This study aimed to explore the mechanism of resveratrol(RES) pretreatment in improving mitochondrial function and alleviating myocardial ischemia-reperfusion(IR) injury by inhibiting stromal interaction molecule 2(STIM2) through microRNA-20 b-5 p(miR-20 b-5 p). Ninety rats were randomly assigned into sham group, IR group, IR+RES(50 mg·kg~(-1) RES) group, IR+RES+antagomir NC(50 mg·kg~(-1) RES+80 mg·kg~(-1) antagomir NC) group, and IR+RES+miR-20 b-5 p antagomir(50 mg·kg~(-1) RES+80 mg·kg~(-1) miR-20 b-5 p antagomir) group, with 18 rats/group. The IR rat model was established by ligation of the left anterior descending coronary artery. Two weeks before the operation, rats in the IR+RES group were intraperitoneally injected with 50 mg·kg~(-1) RES, and those in the sham and IR groups were injected with the same dose of normal saline, once a day. Ultrasonic instrument was used to detect the left ventricular internal diameter at end-diastole(LVIDd) and left ventricular internal diameter at end-systole(LVIDs) of rats in each group. The 2,3,5-triphenyte-trazoliumchloride(TTC) method and hematoxylin-eosin(HE) staining were employed to detect the myocardial infarction area and histopathology, respectively. Real-time quantitative PCR(qRT-PCR) was carried out to detect the expression of miR-20 b-5 p in myocardial tissue. Oxygen glucose deprivation/reoxygenation(OGD/R) was performed to establish an OGD/R model of H9 c2 cardiomyocytes. CCK-8 assay was employed to detect H9 c2 cell viability. H9 c2 cells were assigned into the control group, OGD/R group, OGD/R+RES group(25 μmol·L~(-1)), OGD/R+RES+inhibitor NC group, OGD/R+RES+miR-20 b-5 p inhibitor group, mimic NC group, miR-20 b-5 p mimic group, inhibitor NC group, and miR-20 b-5 p inhibitor group. Flow cytometry was employed to detect cell apoptosis. Western blot was employed to detect the expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-cysteine proteinase 3(cleaved-caspase-3), and STIM2 in cells. The mitochondrial membrane potential(MMP) assay kit, reactive oxygen species(ROS) assay kit, and adenosine triphosphate(ATP) assay kit were used to detect the MMP, ROS, and ATP levels, respectively. Dual luciferase reporter gene assay was adopted to verify the targeting relationship between miR-20 b-5 p and STIM2. Compared with the sham group, the modeling of IR increased the myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and down-regulated the expression of miR-20 b-5 p(P<0.05). These changes were alleviated in the IR+RES group(P<0.05). The IR+RES+miR-20 b-5 p antagomir group had higher myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and lower expression of miR-20 b-5 p than the IR+RES group(P<0.05). The OGD/R group had lower viability of H9 c2 cells than the control group(P<0.05) and the OGD/R+RES groups(25, 50, and 100 μmol·L~(-1))(P<0.05). Additionally, the OGD/R group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved caspase-3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the control group(P<0.05) and the OGD/R+RES group(P<0.05). The OGD/R+RES+miR-20 b-5 p inhibitor group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved-caspase 3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the OGD/R+RES group(P<0.05). miR-20 b-5 p had a targeting relationship with STIM2. The expression of STIM2 was lower in the miR-20 b-5 p mimic group than in the mimic NC group(P<0.05) and lower in the inhibitor NC group than in the miR-20 b-5 p inhibitor group(P<0.05). RES pretreatment can inhibit the expression of STIM2 by promoting the expression of miR-20 b-5 p, thereby improving the function of mitochondria and alleviating myocardial IR damage.
Animals ; Rats ; Adenosine Triphosphate ; Antagomirs/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Glucose/metabolism* ; MicroRNAs/metabolism* ; Mitochondria, Heart/drug effects* ; Myocardial Infarction/drug therapy* ; Myocardial Reperfusion Injury/drug therapy* ; Myocytes, Cardiac ; Oxygen/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism* ; Resveratrol/therapeutic use* ; Stromal Interaction Molecule 2/metabolism*

Liver is the main place of drug metabolism. Mitochondria of hepatocytes are important targets of drug-induced liver injury. Mitochondrial autophagy could maintain the healthy operation of mitochondria in cells and the stable proliferation of cells. Therefore, the use of mitochondrial autophagy to remove damaged mitochondria is an important strategy of anti-drug-induced liver injury. Active ingredients that could enhance mitochondrial autophagy are contained in many traditional Chinese medicines, which could regulate the mitochondrial autophagy to alleviate relevant diseases. However, there are only a few reports on how to accurately and efficiently identify and evaluate such components targeting mitochondria from traditional Chinese medicine. Liquid chromatography-mass spectro-metry(LC-MS) combined with serum pharmacology in vivo can be used to accurately and efficiently find active ingredients of traditional Chinese medicine acting on mitochondrial targets. This paper reviewed the research ideas and methods of traditional Chinese medicine ingredients for increasing the hepatotoxicity of mitochondrial autophagy, in order to provide new ideas and methods for the study of active ingredients of traditional Chinese medicine targeting mitochondria.
Chemical and Drug Induced Liver Injury ; Drug-Related Side Effects and Adverse Reactions ; Drugs, Chinese Herbal/toxicity* ; Humans ; Medicine, Chinese Traditional ; Mitochondria

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

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer's disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP^751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
Amyloid beta-Protein Precursor/physiology* ; Apoptosis/drug effects* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Fluorouracil/pharmacology* ; Humans ; Liver Neoplasms/drug therapy* ; Mitochondria/physiology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; bcl-X Protein/genetics*

The organosulfur compound sulforaphane (SFN; C₆H₁₁NOS₂) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.
Animals ; Antioxidants/pharmacology* ; Apoptosis/drug effects* ; Brain/ultrastructure* ; Carbon Monoxide Poisoning/metabolism* ; Cytoprotection ; Humans ; Isothiocyanates/pharmacology* ; Membrane Potential, Mitochondrial/drug effects* ; Mitochondria/metabolism* ; Sulfoxides

OBJECTIVE: To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids. METHODS: MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting. RESULTS: At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells. CONCLUSIONS In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.
3T3 Cells ; Adenosine Triphosphate ; Animals ; Apoptosis ; Cell Death ; drug effects ; Dexamethasone ; Membrane Potential, Mitochondrial ; Mice ; Microscopy, Electron ; Mitochondria ; ultrastructure ; Necrosis

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

Atractylenolide III (ATL-III), a sesquiterpene compound isolated from Rhizoma Atractylodis Macrocephalae, has revealed a number of pharmacological properties including anti-inflammatory, anti-cancer activity, and neuroprotective effect. This study aimed to evaluate the cytoprotective efficiency and potential mechanisms of ATL-III on corticosterone injured rat phaeochromocytoma (PC12) cells. Our results demonstrate that ATL-III increases cell viability and reduces the release of lactate dehydrogenase (LDH). The results suggest that ATL-III protects PC12 cells from corticosterone-induced injury by inhibiting the intracellular Ca overloading, inhibiting the mitochondrial apoptotic pathway and modulating the MAPK/NF-ΚB inflammatory pathways. These findings provide a novel insight into the molecular mechanism by which ATL-III protected the PC12 cells against corticosterone-induced injury for the first time. Our results provide the evidence that ATL-III may serve as a therapeutic agent in the treatment of depression.
Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Survival ; drug effects ; Corticosterone ; toxicity ; Inflammation Mediators ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactones ; pharmacology ; Mitochondria ; drug effects ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; NF-kappa B ; metabolism ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; Phosphorylation ; drug effects ; Rats ; Sesquiterpenes ; pharmacology ; Signal Transduction ; drug effects

Resistance to cisplatin (DDP)-based chemotherapy is a major cause of treatment failure in human gastric cancer (GC). It is necessary to identify the drugs to re-sensitize GC cells to DDP. In our previous research, Zuo Jin Wan Formula (ZJW) has been proved could increase the mitochondrial apoptosis via cofilin-1 in a immortalized cell line, SGC-7901/DDP. Due to the immortalized cells may still difficult highly recapitulate the important molecular events in vivo, primary GC cells model derived from clinical patient was constructed in the present study to further evaluate the effect of ZJW and the underlying molecular mechanism. Immunofluorescent staining was used to indentify primary cultured human GC cells. Western blotting was carried out to detect the protein expression. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation. Flow cytometry analysis was performed to assess cell apoptosis. ZJW inhibited proliferation and induced apoptosis in primary DDP-resistant GC cells. Notably, the apoptosis in GC cells was mediated by inducing cofilin-1 mitochondrial translocation, down-regulating Bcl-2 and up-regulating Bax expression. Surprisingly, the level of p-AKT protein was higher in DDP-resistant GC cells than that of the DDP-sensitive GC cells, and the activation of AKT could attenuate ZJW-induced sensitivity to DDP. These data revealed that ZJW can increase the chemosensitivity in DDP-resistant primary GC cells by inducing mitochondrial apoptosis and AKT inactivation. The combining chemotherapy with ZJW may be an effective therapeutic strategy for GC chemoresistance patients.
Adult ; Aged ; Aged, 80 and over ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; therapeutic use ; Cofilin 1 ; metabolism ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Mitochondria ; drug effects ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; pathology ; Tumor Cells, Cultured

Ageratina adenophora is a noxious plant and it is known to cause acute asthma, diarrhea, depilation, and even death in livestock (Zhu et al., 2007; Wang et al., 2017). A. adenophora grows near roadsides and degraded land worldwide (He et al., 2015b). In the areas where it grows, A. adenophora is an invasive species that inhibits the growth of local plants and causes poisoning in animals that come in contact with it (Nie et al., 2012). In China, these plants can be found in Yunnan, Sichuan, Guizhou, Chongqing, and other southwestern areas (He et al., 2015a) and they have become a dominant species in these local regions. It threatens the native biodiversity and ecosystem in the invaded areas and causes serious economic losses (Wang et al., 2017). It has been reported that A. adenophora can grow in the northeast direction at a speed of 20 km per year in China (Guo et al., 2009). Because of the damage caused by A. adenophora, it ranks among the earliest alien invasive plant species in China (Wang et al., 2017).
Adenosine Triphosphatases/metabolism* ; Ageratina/toxicity* ; Animals ; Biodiversity ; Chemical and Drug Induced Liver Injury/pathology* ; China ; DNA, Mitochondrial/genetics* ; Ecosystem ; Introduced Species ; Liver/drug effects* ; Mice ; Microscopy, Electron, Transmission ; Mitochondria, Liver/pathology* ; Plant Extracts/toxicity*