ABSTRACT
Mitophagy is the selective degradation of damaged mitochondria, and it is of great significance to maintain the normal quantity and quality of mitochondria to ensure cell homeostasis and survival. Necroptosis is a type of programmed cell necrosis that can be induced by excessive mitophagy. Reactive oxygen species (ROS) are produced mainly by mitochondria and can damage mitochondria. Hyperoxic acute lung injury (HALI) is a serious complication of clinical oxygen therapy, and its pathogenesis is not clear. Existing studies have shown that mitophagy and necroptosis are involved in the occurrence of HALI. There are many mechanisms regulating mitophagy and necroptosis, including tumor necrosis factor-α (TNF-α), E3 ubiquitin protein ligase (PINK1/Parkin) protein pathway encoded by PTEN-induced kinase 1/PARK2 gene, phosphoglycerate mutase 5 (PGAM5), etc. PGAM5 has been proved to be a key factor linking mitophagy and necroptosis. Previous studies of our team found that the mechanism of microRNA-21-5p (miR-21-5p) alleviating HALI was related to its pGAM5-mediated inhibition of mitophagy, but the mechanism of PGAM5-mediated mitophagy and necroptosis remains unclear. Therefore, this paper reviews the targets of PGAM5-mediated mitophagy and necroptosis, in order to find clues of lung protection of pGAM5-mediated mitophagy and necroptosis in HALI, and provide theoretical basis for subsequent basic research.
ABSTRACT
Background Aluminum (Al) can cause irreversible damage to neurons and synapses function, and the mechanism may be connected to mitochondrial damage caused by glycogen synthase kinase-3β (GSK-3β) regulating dynamin-related protein 1 (DRP1), resulting in inhibition of the growth of neuronal protrusions. Objective To investigate the role of GSK-3β regulating DRP1 in the inhibition of primary hippocampal neurite growth induced by Al. Methods Neurons were extracted from the hippocampus of newborn mice (≤24 h old) for primary culture. On day 6, the purity of neurons was detected by immunofluorescence. On day 10, neurons with good growth state were selected for Al exposure and GSK-3β inhibitor SB216763 (SB) intervention. The experiment design included a blank control group, a dimethyl sulfoxide (DMSO) group, an Al (20 μmol·L−1) group, a SB (1 μmol·L−1) group, and a SB (1 μmol·L−1) + Al (20 μmol·L−1) group. After primary hippocampal neurons were treated with Al or SB for 48 h, cell viability was detected by CCK-8 assay, the mitochondrial morphology of primary hippocampal neurons was observed by transmission electron microscopy, the total protrusion length of primary hippocampal neurons was scanned and analyzed by laser confocal imaging, and their complexity was analyzed by Sholl analysis. The expression levels of phospho-GSK-3β, GSK-3β, and DRP1 were detected by Western blotting. Results The immunofluorescent results showed that the purity of primary neurons was higher than 90%. After the Al exposure and the SB intervention for 48 h, compared with the blank control group, there was no obvious difference in cell viability in the DMSO group and the SB group (P>0.05), and the Al group showed reduced cell viability (P=0.006); there was no obvious difference in cell viability between the SB+Al group and the Al group (P>0.05). Compared with the blank control group, there was no obvious difference in the average total length of protrusion in the DMSO group and the SB group (P>0.05), and the Al group showed reduced average total length of neurite (P<0.001); the average total neurite length in the SB+Al group was significantly increased compared with that in the Al group (P=0.001). The results of Sholl analysis revealed that, within 130 μm from the cytosol, the number of intersections of neurons in each group increased with the increase of distance. Above 130 μm from the cytosol, the number of intersections of neurons in each group decreased gradually with increase of distance. At 130 μm and 310 μm from the cytosol, compared with the blank control group, the number of neuronal intersections in the DMSO group and the SB group had no obvious difference (P>0.05), and that in the Al group was significantly reduced (P<0.05); there was no obvious difference in the number of neuronal intersections between the SB+Al group and the Al group (P>0.05). The mitochondrial structure of the blank control group was complete and the crest was clearly visible; there was no apparent variation in the mitochondrial structure in the DMSO group and the SB group; the mitochondria in the Al group were vacuolated and the crista disappeared; the SB+Al group showed clearer crista than the Al group. The difference in GSK-3β phosphorylation level among groups was statistically significant (F=45.841, P<0.001). Compared with the blank control group, the GSK-3β phosphorylation level showed not significantly different in the DMSO group (P>0.05), increased in the SB group (P=0.022), and significantly reduced in the Al group (P<0.001); the GSK-3β phosphorylation level was significantly higher in the SB+Al group than in the Al group (P<0.001). The difference in DRP1 protein level among groups was statistically significant (F=8.389, P=0.003). Compared with the blank control group, the DRP1 protein levels in the DMSO group and the SB group were not significantly different (P>0.05), and significantly increased in the Al group (P=0.001); the DRP1 protein level in the SB+Al group was significantly lower than that in the Al group (P=0.029). Conclusion Al may increase the level of DRP1 protein by activating GSK-3β, causing mitochondrial damage and inhibiting neuronal protrusions growth.
ABSTRACT
Objective:To explore the mechanism of energy changes in the three stages of the formation of coronary heart disease due to blood stasis in rat model from the perspective of mitochondrial fusion-fission dynamic changes. Method:Thirty healthy male rats were divided into the blank control group （<italic>n</italic>=6） and model group （<italic>n</italic>=24） using SPSS 21.0 simple random sampling method. The rats in the blank control group were fed an ordinary diet， while those in the model group a high-fat diet. After seven days of adaptive feeding， the rats were treated with intragastric administration of vitamin D<sub>3</sub> （VitD<sub>3</sub>） at 300 000 U·kg<sup>-1</sup> and then at 200 000 U·kg<sup>-1</sup> 14 d later. The high-fat diet continued for 21 d， and six rats were randomly selected as samples for the pre-stage blood stasis syndrome group， followed by model verification and sampling. The remaining rats continued to receive the high-fat diet for 30 d， and six were randomly selected and categorized into the sub-stage blood stasis syndrome group， followed by model verification and sampling. The rest of rats were classified into the heart blood stasis syndrome group. While continuing the high-fat diet， they were also treated with multipoint subcutaneous injection of isoproterenol （ISO，5 mg·kg<sup>-1</sup>） for three consecutive days. One week later， the electrocardiogram （ECG） was recorded for determining whether the modeling was successful and the samples were taken at the same time. The changes in mitochondrial morphology and quantity were observed under a transmission electron microscope. The expression of mitochondrial dynamics-related proteins was measured by Western blot and the cellular localization of related proteins by immunofluorescence assay. Result:The levels of total cholesterol and low-density lipoprotein cholesterol in the pre-stage and sub-stage blood stasis syndrome groups were significantly increased as compared with those in the blank control group （<italic>P</italic><0.05）. The blood rheology index in the pre-stage blood stasis syndrome group was significantly elevated in contrast to that in the blank control group （<italic>P</italic><0.05）. The three-layered membrane of the aorta in the blank group was intact. However， the tunica media of the pre-stage blood stasis syndrome group began to show obvious calcification， with a small number of inflammatory cells adhering to the intima. The subintima and media smooth muscles in the sub-stage blood stasis syndrome group exhibited cavity structures. The three-layered structure of the arterial wall in the heart blood stasis syndrome group was severely damaged. The ECG of the blank control group revealed the regular appearance of P wave，regular QRS waveform （no broadening or deformity）， and no obvious ST-segment depression or elevation. The ECG of the pre-stage blood stasis syndrome group showed no obvious abnormalities as compared with that of the blank control group. In the sub-stage blood stasis syndrome group， the ECG showed an upward trend of the J point and slight ST-segment elevation， with the elevation≤0.1 mV. The ECG in the heart blood stasis syndrome group displayed significant ST-segment depression （>0.1 mV） and J point depression >0.1 mV. The mitochondria in the blank control group were normal in size and morphology， with clear and dense cristae， whereas those in the pre-stage blood stasis syndrome group were fusiform with sparse cristae. Some mitochondria in the sub-stage blood stasis syndrome group were significantly elongated， and even vacuole-like changes were present. In the heart blood stasis syndrome group， the mitochondria were ruptured. As demonstrated by comparison with the blank control group， the expression levels of mitofusin 2 （Mfn2）， dynamin-related protein 1 （Drp1）， and fission protein 1 （Fis1） in the model group were significantly up-regulated （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the pre-stage blood stasis syndrome group， the heart blood stasis syndrome group exhibited down-regulated Mfn2 （<italic>P<</italic>0.05）. Compared with the blank control group and the pre-stage blood stasis syndrome group， the sub-stage blood stasis syndrome group and the heart blood stasis syndrome group displayed down-regulated optic atrophy 1（OPA1） （<italic>P</italic><0.05，<italic>P</italic><0.01）. The Drp1 and Fis1 protein expression declined significantly in the sub-stage blood stasis syndrome group in comparison with that in the pre-stage blood stasis syndrome group （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of Mfn2 and Drp1 in the heart blood stasis syndrome group were lower than those in the sub-stage blood stasis syndrome group （<italic>P<</italic>0.01）. The comparison with the blank control group showed that Mfn2 and OPA1 were extensively accumulated in mitochondria of both the pre-stage and sub-stage blood stasis syndrome groups， while the red-stained Mfn2 was significantly reduced in the heart blood stasis syndrome group. The Drp1/Fis1 fluorescence was weak in the blank group and the pre-stage blood stasis syndrome group but strong in the sub-stage blood stasis syndrome group and heart blood stasis syndrome group. Conclusion:The cardiomyocyte mitochondria dynamics changes with the change in energy demand of cardiomyocytes. Mfn2 is dominated by fusion effect in the early stage of the formation of coronary heart disease due to blood stasis. With the gradual development of this disease， Mfn2 begins to mediate mitochondrial autophagy. OPA1 plays a role in intimal fusion and cristae integrity. The decreased OPA1 expression is closely related to the accelerated progression of coronary heart disease differentiated into blood stasis syndrome. The process by which Drp1 and Fis1 separate damaged mitochondria to prepare for mitochondrial autophagy contributes to alleviating the imbalance between the energy demand and supply of human body.
ABSTRACT
ObjectiveMild hypothermia was an effective way of cerebral resuscitation after cardiac arrest. The expression of cold-induced RNA binding protein (CIRP) was significantly enhanced when the temperature was lowered. This study was to evaluate the effects and the mechanisms of CIRP inhibition on hippocampal neurological and mitochondria function after mild hypothermia in a rat model of cardiac arrest.MethodsFive male Sprague-Dawley rats were injected with AAV9 in the hippocampus, 1 μL on each side, speeding 0.2 μL/min. The expression of GFP was observed by fluorescence microscopy after 2w. Sixty rats were randomly divided into 5 groups (n= 12 for each group): sham operation group, model group, mild hypothermia group, mild hypothermia + CIRP inhibition group and mild hypothermia + normal control group. Injection of AAV9 was performed on mild hypothermia + CIRP inhibition group, same amount of empty vector on mild hypothermia + normal control group, while normal saline on the other groups. Animal models of global cerebral IR were established by transesophageal cardiac pacing inducing cardiac arrest followed by cardiopulmonary resuscitation at 2w after injection. Cooling to 32-34℃ was initiated and the temperature was maintained for 6h on mild hypothermia groups. NDS score, HE staining and pyramidal cell counting on hippocampal CA1 area were performed at 72h after reperfusion. At 24h after reperfusion, mitochondrial structure of pyramidal cells in hippocampal CA1 was observed under electronic microscope and the expressions of CIRP, dynamin-related protein 1 (Drp1) and cytochrome C (Cyt-C) were detected by Western blot.ResultsThe NDS score of model group was decreased, the number of pyramidal cells was reduced, and the mitochondria were severely damaged. The NDS score of mild hypothermia group was increased, and the number of pyramidal cells was increased (all P<0.05), and mitochondrial damage was reduced compared with model group. In mild hypothermia + CIRP inhibition group, the NDS score was no significant difference compared with mild hypothermia + normal control group and model group, and the number of pyramidal cells was lower than that in mild hypothermia + normal control group [(27.2±4.9) vs (50.2±4.4), P<0.05], similar to model group (25.2±3.8), the damage of mitochondria was severe. After 2 weeks of AAV9 injection, GFP was widely expressed in the hippocampus. The expression of CIRP in mild hypothermia + CIRP inhibition group was respectively small compared with sham operation group [(0.14±0.03) vs (0.03±0.01),P<0.05], which was successfully inhibited by injection of AAV9. The expression of CIRP in model group (0.25±0.05) was significantly higher than that in sham operation group. The expression of CIRP in mild hypothermia group (0.37±0.08) and mild hypothermia + normal control group (0.39±0.04) were higher than that in model group (all P<0.05). The trends of Drp1 and Cyt-C expression were the same, in model group was higher than that in sham operation group, in mild hypothermia group was lower than that in model group, in mild hypothermia + CIRP inhibition group was higher than in mild hypothermia + normal control group (all P<0.05); There were no significant differences between model group and mild hypothermia + CIRP inhibition group, and between mild hypothermia group and mild hypothermia + normal control group.ConclusionInhibition of CIRP expression in hippocampus can weaken the protective effects of mild hypothermia on neurons in a rat model of cardiac arrest. The mechanism of those effects might be association with mitochondrial division.
ABSTRACT
OBJECTIVE: To discuss the effect of ferulic acid(FA) on learning and memory impairment and neuron protection by repairing the imbalance of mitochondrial fission-fusion dynamics in Alzheimer′s disease (AD) mice. METHODS: The KM mice were randomly divided into normal control group (A group, n=10), model group (B group, n=10), positive control group (huperzine A tablets, C group, n=10) and low dose of FA group (D-low group, n=10), high dose of FA group (D-high group, n=10). Mice in B, C, D-low and D-high groups were built as AD model by injecting Aβ1-42 into the lateral ventricle. The learning and memory ability of mice were detected by Morris water maze test. The mRNA of dynamin-related protein 1 (Drp1) were detected by PCR. The AD related pathological proteins and mitochondrial fission-fusion proteins were detected by Western blot. The content and distribution of Aβ was analyzed using immunofluorescence staining. RESULTS: ①The escape latency of mice in D-high group was shorter than B group, but a little longer than A group (P0.05). ②The mean expressions of Drp1, CaN subunit α (CnAα), CnAβ mRNA in D-high group were lower than B group, but higher than A group (P<0.05). ③The mean expressions of amyloid precursor protein (APP), beta-site APP cleaving enzyme 1 (Bace1), Tau46 and pS396 proteins in D-high group were lower than B group, but higher than A group (P<0.05).The mean expressions of Drp1Ser637, CnAα, protein kinase A catalytic subunit c (PKAc), mitofusin gene 2(Mfn2) proteins in D-high group were basically identical with A group. ⑤The levels of Aβ formation and accumulation in mice cortex and hippocampus of D-high group were less than B group. CONCLUSION: It′s suggested that ferulic acid(FA) might repair pathological damage of Alzheimer′s disease by improving the imbalance of mitochondrial fission-fusion dynamics.
ABSTRACT
Objective To investigate the roles of A kinase anchoring protein1(AKAP1)in high-glucose induced mitochondrial fission in podocytes.Methods Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours,and then exposed to different glucose concentration conditions in different time periods.The protein expressions of AKAP1 were observed by immunofluorescence,and AKAP1,dynamin related protein1 (Drp1) and phospho Ser 637-Drp1 (p-Drp1)were analyzed by Western blotting.AKAP1 siRNA was transfected to block AKAP1 expression.Podocytes were then divided into normal control group (5 mmol/L glucose),hypertonic group (30 mmol/L mannitol+5 mmol/L glucose),high glucose group (35 mmol/L glucose),and high glucose+AKAP1 siRNA group.Mitochondrial morphological changes were assessed by mitotracker red staining.Podocyte apoptosis was assessed by flow cytometry.Results Compared with normal group,high-glucose induced more podocytes apoptosis (P ＜ 0.05),more mitochondrial fission with decreased aspect ratio and form factor (all P ＜ 0.05).Upregulated AKAP1 protein level,and increased ratio of p-Drp1/Drp1 (all P ＜ 0.05) in time and concentration dependent manners were also obscrvcd.Compared with high glucose group,transfection of AKAP1 siRNA showed less apoptosis (P ＜ 0.05),less mitochondrial fission with increased aspect ratio and form factor (all P ＜ 0.05),and down-regulated AKAP1 protein level as well as p-Drp1/Drp1 ratio (all P ＜ 0.05).Conclusion High glucose induced mitochondrial fission might be induced through AKAP1-Drp1 pathway.
ABSTRACT
Objective: To observe the apoptosis and mitochondrial fission of human ovarian cancer SKOV3 cells after treated by myricetin and dynamin related protein 1 (DRP1) inhibitor mdivi-1 alone or combined, and to explore the mechanism of myricetin in inducing the apoptosis of SKOV3 cells. Methods: The SKOV3 cells were cultured in vitro and randomly divided into control group, mdivi-1 group, myricetin group and combined group. The cells in mdivi-1 group were treated with 50 μmol · L-1 madivi-1 for 1 h followed by common culture medium for 23 h; the cells in myricetin group were treated with 50 g · L-1 myricetin for 24 h; the cells combined group were treated with 50 jumol · L-1 midiv-1 for 1 h followed by 50 g · L-1 myricetin for 23 h. The survival rates of cells in various groups were detected by MTT assay. The apoptoic rates of cells in various groups were detected by Muse14 apoptosis detection kit. The expression levels of Cyt C, caspase3, DRP1 and FIS1 were observed by Western blotting method. The mitochondrial fission of cells in various groups was observed with MitoTracker® Red. Results; Compared with control group, the survival rate of cells in myricetin group was decreased significantly (P<0. 05); compared with myricetin group, the survival rate of cells in combined group was increased significantly (P<0. 05). Compared with control group, the apoptotic rate of cells in myricetin group was increased (P<0. 05); compared with myricetin group, the apoptotic rate of cells in combined group was decreased (P<0. 05). Compared with control group, the expression levels of Cyt C and caspase3 proteins in the cells in myricetin group were increased (P<0. 05); compared with myricetin group, the expression levels of Cyto C and caspase3 proteins in the cells in combined group were decreased (P<0. 05). Compared with control group, the degree of mitochondrial fission of the cells in myricetin group was increased; compared myricetin group, the degree of mitochondrial fission of the cells in combined group was decreased. Compared with control group, the expression levels of DRP1 and FIS1 proteins in the cells in myricetin group were increased (P<0. 05); compared with myricetin group, the expression levels of DRP1 and FIS1 proteins in the cells in combined group were decreased (P<0. 05). Conclusion: Myricetin can induce the apoptosis of human ovarian cancer SKOV3 cells by promoting the DRPl-dependent mitochondrial fission.
ABSTRACT
Objective To investigate the effect of mild hypothermia combined with mitochondrial divison inhibitor 1 in mitochondrial after cerebral ischemia-reperfusion (IR).Methods Fourty male healthy Sprague-Dawley (SD) rats, weighing 280-320 g, were randomly divided into 5 groups (n=8 each): group Sham, group IR, hypothermia group (group H), Mdivi-1 group (group M) and hypothermia+Mdivi-1 group (group HM).Animal models of global cerebral IR were established by transoesophageal cardiac pacing inducing cardiac arrest followed by cardiopulmonary resuscitation (ischemia 4 min and reperfusion 6 h).The group Sham was similarly treated to group IR except the cardiac arrest and cardiopulmonary resuscitation.In groups H and HM, the core temperature was cooled down to 32-34℃ within 15 min starting from the beginning of reperfusion, and maintained for 6 h.In the other groups, the core temperature was maintained at the normal temperature.In groups M and HM, the animals were given Mdivi-1 (1.2 mg/kg) intravenously at the beginning of the reperfusion and the other groups were given the same Volume of dimethylsnlfone (DMSO).After 6 h of reperfusion, the rats were sacrificed, and bilateral hippocampi were immediately removed for determination the protein level of dynamin-related proten 1 (Drp1) and cytochrome C (Cyt-C) expression by Western blot and obsevation of the mitochondrial structure of pyramidal cell in hippocampal CA1 under electronic microscope.Results Compared with group Sham, the expression of Drp1 and Cyt-C was up-regulated in groups IR, H, M and HM (P＜0.05).Compared with group IR, the expression of Drp1 and Cyt-C was down-regulated in groups H, M and HM (P＜0.05).Compared with groups H and M, the expression of Drp1 and Cyt-C was down-regulated in group HM (P＜0.05).There was no significant difference in the expression of Drp1 and Cyt-C between groups H and M.The mitochondria were rod-shaped with clear and sound structure in group Sham, while mitochondria showed various degree of fission, swollen structures, matrix deposit, vacuoles formation and cristae collapse in other groups.The changes of group HM were relatively slight.Conclusion Mild hypothermia combined with mitochondrial divison inhibitor 1 alleviate mitochondrial damage after global cerebral IR of rats.The combined effect is better than that of any individual application.
ABSTRACT
Necroptosis, or programmed cell death, is a type of cell death with a controllable death signaling pathway and the morphological features similar to necrosis.It is mainly mediated by death receptors or pathogen pattern re-cognition receptors.Among them, tumor necrosis factor receptor 1 (TNFR1)-mediated necroptosis is the most well-studied one.Receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3) are the 2 key kina-ses involved in the formation of complex I & II and necrosome in the process of necroptosis.Phosphoglycerate mutase 5 ( PGAM5) , a member of phosphoglycerate mutase gene family, lacks PGAM activity and possesses the phosphatase activi-ty.PGAM5 is anchored in the mitochondrial membrane and is also called mitochondrial phosphoglycerate mutase 5.It has been shown that PGAM5 involves in the formation of necrosome during necroptosis and it is able to accelerate the fission of mitochondria by dephosphorylation of dynamin-related protein 1 (DRP1), thus promoting cell necroptosis.
ABSTRACT
Objective To investigate the role of DRP1 and OPA1 in the process of apoptotic in hyperoxia induced lung injury of premature rats .Methods 48 premature Wistar rats were divided into control group in which rats were exposed tO 210 ml .L 1 oxy-en ,and hyperoxia group in which Rats were exposed to 950 mL .L 1 oxyen .The lung tissues of 2 groups were gained on the first day ,the second day and the seventh day with which that all mice were killed by cutting neck .Section of lungs were stained with hea-toxylin eosin to observe the pathological changes ,the protein expression in the lung cells which were linked with the change of plas-tosome ,then the premature rat lung tissue apoptosis in each group were evaluated .Results Compored with the contnx group ,the expression level of OPA1 in the hyperoxia group began to decrease on the first day and apparently decreased to the buttom line on the seventh day .On the other hand ,DRP1 in hyperoxia group began to increase on the first day ,while reached the summit on the seventh day .A few of TUNEL positive cells began to increase with time dependence .A lot of TUNEL positive cells could be found in hyperoxia group ,and the apoptotic index reached the peak on the 7 d .There is a significantly positive correlation between the cell apoptosis and the ratio change of DRP1 and OPA1 in high oxygen group(r=0 .725 ,P<0 .01) .Conclusion The classical pathologic characters of lung injury were found out in hyperoxia group ,and it changed obviously with time .
ABSTRACT
Objective To investigate the effect of mitochondrial fission on the function of pancreatic β cells.Methods INS-1 stable cell lines allowing inducible expression of either wild-type dynamin-related protein 1 (Drp-1 WT)or its dominant-negative mutant(Drp-1 K38A)were used.The effect of mitochondrial fission on the function of pancreatic β cells were investigated under different concentrations of glucose.Results There were increased mitochondrial fission and disintegration of the mitochondrial reticulum into multiple punctiform organelles in Drp-1 WT cells induced with doxycycline under high glucose condition.Insulin secretion(P<0.01),mitochondrial membrane potential(P<0.05),and ATP content(P<0.05)were decreased and cytochrome C expression was increased after the expression of Drp-1 WT under high glucose condition while these changes were markedly mild in Drp-1 K38A expression cells.Conclusion The increased mitochondrial fission inhibits pancreatic β cell function.