Effect of Quercetin on the Proliferation and Mitochondrial Transmembrane Potential of CBRH-7919 Cells

[Objective] To investigate the effect of quercetin on the proliferation and mitochondrial transmembrane potential of CBRH-7919 cells. [Method] The CBRH-7919 cells of hepatocarcinoma were cultured in vitro. After treated with different concentrations of quercetin, the OD405 nm of CBRH-7919 cells was detected by using the acid phosphatase assy （APA）; morphologic changes of the cells were observed under inverted microscope; the mitochondrial transmembrane potential （△ψm） intensity changes of CBRH-7919 cells were analyzed by flow cytometry after stained with Rhodamine 123. [Result] Quercetin inhibited the proliferation of CBRH-7919 cells significantly, and the growth inhibitory effect presented time- and dose-dependent relationship. Typical decrease of cell density was observed by optical microscopy on the quercetin-treated cells. With the effect of 10 μg/ml quercetin on CBRH-7919 cells for 12, 24 and 48 h, the percentage of Rhodamine 123 stained hypofluorescence cells increased, while the mitochondrial transmembrane potential（△ψm） intensity of CBRH-7919 cells decreased. [Conclusion] Quercetin could inhibit the proliferation of CBRH-7919 cells in vitro, causing the decrease in mitochondrial transmembrane potential.

Effect of rhTNF-α on Mitochondrial Transmembrane Potential and Motility of Human Sperm in vitro by Flow Cytometry and Computer Aided of Semen Analysis

To evaluate effect of recombined human tumor necrosis factor （rhTNF- α） on mitochondrial transmembrane potential and motility of human sperm in vitro Methods Semen samples for study were obtained from 40 health men （average age 26 ± 1.2 years） with normal semen analysis. Sperm suspension with computer aided of semen analysis （CASA） technique; 2） were stained in the presence of 10 μg/ml Rh123 and PI, mitochondrial transmembrane potential of those was analyzed by flow cytometry （FCM）. Results Significant differences were found between experimental groups and control groups on viability, straight line velocity, curvilinear velocity, average path velocity, progressive motility of human sperm and number of sperm with normal mitochondrial transmembrane potential （P〈0.01） expect final concentration 30 pg/ml group （P〉0. 05）. Sperm motility lowed with increasing rhTNF-α concentration and incubating time （P〈0. 01）. Number of sperm with normal mitochondrial transmembrane potential decreased with increasing rhTNF-α concentration and incubating time （P〈0.01）. Conclusion rh TNF-α can decrease human sperm motility function in vitro, which can interfere the function of human sperm mitochondrial transmembrane potential and may inhibit sperm mitochondrial enzymatic activities.

Restoration of Mitochondrial Structure and Function within Helicobacter pylori VacA Intoxicated Cells

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3

AIM： To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don （S. barbata） and to determine the underlying mechanism of its antiturnor activity in mouse liver cancer cell line H22.METHODS： Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope （EM）. Mitochondrial transmembrane potential was determined under laser scanning confocal microscope （LSCM） with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometrv.RESULTS： M-I-I- assay showed that extracts from S. barbata （ESB） could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phasesof cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G1 phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION： ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

Puerarin prevents high glucose-induced apoptosis of Schwann cells by inhibiting oxidative stress

Oxidative stress may be the unifying factor for the injury caused by hyperglycemia in diabetic peripheral neuropathy. Puerarin is the major isoflavonoid derived from Radix puerariae and has been shown to be effective in increasing superoxide dismutase activity. This study sought to investigate the neuroprotective effect of puerarin on high glucose-induced oxidative stress and Schwann cell apoptosis in vitro. Intracellular reactive oxygen radicals and mitochondrial transmembrane potential were detected by flow cytometry analysis. Apoptosis was confirmed by TUNEL and oxidative stress was monitored using an enzyme-linked immunosorbent assay for the DNA marker 8-hydroxy-2-deoxyguanosine. The expression levels of bax and bcl-2 were analyzed by quantitative real-time reverse transcriptase-PCR, while protein expression of cleaved caspase-3 and -9 were analyzed by means of western blotting. Results suggested that puerarin treatment inhibited high glucose-induced oxidative stress, mitochondrial depolarization and apoptosis in a dose-dependent manner. Furthermore, puerarin treatment downregulated Bax expression, upregulated bcl-2 expression and attenuated the activation of caspase-3 and -9. Overall, our results indicated that puerarin antagonized high glucose-induced oxidative stress and apoptosis in Schwann cells.

Protective paracrine effect of mesenchymal stem cells on cardiomyocytes

Objective： The aim of this study was to test the protective effect of mesenchymal stem cells （MSCs） on cardiomyocytes in vitro and to investigate the anti-apoptotic signaling pathway. Methods： MSCs from Sprague-Dawley （SD） rats were separated and cultured. MSC medium was collected from MSCs （DMEM） under hypoxia. Cultured cardiomyocytes from neonatal cultured in serum-free Dulbecco＇s modified eagle medium SD rats were exposed to hypoxia/reoxygenation （H/R） and treated with MSC medium. The apoptotic cardiomyocytes were stained with Annexin-V-fluorescein isothiocyanate （FITC）, Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL）. The mitochondrial transmembrane potential of cardiomyocytes was assessed using a fluorescence microscope. The expression of Bcl-2, Bax, cyto- chrome C, apoptosis-induced factor （AIF）, and caspase-3 was tested by Western blot analysis. Results： Our data demonstrated that MSC medium reduced H/R-induced cardiomyocyte apoptosis, increased the Bcl-2/Bax ratio, and reduced the release of cyto- chrome C and AIF from mitochondria into the cytosol. Conclusion： MSCs protected the cardiomyocytes from H/R-induced apoptosis through a mitochondrial pathway in a paracrine manner.

Pachymic acid, a novel compound for anti-rejection： effect in rats following cardiac allograft transplantation

Background Pachymic acid （PA）, a natural triterpenoid, is known to significantly reduce cell proliferation and induce apoptosis in vitro through initiation of mitochondria dysfunction. However, its effect on immune cells and anti-rejection following organ transplantation remains unknown. Methods In this study, we investigated PA as a treatment to control acute rejection occurred in rats which had accepted cardiac transplantation. We measured apoptosis of peripheral blood lymphocyte （PBLs）, and CD4^＋ lymphocyte, as well as the number of CD4^＋ and CD8^＋ lymphocytes and the effect of PA on acute rejection in rats 7 days after cardiac transplantation. Results PA treatment might decrease allograft rejection, protect PBLs from apoptosis, and reduce the percentage of CD8^＋ lymphocyte. PA neither regulated the number nor the apoptosis rate of CD4^＋ lymphocyte. Conclusions Our findings indicated that PA has an anti-apoptotic effect acting on PBLs through a novel mechanism involving stabilization of the PBLs mitochondrial transmembrane potential, an anti-rejection effect in rats after cardiac transplantation and an inhibiting effect to CD8^＋ lymphocyte.