Effect Of Chrysophanol On Mitochondrial Transfer In Astrocytes Pretreated With Gap Junction Blocker Gap26

Cerebral ischemia is a common cerebrovascular disease,and its incidence rate increases year by year in our country.Hayakawa K et al.found that in the model of ischemic injury of astrocytes and neurons in vitro,astrocytes could transfer functional mitochondria to neurons,thus enhancing the activity of neurons with ischemic injury.By using Mitotracker Red mitochondrial fluorescence probe to label astrocyte mitochondria,we found that astrocyte mitochondria can transfer to neurons,and Chrysophanol(CHR)can promote the transfer of astrocyte mitochondria to neurons and improve the activity of neurons.gap junction(GJ)is a pathway mediating mitochondrial transfer and plays an important role in mitochondrial transfer.It has been found in the literature that Gap26,a gap junction blocker,can aggravate neuronal damage in co-culture with astrocytes.Therefore,GJ is likely to be an important link in mitochondrial transfer in astrocytes.Whether gap junction blocker Gap26 affects astrocyte mitochondrial transfer and whether it enhances neuronal cell death through GJ in ischemic hypoxia injury is unknown.Previous studies have shown that CHR promotes mitochondrial transfer of astrocytes and attenuates ischemic injury of neurons and astrocytes during ischemia and hypoxia injury.However,it is not known whether the mechanism of action of CHR is accomplished through GJ.Therefore,this experiment will Gap26 pretreatment of astrocytes and neurons in culture,oxygen deprivation of sugar/reoxygenation(oxygen glucose deprivation/reoxygenation,OGD/R)injury.By measuring neuronal survival and Mitotracker Red labeling of astrocyte mitochondria,confocal microscopy was performed to determine fluorescent markers totrack mitochondrial transfer.To investigate the mechanism of CHR on mitochondrial transfer of ischemic injured astrocytes pretreated with Gap26.Ⅰ.The OGD/R model of astrocyte and neuron co-culture was establishedPrimary astrocytes and neurons were extracted from the cerebral cortex of neonatal SD rats and cultured in vitro.Transwell chamber was used to establish a co-culture system for OGD/R damage.Ⅱ.Effects of Gap26 preconditioning and neuron co-culture on mitochondrial transfer of astrocytes during OGD/R injury1.The experiment was divided into four groups: control Ⅰ group(normal oxygen culture of neurons);Model Ⅰ group(neuron OGD/R culture);Co-culture Ⅰ group(OGD/R co-culture of astrocytes and neurons);Gap26 co-culture Ⅰ group(Gap26 pretreated astrocytes and neurons co-culture OGD/R).The results of CCK-8 showed that the survival rate of neurons in group Ⅰ was(47.27±8.20)%,while the survival rate of neurons in group Ⅰ was decreased by(38.24±5.44)% after the addition of Gap26(P < 0.01).OGD/R injury of astrocytes pretreated with Gap26 and cocultured with neurons decreased the survival rate of neurons.2.Effect of Gap26 pretreatment on mitochondrial transfer of astrocytesThe experiment was divided into 2 groups:(1)Co-culture MR Group,Mitotracker Red pre-labeled astrocytes and neurons co-culture OGD/R;(2)In the MR Group co-cultured with Gap26,the astrocytes pretreated with Gap26 were labeled with Mitotracker Red fluorescent probes in advance,and OGD/R was co-cultured with neurons.The fluorescent markers in neurons were determined by confocal microscopy to observe the number of mitochondrial transfer in astrocytes.The fluorescence results showed that the fluorescent markers were red.(1)The visual field under the group of mirrors is visible,and the red color is irregular and dotted,and the arrangement is loose;The visual field of group(2)was visible under the microscope,and the red color was only scattered sporadically,and the number of fluorescent markers was reduced compared with group(1).The results showed that OGD/R injury of astrocytes pretreated with Gap26 and cocultured with neurons could reduce the transfer of astrocyte mitochondria to neurons.Ⅲ.Effect of chrysophanol on mitochondrial transfer in Gap26 pretreated astrocytesThe astrocytes pretreated with Gap26 were added to CHR and co-cultured with neurons for OGD/R injury.Then the survival rate of neurons,ATP content in neurons and mitochondrial morphology were measured.The experiment was divided into 5 groups: control group Ⅱ(normal oxygen culture of neurons);Model group Ⅱ(neuron OGD/R culture);Co-culture group Ⅱ(OGD/R co-culture of astrocytes and neurons);CHR co-culture group Ⅱ(adding OGD/R co-culture of astrocytes and neurons at 10 μ mol · L-1CHR);Gap26+CHR co-culture group Ⅱ(Gap26pretreated astrocytes,adding the concentration of 10 μmol·L-1CHR,and neurons co-culture OGD/R).1.Chrysophanol can improve the survival rate of co-cultured OGD/R neuronsThe results of CCK-8 showed that the survival rate of neurons in co-culture group Ⅱ was(49.41±8.40)%,and the survival rate of neurons added with CHR was(53.39±5.42)%(P < 0.01),indicating that CHR could improve the survival rate of neurons in co-culture OGD/R.Compared with CHR co-culture group Ⅱ,the survival rate of neurons after adding Gap26 was(37.70±3.00)%(P < 0.001).The results showed that the survival rate of neurons after OGD/R co-culture of astrocytes and neurons could be improved by adding Gap26,while GAP26 could counteract the effect of chrysophanol.2.Chrysophanol improves mitochondrial morphology of cocultured OGD/R neuronsImage J analysis showed that the mitochondrial length of cocultured OGD/R neurons in group Ⅱ was(51.45±3.70)%,and that of control group Ⅱ was(68.82 ± 3.17)% after CHR was added(P < 0.01),indicating that CHR could increase the mitochondrial length of cocultured OGD/R neurons.After addition of Gap26,the mitochondrial length of group Ⅱ co-cultured with Gap26+CHR was(44.22±6.33)%(P < 0.001),indicating that chrysophanol can increase the mitochondrial length of neurons after OGD/R,and gradually normalize the mitochondrial morphology.3.Chrysophanol improved ATP content in co-cultured OGD/R neuronsATP content was(43.98±1.66)% in coculture group Ⅱ,and(58.17±7.48)% after CHR was added,ATP content was increased(P < 0.001),indicating that CHR increased ATP content in co-culture OGD/R neurons.After addition of Gap26,the ATP content was(37.81±3.11)%,which was lower than that of CHR co-culture group Ⅱ(P < 0.001).The results showed that chrysophanol could increase the ATP content in neurons after OGD/R injury,while the addition of Gap26 could counteract the effect of chrysophanol.