| Objectives:To establish the model of cerebral ischemia reperfusion injury in mice by Middle Cerebral Artery Occlusion(MCAO); To investigate the variance of TLR4,MyD88,VEGF-A,IL-10,TGF-β1 in brain, the variance of TLR4,VEGF-A,IL-10,TGF-β1,Treg cells in spleen, the variance of TLR4 in thymus after cerebral ischemia injury early, meanwhile to observe the influence of human serum albumin or zinc chloride on these inflammatory factors in cerebral ischemia reperfusion injury early. At first, to clarify changes of immune inflammatory reaction in cerebral ischemia reperfusion damage early; Secondly, from the perspective of immunization, to study of albumin molecular mechanism of brain protection; Moreover, to study the effection of zinc chloride and the influence of zinc chloride on TLR4 signaling pathway in cerebral ischemia reperfusion injury.Methods:(一) Treatment of albumin on cerebral ischemia reperfusion injury.1. Neurological deficit assessment was performed by 5-point scoring system,at 24h after cerebral ischemia reperfusion injury.2. The serum albumin level was measured by bromocresol green assay, at 6h,24h after cerebral ischemia reperfusion injury.3. PCR were used to analyse the level of TLR4 mRNA,MyD88 mRNA,VEGF-A mRNA,IL-10 mRNA,TGF-β1 mRNA in brain, at 6h,24h after cerebral ischemia reperfusion injury.4. PCR were used to analyse the level of TLR4 mRNA,VEGF-A mRNA,IL-10 mRNA,TGF-β1 mRNA in spleen, at 6h,4h after cerebral ischemia reperfusion injury.5. PCR were used to analyse the level of TLR4 mRNA in thymus, at 6h,24h after cerebral ischemia reperfusion injury.6. The expression of TLR4 protein in brain,spleen and thymus was detected by immunohistochemistry, at 24h after cerebral ischemia reperfusion injury.7. The level of four groups of T cells in spleen cells was detected by flow cytometry, at 24h after cerebral ischemia reperfusion injury.(二) Treatment of zinc chloride on cerebral ischemia reperfusion injury.1. Neurological deficit assessment was performed by 5-point scoring system,at 6h after cerebral ischemia reperfusion injury.2. PCR were used to analyse the level of TLR4 mRNA,MyD88 mRNA in brain, at 6h,24h after cerebral ischemia reperfusion injury.3. The expression of TLR4 protein in brain,spleen and thymus was detected by immunohistochemistry, at 24h after cerebral ischemia reperfusion injury.4. The level of four groups of T cells in spleen cells was detected by flow cytometry, at 24h after cerebral ischemia reperfusion injury.Results:(一) Treatment of albumin on cerebral ischemia reperfusion injury.1. The albumin group significantly improved neurological deficit, at 24h after cerebral ischemia reperfusion injury(p<0.01).2. Compared with the sham group, the saline group decreased serum albumin level (P<0.01), the serum albumin level in albumin group was higher(P<0.01), compared with the saline group, serum albumin level in the albumin group was significantly higher(p<0.01), at 6h,24h after cerebral ischemia reperfusion injury.3. Compared with the sham group, the expression levels of TLR4 mRNA, MyD88 mRNA, VEGF-A mRNA, IL-10 mRNA, TGF-β1 mRNA in brain of the saline and albumin group were increased(p<0.01), compared with the saline group, the expression levels of TLR4 mRNA, MyD88 mRNA, VEGF-A mRNA in brain were decreased(p<0.01),while the expression levels of IL-10 mRNA,TGF-β1 mRNA in brain were increased(p<0.01) of the albumin group, at 6h,24h after cerebral ischemia reperfusion injury.4. Compared with the sham group, the expression levels of TLR4 mRNA,VEGF-A mRNA,IL-10 mRNA,TGF-β1 mRNA in spleen of the saline and albumin group were increased(p<0.01), compared with the saline group, the expression levels of TLR4 mRNA,VEGF-A mRNA in spleen were decreased(p<0.01),while the expression levels of IL-10 mRNA,TGF-β1 mRNA in spleen were increased(P<0.01) of the albumin group, at 6h,24h after cerebral ischemia reperfusion injury.5. Compared with the sham group, the expression level of TLR4 mRNA in thymus tissue of the saline and albumin group was increased(p<0.01), compared with the saline group, the expression level of TLR4 mRNA in thymus was decreased(p<0.01) of the albumin groups, at 6h,24h after cerebral ischemia reperfusion injury.6. Compared with the sham group, the intensity of TLR4 protein in brain,spleen,thymus was significantly stronger of the saline group, compared with the saline group, the intensity of TLR4 protein was significantly weaker in brain,spleen,thymus of the albumin group, at 24h after cerebral ischemia reperfusion injury.7. With regard to the spleen CD4+CD25-FoxP3+T cells,between the saline group and the sham group, the difference was not statistically significant(p>0.05); compared with the sham group, the level of CD4+CD25-FoxP3+T cells in the albumin group was significantly higher(p<0.01), compared with the saline group, the level of CD4+CD25-FoxP3+T cells in the albumin group was significantly higher(p<0.01), at 24h after cerebral ischemia reperfusion injury.With regard to the spleen CD4+CD25-FoxP3-T cells, compared with the sham group, the level of CD4+CD25-FoxP3-T cells in the albumin and saline group was significantly lower (p<0.01), compared with the saline group, the level of CD4+CD25-FoxP3-T cells in the albumin group was significantly lower(p<0.01), at 24h after cerebral ischemia reperfusion injury.With regard to the spleen CD4+CD25+FoxP3+T(Treg) cells, compared with the sham group, the level of CD4+CD25+FoxP3+T cells in the albumin and saline group was significantly higher(p<0.01), compared with the saline group, the level of CD4+CD25+FoxP3+T cells in the albumin group was significantly higher(p<0.01), at 24h after cerebral ischemia reperfusion injury.With regard to the spleen CD4+CD25+FoxP3-T cells, compared with the sham group, the level of CD4+CD25+FoxP3-T cells in the albumin and saline group was significantly higher(p<0.01), compared with the saline group, the level of CD4+CD25+FoxP3-T cells in the albumin group was significantly higher(p<0.01), at 24h after cerebral ischemia reperfusion injury.(二) Treatment of zinc chloride on cerebral ischemia reperfusion injury. 1. The zinc chloride group significantly improved neurological deficit, at 24h after cerebral ischemia reperfusion injury(p<0.01).2. Compared with the sham group, the levels of TLR4 mRNA,MyD88 mRNA in brain of the saline and zinc chlorine groups were increased(p<0.05), compared with the saline group, the levels of TLR4 mRNA,MyD88 mRNA in brain of zinc chlorine group did not change significantly(p>0.05), at 6h,24h after cerebral ischemia reperfusion injury.3. Compared with the sham group, the intensity of TLR4 protein in brain,spleen,thymus was significantly stronger of the saline group and zinc chloride group, compared with the saline group, the intensity of TLR4 protein was no change in brain,spleen,thymus tissue of the zinc chloride group, at 24h after cerebral ischemia reperfusion injury.4. With regard to the spleen CD4+CD25-FoxP3+T cells, among the Sham group,the saline group,the zinc chloride group, the difference was not statistically significant(p>0.05), at 24h after cerebral ischemia reperfusion injury. With regard to the spleen CD4+CD25"FoxP3-T cells, compared with the sham group, the level of CD4+CD25-FoxP3-T cells in the zinc chloride and saline group was significantly lower(P<0.01), compared with the saline group, the level of CD4+CD25-FoxP3-T cells in the zinc chloride group was significantly higher(p <0.01), at 24h after cerebral ischemia reperfusion injury. With regard to the spleen CD4+CD25+FoxP3+T cells, compared with the sham group, the level of CD4+CD25+FoxP3+T cells in the zinc chloride and saline group was significantly higher(p<0.01), beween the zinc chloride and saline group, there was no significantly difference(p>0.05). With regard to the spleen CD4+CD25+FoxP3-T cells, compared with the sham group, the level of CD4+CD25+FoxP3-T cells in the zinc chloride and saline group was significantly higher(p<0.01), beween zinc chloride and saline group,there was no significantly difference(p>0.05).Conclusions: At 6h,24h after cerebral ischemia reperfusion, the TLR4 signal was activated in brain,spleen,thymus, the levels of inflammatory cytokine VEGF-A,anti-inflammatory cytokines IL-10,TGF-β1 in brain,spleen and regulatory T cells in spleen cells were increased; Albumin treatment could inhibit TLR4 signal of brain,spleen,thymus, decreased the level of VEGF-A, enhanced the level of IL-10,TGF-β1 of brain,spleen and regulatory T cells of spleen cells, it improved neurological function after cerebral ischemia in mice, these revealed that albumin of brain protection might be through interference immune inflammatory reaction; Zinc chloride treatment had no effect on the expression of TLR4 in brain,spleen,thymus, but it improved neurological deficit in mice of cerebral ischemia. |
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