| Intracerebral hemorrhage(ICH)is the most common cerebrovascular disease in human beings.It is also the most difficult subtype to treat hemorrhagic cerebral apoplexy,which accounts for about 13%-15% of all types of stroke.Overall spontaneous brain hemorrhage,based on its location,can be categorized into intracerebral,sub-arachnoid,epidural and subdural.Intracranial hemorrhage(ICH)is more widely distributed in the population,so researchers pay more attention to it as the focus.Cerebral hemorrhage(ICH)is due to ruptured blood vessels in the brain,resulting in blood leakage.Uncontrolled hypertension is a major modifiable risk factor and the leading cause of ICH with age increasing the incidence progressively.Studies have shown that neurological impairment after intracerebral hemorrhage is usually caused by nerve inflammatory reaction,neuronal apoptosis and cytotoxic reaction.About 80% of the rehabilitative patients will not be considered ‘‘independent’’ by 6 months after ICH.These patients will suffer from neurological deficits for life.In addition to motor dysfunction after cerebral hemorrhage,cognitive impairment is also the most intractable.The most common location of cerebral hemorrhage is basal ganglia(striatum).It is reported that lesions in this part can cause significant learning and memory deficits.Osteopontin(OPN)is a glycosylated phosphoprotein,which exists in all body fluid and mineralized tissue protein matrix.It itself contains arginine glycine aspartate(Rgd)domain,which combines various integrin receptors and CD 44 variants.Therefore,it can be used as cell attachment protein as well as cytokine.It can transmit signals to cells through multiple receptors including integrins and CD44.It has been found that the level of OPN expression in the central nervous system can be significantly decreased after focal cerebral ischemia,subarachnoid hemorrhage,spinal cord injury,and mechanical brain injury.In the in vitro experiment of cerebral ischemia injury,OPN shows a good protective effect on the cultured neurons.In the experiment of cerebral ischemia,OPN can reduce the area of cerebral infarction in the ischemic stroke brain mice by reducing the apoptosis of neural cells.In mechanical brain damage,OPN can regulate the expression of β1 integrin protein and CD 44 in neurons and neuroglia cells,and ultimately repair synapses in the hippocampus,and have a positive effect on synaptic reorganization and functional recovery.In conclusion,OPN as a neuroprotective agent has a very good application prospect and plays an important role in the nervous system.On the basis of a large number of literature and preliminary experimental results,we speculate that intracerebral injection of r-OPN can effectively improve the recovery of neural dysfunction in ICH rats,reduce the death of nerve cells after injury,positively affect the synaptic plasticity,and improve the cognitive impairment of rats after injury.The mechanism may be related to the PI3K-Akt-GS3Kβ pathway.In order to prove our hypothesis,this topic will be divided into three parts of the experimental form to elaborate.Part one Neuroprotection of recombinant osteopontin in rats with intracerebral hemorrhageObjective: It is proved that recombinant osteopontin alleviated neurological dysfunction after intracerebral hemorrhage in rats and exerts neuroprotective effect.Methods: 90 male adult SD rats were randomly divided into three groups: 1)sham operation group(Sham group);2)cerebral hemorrhage group(ICH group);3)r-OPN treatment group of cerebral hemorrhage(ICH+r-OPN).The model was made by injecting 100 ml of autologous blood into the rat basal ganglia.Ten minutes after the successful modeling,drug r-OPN was injected into the lateral ventricle of ICH rats through a stereotaxic apparatus.After that,we observed the difference between 3 different groups through different neurological assessment methods,and finally determined the final therapeutic effect.The test indexes were as follows: 1)HE staining was used to observe the pathological changes of brain tissue and the survival of nerve cells after 24 h injury after ICH;2)the water content of the brain tissue was measured for 1-3 days after ICH,the degree of edema in the cerebral tissue of the injured hemisphere;3)the mNSS score was evaluated for 1-5 days after ICH;and 4)the rotating rod test was used to estimate the recovery of neural movement after injury in rats.5)Morris water maze test was used to determine the learning cognitive ability of rats after injury;6)the space exploration experiment in the Morris water maze was used to evaluate the early memory ability of the rats after ICH injury.Image Lab5.1 and Image J analysis systems were used to determine the experimental results.The experimental results were written in the form of mean±standard deviation and analyzed with statistical software SPSS 17.0.The difference was statistically significant when P<0.05.Results:1: The preparation of rat ICH model: The model rats were observed after ICH.When observing the injection point and coronal surface of the rat brain hematoma,it was found that an obvious hematoma was adjacent to the caudate nucleus of the basal ganglia,and the local brain tissue was damaged in the surrounding area of the hematoma.HE staining was used to observe the morphological changes of brain tissue in 24 hours after injury.In Sham group,the number of neurons in the brain tissue of rats was rich and well-balanced,with normal interstitial space,homogeneous cytoplasm,and clearly visible nuclei.There is neither infiltrating inflammatory cells nor expanded blood vessels.In the ICH model group,a large number of neuronal nuclear shrinkage necrosis and cytoplasmic eosinophilia were found.It can be observed that there is obvious edema in the tissue,uneven distribution of nerve cells,widening of the gap,and vasodilatation,accompanied by a large number of inflammatory cell infiltration.After r-OPN treatment,the number of neurons in brain tissue was significantly reduced,and the edema of tissues was significantly reduced.2: Water content of brain tissue: The water content of brain tissue in group Sham did not change within 1-3 days.The water content of the brain tissue in group ICH increased significantly at the 1-3 day after injury and reached the peak in second days after injury(P < 0.05).The water content in brain tissue after r-OPN treatment was significantly lower than that in group ICH(P < 0.05).3: Neural function assessment after ICH: The mNSS score in group ICH increased significantly on the 1-5 day after injury compared with that in Sham group,but decreased slightly with time after injury,but the decrease was not obvious(P < 0.05).Compared with the ICH group,the mNSS scores of rats treated with r-OPN were decreased,and the differences were significant(P < 0.05).The rotarod test found that the rats in the ICH group had significantly shorter dwell time on the rotarod than those in the Sham group on days 1-5 after injury(P < 0.05).Compared with the ICH group,the rats in the r-OPN group had a significantly longer dwell time on the rotarod,which was statistically significant(P < 0.05).4: Evaluation of cognitive and memory function after ICH: In the navigation experiment of Morris water maze,the rats in ICH group were significantly longer than the rats in Sham group at 1,3,7 days after injury,and the difference was significant(P < 0.05).The time of escape latency in r-OPN group was significantly shorter than that in ICH group,with statistical significance(P < 0.05).In the space exploration experiment of the Morris water maze,the number of rats crossing the platform in the ICH group was significantly less than that in the Sham group within 2 minutes(P < 0.05).In the r-OPN rats,the number of crossing platforms was significantly higher than that of ICH,which was statistically significant.Summary: Rat ICH model was successfully established using autologous blood injection.Brain edema,neuromotor deficits,and cognitive and memory impairments occur after ICH in rats.After r-OPN treatment,it can play a neuroprotective effect.It can reduce brain tissue damage around the hematoma,improve brain edema and neurological deficits and cognitive memory impairment in ICH rats.Part two Recombinant osteopontin reduces neuronal dysfunction and apoptosis after intracerebral hemorrhage by acting on the PI3K-Akt-GSK-3β signaling pathwayObjective: To investigate whether r-OPN can reduce neural dysfunction and apoptosis of brain tissue through PI3K-Akt-GSK-3β pathway after ICH in rats,by using the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin.Methods: 120 male SD rats were randomly divided into four groups: 1)the sham operation group(Sham group);2)the cerebral hemorrhage group(ICH group);3)the cerebral hemorrhage +r-OPN treatment group(r-OPN group);4)the cerebral hemorrhage +r-OPN + Wortmannin intervention group(Wort group).The modeling process is the same as described in the first section.The following indexes were detected: 1)mNSS score and rod test were used to observe the neurological function of rats;2)terminal deoxynucleotidyl transferase mediated(DUTP)nick end labeling(TUNEL)method to detect cell apoptosis;3)immunofluorescence was used to observe the pathway factor p-Akt and NeuN in the damaged brain,GSK-3β and NeuN confocal results;4)the expression of related proteins Akt,p-Akt,GSK-3β,p-GSK-3β,apoptosis related protein Bcl-2,Bax,cleaved caspase-3(CC3),caspase-3 were detected by Western-blot after ICH.The measurement and statistical analysis of the experimental results are the same as those in the first part.Results:1: The results of neural function assessment: After ICH,the escape latency time of rats was significantly prolonged,and the residence time of rods was significantly shorter than that of Sham group.The difference was significant(P < 0.05).When treated with r-OPN,r-OPN could significantly reduce the neuromotor function damage caused by ICH,shorten the escape latency and prolong the stay time obviously(P < 0.05).When Wortmannin,a PI3K-Akt-GSK-3β pathway inhibitor,was administered,it was observed that the escape latency was prolonged compared with r-OPN rats.The time spent for rotating the rods was shortened in ICH group and the difference was significant(P < 0.05).Wortmannin reverses the ability of r-OPN to improve neurological dysfunction;2: TUNEL/DAPI fluorescence showed positive apoptosis cells around the hematoma.TUNEL positive cells were labeled with green fluorescence and blue fluorescence labeled DAPI nuclei.In Sham group,the expression of green fluorescence was less,suggesting that there were fewer apoptotic cells.In ICH group,a large number of green fluorescence positive cells were found in the field of vision,which was significantly increased compared with Sham group(P < 0.05).The green fluorescence in r-OPN group was significantly reduced,and the apoptotic cells in ICH group were less obvious.When the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was given,the green fluorescence increased significantly,and the apoptotic positive cells increased significantly(P < 0.05).3: Immunofluorescence copolymerization of p-Akt and neuron marker NeuN: NeuN was labeled with green fluorescence,p-Akt was labeled with red fluorescence,and DAPI was labeled by blue fluorescence.Copolymerization was observed.Sham Group showed more green fluorescence expressed with a certain amount of red fluorescence expression.The expression of orange overlapping portion copolymerizated by red and green fluorescence was less,suggesting that ICH can reduce the expression of p-Akt.Red fluorescence increased significantly in group r-OPN,and a large number of orange overlaps appeared after copolymerization,suggesting that r-OPN could activate p-Akt.When the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was given,it was found that the red fluorescence decreased significantly.Meanwhile,the orange fluorescence of the three colour copolymers decreased significantly,suggesting that the expression of p-Akt was inhibited.4: Immunofluorescence copolymerization of p-GSK-3β and neuronal marker NeuN: Green fluorescence labeled NeuN,red fluorescence labeled p-GSK-3β and blue fluorescence labeled DAPI nucleus.Copolymerization were observed.More green fluorescence expression was observed in the Sham group and very little red fluorescence expression was observed.A small amount of orange overlap was observed after copolymerization.In ICH group,red fluorescence and oranges fluorescence copolymerizated by red and green fluorescence increased significantly,suggesting that ICH increased the expression of p-GSK-3β.In group r-OPN,red fluorescence decreased significantly,and only a small amount of orange yellow overlapped after copolymerization,suggesting that r-OPN could inhibit p-GSK-3β activation.When the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was given,red fluorescence was significantly enhanced.Meanwhile,the orange fluorescence of the three colour copolymers increased significantly.It is suggested that p-GSK-3β can be reactivated.5: The results of Western blot analysis of p-Akt: There was a certain amount of p-Akt protein expression in sham group,and the ratio of p-Akt /Akt was lower than that in Sham group after ICH,and the difference was significant(P < 0.05).After treatment with r-OPN,it was found that the ratio of p-Akt /Akt was significantly higher than that in group ICH(P < 0.05).However,when the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was given,it was found that the ratio of p-Akt /Akt protein was significantly lower than that in the r-OPN group(P < 0.05).6: The results of Western blot analysis of p-GSK-3β: The expression of p-GSK-3β protein in sham group was less,and the ratio of p-GSK-3β/ GSK-3β was higher than that of Sham after ICH(P < 0.05).After treatment with r-OPN,the ratio of p-GSK-3β/GSK-3β was found to be significantly lower than that of ICH group(P < 0.05).However,when Wortmannin,a PI3K-Akt-GSK-3β pathway inhibitor,was administered,it was found that the ratio of p-GSK-3β/GSK-3β protein was significantly higher than that of r-OPN treatment group(P < 0.05).7: Western blot analysis of Bax/Bcl-2: The ratio of Bax/Bcl-2 in ICH group was significantly higher than that in Sham group(P < 0.05).When treated with r-OPN,the ratio of Bax/Bcl-2 was found to be significantly lower,which was statistically significant relative to the ICH group.However,when Wortmannin,a PI3K-Akt-GSK-3β pathway inhibitor,was administered,compared with r-OPN group,the Bax/Bcl-2 ratio increased,and the difference was significant(P < 0.05).8: Western blot analysis of CC3(cleaved caspase-3)and caspase-3: The CC3 protein expression in the Sham group was very low.After ICH,it was observed that the expression of CC3 protein increased significantly compared with Sham group(P < 0.05).Treatment with r-OPN showed a significant decrease in the expression of CC3 protein compared with the ICH group(P < 0.05).However,when the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was administered,compared with the r-OPN group,the expression of CC3 protein increased,and the difference was significant(P < 0.05).There was no significant difference in caspase-3 protein expression between the groups(P < 0.05).Summary: The cells in the damaged brain tissue surrounding the hematoma region after ICH undergo apoptosis and the PI3K-Akt-GSK-3β pathway is inhibited.When treated with r-OPN and the inhibitor Wortmannin,it was able to confirm that r-OPN plays a role in improving neurological dysfunction by inhibiting p-GSK-3β by activating p-Akt.It ultimately reduces the expression of important apoptosis-related proteins and reduces neuronal apoptosis.Part three Recombinant osteopontin improves synaptic plasticity by acting on PI3K-Akt-GSK-3β signaling pathway to protect cognitive memory impairment after cerebral hemorrhageObjective: After r-OPN treatment,corresponding changes in synaptic plasticity-related proteins in the hippocampus were observed.Wortmannin,a PI3K-Akt-GSK-3β pathway inhibitor,was administered to determine whether r-OPN could improve synaptic plasticity through this pathway after ICH,ultimately improving the cognitive and memory abilities of ICH rats.Methods: Ninety male SD rats were randomly divided into four groups: 1)Sham group;2)Intracerebral hemorrhage group(ICH group);3)Cerebral hemorrhage + r-OPN group(r-OPN group);4)Cerebral hemorrhage + r-OPN + Wortmannin intervention group(Wort group).The following indicators were tested postoperatively: 1)The cognitive learning ability of rats after injury was judged by positioning navigation experiments in the Morris water maze,and spatial exploration experiments in the Morris water maze were used to evaluate the early memory capacity of rats after ICH injury.2)Western-blot was used to detect Akt,p-Akt,GSK-3β,p-GSK-3β,synaptoplasty-related proteins such as synaptophysin(Syn),PSD-95 and microtubule-associated protein 2(MAP-2)in damaged brain tissue at 1,3,and 7 days after ICH.3)Immunofluorescence was used to detect the expression of synaptic plasticity related factors Syn and PSD-95 in hippocampal CA1 region on the third day after injury.The method of determination,recording and statistical analysis of the experimental results are the same as those in the first part.Results:1: The results of the Morris water maze: After 1-3 days of ICH,the escape latency of rats was significantly prolonged,and the number of times of crossing platforms decreased significantly compared with Sham group compared to the Sham group(P < 0.05).After r-OPN treatment,the escape latency time was significantly reduced,and the number of times of crossing platform increased significantly,with significant difference(P < 0.05).When Wortmannin,a PI3K-Akt-GSK-3β pathway inhibitor,was administered,the escape latency was prolonged and the number of times of crossing platforms was reduced compared with rats in r-OPN,showing significant differences(P < 0.05).Wortmannin weakens the ability of r-OPN to improve cognitive memory in neurological ICH rats.2: Immunoblot analysis of p-Akt and Akt in the hippocampus: The expression of p-Akt and Akt in different groups was observed on 1,3,and 7 days after injury.The sham group had a certain amount of p-Akt protein expression,and the ratio of p-Akt /Akt was lower than that of Sham group after ICH(P < 0.05).After treatment with r-OPN,it was found that the ratio of p-Akt/Akt was significantly higher than that of the ICH group,and it was significantly higher on the third day(P < 0.05).However,when the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was administered,it was found that the ratio of p-Akt/Akt protein was significantly lower than that of r-OPN group(P < 0.05).3: Western blot analysis of p-GSK-3β,GSK-3β in the hippocampus: The expression of p-GSK-3β and GSK-3β in different groups was observed on the 1st,3rd,and 7th days after injury.The expression of p-GSK-3β protein was lower in sham group,and the ratio of p-GSK-3β/GSK-3β was higher after ICH.The difference was significant(P < 0.05).After treatment with r-OPN,it was found that the ratio of p-GSK-3β/GSK-3β was decreased compared with the ICH group,with the lowest ratio on the third day(P<0.05).However,when the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was administered,the ratio of p-GSK-3β/GSK-3β protein was found to be significantly higher than that of r-OPN treatment group(P < 0.05).4: Western blot analysis of MAP-2 protein in hippocampus: The expression of MAP-2 in different groups was observed on 1,3,and 7 days after injury.The MAP-2 protein was expressed to a certain extent in the Sham group.After ICH,the expression of MAP-2 protein was significantly reduced,which was significantly different from that of Sham group(P < 0.05).After treatment with r-OPN,the expression of MAP-2 protein was significantly increased and the highest expression was observed on the third day,which was statistically significant compared with ICH group(P < 0.05).However,when the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was administered,compared with the r-OPN group,the expression of MAP-2 protein was decreased,and the difference was significant(P < 0.05).5: Immunohistochemical analysis of Syn protein in hippocampus: The expression of Syn was observed in different groups on 1,3,and 7 days after injury.The Sham group has a certain level of expression of Syn protein.After ICH,it was observed that the expression of Syn protein was significantly reduced,which was significantly different from that of Sham group(P < 0.05).The expression of Syn was significantly increased after r-OPN treatment,and the highest expression was observed on the third day,which was statistically significant compared with the ICH group(P < 0.05).However,when the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was administered,the expression level of Syn was decreased compared with r-OPN group(P < 0.05).6: Western blot analysis of PSD-95 protein in hippocampus: The expression of PSD-95 was observed in different groups on 1,3,and 7 days after injury.Sham group PSD-95 protein has a certain degree of expression.After ICH,it was observed that the expression of PSD-95 protein was significantly reduced compared with the Sham group(P < 0.05).Treatment with r-OPN showed a significant increase in Syn expression,with the highest expression on the third day.Compared with the ICH group,the expression level was statistically significant in the r-OPN group(P < 0.05).However,when the PI3K-Akt-GSK-3β pathway inhibitor Wortmannin was administered,the expression of PSD-95 protein was decreased compared with r-OPN group(P < 0.05).7: Syn/DAPI fluorescence in hippocampus: The localization of synaptic plasticity in hippocampal CA1 region was detected on the 3rd day after ICH injury.Red fluorescence represents synaptic plasticity-related factor Syn and blue fluorescence-labeled DAPI cell nuclei.The Sham group had a certain amount of red fluorescence expression.However,it was observed that red fluorescence was significantly reduced after injury in the ICH group,demonstrating that Syn was significantly reduced in the hippocampal CA1 area.The red fluorescence in the r-OPN group was significantly increased,suggesting that the expression of Syn was significantly increased after r-OPN treatment.When Wortmannin,a PI3K-Akt-GSK-3β pathway inhibitor,was administered,the expression level of red fluorescence was lower and the expression of PSD-95 was decreased.When the pathway was inhibited,r-OPN could not improve synaptic plasticity.8: The PSD-95/DAPI fluorescence in the hippocampus: Localization and expression of synaptic plasticity related protein in hippocampal CA1 region were observed on the 3rd day after ICH injury.Red fluorescence represents synaptic plasticity-related factor PSD-95,blue fluorescence-labeled DAPI cell nuclei.The Sham group had a certain amount of red fluorescence expression.However,it was observed that red fluorescence was significantly reduced after injury in the ICH group,demonstrating that PSD-95 is significantly reduced in the hippocampal CA1 area.The red fluorescence in the r-OPN group was significantly increased,suggesting that r-OPN treatment can significantly increase the expression of PSD-95.When Wortmannin,a PI3K-Akt-GSK-3β pathway inhibitor,was administered,the red fluorescence level was lower and PSD-95 expression was decreased.It was suggested that r-OPN could not improve synaptic plasticity when the pathway was inhibited.Summary:r-OPN can protect cognitive memory dysfunction post ICH.It may be related to improve synaptic plasticity by activation of PI3K-Akt-GSK-3β pathwayConclusion: r-OPN can exert neuroprotective effect and improve cognitive dysfunction after ICH.It may be related to the activation of PI3K-Akt-GSK-3β pathway to reduce neuronal apoptosis and improve synaptic plasticity. |
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