This paper explored the specific mechanism of ginsenoside Rg_1 in regulating mitochondrial fusion through the neurogenic gene Notch homologous protein 1(Notch1) pathway to alleviate hypoxia/reoxygenation(H/R) injury in HL-1 cells. The relative viability of HL-1 cells after six hours of hypoxia and two hours of reoxygenation was detected by cell counting kit-8(CCK-8). The lactate dehydrogenase(LDH) activity in the cell supernatant was detected by the lactate substrate method. The content of adenosine triphosphate(ATP) was detected by the luciferin method. Fluorescence probes were used to detect intracellular reactive oxygen species(Cyto-ROS) levels and mitochondrial membrane potential(ΔΨ_m). Mito-Tracker and Actin were co-imaged to detect the number of mitochondria in cells. Fluorescence quantitative polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels of Notch1, mitochondrial fusion protein 2(Mfn2), and mitochondrial fusion protein 1(Mfn1). The results showed that compared with that of the control group, the cell activity of the model group decreased, and the LDH released into the cell culture supernatant increased. The level of Cyto-ROS increased, and the content of ATP decreased. Compared with that of the model group, the cell activity of the ginsenoside Rg_1 group increased, and the LDH released into the cell culture supernatant decreased. The level of Cyto-ROS decreased, and the ATP content increased. Ginsenoside Rg_1 elevated ΔΨ_m and increased mitochondrial quantity in HL-1 cells with H/R injury and had good protection for mitochondria. After H/R injury, the mRNA and protein expression levels of Notch1 and Mfn1 decreased, while the mRNA and protein expression levels of Mfn2 increased. Ginsenoside Rg_1 increased the mRNA and protein levels of Notch1 and Mfn1, and decreased the mRNA and protein levels of Mfn2. Silencing Notch1 inhibited the action of ginsenoside Rg_1, decreased the mRNA and protein levels of Notch1 and Mfn1, and increased the mRNA and protein levels of Mfn2. In summary, ginsenoside Rg_1 regulated mitochondrial fusion through the Notch1 pathway to alleviate H/R injury in HL-1 cells.
Ginsenosides/pharmacology* ; Receptor, Notch1/genetics* ; Signal Transduction/drug effects* ; Mice ; Animals ; Mitochondrial Dynamics/drug effects* ; Mitochondria/metabolism* ; Cell Line ; Reactive Oxygen Species/metabolism* ; Oxygen/metabolism* ; Cell Hypoxia/drug effects* ; Cell Survival/drug effects* ; Membrane Potential, Mitochondrial/drug effects* ; Humans

In this report, the protective effect and molecular mechanism of Chaihu Shugan San-containing serum on corticosterone(CORT)-induced mitochondrial damage in pheochromocytoma(PC12) cells was studied based on CORT-induced rat PC12 cell model. The cultured cells were divided into five groups: blank control group, CORT group(400 μmol·L~(-1) CORT), Chaihu Shugan San-containing serum group(400 μmol·L~(-1) CORT + 10% Chaihu Shugan San-containing serum), control serum group(400 μmol·L~(-1) CORT + 10% control serum), and fluoxetine group(400 μmol·L~(-1) CORT + 10% fluoxetine-containing serum). The study was carried out by cell activity detection, mitochondrial morphology observation, membrane potential measurement, energy metabolism analysis, and mitochondrial dynamics-related protein detection. The results showed that CORT treatment significantly reduced the survival rate of PC12 cells, altered mitochondrial morphology, and decreased mitochondrial membrane potential and adenosine triphosphate(ATP) synthetic rate. Both Chaihu Shugan San-and fluoxetine-containing serum significantly increased the survival rate of CORT-treated PC12 cells and the ATP synthetic rate in the mitochondria. Unlike fluoxetine, Chaihu Shugan San-containing serum significantly inhibited the decrease in mitochondrial membrane potential caused by CORT and increased the oxygen consumption rate(OCR) values of both mitochondrial maximum respiration and reserve respiration capacity. Western blot analysis showed that CORT induced upregulated protein expressions of dynamin-related protein 1(Drp1) and peroxisome proliferator-activated receptor gamma co-activator 1α(PGC-1α) in PC12 cells and specific protein expression of optic atrophy protein 1(OPA1), yet it repressed the protein expressions of silent information regulator 1(SIRT1) and mitochondrial fusion protein 1(Mfn1) in PC12 cells. Both Chaihu Shugan San-and fluoxetine-containing serum significantly inhibited the protein expression of Drp1. However, only Chaihu Shugan San-containing serum could significantly inhibit the CORT-induced upregulation protein of PGC-1α. RESULTS:: herein suggest that Chaihu Shugan San-containing serum can alleviate CORT-induced damage in PC12 cells, which may be related to the mitochondrial fragmentation/lipid peroxidation protection by Drp1 inhibition, as well as mitochondrial dynamics and energy metabolism mediated by PGC-1α/SIRT1 signaling pathway.
Animals ; PC12 Cells ; Rats ; Mitochondrial Dynamics/drug effects* ; Mitochondria/metabolism* ; Corticosterone/adverse effects* ; Membrane Potential, Mitochondrial/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Protective Agents/pharmacology* ; Cell Survival/drug effects*

Astragali Radix (AR) and Notoginseng Radix et Rhizoma (NR) are frequently employed in cardiovascular disease treatment. However, the efficacy of the AR-NR medicine pair (AN) in improving cardiac remodeling and its underlying mechanism remains unclear. This study aimed to evaluate AN's cardioprotective effect and potential mechanism on cardiac remodeling using transverse aortic constriction (TAC) in mice and angiotensin II (Ang II)-induced neonatal rat cardiomyocytes (NRCMs) and fibroblasts in vitro. High-performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS) characterized 23 main components of AN. AN significantly improved cardiac function in the TAC-induced mice. Furthermore, AN considerably reduced the serum levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), cardiac troponin T (CTn-T), and interleukin-6 (IL-6) and mitigated inflammatory cell infiltration. Post-AN treatment, TAC-induced heart size approached normal. AN decreased cardiomyocyte cross-sectional area and attenuated the upregulation of cardiac hypertrophy marker genes (ANP, BNP, and MYH7) in vivo and in vitro. Concurrently, AN alleviated collagen deposition in TAC-induced mice. AN also reduced the expression of fibrosis-related indicators (COL1A1 and COL3A1) and inhibited the activation of the transforming growth factor-β1 (TGF-β1)/mothers against decapentaplegic homolog 3 (Smad3) pathway. Thus, AN improved TAC-induced cardiac remodeling. Moreover, AN downregulated p-dynamin-related protein (Drp1) (Ser616) expression and upregulated mitogen 2 (MFN-2) and optic atrophy 1 (OPA1) expression in vivo and in vitro, thereby restoring mitochondrial fusion and fission balance. In conclusion, AN improves cardiac remodeling by regulating mitochondrial dynamic balance, providing experimental data for the rational application of Chinese medicine prescriptions with AN as the main component in clinical practice.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Myocytes, Cardiac/metabolism* ; Mice ; Rats ; Male ; Mitochondrial Dynamics/drug effects* ; Ventricular Remodeling/drug effects* ; Astragalus Plant/chemistry* ; Mice, Inbred C57BL ; Rhizome/chemistry* ; Panax notoginseng/chemistry* ; Rats, Sprague-Dawley ; Natriuretic Peptide, Brain/genetics* ; Humans ; Angiotensin II ; Astragalus propinquus

The aim of this study was to investigate the effect of edaravone (Eda) on the balance of mitochondrial fusion and fission in Parkinson's disease (PD) cell model. A cell model of PD was established by treating PC12 cells with 500 μmol/L 1-methyl-4-phenylpyridinium (MPP). Thiazole blue colorimetry (MTT) was used to detect the effect of different concentrations of Eda on the survival rate of PC12 cells exposed to MPP. The mitochondrial morphology was determined by laser confocal microscope. Western blot was used to measure the protein expression levels of mitochondrial fusion- and fission-related proteins, including OPA1, MFN2, DRP1 and Fis1. The results showed that pretreatment with different concentrations of Eda antagonized MPP-induced PC12 cell damage in a dose-dependent manner. The PC12 cells treated with MPP showed mitochondrial fragmentation, up-regulated DRP1 and Fis1 protein expression levels, and down-regulated MFN2 and OPA1 protein expression levels. Eda could reverse the above changes in the MPP-treated PC12 cells, but did not affect Fis1 protein expression. These results suggest that Eda has a protective effect on the mitochondrial fusion disruption induced by MPP in PC12 cells. The mechanism may be related to the up-regulation of OPA1/MFN2 and down-regulation of DRP1.
1-Methyl-4-phenylpyridinium ; Animals ; Dynamins ; Edaravone ; pharmacology ; GTP Phosphohydrolases ; Mitochondria ; drug effects ; Mitochondrial Dynamics ; Mitochondrial Proteins ; PC12 Cells ; Parkinson Disease ; Rats ; Up-Regulation

OBJECTIVE: To examine the effects of ethyl pyruvate (EP) on mitochondrial dynamics and cell apoptosis in lipopolysaccharide (LPS)-induced human kidney-2 (HK-2) cells. METHODS: HK-2 cells were divided into three groups: HK-2 cells were challenged with LPS (800 μg/L) for 24 hours as LPS group, or LPS mixed with EP (0.25 mmol/L) for 24 hours as EP group. Cells were incubated with normal saline for 24 hours as control group. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intracellular adenosine triphosphate (ATP) were detected by enzyme linked immunosorbent assay (ELISA). JC-1 staining and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assays were used to evaluate mitochondrial membrane potential and cell apoptosis, respectively. Western Blot was used to evaluate the protein expressions of mitochondrial dynamics, including death-associated protein kinase 2 (DAPK-2), mitofusin (Mfn-1 and Mfn-2), and apoptotic associated biomarkers, including caspase-3, caspase-9, Bcl-2, Bcl-xL, cytochrome C (Cyt C), and DNA repair enzyme poly ADP-ribose polymerase (PARP). RESULTS: Compared with the NC group, MDA, IL-6, TNF-α of LPS group were significantly increased, the expression of SOD, mitochondrial membrane potential and ATP level were significantly decreased, the expression of mitochondrial fission protein DAPK-2 was significantly increased, and mitochondrial fusion proteins Mfn-1 and Mfn-2 were significantly decreased, cell apoptosis and apoptotic protein caspase-3, caspase-9 and Cyt C were increased, and anti-apoptotic protein Bcl-2, Bcl-xL, PARP were significantly decreased. Compared with the LPS group, the oxidative activities and inflammatory factors above were inhibited in EP group [MDA (μmol/L): 12.35±2.21 vs. 45.95±1.76, SOD (kU/L): 54.68±1.42 vs. 40.73±1.60, IL-6 (ng/L): 67.87±2.61 vs. 338.92±20.91, TNF-α (ng/L): 19.23±1.80 vs. 180.69±6.51], mitochondrial membrane potential and ATP level were significantly increased [mitochondrial membrane potential: (99.43±0.25)% vs. (69.40±0.75)%, ATP (×10⁶ RLU): 0.19±0.01 vs. 0.12±0.05], the expression of mitochondrial fission protein was significantly decreased (DAPK-2/β-actin: 0.03±0.01 vs. 0.61±0.02), mitochondrial fusion proteins were significantly increased (Mfn-1/β-actin: 0.43±0.04 vs. 0.17±0.01, Mfn-2/β-actin: 0.201±0.004 vs. 0.001±0.001), percentage of cell apoptosis was significantly decreased [(5.25±0.17)% vs. (34.42±0.64)%], the expressions of apoptotic proteins were significantly decreased (caspase-3/β-actin: 0.25±0.15 vs. 1.76±0.01, caspase-9/β-actin: 0.09±0.02 vs. 1.52±0.12, Cyt C/β-actin: 0.001±0.001 vs. 0.350±0.030), and the expressions of anti-apoptotic proteins and PARP were significantly increased (Bcl-2/β-actin: 0.500±0.010 vs. 0.009±0.004, Bcl-xL/β-actin: 0.550±0.010 vs. 0.009±0.001, PARP/β-actin: 0.94±0.01 vs. 0.16±0.13), with statistically significant differences (all P < 0.05). CONCLUSIONS There are enhanced mitochondrial fission and diminished mitochondrial fusion in LPS-induced HK-2 cells. EP can protect mitochondria functions by regulate mitochondrial dynamics, and reducethe apoptosis of LPS-induced HK-2 cells.
Apoptosis ; Humans ; Kidney ; Lipopolysaccharides ; Mitochondrial Dynamics/drug effects* ; Protective Agents/pharmacology* ; Pyruvates/pharmacology*

Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.
Adenine ; administration & dosage ; analogs & derivatives ; Apoptosis ; drug effects ; Autophagy ; drug effects ; genetics ; CDC2 Protein Kinase ; Cell Cycle ; drug effects ; genetics ; Cell Division ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin B ; biosynthesis ; Cyclin-Dependent Kinases ; Gene Expression Regulation ; drug effects ; Humans ; Membrane Proteins ; biosynthesis ; Microtubule-Associated Proteins ; biosynthesis ; Mitochondrial Dynamics ; drug effects ; genetics ; Mitochondrial Proteins ; biosynthesis ; Neuroblastoma ; Signal Transduction ; drug effects

OBJECTIVETo investigate the changes of mitochondrial distribution in axon/soma and the expression of mitochondrial fission 1 (Fis1) protein in the cortical neurons of rats with chronic fluorosis.

METHODSSixty SD rats were divided into 3 groups (20 each) according to weight hierarchy and fed with different concentrations of fluoride in drinking water (0, 10 and 50 mg/L, respectively) for 6 months. Images of mitochondria and tubulin labeled by immunofluorescence COXIV and tubulin-α were captured with fluorescence microscope. Fis1 protein expression in cortical neurons was analyzed with immunohistochemistry and Western blot. The expression of Fis1 mRNA was detected with real-time PCR.

RESULTSVarying degrees of dental fluorosis and increased fluoride contents in urine were observed in the rats receiving additional fluoride in drinking water. In the cortical neurons of rats fed with 10 mg/L and 50 mg/L fluoride, the numbers of neuronal soma stained with COXIV(34.8 ± 4.7 and 39.3 ± 3.0, respectively), and the expression of Fis1 protein (immunohistochemistry: 54.0 ± 3.6 and 51.3 ± 4.1, respectively; Western blot: 2.9 ± 0.4 and 2.6 ± 0.6, respectively) and mRNA (3773 ± 1292 and 1274 ± 162, respectively) was markedly increased as compared with controls (4.4 ± 2.3, 25.2 ± 2.5, 1.8 ± 0.2 and 277 ± 73) over the experimental period of 6 months.

CONCLUSIONSExcessive intake of fluoride results in an altered mitochondrial distribution in axon and soma in cortical neurons (i.e., the increase in soma and the decrease in axon), increased expression of Fis1 gene and enhanced mitochondrial fission. The altered mitochondrial distribution may be related to the high expression level of Fis1 and a functional disorder of mitochondria.

Animals ; Axons ; pathology ; Cerebral Cortex ; metabolism ; Drinking Water ; adverse effects ; chemistry ; Electron Transport Complex IV ; metabolism ; Female ; Fluorides ; adverse effects ; urine ; Fluorosis, Dental ; etiology ; metabolism ; pathology ; Male ; Mitochondria ; pathology ; Mitochondrial Dynamics ; drug effects ; Mitochondrial Proteins ; genetics ; metabolism ; Neurons ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tubulin ; metabolism