| Objective: To explore the behavior ability,dentate gyrus(DG)structure and hippocampal bone morphogenetic protein 4(BMP4),bone morphogenetic protein 6(BMP6)of Noggin protein injected into the lateral ventricle of mice after cerebral ischemia reperfusion injury(CIRI),and the influence of inflammatory response,oxidative stress,apoptosis and other pathogenesis after CIRI,in order to provide new strategies for the prevention and treatment of clinical ischemic cerebrovascular diseases.Methods:(1)324 Kunming male mice were randomly divided into: sham operation group(sham,n=108),ischemia-reperfusion injury group(I/R,n=108),ischemia Perfusion injury + Noggin treatment group(Noggin,n=108),each group is divided into 4 subgroups of 1d,3d,7d,and 14d;Noggin group and I/R group were prepared with modified thread embolization method for the right CIRI model 30 min after successful modeling,the lateral ventricle was given 2?l Noggin protein solution and 0.9% sodium chloride injection 2?l once.In the Sham group,blood vessels were separated according to the model construction method,and middle cerebral artery occlusion(MCAO)was not embolized.2 ?l of 0.9% sodium chloride injection was injected into the lateral ventricle once.(2)The mice in each group were tested with Y-shaped electric maze stimulator and balance beam experiment 1 day before the materials were taken to detect the learning and memory ability and balance ability of the mice;Then take the material to detect the content of superoxide desmutase(SOD)and maleic dialdehyde(MDA)in ischemic brain tissue content;ELISA method was used to detect the content of tumornecrosis factor-?(TNF-?)and interleukin-6(IL-6);Western blot detection and quantitative analysis of the expression changes of Noggin-related antagonist proteins BMP4 and BMP6;Finally,the 2,3,5-triphenyl tetrazolium chloride(TTC)staining method was used to detect the area of cerebral infarction;Nissl staining and immunofluorescence were used to observe the changes of DG neurons and GFAP positive cells;TUNEL staining was used to observe and count the apoptosis of DG neurons;Transmission electron microscopy to observe the fine structure changes of DG neurons.Results:(1)Successfully constructed mouse model of cerebral ischemia-reperfusion injury.(2)Nerve function score,balance beam test and Y-maze test results: Compared with the Sham group,the nerve function score,balance ability and learning and memory ability of the I/R group and the Noggin group all decreased(P<0.05);And I/R compared with the group,the Noggin group's neurological function score,balance ability and learning and memory ability were improved(P<0.01).(3)SOD and MDA test results: Compared with the Sham group,the SOD activity of the I/R group and the Noggin group decreased,and the MDA content increased(P<0.001);While the Noggin group compared with the I/R group,the SOD content increased,MDA content decreased(P<0.001).(4)IL-6 and TNF-? test results: Compared with the Sham group,the levels of IL-6 and TNF-? in the I/R group and the Noggin group increased(P<0.001);while the Noggin group was more time-sensitive than the I/R group The content of IL-6 and TNF-? decreased(P<0.001).(5)Western blot detection results: Compared with the Sham group,the BMP4 protein expression increased in the I/R group and the Noggin group,while the BMP6 protein expression decreased(P<0.001);Noggin decreased the BMP4 protein expression at each time point compared to the I/R group,BMP6 Protein expression increased(P<0.001).(6)TTC staining results: Compared with the Sham group,the ratio of cerebral infarct area in the I/R group and the Noggin group increased(P<0.001);compared with the I/R group,the ratio of the cerebral infarct area in the Noggin group decreased(P< 0.001).(7)Nissl staining results: under light microscope,the structure of DG neurons in the Sham group was not abnormal;the DG neurons in the I/R group were obviously damaged,the cells were edema,the arrangement was loose,and the Nissl body was stained deeply;Among them,the 7d subgroup The damage of DG neurons was the most serious.The gaps of cells widened and the nucleus contraction was obvious.In the Noggin group,the damage of DG neurons was significantly improved,the staining of Nissl body became lighter,and the number of cells increased(P<0.01).(8)Transmission electron microscopy observation: The ultrastructure of DG neurons was observed under the electron microscope of the Sham group without obvious abnormalities and rich in organelles;While the neurons in each subgroup of I/R were damaged to varying degrees,the nuclear membrane was incomplete,and chromatin In Bianji,part of the organelles dissolved and disappeared,and the 7d subgroup was the most severely injured;after Noggin treatment,the neuronal damage was alleviated compared with the I/R group.(9)GFAP immunofluorescence test results: Compared with the Sham group,the number of GFAP positive cells in the I/R group and the Noggin group increased significantly(P<0.001);In the four subgroups of I/R,the number of GFAP positive cells gradually increased from 1d to 7d The peak reached a star shape,the protrusions increased and became thicker,and decreased at 14 days.The number of GFAP positive cells in Noggin at each time point was reduced compared with the I/R group(P<0.01).The cell body became smaller and the protrusions became thinner.(10)TUNEL test results: No obvious apoptotic cells were observed in the Sham group;The number of apoptotic cells in the I/R group and the Noggin group increased significantly,and reached a peak at 7d subgroup,compared with the Sham group(P<0.01),the number of DG apoptotic cells in each subgroup of Noggin was significantly reduced compared with the I/R group(P<0.001).Conclusion: Noggin protein improves the nerve function of cerebral ischemia-reperfusion injury in mice,improves balance ability and learning and memory ability,enhances anti-oxidation,anti-inflammatory and anti-apoptosis ability,down-regulates Noggin antagonist protein BMP4 and up-regulates BMP6 protein Express,it may become a new way to prevent and control CIRI. |
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