scholarly journals Neuroprotective Effects of Idebenone on hydrogen peroxide (H2O2)-induced Oxidative Damage in Retinal ganglion cell-5 (RGC-5) Cells

2019 ◽  
Author(s):  
Yuping Wang ◽  
Jing Wang ◽  
Xi Zhang ◽  
Yifan Feng ◽  
Yuanzhi Yuan
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Oxidative Damage ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Beclin 1 ◽  
Control Group ◽  
Related Protein ◽  
H2o2 Treatment ◽  
Cyt C

Abstract Purposes To investigated the neuroprotective effect of Idebenone against H2O2-induced oxidative damage in RGC-5 cells. Methods RGC-5 cells were treated with different concentrations (5, 10, 20μM) of idebenone for 12h prior to addition of 300µM H2O2 for 12 h. The apoptosis of RGC-5 cells were detected by flow cytometry. The changes of mitochondrial membrane potential were detected by JC-1 staining. The autophagy in RGC-5 cells was observed by transmission electron microscopy, and the expression level of autophagy-related protein light chain3, Beclin-1 and mitochondrial membrane potential-related protein Cyt-c in RGC-5 cells were measured by Western blot analysis. Results Flow cytometry showed that the apoptosis rates in control group, H2O2 group and H2O2-treatment with Idebenone pretreatment groups were (6.48±0.55)%, (27.34±0.51)%, (22.88±0.52)%, (15.45±0.81)%, (12.59±0.58)%, respectively(F = 559.7, P <0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, while which was decreased in H2O2-treatment with Idebenone pretreatment groups. After incubation of RGC-5 cells with H2O2, the mitochondrial membrane potential was significantly decreased, while idebenone could prevent the decrease of MMP. Contrast with control group, LC3 II /I, the expression levels of Beclin-1 and Cyt-c in H2O2 group increased significantly(P<0.05); while contrast with H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c in H2O2-treatment with Idebenone pretreatment groups was significantly decreased(P<0.05). Conclusion Idebenone may have protective effects on RGC-5 cells suffering from oxidative damage induced by H2O2 through improving antioxidant capacity, reducing mitochondrial membrane potential decline and the activity of autophagy.

Beclin 1 Influences Cisplatin-Induced Apoptosis in Cervical Cancer CaSki Cells by Mitochondrial Dependent Pathway

2012 ◽  
Vol 22 (7) ◽  
pp. 1118-1124 ◽  
Author(s):  
Yang Sun ◽  
Jia-hua Liu ◽  
Long Jin ◽  
Ling Pan ◽  
Yu-xia Sui ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Messenger Rna ◽  
Mitochondrial Membrane ◽  
Beclin 1 ◽  
Control Group ◽  
Induced Apoptosis ◽  
Dependent Pathway

PurposeTo investigate the role of Beclin 1 expression on the cisplatin-induced apoptosis in cervical cancer CaSki cells and to explore the potential mechanism underlying this effect.Materials and MethodsAfter overexpression or partial silencing of Beclin 1 in cervical cancer CaSki cells, the transfected group and the control group were treated with cisplatin for 24 hours. The percentage of apoptotic cells were assessed by flow cytometry. The mitochondrial membrane potential and activities of caspase-8/9/3 were detected by JC-1 fluorescence staining and colorimetry. The expression of cytochrome c was measured using a Western blot. The messenger RNA expression of Bax and Bcl-2 were detected by real-time quantitative reverse transcription polymerase chain reaction.ResultsExpression of Beclin 1 protein was up-regulated in overexpressed transfectants of CaSki cells. After treatment with cisplatin, the Beclin 1 overexpression group led to the decrease of mitochondrial membrane potential and increase of activities of caspase-9 and caspase-3, and showed a greater increase in apoptosis than did the nontransfected group. Furthermore, Beclin 1 overexpression resulted in increased cytoplasmic cytochrome c and Bax expression and decreased mitochondrial cytochrome c and Bcl-2 expression.ConclusionOverexpression of Beclin 1 in CaSki cells may influence cisplatin-induced apoptosis by mitochondrial dependent pathway.

scholarly journals Studies on the Mechanism of Alloimperatorin on the Proliferation and Apoptosis of HeLa Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Yingying Bai ◽  
Lijuan Yang ◽  
Chaihong Zhang ◽  
Yongxiu Yang
Keyword(s):  
Membrane Potential ◽  
Molecular Mechanism ◽  
Mitochondrial Membrane Potential ◽  
Hela Cells ◽  
Mitochondrial Membrane ◽  
Control Group ◽  
Cervical Cancer Cells ◽  
Cyt C ◽  
Apoptotic Pathways ◽  
Induced Apoptosis

Alloimperatorin is a compound extracted from the traditional Chinese medicine (Angelica dahurica), which has exhibited anticancer activity. However, its precise molecular mechanism of anticancer remains unclear. Alloimperatorin-induced apoptosis of cervical cancer cells and its molecular mechanism were investigated in the present study. Cholecystokinin octapeptide (CCK-8) was employed to evaluate the cytotoxicity of alloimperatorin on HeLa, SiHa, and MS-751 cells. Flow cytometry was used to assess apoptosis induced by alloimperatorin. The mechanism of apoptosis was verified by mitochondrial membrane potential, Western blotting, and fluorescent PCR. The results of the study showed that alloimperatorin reduced the activity of HeLa cells. The calculated IC50 at 48 hours was 116.9 μM. Compared with the control group, alloimperatorin increased the apoptotic rate of HeLa cells and reduced the mitochondrial membrane potential of HeLa cells. The Western blot results showed that alloimperatorin promotes the expression of caspase3, 8, 9 and that Bax apoptotic proteins reduce PARP expression, procaspase3, 8, 9, and BCL-2 proteins and reduces the cyt-c in the mitochondria expression. The results demonstrated that alloimperatorin can induce HeLa cell apoptosis through mitochondria and extrinsic apoptotic pathways.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

Supplementation of kaempferol to in vitro maturation medium regulates oxidative stress and enhances subsequent embryonic development in vitro

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 59-64
Author(s):  
Yuhan Zhao ◽  
Yongnan Xu ◽  
Yinghua Li ◽  
Qingguo Jin ◽  
Jingyu Sun ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Treated Group ◽  
Control Group ◽  
Oxygen Species

SummaryKaempferol (KAE) is one of the most common dietary flavonols possessing biological activities such as anticancer, anti-inflammatory and antioxidant effects. Although previous studies have reported the biological activity of KAE on a variety of cells, it is not clear whether KAE plays a similar role in oocyte and embryo in vitro culture systems. This study investigated the effect of KAE addition to in vitro maturation on the antioxidant capacity of embryos in porcine oocytes after parthenogenetic activation. The effects of kaempferol on oocyte quality in porcine oocytes were studied based on the expression of related genes, reactive oxygen species, glutathione and mitochondrial membrane potential as criteria. The rate of blastocyst formation was significantly higher in oocytes treated with 0.1 µm KAE than in control oocytes. The mRNA level of the apoptosis-related gene Caspase-3 was significantly lower in the blastocysts derived from KAE-treated oocytes than in the control group and the mRNA expression of the embryo development-related genes COX2 and SOX2 was significantly increased in the KAE-treated group compared with that in the control group. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased after KAE treatment. Mitochondrial membrane potential (ΔΨm) was increased and the activity of Caspase-3 was significantly decreased in the KAE-treated group compared with that in the control group. Taken together, these results suggested that KAE is beneficial for the improvement of embryo development by inhibiting oxidative stress in porcine oocytes.

The Inhibitor of Cyclin-Dependent Kinase4a (INK4a) Signaling Pathway Induces Aging in Human Skeletal Muscle Myoblasts and Decreases the Mitochondrial Membrane Potential

2019 ◽  
Vol 9 (9) ◽  
pp. 1192-1198
Author(s):  
Dong Yan ◽  
Xiang Liao ◽  
Li-Xia Zhao ◽  
Chang-Hong Xiao
Keyword(s):  
Skeletal Muscle ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Signaling Pathway ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Human Skeletal Muscle ◽  
Lentiviral Vector ◽  
Accelerated Aging ◽  
Senescence Phenotype

The effect of the inhibitor of cyclin-dependent kinase4a (INK4a) signaling pathway on myoblastic aging was studied in this paper. Human skeletal muscle myoblasts were transfected with a recombinant lentiviral vector, pLVX-p16INK4a, encoding the p16INK4a gene, and RT-qPCR and western blotting were used to identify p16INK4a gene transcription and protein expression. The degree of cell senescence was assessed using Senescence-associated β-galactosidase staining. flow cytometry and JC-1 staining was used to analyze the mitochondrial membrane potential (MMP). The senescence phenotype was observed in myoblasts transfected with p16INK4a, the MMP was significantly decrease in p16INK4a-transfected myoblasts, while the MMP was decreased only slightly in control cells. Upregulation of the INK4a signaling pathway directly induced aging in human skeletal muscle myoblasts. Moreover, INK4a signaling pathway activated the mitochondrial pro-aging pathway by reducing the MMP, which indirectly accelerated aging in myoblasts.

scholarly journals Neuroprotective Effects of Hesperidin, a Plant Flavanone, on Rotenone-Induced Oxidative Stress and Apoptosis in a Cellular Model for Parkinson’s Disease

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Kuppusamy Tamilselvam ◽  
Nady Braidy ◽  
Thamilarasan Manivasagam ◽  
Musthafa Mohamed Essa ◽  
Nagarajan Rajendra Prasad ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Neuroprotective Effect ◽  
Neuroblastoma Cell ◽  
Ros Generation ◽  
Enzymatic Antioxidants ◽  
Neuroprotective Effects ◽  
Human Neuroblastoma ◽  
Cyt C

Rotenone a widely used pesticide that inhibits mitochondrial complex I has been used to investigate the pathobiology of PD bothin vitroandin vivo. Studies have shown that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species (ROS), leading to neuronal apoptosis. The current study was carried out to investigate the neuroprotective effects of hesperidin, a citrus fruit flavanol, against rotenone-induced apoptosis in human neuroblastoma SK-N-SH cells. We assessed cell death, mitochondrial membrane potential, ROS generation, ATP levels, thiobarbituric acid reactive substances, reduced glutathione (GSH) levels, and the activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) using well established assays. Apoptosis was determined in normal, rotenone, and hesperidin treated cells, by measuring the protein expression of cytochrome c (cyt c), caspases 3 and 9, Bax, and Bcl-2 using the standard western blotting technique. The apoptosis in rotenone-induced SK-N-SH cells was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, the depletion of GSH, enhanced activities of enzymatic antioxidants, upregulation of Bax, cyt c, and caspases 3 and 9, and downregulation of Bcl-2, which were attenuated in the presence of hesperidin. Our data suggests that hesperidin exerts its neuroprotective effect against rotenone due to its antioxidant, maintenance of mitochondrial function, and antiapoptotic properties in a neuroblastoma cell line.

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