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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.
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Ischemia-reperfusion injury (IRI) is one of the main causes of early graft dysfunction after renal transplantation. In China, organ transplantation has entered into the era of organ donation after citizen's death. The increased risk of cardiopulmonary resuscitation, prolonged hypoperfusion time and warm ischemia time of donors may lead to IRI of the graft, and affect the short- and long-term clinical prognosis of the recipient and graft. Under IRI and other stress conditions, the mechanism of mitochondrial dynamics, mainly manifested by dynamic regulation of mitochondrial division and fusion, exert critical effect upon the biological function of mitochondria. Cell apoptosis caused by mitochondrial injury is the key event leading to acute kidney injury, which is mainly manifested by the imbalance of the regulatory mechanism of mitochondrial dynamics. In this article, the research progress on the regulatory mechanism of mitochondrial dynamics on renal IRI was reviewed, aiming to provide reference for improving the clinical outcomes of renal transplantation.
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Objective @# To investigate the effect of mitochondrial fission protein 1 (FIS1) on apoptosis and cisplatin resistance in tongue squamous cell carcinoma (TSCC) cells.@*Methods @#The squamous cell carcinoma cell lines SCC9 and CAL27 were used to detect the mRNA and protein levels of FIS1 after cisplatin treatment, the knockdown and overexpression of FIS1 of SCC9 and CAL27 with or without cisplatin treatment were accomplished through small interfering RNA (siRNA) and plasmid, respectively. The mitochondrial division state in cells was detected by mitochondrial staining, and the apoptosis state of cells was detected by TUNEL, flow cytometry and Caspase 3/7.@*Results@#FIS1 protein expression in tongue squamous carcinoma cells treated with cisplatin was increased, but the mRNA level did not change. Silencing of FIS1 expression reduced mitochondrial division and apoptosis in squamous cell carcinoma cells treated with cisplatin, whereas overexpression of FIS1 exhibited the opposite effects. The percentage of dividing mitochondria, the number of apoptotic cells and the activity of Caspase 3/7 in SCC9 and CAL27 cells were significantly different before and after modulation of FIS1 expression (P < 0.05). @*Conclusion@#FIS1 is involved in the regulation of cisplatin chemotherapy sensitivity in tongue squamous cell carcinoma and can be used as a new target for improving the sensitivity of cisplatin chemotherapy in oral squamous cell carcinoma.
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Objective To investigate the influence of inhibited gene expression of fisson 1 (Fis1) gene on the level of Fis1,mitofusin 1 (Mfn1) and mitochondrial membrane potential in SH-SY5Y cells with fluorine,to study the role of mitochondrial dynamic balance in the pathogenesis of chronic fluorosis.Methods SH-SY5Y cells were cultured in vitro,when adherent cells entered the logarithmic phase,using a group design,they were divided into four groups:blank control group (control),fluoride group [2 mmol/L sodium fluoride (NaF)],fluoride negative control group (2 mmol/L NaF + non-specific siRNA) and the gene-silencing group (2 mmol/L NaF + specific siRNA-Fis1).The protein expression levels of Fis1 and Mfn1 were measured by Western blotting;the mRNA expression levels of Fis1 and Mfn1 were measured by Real-time PCR;and the levels of the mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit.Results Compared with control (1.37 ± 0.18,1.00 ± 0.04;1.57 ± 0.19,1.00 ± 0.04;1.00 ± 0.10),the expression levels of Fisl protein (1.72 ± 0.04) and mRNA (1.48 ± 0.13) in fluoride group were increased,the expression levels of Mfn1 protein (0.87 ± 0.02) and mRNA (0.69 ± 0.07) in fluoride group were decreased,the level of mitochondrial membrane potential (0.76 ± 0.13) was decreased (P ＜ 0.05).Compared with control,the expression levels of Fis1 protein (0.79 ± 0.07) and mRNA (0.06 ± 0.03) in gene-silencing group were decreased,the expression levels of Mfn1 protein (1.71 ± 0.04) and mRNA (1.52 ± 0.05) in gene-silencing group were increased (P ＜ 0.05),the level of mitochondrial membrane potential (0.94 ± 0.01) was decreased.Compared with fluoride group,the expression levels of Fis1 protein and mRNA in gene-silencing group were decreased,the expression levels of Mfn1 protein and mRNA in gene-silencing group were increased,the level of mitochondrial membrane potential in gene-silencing group was increased (P ＜ 0.05).Conclusion Gene expression inhibition of Fis1 gene can reduce the mitochondrial division and damage of mitochondrial membrane potential in SH-SY5Y cells induced by fluoride.
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Objective To evaluate the influence of fluoride on mitochondrial membrane potential of neuroblastoma SH-SY5Y cells,and on the expression levels of mitochondrial proteins mitofusion 1 (Mfn1) and fission 1 (Fis1).Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentration of sodium fluoride [0.0 (control),0.4,2.0 and 4.0 mmol/L],and various periods exposure of 6,12,24,48 h;the mitochondrial membrane potential of SH-SY5Y cells was detected by mitochondrial membrane potential assay kit (JC-1);and the expression levels of Mfn1 and Fis1 proteins were detected by Western blotting.Results Compared with the control group (1.63 ± 0.18,1.13 ± 0.15,1.30 ± 0.02) for various periods exposure (6,12,48 h),the red/green fluorescence ratios of the mitochondrial membrane potential of SH-SY5Y cells exposed to 2.0 and 4.0 mmol/L of sodium fluoride were decreased significantly (1.01 ± 0.10,0.80 ± 0.04;0.75 ± 0.13,0.62 ± 0.10;0.82 ± 0.01,0.56 ± 0.04,P ＜ 0.05);compared with the control group (0.93 ± 0.03,1.05 ± 0.07,1.17 ± 0.04) for various periods exposure,the expression levels of mitochondrial Mfn1 protein were decreased significantly in 0.4,2.0,4.0 mmol/L sodium fluoride groups (6,12,48 h:0.75 ± 0.02,0.65 ± 0.05,0.57 ± 0.06;0.83 ± 0.06,0.79 ± 0.06,0.69 ±0.06;0.98 ± 0.05,0.73 ± 0.07,0.62 ± 0.09,P ＜ 0.05).Compared with the control group (0.90 ± 0.05) for exposure time 12 h,the expression levels of Fis1 protein were increased significantly in 2.0,4.0 mmol/L sodium fluoride groups (1.14 ± 0.06,1.23 ± 0.06,P ＜ 0.05).Conclusions The mitochondrial membrane potential and the expression levels of mitofusion 1 and fission 1 of SH-SY5Y cells treated with fluoride are abnormal,which might be associated with the theory of nerve cell damage from high oxidative stress.
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Objective To observe the effects of acupuncture on the behaviors and the expressions of Fis1 and OPA1, as well as mitochondrial ultrastructure in the hippocampus of mice with Alzheimer disease (AD); To explore the mechanism of action of acupuncture for AD.Methods Forty male SAMP8 mice were randomly divided into acupuncture group and model group, with 20 mice in each group. Another 20 male natural aging mice with the same age (SAMR1 mice) were set as the normal group. Acupuncture group chose Shenshu, Baihui, Xuehai and Geshu for intervention. 8 weeks later, Morris water maze was used to test the mice behaviors, and then hippocampus organization was taken. Western blot was used to detect the expressions of Fis1 and OPA1 and mitochondrial ultrastructure in hippocampal neurons of mice was observed by transmission electron microscopy.Results Compared with the model group, the escape latency of acupuncture group was significantly shortened (P<0.05), and the stay time in the former platform quadrant and former platform crossing times were significantly increased (P<0.05). The expression of Fis1 in the hippocampus of acupuncture group decreased significantly (P<0.05), while the expression of OPA1 increased significantly (P<0.05). The mitochondrial ultrastructure in hippocampus in the acupuncture group was effectively improved, and the mitochondrial surface density and body density were both increased in the acupuncture group compared with the model group (P<0.05).Conclusion Acupuncture may play a potential therapeutic role in AD by decreasing the expression of Fis1, increasing the expression of OPA1, recovering the injury of mitochondrial ultrastructure.
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Objective To investigate the effects of CORM-2 via p38 mitogeu-activated protein kinase (p38MAPK) signaling pathway on the expression of the mitochondrial fission protein 1 (Fisl) in lipopolysaccharide (LPS)-induced mouse pulmonary macrophages.Methods The rat subculture alveolar macrophages were seeded on 96 well plates with 2 × 105/ml densities.After 24 hours of culture,it was divided into 4 groups by random number table method:normal control group (group C),group LPS (group L),CO releasing agent CORM-2 + LPS group (group LC),p38MAPK inhibitor SB203580 + CORM-2 + LPS group (group LCS).When the cells were incubated for 24 hours,the mitochondrial MDA content and SOD activity were determined by ELISA kit,the levels of HO-1、mitochondrial fission protein Fis1 and p38 were determined by Western blot,the expressions of HO-1 and mitochondrial fission protein Fis1 were detected by RT-PCR.Results Compared with the C group,the levels of MDA [(2.43 ±0.12) vs.(3.59 ±0.07)],HO-1 [(1.31±0.27) vs.(1.65±0.41)],Fis1 [(1.27±0.23) vs.(1.65±0.41)] andp38 [(1.01 ±0.24) vs.(1.36 ±0.17)] in group L were increased,and the activity of SOD [(81.7 ± 1.62) vs.(54.7 ± 1.62)] was decreased (P ＜ 0.05);Compared with the group L,the MDA content [(3.59 ± 0.07) vs.(3.08 ±0.52)] and the level of Fis1 [(2.01 ±0.35) vs.(1.48 ±0.39)] in group LC were down-regulated,and the levels of SOD [(54.7 ± 1.62) vs.(67.4 ± 1.32)]、and the expressions of HO-1 [(1.65±0.41)vs.(2.25±0.18)] andp38 [(1.36±0.17) vs.(1.78±0.23)] wereup-regulated (P ＜0.05).Compared with the group LC,the MDA content [(3.08 ±0.52) vs.(4.16 ±0.19)] and the expression of Fis1 [(1.48 ±0.39) vs.(1.96 ±0.31)] in group LCS were increased,and the level of SOD [(67.4±1.32)vs.(45.9±1.52)]、and the expressions of HO-1 [(2.25±0.18)vs.(1.78± 0.19)] and p38 [(1.78 ±0.23) vs.(1.12 ±0.29)] were decreased (P ＜0.05).Conclusions HO-1/CO system inhibits the expression of Fis1 in LPS-induced lung macrophages,which may be regulated by p38MAPK signaling pathway.
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Objective To observe the expression of mitochondrial fission protein locus Fis1 and ultrastructural changes in the renal cells of rats with chronic fluorosis,and to reveal the mechanism in mitochondrial damage of the renal cells.Methods Sixty SD rats were randomly divided into 3 groups according to sex and body mass(20 in each group):control group,lower fluoride group and higher fluoride group.All the rats were fed with different doses of sodium fluoride in drinking water(0,10 and 50 mg/L,respectively).Six-month later,the expression of Fisl in renal cells was determined by real-time fluorenscence quantitative PCR and immunohistochemistry technology,the mitochondrial morphology of renal cells was observed under transmission electron microscopy (TEM).Results As compared with the control group(28.70 ± 12.41),Fis1 mRNA levels(91.48 + 34.83 and 582.09 ± 184.69) in renal cells of the lower fluoride and the higher fluoride groups were increased(all P ＜ 0.05).As compared with the control group(10.49 ± 7.66),Fisl protein levels(16.33 ± 10.26 and 21.50 ± 5.24) in renal cells of the lower fluoride and the higher fluoride groups showed a trend of increasing,the higher fluoride group was higher than that of the control group(P ＜ 0.05).By TEM,mitochondrial crest in renal cells of the lower fluoride and the higher fluoride groups was vague or disappeared,mitochondrial division section appeared.Conclusions Fluoride is a kind of toxicant that can cause damage to mitochondrion of renal cells,induce the expression of Fis1 in transcriptional and protein level,and lead to the obstacles of mitochondrial fusion-fission and ultrastructural abnormality of mitochondrion,which may play an important role in mechanism of mitochondrial damage in the renal cells of rats with chronic fluorosis.