NF-κB p65 pathway might be a molecular mechanism that could aggravate the damage of nervous system after cerebral infarction.Astrocytes in the cortex of rats with cerebral ischemia-reperfusion were significantly activated,which induced a significant increase of the IL-6 and pSTAT3 portein expression.After the folic acid deficiency intervation,IL-6 and pSTAT3 protein expression in astrocytes was further raised,and caspase3 protein expression was increased.In vitro,folic acid deficiency interfered with the production of IL-6 in primary astrocytes,which promoted the the phosphorylation of STAT3.In turn,the expression of pSTAT3 further aggravated the IL-6 production.There may be an interaction between IL-6 expression and pSTAT3 in the astrocytes. |
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