| Objectives Propofol is a commonly used drug for pediatric anesthesia.However,propofol interferes with the mitochondrial electron transport chain,causing an imbalance in the energy supply of developing neurons,resulting in neurotoxicity.The purpose of this study was to explore the effect of propofol on the release of extracellular vesicles from neurons and astrocytes,and to investigate the effect of co-culture with astrocytes or exogenous mitochondrial transplantation on propofol-induced neurotoxicity.By sequencing the neuronal exosomal RNAs after propofol incubation,this study will explore and speculate the mechanism of transcellular information transmission and functional regulation between neurons and astrocytes.Methods 1.Cortical neurons and astrocytes of neonatal 1-day rats were isolated.Neurons were cultured for 7 days and divided into NCM group and NPM group.Astrocytes were cultured and purified and divided into ACM group and APM group;The NCM group and ACM group were normal cultured control groups,and the NPM group and APM group were both experimental groups incubated with propofol 100μM for 6 h.The above 4 groups were simultaneously replaced with fresh medium and incubated for 24 h,and then the medium was collected.The conditioned medium is concentrated by high-speed centrifugation and the particles are collected.The subcellular structures in the pellets were observed by electron microscopy;mitochondrial marker proteins were detected by immunoblotting.Flow cytometry was used to detect the number of mitochondria and membrane potential in the medium of each group;Cell Titer-Glo ATP reagent was used to detect the content of ATP in the medium.2.Cortical neurons and astrocytes of neonatal 1-day rats were isolated.Cultured neurons up to 7 days and purified astrocytes were divided into 8 groups.4 Group were cultured alone.NC group was a neuron control group;NP group was a neuron experimental group incubated with 100μM propofol for 6 h;AC group was astrocyte control group;AP group was a astrocyte experimental group incubated with 100μM propofol for 6 h.In the other 4 groups,astrocytes and neurons were co-cultured in Transwell at a ratio of 1:4.The astrocytes in the upper chamber are the NAC group and the NAP group,and the neurons in the upper chamber are the ANC group and the ANP group.The NAC group and ANC group were normal cultured control groups,and the NAP group and ANP group were the experimental groups incubated with propofol 100μM for 6 h.The cells in the 8 groups were replaced with fresh medium and incubated for 24h after the intervention,and then tested.The mitochondria of the two types of cells were labeled in advance,and the mitochondrial transfer was observed by confocal microscope after 24 hours of co-culture.The apoptosis rate of each group was detected by flow cytometry;the mitochondrial membrane potential of each group was detected by JC-1,TMRM and double staining;Flow cytometry was used to detect the ROS content of cells in each group;Hoechst/PI double staining was used to detect the cell mortality of each group;CCK-8 and Alamar blue were used to detect the cell viability in each group;LDH kit was used to detect the release of LDH in each group of cells;Cell Titer-Glo ATP reagent was used to detect the content of ATP in each group of cells.Western blot was used to detect the expression of related proteins in AC group and AP group.3.Cortical neurons of neonatal 1-day rats were isolated,cultured for 7 days and divided into 4 groups.Group C was the control group.Group P,PM and PMD were incubated with propofol 100μM for 6 hours.Afterwards,the medium of group P was changed and cultured for 24 hours.The PM group changed the medium and added concentrated astrocyte-derived healthy extracellular mitochondria for 24 hours.The PMD group changed the medium and added concentrated astrocyte-derived dysfunctional extracellular mitochondria for 24 hours.Confocal microscopy was used to observe the transfer of pre-labeled concentrated mitochondria to neurons and the morphology of mitochondrial network.The apoptosis rate of neurons in each group was detected by flow cytometry;the mitochondrial membrane potential of neurons in each group was detected by JC-1,TMRM and double staining methods;the content of ROS in neurons in each group was detected by flow cytometry;Mito SOX Red was used to detect the content of superoxide in neurons in each group;Hoechst/PI double staining was used to detect the mortality of neurons in each group;CCK-8 and Alamar blue were used to detect the activity of neurons in each group;LDH kit was used to detect the release of LDH in neurons of each group;Cell Titer-Glo ATP reagent was used to detect the content of neuronal ATP in each group;Western blot was used to detect the expression of neuron-related proteins in each group.4.Cortical neurons of neonatal 1-day rats were isolated.Cultured for 7 days and divided into control group and propofol group.The control group was cultured normally,and the propofol group was incubated with propofol 100μM for 6 hours.Afterwards,the two groups of cells were cultured for 24 hours by changing the medium.The culture medium was collected,and the exosomes of the two groups of neurons were extracted by ultracentrifugation.Exosomes were identified using NTA,transmission electron microscopy,and immunoblotting.RNAs in exosomes were extracted for high-throughput and array chip sequencing.Results 1.The results of transmission electron microscopy showed that the conditioned medium contained extracellular mitochondria,which were mainly free,and a small amount was encapsulated by microvesicles.The results of immunoblotting showed that ANT1,TOMM40,COX IV and PCG-1αproteins were contained in the concentrated microparticles of the conditioned medium.The results of flow cytometry showed that compared with the ACM group and the NCM group,the number of mitochondria in the APM group and the NPM group was significantly increased(P<0.05).There was no significant difference in the number of mitochondria between ACM group and NCM group.Compared with the ACM group and the NCM group,the mitochondrial membrane potential of the APM group and the NPM group was significantly decreased(P<0.05).There was no significant difference in membrane potential between ACM group and NCM group.The results of ATP detection showed that compared with the ACM group,the ATP content of the APM group was significantly decreased(P<0.05).Compared with the NCM group,the ATP content in the APM group and NPM group was significantly decreased(P<0.05).There was no significant difference between ACM group and NCM group.2.The astrocytes and neurons co-cultured by Transwell were observed by confocal microscopy,and it was found that mitochondria were transferred between the two cells.The results of apoptosis rate showed that compared with the NC group,the apoptosis rate of the NP group and the NAP group was significantly increased(P<0.05);compared with the NAC group,the apoptosis rate of the NP group and the NAP group was significantly increased(P<0.05);there was no significant difference in apoptosis rate between NC group and NAC group;there was no significant difference in apoptosis rate between AC group,AP group,ANC and ANP group.The results of mitochondrial membrane potential(JC-1,TMRM and double staining)showed that compared with the NC group,the neuronal membrane potential of the NP group and the NAP group was significantly decreased(P<0.05).Compared with the NAC group,the neuronal membrane potential of the NP group and the NAP group was significantly decreased(P<0.05).There was no significant difference in membrane potential between the NC group and the NAC group.There were no significant differences in astrocyte membrane potential between AC,AP,ANC,and ANP groups.Detection of ROS content:Compared with the NC group,the neuronal ROS content of the NP group and the NAP group was significantly increased(P<0.05).Compared with the NAC group,the neuronal ROS content in the NP group and the NAP group was significantly increased(P<0.05).There was no significant difference in ROS content between the NC group and the NAC group.There was no significant difference in astrocyte ROS content among AC,AP,ANC and ANP groups.Results of cell mortality:Compared with the NC group,the neuronal mortality of the NP group and the NAP group was significantly increased(P<0.05).Compared with the NAC group,the neuronal mortality in the NP group and the NAP group was significantly increased(P<0.05).There was no significant difference in mortality between the NC and NAC groups.There was no significant difference in the PI positive ratio of astrocytes among AC,AP,ANC and ANP groups.Cell viability test(CKK-8,alamar blue)results:Compared with the NC group,the neuronal cell viability of the NP group and the NAP group was significantly decreased(P<0.05);Neuronal cell viability was significantly decreased(P<0.05);there was no significant difference in cell viability between NC group and NAC group;there was no significant difference in astrocyte viability between AC group,AP group,ANC and ANP group.Results of ATP content:Compared with the NC group,the ATP content of neurons in the NP group and the NAP group was significantly decreased(P<0.05);compared with the NAC group,the neuronal ATP content in the NP group and the NAP group was significantly decreased(P<0.05).);there was no significant difference in ATP content between NC group and NAC group;there was no significant difference in astrocyte ATP content between AC group,AP group,ANC group and ANP group.Western blotting results showed that compared with AC group,AP group had no significant difference in the expression of apoptosis-related proteins BAX,Bcl-2,Cleaved caspase 3,Cleaved caspase 9 and NF-κB.Compared with the AC group,the expressions of pyroptosis-related proteins NLRP3 and Cleaved caspase 1were not significantly different in the AP group.Compared with AC group,the expression of transmembrane protein CD38 in AP group was significantly increased(P<0.05).Compared with AC group,the expression of transmembrane protein CX43 in AP group was significantly decreased(P<0.05).There were no significant differences in the expression of mitochondrial proteins TOMM40,ANT1 and PGC-1αin AP group compared with AC group.Compared with the AC group,the expression of BDNF in the AP group was significantly decreased(P<0.05);Using mitochondrial protein COX IV as an internal control,there was no significant difference in the phosphorylation rate of the split protein p DRP1(ser616)in the AP group compared with the AC group.3.Transfer of enriched astrocyte extracellular mitochondria into neurons was visualized by confocal microscopy.The mitochondrial mitochondrial network in group C and PM group was long tubular,and group P and PMD group showed short spherical shape.The mitochondrial membrane potential(JC-1,TMRM and double staining)results showed that compared with the C group,the neuron membrane potential of the P group and the PMD group was significantly decreased(P<0.05).Compared with PM group,the neuron membrane potential of P group and PMD group was significantly decreased(P<0.05).There was no significant difference in membrane potential between the C group and the PM group.Superoxide detection results:Compared with C group,the superoxide content of neurons in P group,PM group and PMD group was significantly increased(P<0.05).Compared with the PM group,the mean fluorescence intensity of superoxide content in neurons in the P group and PMD group was significantly increased(P<0.05).Results of neuronal mortality:Compared with the C group,the neuronal mortality in the P,PM and PMD groups was significantly increased(P<0.05).Compared with the PM group,the neuronal mortality in the P and PMD groups was significantly increased(P<0.05).Results of ATP content:Compared with the C group,the neuronal ATP content in the P and PMD groups was significantly decreased(P<0.05).Compared with PM group,the neuronal ATP content in P group and PMD group was significantly decreased(P<0.05).Western blotting results:Compared with the C group,the expression levels of BAX,Cleaved Caspase 3 and Cleaved Caspase 9 proteins in the P and PMD groups were significantly increased(P<0.05).Compared with PM group,the expressions of BAX,Cleaved Caspase3 and Cleaved Caspase 9 proteins in P group and PMD group were significantly increased(P<0.05).Compared with the C group,the expressions of Bcl-2 and NF-κB proteins in the P and PMD groups were significantly decreased(P<0.05).Compared with the PM group,the Bcl-2 and NF-κB proteins in the P and PMD groups.The expression level was significantly decreased(P<0.05).Compared with the C group,the expressions of NLRP3 and Cleaved Caspase 1 proteins in the P and PMD groups were significantly increased(P<0.05).Compared with the PM group,the expressions of NLRP3 and Cleaved Caspase 1 proteins in the P and PMD groups were significantly increased(P<0.05).There was no significant difference in the expression of CD38 protein among the four groups of neurons.Compared with group C,the expression of CX43 protein in group P,PM and PMD was significantly decreased(P<0.05).Compared with group C,the expressions of ANT1,TOMM40 and PGC-αproteins in PM group and PMD group were significantly increased(P<0.05).Compared with PM group,the expressions of ANT1,TOMM40 and PGC-αproteins in P group were significantly decreased(P<0.05).Compared with group C,the expression of BDNF protein in group P and PMD was significantly decreased(P<0.05).Compared with PM group,the expression of BDNF protein in P group and PMD group was significantly decreased(P<0.05).Mitochondrial protein COX IV was used as an internal reference.Compared with the C group,the phosphorylation rate of the split protein p DRP1(ser616)in the P and PMD groups was significantly increased(P<0.05).Compared with the PM group,the phosphorylation rate of the split protein p DRP1(ser616)in the P and PMD groups was significantly increased(P<0.05).4.Circular,disc-shaped,and cap-shaped exosomes were observed by transmission electron microscopy.The average particle size of exosomes measured by NTA was 139.83±1.10 nm,and the average particle concentration was 4.57×10~9/ml.The expression of CD63 protein was detected by Western blotting.miRNA high-throughput sequencing results:Compared with the control group,the expression of 36 miRNAs in the propofol group was significantly up-regulated(1.5 times),the expression of 30 was significantly down-regulated,and the expression of the remaining 456 miRNAs had no significant difference.Array chip mRNA sequencing results:Compared with the control group,109mRNAs in the propofol group were significantly up-regulated(1.5 times),494were significantly down-regulated,and the remaining 27,309 had no significant difference.Array chip LncRNA sequencing results:Compared with the control group,the expression of 40 LncRNAs in the propofol group was up-regulated(1.5 times),129 were down-regulated,and the remaining 9941 had no difference in expression.The results of differential expression of RNAs in exosomes were verified by PCR in line with the trend obtained by sequencing.Conclusions 1.Propofol increased the release of extracellular mitochondria from astrocytes and neurons,but the released extracellular mitochondria decreased the membrane potential and ATP content.2.Under non-contact co-culture,astrocytes and neurons conduct mitochondrial transfer by releasing extracellular mitochondria.However,the extracellular mitochondria of astrocytes in this mode could not alleviate the neurotoxicity of propofol.Propofol is not significantly toxic to astrocytes,but may affect their outward mitochondrial transfer.Propofol has no obvious toxicity to astrocytes.3.Transplantation of a large number of extracellular mitochondria from astrocytes into neurons can alleviate the neurotoxicity induced by propofol.4.In neuronal exosomes,significantly differentially expressed non-coding RNAs such as miR-291a-3p,miR-17-3p,miR-203a-3p,and mRNAs such as Pik3cg and Akt3are the regulatory signals,which may affect mitochondrial transfer,transmitted from neurons to astrocytes. |
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