The role of Drp1 mediated mitochondrial dynamics balance in sepsis induced cardiomyopathy / 中华急诊医学杂志

Objective:To analyze the effect of inhibiting excessive mitochondrial fission mediated by dynamic related protein 1 (Drp1) on the function of injured cells and mitochondria in the septic myocardium, and to explore the protective effect of maintaining mitochondrial dynamic balance in the pathogenesis of sepsis induced cardiomyopathy(SIC).Methods:Rat H9C2 cardiomyocytes were cultured and stimulated with lipopolysaccharide (LPS) to establish a model of SIC. Mitochondrial division inhibitor 1 (Mdivi-1) was given 30 min before LPS stimulation. They were divided into the control group, LPS stimulated group (LPS), Mdivi-1 control group (Mdivi-1), and LPS+Mdivi-1 intervention group (LPS+Mdivi-1). CCK-8 was used to detect the cell viability, and lactate dehydrogenase (LDH) was used to detect cellular damage. A MitoTracker probe was used to observe mitochondrial morphology by laser scanning confocal microscopy, JC-1 staining was used to detect mitochondrial membrane potential level, a DCFH-DA probe was used to detect total ROS level, and an AnnexinV-FITC/PI probe was used to detect the cell apoptosis ratio. The expression levels of mitochondrial fission protein Drp1 and fusion proteins Optic Atrophy 1(Opa1) and Mitofusin2 (Mfn2) were detected by real-time PCR and Western blot. One-way ANOVA was used to compare the differences between groups, and the LSD- t test was used for pairwise comparisons between groups. Results:Compared with the control group, cell viability, the average length of mitochondria and the mitochondrial membrane potential were decreased, and ROS production, the cell apoptosis rate and LDH were increased in the LPS group (all P<0.05). After Mdivi-1 intervention, compared with the LPS-stimulated group, the cell viability was increased, myocardial cell damage was reduced, the average length of mitochondria was prolonged, mitochondrial dysfunction was alleviated, and the cell apoptosis rate was inhibited in the LPS+Mdivi-1 group (all P<0.05). Conclusions:Mdivi-1 might inhibit mitochondrial fission mediated by Drp1, maintain mitochondrial dynamic balance, alleviate mitochondrial dysfunction and protect myocardial cells from LPS-induced injury.

Mitochondrial Dynamics Mediates Myocardial Energy Metabolism in Coronary Heart Disease due to Blood Stasis / 中国实验方剂学杂志

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 D₃ （VitD₃） at 300 000 U·kg^-1 and then at 200 000 U·kg^-1 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^-1） 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.

Melatonin receptor agonist neu-pll improves insulin resistance in T2DM rats / 中国药理学通报

Aim To study the effect of melatonin receptor agonist Neu-pl 1 on IR in T2DM rats and to predict its possible mechanism. Methods Long-term high-fat feeding plus low-dose streptozotocin (STZ, 30 mg • kg"1, IP) were used to induce an animal model of type 2 diabetes in female rats. After 4 weeks of intragastric administration, liver tissue was sacrificed. Hie mRNA expression levels of Sirtl, Nrf-1/2 and ERRa in rats were determined by RT-PCR. The relative expressions of PGC-la and Mfn2 were determined by Western blot. Rat INS was measured and HOME-IR and ISI were calculated. The liver ATP content and ATPase activity were measured. Results Compared with normal group, T2DM rats developed IR, PGC-la, Mfn2 protein expression increased, Sirtl and other mRNA expression decreased significantly, ERRa mRNA expression increased, ATP content decreased significantly, and ATPase activity decreased. Compared with model group, the Neu-pll low-dose group reduced INS in T2DM rats, improved IR in T2DM rats, up-regulated rat liver protein and mRNA expression, down-regulated ERRa mRNA expression, and increased rat liver ATP content and ATPase activity. Conclusions The melatonin receptor agonist Neu-pl 1 may improve IR by regulating the mitochondrial bal-ance-related factors in liver of T2DM rats and maintaining normal mitochondrial function.

PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation

A change in the metabolic flux of glucose from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis is regarded as one hallmark of cancer. However, the mechanisms underlying the metabolic switch between aerobic glycolysis and OXPHOS are unclear. Here we show that the M2 isoform of pyruvate kinase (PKM2), one of the rate-limiting enzymes in glycolysis, interacts with mitofusin 2 (MFN2), a key regulator of mitochondrial fusion, to promote mitochondrial fusion and OXPHOS, and attenuate glycolysis. mTOR increases the PKM2:MFN2 interaction by phosphorylating MFN2 and thereby modulates the effect of PKM2:MFN2 on glycolysis, mitochondrial fusion and OXPHOS. Thus, an mTOR-MFN2-PKM2 signaling axis couples glycolysis and OXPHOS to modulate cancer cell growth.

Effect of butylphthalide combine edaravone on the dynamic change of Drp1 and Mfn2 in rats with focal cerebral ischemia and its protection mechanism / 医学研究生学报

Objective Apoptosis was induced by oxidative stress in nerve cells after cerebral ischemia. It further breaks the dy-namic balance of mitochondrial division of fusion. This study aimed to investigate the effect of butylphthalide combined with edaravone treat-ment on the dynamic change of Drp1 and Mfn2 in rats with focal cere-bral ischemia cells and its protection mechanism. Methods 96 rats were divided into 4 groups according to random number table. The 4 groups were ischemia group,butylphthalide group,edaravone group and butylphthalide combined with edaravone groups(combine group),each group divided into three subgroups(3 d,7 d,14 d). Longa-Zea suppository method is adopted to establish the middle cerebral artery occlusion(MCAO)model.Butylphthalide group,edar-avone group and combine group were injected butylphthalide(0.4 g/kg)and/or edaravone(10 mL/kg)peritoneally 2 hours before surgery and 1 day after surgery. The same volume of isotonic saline was given at the same time of the other 3 groups in ischemia group. The cerebral cortex of the left ischemic region was obtained at the day 3,7,14. The evaluation of the curative effect was evaluated with neurological function score.HE staining was used to observe the cerebral cortex neuron morphological structure,protein and mRNA lev-el of Drp1 and Mfn2 was measured by western blot and RT-PCR. Results At the day 3,7,and 14,the neurological function score was higher in ischemia group than the other 3 groups(P<0.05). Compared with the combine group at day 3,7,and 14[(1.06± 0.18),(0.82±0.13),(0.57±0.10)],the neurological function score was elevated in butylphthalide group[(2.02±0.18),(1.23± 0.13),(0.86±0.10)]and edaravone group[(2.08±0.17),(1.23±0.13),(0.85±0.12)](P<0.05). At the same time point,com-pared with the ischemia group,Drp1 protein and mRNA levels were lower in the other 3 groups(P < 0.05)while Mfn2 protein and mRNA levels were elevated(P<0.05). Compared with the butylphthalide group and edaravone group,Drp1 protein and mRNA levels were lower in combine group(P<0.05)while Mfn2 protein and mRNA levels were elevated(P<0.05). Conclusion The protective effect of edaravone combined with butylphthalide is better than single use. Its mechanism may be related to the removing of oxygen free radicals and reducing oxidative stress by edaravone,and the protection of the mitochondria by butylphthalide.

Association between MFN2 gene polymorphisms and the risk and prognosis of acute liver failure: a case-control study in a Chinese population

This study aimed to determine the role of mitofusin 2 (MFN2) gene polymorphisms in the risk and prognosis of acute liver failure (ALF). A total of 298 blood samples were collected from 138 ALF patients (case group) and 160 healthy participants (control group). Coagulation function, glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), total bilirubin (TB), blood ammonia and lactic acid (LA) were measured. The predictive evaluation of MFN2 gene polymorphisms in the risk and prognosis of ALF patients was estimated using Kaplan-Meier survival analysis, haplotype analysis, binary logistic regression analysis and Cox regression analysis. Higher levels of GPT, GOT, TB, blood ammonia and LA were observed in ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 than in those with the CC genotype of these two SNPs. The GTACAGC and GTGTGGC haplotypes were a protective factor and a risk factor for ALF, respectively. Blood ammonia and LA levels were independent risk factors and the CC genotype of rs873457 and the CC genotype of rs4846085 were protective factors for ALF. ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 had a lower survival rate than those with other genotypes of these two SNPs. The rs4846085 and rs873457 polymorphisms were both independent factors affecting the prognosis of ALF patients. MFN2 gene polymorphisms (rs873457, rs2336384, rs1474868, rs4846085 and rs2236055) may be associated with ALF and the rs873457 and rs4846085 polymorphisms are correlated with the risk and prognosis of ALF.

Association between Mitofusin 2 Gene Polymorphisms and Late-Onset Alzheimer's Disease in the Korean Population

OBJECTIVE: Mitochondrial dysfunction is a prominent and early feature of Alzheimer's disease (AD). The morphologic changes observed in the AD brain could be caused by a failure of mitochondrial fusion mechanisms. The aim of this study was to investigate whether genetic polymorphisms of two genes involved in mitochondrial fusion mechanisms, optic atrophy 1 (OPA1) and mitofusin 2 (MFN2), were associated with AD in the Korean population by analyzing genotypes and allele frequencies. METHODS: One coding single nucleotide polymorphism (SNP) in the MFN2, rs1042837, and two coding SNPs in the OPA1, rs7624750 and rs9851685, were compared between 165 patients with AD (83 men and 82 women, mean age 72.3±4.41) and 186 healthy control subjects (82 men and 104 women, mean age 76.5±5.98). RESULTS: Among these three SNPs, rs1042837 showed statistically significant differences in allele frequency, and genotype frequency in the co-dominant 1 model and in the dominant model. CONCLUSION: These results suggest that the rs1042837 polymorphism in MFN2 may be involved in the pathogenesis of AD.

Effects of mitochondrial fission in proliferation, apoptosis and invasion of thyroid squamous carcinoma cell line SW579 / 天津医药

Objective To detect the expression of mitochondrial dynamics proteins (Mfn2 and Drp1) in thyroid squa?mous carcinoma cell line SW579 and the effects of Mitochondrial division inhibitor, Mdivi-1, on proliferation, apoptosis and invasion of SW579. Methods In SW579 and Nthy-ori 3-1 cell lines, the expression levels of Mfn2 and Drp1 were deter?mined by western blot while the transcription level of Mfn2 and Drp1 mRNA were measured by RT-PCR. Then, SW579 cells were divided into control group (DMSO, 0.1%) and Mdivi-1 low, medium and high dose groups (Mdivi-1 of 15,30 and 45μmol/L were incubated with cells for 16 hours respectively). Then the ability of cell proliferation was detected using MTT assay, the mitochondrial membrane potential was determined by fluorescence spectrophotometer, the expression levels of cy?tochrome C and Caspase-3 were quantified by Western blot and the transcription level of the Cyt C and Caspase-3 mRNA were determined by RT-PCR. The ability of invasion in each group was measured with Transwell assays. Results Com?pared with Nthy-ori 3-1, the mRNA transcription and protein expression levels of the Mfn2 was remarkably decreased, while the mRNA transcription and protein expression of the Drp1 was significantly increased in SW579 cells (P<0.01). Compared with control group, the cell survival rates and mitochondrial membrane potential of SW579 were decreased dramat?ically (P<0.01). The mRNA transcription and protein expression of the cytochrome C and Caspase-3 were increased dra?matically (P<0.01) and the capability of invasion was markedly decreased in all the Mdivi-1 groups in a dosage dependent manner compared with those in control groups (P<0.01). Conclusion Abnormal mitochondrial dynamics may be involved in thyroid squamous cell carcinoma SW579 cells;Mdivi-1 can inhibit the cell proliferation and invasion as well as induce apoptosis.

Expression of Mitofusin-2 (mfn2) and nm23 in Different Bladder Tissues / 天津医药

Objective To research the expression of mitofusin-2 (mfn2) and tumor metastasis suppressor genes (nm23)in bladder cancer cells and its correlation with clinical pathological feature. Methods Immunohistochemistry was used to measure the expression of mfn2 and nm23.Sixty-five cases of bladder cancer were sampled,which include fif-ty cases of male and fifteen cases of female. TNM stage:Forty-seven cases were in stage I;Ten cases were in stage II; Five cases were in stageⅢ; Three cases were in stageⅣ. Other fifteen cases were sampled from normal bladder or benign tumor of bladder as control . All cases were collected from department of pathology,affiliated Hospital of hebei union university. Results The positive expression rate of mfn2 in bladder cancer tissues was significantly higher than those in normal blad-der tissues and benign tumor of bladder(χ2=32.528,P<0.05);The positive expression rate of nm23 in bladder cancer tis-sues was significantly lower than those in normal bladder tissues and benign tumor of bladder (χ2=19.719,P<0.05);the high expression level of mfn2 in bladder cancer was associated with tumor differentiation and TNM stage(P<0.05),but not corre-lated with age,sex,lymph node metastasis and clinical grade. The low level of nm23 was associated with TNM stage,clinical grade and LN metastasis. Conclusion The positive expression rate of mfn2 was increased in bladder cancer. It indicated that there was a close relationship between mfn2 and the occurrence and development of bladder cancer;The expression rate of nm23 was decreased in bladder cancer,it may be a predictor for metastasis and prognosis of bladder cancer.