| Objective:Intracerebral hemorrhage is a common type of stroke.The secondary injuries of intracerebral hemorrhage,such as local inflammatory reaction and destruction of blood-brain barrier,are the main causes of death in the acute stage of intracerebral hemorrhage.Microglia cells are specific immune cells in the nervous system.Fe2+released by red blood cells after intracerebral hemorrhage can activate microglia cells to promote or reduce inflammation.Resveratrolis a natural polyphenol plant antitoxin.Studies have confirmed that resveratrol is beneficial to improve the prognosis of intracerebral hemorrhage.This study aims to observe the effect of Fe2+released by red cell lysis in the early stage of intracerebral hemorrhage on the function of microglia cells and the effect of resveratrol intervention,and preliminarily explore its possible molecular mechanism.Methods:（1）Animal test:27 SD rats aged 12 weeks,They were randomly divided into sham-operated group（Sham group）,Intracerebral hemorrhage group（ICH group）,and Resveratrol treatment group（Res group）,and each group was randomly divided into 3 subgroups at 6h,24h and 72h,with 3 rats in each subgroup.The nervous system function was evaluated by m NSS score in each group.The morphological changes of brain cells were observed by HE staining.The damage of neurons was studied by Nith staining and Tunel staining.The protein expressions of TRL4,CD36,HO-1 and Nrf2 were observed by immunofluorescence staining.（2）Cell experiment:BV2 microglia were divided into control group（normal culture）,Fe2+intervention group（adding Fe SO4solution to the concentration of 10μmol/L）,Fe2++resveratrol group（adding Fe SO4solution to the concentration of 10μmol/L）,and Fe2++resveratrol group（adding Fe SO4solution to the concentration of 10μmol/L）.In addition,resveratrol was added to the concentration of 25μmol/L and Fe2++resveratrol（50μM）treatment group（Fe SO4solution was added to the concentration of 10μmol/L and resveratrol was added to the concentration of50μmol/L）.When cultured to 6h,24h and 72h,each group of cells was photographed by light microscope.The total number of cells was counted,and the expression levels of TLR4,CD36,Nrf2,p-Nrf2 and HO-1 were detected by Wesern Blot at 24h and 72h,respectively.The Nrf2 protein was localization observed by cellular immunofluorescence.Results:（1）Animal tests:mNSS score showed that no significant neurological dysfunction was observed in rats of Sham group at all time points.Compared with the Sham group,the ICH group showed significant neurological dysfunction,and the neurological dysfunction became more obvious with the passage of time,and resveratrol could reduce the degree of ICH neurological dysfunction.HE staining of the brain tissue of rats in the Sham group indicated that the structure of the brain tissue of rats in the Sham group was complete at three time points,no erythrocyte infiltration was observed,and inflammatory cell infiltration was not obvious.The infiltration of red blood cells and inflammatory cells in ICH group was significantly increased compared with that in Sham group,and resveratrol could reduce the infiltration of red blood cells and inflammatory cells in ICH group.The staining of the brain tissue of rats in the Sham group indicated that polygonal neurons,nuclei in the center,and cytoplasm were abundant in blue patch-nishi bodies were observed at all three time points.The dissolution of Nishi bodies in ICH group was higher than that in Sham group,and with the passage of time,the dissolution was increased.Resveratrol could reduce the dissolution of Nishi bodies in ICH nerve cells.Tunel staining showed that no obvious apoptosis was observed in the brain tissues of the Sham group at three time points.Compared with Sham group,the apoptosis index of nerve cells in ICH group was significantly increased at all time points（P<0.001）,and the apoptosis index decreased with time within 72h.Resveratrol could improve the apoptosis of nerve cells in brain tissue.Immunofluorescence showed that TLR4,CD36,Nrf2and HO-1 proteins were less expressed in brain tissue of rats in Sham group.The expression of Nrf2 protein in ICH group was significantly higher than that in Sham group（P<0.001）,but the expression of Nrf2 protein showed a downward trend over time.The expression of Nrf2 protein increased significantly after resveratrol injection.The expression of HO-1 protein in brain tissue of ICH group was higher than that of Sham group,with statistical significance only at 24h time point（P<0.05）.After resveratrol injection,the expression of HO-1 protein in brain tissue increased significantly.The expression of TLR4 protein in the brain tissue of ICH group was significantly higher than that of Sham group（P<0.001）,and the expression of TLR4 protein showed a gradual decline over time.After resveratrol injection,the expression of TLR4 protein in the brain tissue of ICH group was significantly decreased（P<0.001）.The expression of CD36 protein in brain tissue of ICH group was higher than that of Sham group（P<0.05）,and the expression of CD36 protein in brain tissue cells of ICH rats was increased by injecting resveratrol（P<0.001）.（2）Cell experiments:The total number of cells in control group,Fe2+intervention group,Fe2++resveratrol（25μM）and Fe2++resveratrol（50μM）groups increased with the extension of culture time,the total number of microglia cells decreased after Fe2+intervention,and increased after adding resveratrol.The proteins of TLR4,CD36,Nrf2,p-nr F2 and HO-1 were detected at 24h and 72h.The results indicated that compared with the control group,the five proteins of Fe2+intervention group were increased at the same time point（P<0.05）,and those of 72h group were higher than those of 24h group.Compared with Fe2+intervention group,TLR4 protein expression in Fe2++resveratrol treatment group was significantly decreased at the same time point（P<0.05）,while the protein expression of CD36,Nrf2,P-nr F2 and HO-1 in72h group was significantly increased（p<0.05）,and that in 72h group was higher than that in 24h group.This trend was more obvious when the resveratrol concentration was 50μmol/L.The results of cell fluorescence in each group suggested:Compared with the control group,the positive rate of Nrf2 protein in the nucleus of Fe2+intervention group was significantly increased（P<0.05）.After the addition of resveratrol intervention,the positive rate of Nrf2 protein in the nucleus of Fe2+intervention group was significantly increased（P<0.05）,and the positive rate in 72h group was higher than that in24h group,and the high concentration of resveratrol group was more significantly increased than that in low concentration group.Conclusion:1.Resveratrol can improve the neurological function of rats after intracerebral hemorrhage;2.Fe2+can activate microglia after cerebral hemorrhage in rats,and the activated microglia mainly show pro-inflammatory function within 72h.3.Resveratrol can transform the function of activated microglia cells from pro-inflammatory to anti-inflammatory in a short period of time,and this transformation may be achieved by regulating Nrf2/HO-1signaling pathway. |
