Effect of Yixintai on Mitochondrial Fission Proteins Fis1 and Mff in Rat Model of Chronic Heart Failure / 中国实验方剂学杂志

ObjectiveTo study the effect and mechanism of Yixintai on mitochondrial fission proteins in the rat model of chronic heart failure. MethodTen of 60 SD rats were randomly selected as the sham operation group， and the remaining 50 rats were subjected to ligation of the left anterior descending coronary artery for the modeling of heart failure post myocardial infarction. The successfully modeled rats were randomized into model， low-， medium-， and high-dose （1.4， 2.8， and 5.6 g·kg-1， respectively） Yixintai， and trimetazidine （10 mg·kg-1） groups. The rats were administrated with corresponding doses of drugs by gavage， and the rats in the model group and sham operation group were given an equal volume of normal saline by gavage for 28 consecutive days. Enzyme-linked immunosorbent assay （ELISA） was then employed to measure the levels of amino-terminal pro-B-type natriuretic peptide （NT-pro BNP）， B-type natriuretic peptide （BNP）， and adenosine triphosphate （ATP） in the serum. Color Doppler ultrasound imaging was conducted to examine the cardiac function indicators. Hematoxylin-eosin staining and Masson staining were conducted to observe the pathological changes in the heart， and Image J was used to calculate collagen volume fraction （CVF）. Transmission electron microscopy was employed to observe the ultrastructural changes of myocardial cells. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to measure the apoptosis rate of myocardial cells. Western blot was employed to determine the protein levels of mitochondrial fission protein 1 （Fis1） and mitochondrial fission factor （Mff） in the outer mitochondrial membrane of the myocardial tissue. ResultCompared with the sham operation group， the model group showed elevated levels of NT-pro BNP and BNP in the serum， decreased ATP content， left ventricular ejection fraction （LVEF）， and left ventricular fraction shortening （LVFS）， increased left ventricular end-diastolic diameter （LVIDd） and left ventricular end-systolic diameter （LVIDs）， disarrangement of myocardial cells， inflammatory cell infiltration， increased collagen fibers and CVF， damaged myocardium and mitochondria， and increased apoptosis rate of myocardial cells， and up-regulated expression of Fis1 and Mff in the cardiac tissue （P<0.01）. Compared with the model group， different doses of Yixintai and trimetazidine lowered the serum levels of NT-pro BNP and BNP （P<0.05）， increased the ATP content （P<0.05）， increased LVEF and LVFS （P<0.01）， decreased LVIDd and LVIDs （P<0.01）. Moreover， the drugs alleviated the myocardial inflammatory damage and fibrosis， reduced CVF （P<0.01）， repaired the myocardial mitochondrial structure， and decreased the apoptosis rate of myocardial cells （P<0.01）. Medium- and high-dose Yixintai and trimetazidine down-regulated the expression of Fis1 and Mff in the myocardial tissue （P<0.05）. ConclusionYixintai can improve mitochondrial structure， reduce myocardial cell apoptosis， and improve cardiac function by inhibiting the expression of Fis1 and Mff in the myocardial tissue.

Effect of Linggui Zhugantang on Mitochondrial Fusion-fission and Sirt3/ AMPK Signaling Pathway in Chronic Heart Failure Rats After Myocardial Infarction / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg-1） and Captopril group （0.002 57 g·kg-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.

Mutual promotion of mitochondrial fission and oxidative stress contributes to mitochondrial-DNA-mediated inflammation and epithelial-mesenchymal transition in paraquat-induced pulmonary fibrosis / 世界急诊医学杂志（英文）

@#BACKGROUND: Pulmonary fibrosis (PF) is one of the main causes of death in patients with paraquat (PQ) poisoning. This study aimed to evaluate the relationship between mitochondrial fission and oxidative stress in PQ-induced epithelial-mesenchymal transition (EMT) and PF. METHODS: C57BL/6 mice and MLE-12 cells were exposed to PQ to construct a PF model in vivo and in vitro. Histological changes in the lungs were examined by hematoxylin and eosin (H&E) staining. Mitochondrial morphology was detected by MitoTracker® Deep Red FM or transmission electron microscopy (TEM). Western blotting and immunofluorescence were used to determine the expression of protein. The migration ability of the cells was detected by the cell scratch test. Mitochondrial DNA (mtDNA) levels were assessed by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect cytokine levels. Superoxide dismutase (SOD) activity and the levels of glutathione (GSH) and malondialdehyde (MDA) were detected by chemichromatometry. RESULTS: PQ exposure caused EMT and PF in vivo and in vitro. PQ destroyed mitochondrial structure and enhanced the expression of dynamin-related protein 1 (Drp1), which were accompanied by oxidative stress. Inhibiting mitochondrial fission using mitochondrial division inhibitor-1 (Mdivi-1), a selective inhibitor of Drp1, attenuated PQ-induced EMT and oxidative damage. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, reduced Drp1 expression, attenuated mitochondrial structure damage and inhibited PQ-induced EMT and PF. Both Mdivi-1 and NAC treatment markedly suppressed mtDNA release, the expression of Toll-like receptor 9 (TLR9) and phosphorylation (P)-NF-κB p65 as well as cytokines (interleukin 6 [IL-6], interleukin-1β [IL-1β], and tumor necrosis factor-α [TNF-α]) production. CONCLUSION: Mutual promotion of mitochondrial fission and oxidative stress contributes to EMT in PQ-induced PF, which is associated with the mtDNA/TLR9/NF-κB pathway.

Piroctone olamine disrupts mitochondrial dynamics in glioma cells through the PI3K/AKT pathway / 南方医科大学学报

OBJECTIVE@#To investigate the growth-inhibitory and pro-apoptotic effects of piroctone olamine (PO) on glioma cells and explore the underlying mechanism.@*METHODS@#Human glioma cell lines U251 and U373 were treated with PO and the changes in cell proliferation were detected using CCK-8 assay and EdU assay. Clone formation assay and flow cytometry were used to examine the changes in clone formation ability and apoptosis of the treated cells. Mitochondrial membrane potential of the cells and morphological changes of the mitochondria were detected using JC-1 staining and a fluorescence probe, respectively. The expressions of mitochondrial fission protein DRP1 and the fusion protein OPA1 were determined with Western blotting. Transcriptome sequencing and differential gene enrichment analysis was performed, and the expression levels of PI3K, AKT and p-AKT in the treated cells were verified using Western blotting.@*RESULTS@#CCK-8 assay showed that PO significantly inhibited the proliferation of U251 and U373 cells in a time- and dose-dependent manner (P < 0.001). EdU test showed that the proliferative activity of PO-treated cells was significantly decreased, and the number of cell colonies also decreased significantly (P < 0.01). PO treatment significantly increased apoptotic rates (P < 0.01), decreased mitochondrial membrane potential and caused obvious changes in mitochondrial morphology of the cells. Pathway enrichment analysis showed that the down-regulated genes were significantly enriched in the PI3K/AKT pathway, which was verified by Western blotting showing significantly down-regulated expression levels of PI3K, AKT and p-AKT in PO-treated cells (P < 0.05).@*CONCLUSION@#PO interferes with mitochondrial fusion and fission function through the PI3K/AKT pathway, thereby inhibiting the proliferation and increasing apoptosis of glioma cells.

Mitofusin 2 and ischemia-reperfusion injury / 器官移植

Mitochondria is one of the important organelles, which is composed of outer mitochondrial membrane and inner mitochondrial membrane. Mitochondrial structure and function are regulated by mitochondrial dynamics. Mitochondrial fusion- and fission-related proteins may participate in the process of mitochondrial fusion and fission, mediate mitochondrial dynamics, thereby regulating cell structure, function and energy metabolism. Mitofusin (MFN) 2, a protein located on the outer mitochondrial membrane of mammalian, has guanosine triphosphatase activity, which may mediate mitochondrial fusion, participate in mitophagy, formation of mitochondria-associated endoplasmic reticulum membrane and apoptosis, and significantly affect the incidence and development of ischemia-reperfusion injury (IRI). In this article, the structure, regulation, function of MFN2 and its role in IRI were reviewed, and the relationship between MFN2 and IRI and underlying mechanism were investigated, aiming to provide novel targets and ideas for the prevention and treatment of IRI.

Changes of mitochondrial fission and fusion after myocardial injury in cardiac arrest rats / 中华急诊医学杂志

Objective:To investigate the dynamic changes of mitochondrial fission and fusion in the heart of cardiac arrest (CA) rats after return of spontaneous circulation (ROSC), and to explore the role of mitochondrial fission and fusion in the myocardial injury after ROSC.Methods:Healthy male SD rats were randomly random number assigned into the post-resuscitation (PR) 4 h ( n=12), PR 24 h ( n=12), PR 72 h ( n=12), and sham groups ( n=6). The rat CA model was induced by asphyxia, and cardiopulmonary resuscitation (CPR) was performed 6 min after CA. The protein expressions of mitochondrial Drp1, Fis1, Mfn1, and Opa1 were determined by Western blot in each group at 4, 24 and 72 h after ROSC. The mRNA expressions of Drp1, Fis1, Mfn1, and Opa1 were determined by RT-PCR. Myocardial ATP content and mitochondrial respiratory function were measured. The histopathologic changes of myocardial tissue were observed under light microscope. One-way analysis of variance (ANOVA) was use to compare quantitative data, and LSD- t test was used for comparison between groups. Results:Compared with the sham group, the protein and mRNA expressions of Drp1 and Fis1 were increased (all P<0.05) and the protein and mRNA expressions of Mfn1 and Opa1 were decreased (all P<0.05) in the PR 4 h and PR 24 h groups. However, there were no statistical differences in the protein and mRNA expressions of Drp1, Fis1, Mfn1, and Opa1 in the PR 72 h group compared with the sham group (all P>0.05). Compared with the sham group, the levels of ATP content [(4.53±0.76) nmol/g protein vs. (8.57±0.44) nmol/g protein and (5.58±0.58) nmol/g protein vs. (8.57±0.44) nmol/g protein] and mitochondrial respiratory control rate [(2.47±0.38) vs. (3.45±0.32) and (2.97±0.24) vs. (3.45±0.32)] were obviously decreased in the PR 4 h and PR 24 h groups (all P<0.05). There were no statistically significant differences in the ATP content [(7.73±0.95) nmol/g protein vs. (8.57±0.44) nmol/g protein] and mitochondrial respiratory control ratio [(3.39±0.34) vs. (3.45±0.32)] between the PR 72 h group and the sham group (all P>0.05). The pathological damage of myocardial tissue was obvious in the PR 4 h group, and was improved significantly in the PR 72 h group. Conclusions:The imbalance of mitochondrial fission and fusion is probably involved in the pathological process of myocardial injury after ROSC, which may be related to mitochondrial dysfunction.

Astaxanthin attenuates contrast-induced acute kidney injury in rats by inhibiting dynamin-related protein 1-mediated mitochondrial fission through activating SIRT1 pathway / 中华肾脏病杂志

Objective:To investigate whether astaxanthin (AST) down-regulates dynamin-related protein 1 (Drp1) through activating the silent mating type information regulation 2 homolog-1 (SIRT1) signaling pathway, thereby attenuating contrast-induced acute kidney injury.Methods:Forty adult male Sprague-Dawley rats weighing 160-180 g were randomly divided into five groups: sham surgery group (Sham group), contrast medium injury group (CM group), astaxanthin-intervention group (AST+CM group), SIRT1 inhibitor Ex527 intervention group (Ex527+CM group), and astaxanthin combined with Ex527 intervention group (AST+Ex527+CM group). After 72 hours of modeling, heart blood was removed and kidney tissues were collected for follow-up testing. Serum creatinine (Scr), blood urea nitrogen (BUN), and oxidative stress-related indexes total superoxide dismutase (T-SOD) and malondialdehyde (MDA) were measured by biochemistry; hematoxylin and eosin staining was performed to observe the pathological changes in the kidney; mitochondrial morphology and number were observed by transmission electron microscopy; reactive oxygen species (ROS) levels were detected by ROS staining in frozen sections; TUNEL staining was performed to detect apoptosis level. The expression levels of SIRT1, p53, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), Drp1 and apoptosis-related proteins Bcl-2 and Bax were detected by Western blotting.Results:(1) Compared with the CM group, Scr and BUN level were significantly lower, T-SOD level was higher and MDA level was lower in the AST+CM group, while T-SOD level decreased and MDA level increased after the combination of Ex527 (all P<0.05). (2) ROS expression was lower in the AST+CM group compared to the CM group and higher after the combination of Ex527 (both P<0.05). (3) The number of apoptotic cells was significantly reduced in the AST+CM group compared to the CM group and increased after the combination of Ex527 (both P<0.05). (4) The protein expression levels of SIRT1, PGC-1α and Bcl-2 were increased and the protein expression levels of p53, Drp1 and Bax were decreased (all P<0.05) in the AST+CM group compared with the CM group, and the protein expression levels of SIRT1, PGC-1α and Bcl-2 were decreased and the protein expression levels of p53, Drp1 and Bax were increased when Ex527 was combined (all P<0.05). Conclusion:Astaxanthin can inhibit Drp1-mediated mitochondrial fission by activating the SIRT1 pathway, thereby reducing contrast-induced acute kidney injury in rats.

Effect of Zishen Huoxue Prescription on Regulation of Mitophagy and Neurogenesis in Hippocampal CA1 Region of Vascular Dementia Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Zishen Huoxue prescription on promoting neurogenesis in hippocampal CA1 region of vascular dementia （VD） rats by regulating mitophagy. MethodThe 2-VO method was used to establish the VD rat model and 60 SD rats were randomly divided into sham operation group， model group， donepezil hydrochloride group， and Zishen Huoxue prescription low-dose（8.9 g·kg-1）， medium-dose（17.8 g·kg-1） and high-dose（35.6 g·kg-1） groups. Morris water maze test was performed to detect the escape latency and the number of crossing platform in each group. The expression of phosphatase and tensin homology-induced kinase 1 （PINK1） and Parkinson protein （Parkin） mRNA in hippocampal CA1 region was detected by Real-time fluorescent quantitative polymerase chain reaction（Real-time PCR）. Western blot was used to determine the expression of mitochondrial autophagy signaling pathway-related proteins Parkin， prohibitin 2 （PHB2）， mitofusin 2 （Mfn2） and dynamin-related protein 1 （Drp1） in hippocampal CA1 region. The neurogenesis in hippocampal CA1 region was tested by Brdu method. ResultCompared with the conditions in the sham operation group， the learning and spatial memory abilities of the model group were decreased （P<0.05）， with damaged mitochondrial structure and autolysosome formation in the hippocampal CA1 region. The expressions of Parkin， Pink1 mRNA and Parkin， PHB2， and Drp1 proteins were up-regulated （P<0.05）， while the expression of Mfn2 protein and the neuronal regeneration in hippocampal CA1 region were reduced （P<0.05， P<0.05）. Compared with the conditions in the model group， Zishen Huoxue prescription enhanced the learning and spatial memory abilities of VD rats （P<0.05）， increased the number of autophagosomes in hippocampal CA1 region and improved the mitochondrial structure. The expression of Parkin， Pink1 mRNA and Parkin， PHB2， and Drp1 proteins in hippocampal CA1 region was up-regulated （P<0.05， P<0.01）while the expression of Mfn2 protein was down-regulated（P<0.05， P<0.01）. The number of new neurons in hippocampal CA1 region was also increased（P<0.05， P<0.01）. ConclusionThe promoting effect of Zishen Huoxue prescription on the neurogenesis in hippocampal CA1 region of VD rats was related to the mitophagy mediated by Pink1/Parkin signaling pathway.

Endogenous HMGB1 Modulates Fatty Acid Metabolism of Hypoxic and Nutrient-poor Pancreatic Cancer Cells and Related Mechanism / 肿瘤防治研究

Objective To explore the role and mechanism of HMGB1 in the fatty acid metabolism reprogramming and mitochondrial fusion/fission of hypoxic and nutrient-poor pancreatic cancer cells. Methods The correlation between the expression level of HMGB1 in pancreatic cancer tissue and the survival rate of pancreatic cancer patients were analyzed by GEPIA database. CCK-8 assay was used to measure cell proliferation rate, and scratch test and Transwell chamber method were carried out to detect the effects of endogenous HMGB1 on the invasion and migration abilities of human pancreatic cancer cell line Patu8988 after hypoxic and nutrient-poor treatment. Laser confocal microscope was used to observe the changes of mitochondrial morphology of Patu8988 cells. Western blot was used to detect the expression levels of mitochondrial fusion/fission and de novo fatty acid synthesis-related proteins. Results GEPIA database analysis results showed that HMGB1 was highly expressed in pancreatic cancer tissues (P < 0.01), and the expression level was negatively correlated with the survival time of pancreatic cancer patients (P=0.00097). Knockdown of HMGB1 expression could inhibit the proliferation, invasion and migration abilities of Patu8988 cells under hypoxic and nutrient-poor conditions. However, mitochondrial fission in patu8988 cells was increased. Knockdown of HMGB1 in Patu8988 cells increased the expression of fission-related protein FIS1 while decreased the expression of p-DRP1(Ser637) and fusion-related protein MFN1 and MFN2 in hypoxic and nutrient-poor environment; ACLY, p-ACLY and FASN protein expression levels were down-regulated. Conclusion Endogenous HMGB1 can promote the fusion and inhibit the fission of mitochondria in hypoxic and nutrient-poor Patu8988 cells, maintain mitochondrial morphology and function, and thereby up-regulate ACLY protein expression and phosphorylation level, promote FA synthesis, and maintain the proliferation, invasion and migration abilities of pancreatic cancer cells.

Low-Temperature Extracts of Purple Blossoms of Basil (Ocimum Basilicum L) Intervened Mitochondrial Translocation Contributes Prompted Apoptosis in Human Breast Cancer Cells

BACKGROUND: The preventive and therapeutic medical utilization of this plant is an age-long practice across the globe. This study aimed to validate the impact of dark purple blossoms of basil (Ocimum basilicum L.) aqueous extract at low temperature (0 °C) mediated mitochondrial fission contributed to induced apoptosis in human breast cancer cells. METHODS: Fresh blossoms were extracted at low temperature (0 °C) using a watery solvent. Human MCF7 breast cancer cells were then treated with 3 separate fluctuated concentrations of 0, 50, 150 and 250 µg/mL for 24 and 48 h. RESULTS: The outcomes demonstrated the presence of anthocyanins, anthraquinones, tannins, reducing sugars, glycosides, proteins, amino acids, flavonoids and volatile oils and nonappearance of Terpinoids and alkaloids. Contrastingly, frail presence of steroids in basil blossoms aqueous concentrate was noted. In addition, the results from a phytochemical subjective examination of basil (Ocimum basilicum L.) blossoms aqueous extract demonstrated that most of the credited natural impacts containing more remarkable contents of antioxidants and anticancer compounds in basil blossoms aqueous extract. Moreover, the restraint of glucose take-up was alleviated mediated by a dose-dependent manner in MCF7 cells with basil (Ocimum basilicum L.) blossoms aqueous extract inducted for 24 h, resulting in mitochondrial fission. CONCLUSION: This is the first study that shows the impact of the aqueous extract of basil (Ocimum basilicum L.) blossoms was extracted at low temperature (0°C/6 h) underlined high amounts of flavonoids and phenolic compounds bearing more anticancer and antioxidant activities compared to another aqueous extract (using boiled water solvent) and alcoholic extracts.

Effect of Ferulic Acid on Learning and Memory Impairment by the Repairing of Mitochondrial Fission-Fusion Imbalance in AD Mice / 中国药学杂志

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.

LRRK2 Kinase Activity Induces Mitochondrial Fission in Microglia via Drp1 and Modulates Neuroinflammation

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD). LRRK2 contains a functional kinase domain and G2019S, the most prevalent LRRK2 pathogenic mutation, increases its kinase activity. LRRK2 regulates mitochondria morphology and autophagy in neurons. LPS treatment increases LRRK2 protein level and mitochondrial fission in microglia, and down-regulation of LRRK2 expression or inhibition of its kinase activity attenuates microglia activation. Here, we evaluated the direct role of LRRK2 G2019S in mitochondrial dynamics in microglia. Initial observation of microglia in G2019S transgenic mice revealed a decrease in mitochondrial area and shortage of microglial processes compared with their littermates. Next, we elucidated the molecular mechanisms of these phenotypes. Treatment of BV2 cells and primary microglia with LPS enhanced mitochondrial fission and increased Drp1, a mitochondrial fission marker, as previously reported. Importantly, both phenotypes were rescued by treatment with GSK2578215A, a LRRK2 kinase inhibitor. Finally, the protein levels of CD68, an active microglia marker, Drp1 and TNF-α were significantly higher in brain lysates of G2019S transgenic mice compared with the levels in their littermates. Taken together, our data suggest that LRRK2 could promote microglial mitochondrial alteration via Drp1 in a kinase-dependent manner, resulting in stimulation of pro-inflammatory responses. This mechanism in microglia might be a potential target to develop PD therapy since neuroinflammation by active microglia is a major symptom of PD.

Expression of MFF and its biological effects in hepatocellular carcinoma / 国际肿瘤学杂志

Objective To evaluate the expression of mitochondrial fission factor (MFF) and its biological effects in the progression of hepatocellular carcinoma (HCC).Methods ①Quantitative real-time PCR (qPCR),Western blotting and immunohistochemistry analysis were used to detect the expression levels of MFF in HCC tumor tissues and cell lines.②The effect of MFF knockdown on proliferation of HCC cells was analyzed by methyl thiazolyl tetrazolium (MrTT) and colony formation assays in siCtrl,si-MFF#1,si-MFF#2 groups.③The effect of MFF knockdown on apoptosis of HCC cells was analyzed by apoptosis assay with Annexin Ⅴ-FITC and PI.Results ①The MFF expression was higher in tumor tissues compared with tumor-adjacent normal tissues [mRNA level M(QR):0.292 (0.443) vs.0.235(0.333),Z=-4.166,P＜0.001;protein level M(QR):5.414 (4.545) vs.3.120 (3.955),Z =-3.961,P ＜ 0.001)].The MFF expression was higher in HCC cell lines compared with normal liver cell line.②RNA interference-mediated knockdown of MFF inhibited proliferation of HCC cells (siCtrl vs.si-MFF#1:5.29 ± 0.34 vs.3.34 ± 0.37,P =0.014;siCtrl vs.si-MFF#2:5.29 ± 0.34 vs.3.09 ± 0.40,P =0.010).RNA interference-mediated knockdown of MFF inhibited colony formation of HCC cells (siCtrl vs.si-MFF#1:95.35 ± 21.20 vs.37.56 ± 10.61,P =0.003;siCtrl vs.si-MFF#2:95.35 ± 21.20 vs.41.23 ± 10.82,P =0.004).③RNA interference-mediated knockdown of MFF induced apoptosis of HCC cells (siCtrl vs.si-MFF#1:9.56％ ± 1.70％ vs.20.08％ ± 2.03％,P ＜ 0.001;siCtrl vs.si-MFF#2:9.56％ ± 1.70％ vs.21.14％ ± 1.38％,P ＜ 0.001).Conclusion MFF is overexpressed in HCC,which accelerates cell proliferation and suppresses apoptosis,indicating that MFF can serve as a potential oncogene and drug target in HCC treatment.

Induction effect of myricetin on apoptosis of human ovarian cancer SKOV3 cells by promoting DRP1-dependent mitochondrial fission / 吉林大学学报(医学版)

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.

Effects of electroacupuncture on mitochondrial fusion-fission during endotoxin-induced acute lung injury in rabbits / 中华麻醉学杂志

Objective To evaluate the effect of electroacupuncture (EA) on mitochondrial fusionfission during endotoxin-induced acute lung injury (ALI) in rabbits.Methods Sixty clean-grade New Zealand white rabbits,aged 2 months,weighing 1.5-2.0 kg,were divided into 4 groups (n=15 each) using a random number table:control group (group C),ALI group,EA at non-acupoint plus ALI group (group SEAM+ALI) and EA at acupoint plus ALI group (group EAM+ALI).Endotoxin-induced ALI was induced by injecting lipopolysaccharide 5 mg/kg (in 2 ml of 0.9％ normal saline) via the auricular vein in ALI,SEAM+ALI and EAM+ALI groups.At 4,3,2 and 1 days and 30 min before lipopolysaccharide injection,bilateral Zusanli (ST36) and Feishu (BL13) acupoints were stimulated with an electric stimulator for 30 min in group EAM+ALI,and EA was performed for 30 min at the points 1 cm lateral to the acupoints of Zusanli and Feishu in group SEAM+ALI.The animals were sacrificed at 6 h after lipopolysaccharide injection and the lungs were removed for microscopic examination of the pathological changes (with a light microscope) and mitochondrial ultrastructure (with an electron microscope) and for determination of ROS and ATP contents and expression of Mfn1,Mfn2,optic atrophy 1 (OPA1) and dynamin-related protein 1 (Drp1) protein and mRNA in lung tissues (by real-time polymerase chain reaction or Western blot).Results Compared with group C,ATP content was significantly decreased,ROS content was increased,the expression of Mfn1,Mfn2 and OPA1 protein and mRNA was down-regulated,and the expression of Drp1 protein and mRNA was up-regulated in ALI,SEAM +ALI and EAM +ALI groups (P＜ 0.05).Compared with group ALI,ATP content was significantly increased,the expression of Mfn1,Mfn2 and OPA1 protein and mRNA was up-regulated,and the expression of Drp1 protein and mRNA was down-regulated in SEAM+ ALI and EAM+ALI groups,and ROS content was decreased in group EAM+ALI (P＜0.05).Compared with group SEAM+ALI,ATP content was significantly increased,ROS content was decreased,the expression of Mfn1,Mfn2 and OPA1 protein and mRNA was up-regulated,and the expression of Drp1 protein and mRNA was down-regulated in group EAM+ALI (P＜0.05).Conclusion The mechanism by which EA mitigates endotoxin-induced ALI may be related to promoting mitochondrial fusion and inhibiting mitochondrial fission in rabbits.

Relationship between p38MAPK signaling pathway and lipopolysaccharide-induced mitochondrial fission in alveolar epithelial cells: an in vitro experiment / 中华麻醉学杂志

Objective To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) signaling pathway and lipopolysaccharide (LPS)-induced mitochondrial fission in alveolar epithelial cells using an in vitro experiment.Methods The cultured alveolar epithelial cells were subcultured and seeded in 96-well plates at the density of 2 × 105 cells/ml (200 μl/well).The cells were divided into 4 groups (n=10 each) when cell confluence reached 80％ using a random number table method:control group (group C),LPS group,LPS+SB203580 group (group LPS+SB) and LPS+dimethyl sulfoxide (DMSO) group.Cells were incubated with LPS 10 μg/ml for 24 h in group LPS.Cells were incubated with p38MAPK inhibitor SB203580 10 μmol (dissolved in DMSO) for 1 h and then with LPS 10 μg/ml for 24 h in group LPS+SB.Cells were incubated with the equal volume of DMSO for 1 h and then with LPS 10 μg/ml for 24 h in group LPS+DMSO.Malonaldehyde (MDA) content and superoxide dismutase (SOD) activity were measured.The expression of phosphorylated p38MAPK (p-p38MAPK),heme oxygenase-1 (HO-1),dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1) was determined by Western blot.Results Compared with group C,the MDA content was significantly increased,the SOD activity was decreased,and the expression of p-p38MAPK,HO-1,FIS1 and DRP1 was up-regulated in LPS,LPS+SB and LPS+ DMSO groups (P＜0.05).Compared with group LPS,the MDA content was significantly increased,the SOD activity was decreased,the expression of p-p38MAPK and HO-1 was down-regulated,the expression of FIS1 and DRP1 was up-regulated in group LPS+SB (P＜0.05),and no significant change was found in the parameters mentioned above in group LPS+DMSO (P＞0.05).Conclusion The p38MAPK signaling pathway activation can up-regulate the expression of HO-1,thus reducing LPS-induced mitochondrial fission in alveolar epithelial cells.

Induction effect of myricetin on autophagy in SKOV3 cells and promoting effect on mitochondrial fission / 吉林大学学报(医学版)

Objective:To observe the effects of myricetin on autophagy and mitochondrial fission in the human ovarian cancer SKOV3 cells,and to explore its induction effect on autophagy and promoting effect on mitochondrial fission.Methods: The SKOV3 cells were cultured in vitro.0,20,40,and 60 g·L-1 myricetin were used in control group and low, middle, high doses of myricetin groups for 12 h.The changes of mitochondrial membrane potential were detected by flow cytometry;the morphology of mitochondria was observed by MitoTracker Red;the expression levels of dynamin related protein1 (Drp1) and fission 1 (Fis1) in various groups were detected by Western blotting method;and the expression levels of autophagy related protein LC3 were detected by both Western blotting method and immunofluorescence method.Results:Compared with control group, the ratios of decreased mitochondria in different doses of myricetin groups were significantly increased(t=3.27, t=6.85, t=5.49,P<0.05).Compared with control group, the numbers of mitochondria in different doses of myricetin groups were increased, and the mitochondria looked more like gravel.Compared with control group,the expression levels of Drp1 in different doses of myricetin groups were significantly increased (t=4.35, t=3.28, t=6.17,P<0.05), and the expression levels of Fis1 were increased also(t=8.32, t=6.74, t=9.27).The immunofluorescence results showed that the expression levels of LC3 in different doses of myricetin groups were significantly increased with the increase of myricetin dose compared with control group.The Western blotting results showed that the ratios of LC3-Ⅱ/LC3-Ⅰ in middle and high doses of myricetin groups were significantly increased compared with control group(t=3.28, t=4.21,P<0.05).Conclusion: Myricetin can induce the autophagy of SKOV3 cells, and it can also promote the mitochondrial fission.

Effects of CORM-2 on mitochondrial fission protein Fis1 in the LPS-activated lung macrophages of rats through p38MAPK signaling pathway / 中华急诊医学杂志

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.

MicroRNA-484 regulates liver fibrosis course through targeting Fisl in hepatic stellate cells / 第二军医大学学报

Objective To explore the effects of microRNA-484 (miR-484) on hepatic stellate cells (HSC's), the key cell in the occurrence and development of liver fibrosis, and to investigate the functions. Methods On the basis of our previous microarray analysis results, we transfected HSC-T6 cell lines with miR-484 inhibitor to intervene the expression of miR-484 in vitro. The expressions of mRNA and proteins related to liver fibrosis and apoptosis were detected by qPCR and Western blotting, respectively. The cell apoptosis with Annexin V-FITC/PI double staining was determined by flow cytometry. Results Compared with the control group, the mRNA and protein expression of miR 484-targeted gene Fisl and proapoptotic factor caspase-3 were both significantly up-regulated (P<0. 05), and apoptosis inhibitory factor Bcl-2 was significantly down-regulated (P

Protective effect of PEDF on H9C2 myocardial cells under hypoxia condition / 重庆医学

Objective To investigate the protective effects and possible mechanism of pigment epithelium derived faetor (PEDF) on myocardial cells H9C2 under hypoxia and serum-free condition.Methods H9C2 cells were culture in vitro and performed the hypoxia and serum-free processing.The cells were divided into the control group (H9C2),hypoxia group (hypoxia + H9C2),PEDF group(hypoxia+H9C2 +PEDF) and mitochondrial fission inhibitor(Mdivi-1) group(hypoxia+h9C2+Mivi-1).The apoptotic rate was detected by TUNNEL staining.The proteins levels of dynamin related peptide1 (Drp1) and cleaved-caspase 3 were measured by Western blot.Electron Microscopy and MitoTracker Red were used to detect the mitochondria morphology,the mitochondrial membrane potential was evaluated by cationic dye JC-1.MitoSOXTM was used to detect mitochondrial reactive oxygen species (ROS).Results Hypoxia induced mitochondrial fission(P＜0.05).The hypoxia group (6 h) and control group had statistical difference(P＜0.05).PEDF reduces mitochondrial fission under hypoxia condition(P＜0.05),which had statistical difference between the PEDF group and hypoxia group (6 h)(P＜0.05).PEDF and Mdivi-1 could decrease cell apoptosis under hypoxia condition(24 h),compared with the hypoxia group,the difference was statistically significant(P＜0.05).Conclusion PEDF decrease cell apoptosis by inhibiting H9C2 cells mitochondrial fission under hypoxia condition.