| BackgroundIntracerebral hemorrhage(ICH)is a serious hazard to human health,with an annual incidence of 10-30/100,000 and approximately 10% to 30% of all types of stroke Bleeding in the brain develops in basal ganglia area.The formation of hematoma and adjacent tissue damage,leading to severe brain injury,involves a series of molecular processes,including inflammation,apoptosis and cerebral blood brain barrier damage.Apoptosis is the main form of cell death around hematoma area.Existing studies show that apoptosis is closely related to the neurological damage.It is well known that early rehabilitation is effective for functional recovery after ICH.However,these mechanisms have not been well described.Further study of the mechanisms by which rehabilitation training improves neurological function after ICH may provide new ideas for the treatment of such diseases.Serine-threonine kinase(Akt)plays an important role in ICH rehabilitation.Akt plays an important role in a variety of physiological and pathogenesis processes,including inflammation and apoptosis.Activated Akt regulates several downstream targets of survival and apoptotic pathways to inhibit apoptosis.The most important of which is glycogen synthase kinase 3β(GSK3β),activated GSK3β promotes cell damage and increases cleaved caspase 3(CC3)activity,which is an early step in the execution phase of apoptosis.Methylene blue(MB)has neuroprotective effects on subarachnoid hemorrhage(SAH)by activating the Akt/GSK3β signaling pathway.Swimming reduced individuals’ vulnerability to stress and contributes to the Akt/GSK-3β/CRMP-2 pathway and microtubule dynamics mediated protection against neuroplasticity.Our results demonstrated that the expression levels of p-Akt/Akt and p-GSK3β/GSK3β proteins were significantly decreased after ICH in mice,and the neurological function and motor ability were significantly decreased.Swimming training intervention promoted the expression of p-Akt/Akt and p-GSK3β/GSK3β proteins,and improved the neurological function and motor ability of ICH mice.However,the use of Akt enzyme activity inhibitor GSK690693 reversed this effect.Intervention of Akt/GSK3βsignaling pathway during rehabilitation training may help improve neurological function in patients with hemorrhagic stroke.ObjectiveTo investigate the effect of early swimming training on neurological function,motor ability and Akt signalling in ICH mice.The Akt kinase inhibitor GSK690693(Inhibitor of Akt activity)was used to inhibit Akt activity,and to explore the mechanism of swimming training in improving neurological function in mice with intracerebral hemorrhage.MethodsIn this study,98 adult SPF male C57BL/6 mice(10-12 weeks of age,25-30 g)were injected with type Ⅳ collagenase into their brains by stereotaxic instrument for intracerebral hemorrhage modeling or injection of normal saline for sham operation.Mice were randomly assigned to two separate experiments based on m NSS and MRI T2-weighted imaging.Experiment 1: Animals were randomly divided into 5 groups: Sham(n=10),ICH7d(n=10),ICH+7d ST(n=10),ICH14d(n=10),and ICH+14d ST(n=10)group.ST intervention was not performed in the sham group,the ICH7 d group or the ICH14 d group.The ICH+7d ST group and ICH+14d ST group were given 7 days and 14 days of ST intervention,respectively.Experiment 2: The Experiment 1 results showed that ST ameliorated neurological deficits in ICH mice and promoted the activation of Akt/GSK3β pathway.In Experiment 2,the Akt kinase inhibitor GSK690693 was used to further explore whether the Akt/GSK3β pathway mediates the protective effect of ST on ICH injury.Animals were randomly divided into 4groups: sham(n=12),ICH7d(n=12),ICH+inhibitor+7d ST(n=12),and ICH+7d ST(n=12).ST intervention was not performed in the sham group or the ICH7 d group.The ICH+inhibitor+7d ST group and ICH+7d ST group were given ST intervention for 7 days.Open field Test was used to evaluate spontaneous locomotor activity and anxiety-related behavior of mice after swimming training(including total distance,average velocity and central total distance/total distance of open field test).Diffusion tensor imaging(DTI)were used to measure the Fractional anisotropy(FA)and mean dispersion rate(MEAN)of white matter fibers in motor cortex of mice.Magnetic resonance spectroscopy(MRS)was used to measure the relative levels of creatine,choline,N-acetylaspartic acid,inositol,glutamate and lactate in basal ganglia of striatum.Western blot were used to detect the expression levels of p-Akt,Akt,p-GSK3β,GSK3β,Bax and Bcl-2 around the haematoma of mice in each group.Immunohistochemistry was used to detect the activation of glial cells around the haematoma of mice in each group: the expression levels of Iba1 and GFAP.Observed and evaluated the effects of early swimming training on the neurological function and motor ability of ICH mice.To investigate the role of Akt/GSK3β signaling pathway in improving neurological function and motor ability in intracerebral hemorrhage mice after swimming training.Results1.ST significantly improved the neurological function of ICH mice: compared with the Sham group,the m NSS of model group was significantly increased and the correct alternation rate of Y maze was significantly decreased(P < 0.01).Compared with the ICH7 d group,the m NSS of the ICH+7d ST group was significantly decreased(P < 0.05).However,compared with the ICH14 d group,the m NSS score in the ICH+14d ST group was decreased,but the difference was not significant(P > 0.05).Compared with the model group,the correct alternation rate of Y maze in the ICH+7d ST and ICH+14d ST groups were significantly increased(P < 0.01).Compared with the ICH7 d group,both m NSS and correct alternation rate of Y maze were improved in ICH14 d group,but the differences were not significant(P >0.05).2.ST significantly improved the exercise ability of ICH mice: compared with the Sham group,the exercise ability(total distance,average velocity of the open field test)of the model group was significantly decreased(P < 0.01).However,after 7 days or 14 days of ST,the exercise ability(total distance,average velocity of the open field test)of the ICH+7d ST group and ICH+14d ST group was significantly increased(P < 0.01).Compared with the ICH7 d group,the exercise ability(total distance,average velocity of the open field test)was increased in the ICH14 d group,but the difference was not significant(P > 0.05).There was no anxiety or depression behavior in mice,and the ratio of central distance to total distance of open field test showed no significant difference in each group(P > 0.05).suggesting that ST plays a protective role in ICH.In addition,ST did not increase the mortality in the early stages of ICH.3.ST affected the relative metabolism levels of Cho,NAA,MI,Glu and Lac in ICH mouse brain: After ICH,the relative metabolism levels of Cho/Cr and NAA/Cr decreased significantly(P < 0.05 or P < 0.01),While ST significantly increased the relative metabolism levels of Cho/Cr and NAA/Cr(P < 0.01).The relative metabolism levels of MI/Cr,Glu/Cr and Lac/Cr increased in the model group(P < 0.05 or P < 0.01),and ST significantly reversed this trend(P < 0.05 or P < 0.01).It is suggested that ST can improve brain metabolism and reduce brain injury,which is consistent with behavioral test results.4.ST intervention reduced the damage of white matter fiber in ICH mice: Compared with the Sham group,the FA(Ipsilateral/contralateral)values of the ICH7 d group was significantly decreased(P < 0.01).The FA values of the ICH+7d ST group was significantly increased compared with the ICH7 d group.ST intervention promoted the repair of damaged white matter(P < 0.01).Compared with the ICH7 d group,the FA values of the ICH14 d group was also significantly increased(P < 0.05).Compared with the Sham group,the MD(Ipsilateral/contralateral)values of the model group was significantly increased,and white matter fibers were severely damaged(P < 0.01).Compared with the model group,ST intervention significantly inhibited the damage of white matter fibers,and the MD values of the ICH+7d ST and ICH+14d ST group were significantly reduced(P < 0.05).5.ST promoted the expression of p-Akt/Akt protein: Compared with the Sham group,the expression of p-Akt /Akt in the model group was significantly decreased(P < 0.01).Compared with the ICH7 d group,the expression of p-Akt/Akt in the ICH+7d ST group was significantly increased(P < 0.05).Compared with the ICH14 d group,the expression of p-Akt/Akt in the ICH+14d ST group was increased,but the difference was not significant(P >0.05).6.ST inhibited the activation of glial cells: microglia and astrocytes were activated in large quantities after ICH in mice,and the expression levels of Iba1 and GFAP were significantly increased(P < 0.05 or P < 0.01).However,after ST intervention,the expression levels of Iba1 and GFAP were significantly decreased(P < 0.05 or P < 0.01).7.ST improved neurological function in ICH mice by activating the Akt/GSK3βsignalling pathway: Compared with the Sham group and ICH+7d ST group,Y maze alternation rate in the ICH group was decreased,and that in the ICH+inhibitor+7d ST group was significantly decreased(P < 0.01).Compared with the sham group and ICH+7d ST group,ICH group had higher m NSS score and ICH+inhibitor+7d ST group had the highest m NSS score(P < 0.05 or P < 0.01).8.ST improved motor function in ICH mice by activating the Akt/GSK3β signaling pathway: compared with the Sham group and ICH+7d ST group,the open field track of the ICH group was significantly decreased,Open field track was significantly decreased in the ICH+inhibitor+7d ST group.Compared with the Sham group,the ICH+inhibitor+7d ST group had shorter total distance and lower average velocity of open field test(P < 0.01).The total distance and average velocity of open field test were significantly increased in the ICH+7d ST group,Compared with the ICH7 d group and the ICH+inhibitor+7d ST group(P < 0.01).The use of Akt inhibitor reversed the effect of ST intervention.There was no anxiety or depression behavior in mice,and the ratio of central total distance to total distance of open field test showed no significant difference in each group.9.ST improved neurometabolism in ICH mice by activating the Akt/GSK3β signaling pathway: the results showed that the relative levels of NAA/Cr and Cho/Cr were significantly decreased in the model group(P < 0.05 or P < 0.01),while ST significantly increased the relative levels of NAA/Cr and Cho/Cr(P < 0.01),and the use of Akt inhibitor reversed the promoting effect of ST on NAA/Cr and Cho/Cr.The relative levels of MI/Cr,Glu/Cr and Lac/Cr increased in the model group(P < 0.01)and ST significantly reversed this trend.However,the inhibition of ST on the relative levels of MI/Cr and Lac/Cr was reversed by using Akt inhibitor(P < 0.01),suggested that ST could improve brain metabolism and alleviate brain injury,while the Akt inhibitor reversed the effect of ST,which was consistent with the results of behavioral test.10.ST inhibited neuronal apoptosis in ICH mice by activating Akt/GSK3β signaling pathway: the expression levels of p-Akt /Akt and p-GSK3β/GSK3β in the model group were significantly decreased(P < 0.05),and the expression levels of p-Akt /Akt and p-GSK3β/GSK3β in the ST group were higher than those in the Akt inhibition group(P <0.01).It is suggested that the neuroprotective effect of ST is related to the Akt/GSK3βpathway.The expression of Bax/Bcl-2 is significantly increased in the model group(P < 0.01),and ST significantly inhibited the expression of Bax/Bcl-2(P < 0.05).Akt inhibitor could up-regulate the expression of Bax and inhibit the expression of Bcl-2.These results suggested that early ST inhibited apoptosis and plays a neuroprotective role in ICH mice through Akt/GSK3β pathway.11.ST inhibited glial cell activation in ICH mice by activating the Akt/GSK3β signaling pathway: microglia and astrocytes were activated in the model group,the expression levels of Iba1 and GFAP were significantly increased(P < 0.01).The expression levels of Iba1 and GFAP were significantly decreased after ST intervention(P < 0.05 or P < 0.01),suggesting that ST intervention could inhibit the activation of microglia and astrocytes.The use of Akt inhibitor reversed the inhibitory effect of ST on microglia and astrocytes activation(P < 0.05 or P < 0.01).ConclusionThese results showed that ST significantly improved the neurological function and motor ability of ICH mice,and promoted the expression of p-Akt/Akt protein.ST inhibited the activation of microglia and astrocytes in ICH mice,inhibited apoptosis,and improved neurological metabolism,Akt/GSK3β signaling pathway involved in the neuroprotection of ST. |
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