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Ginkgo biloba extract on H2O2-induced lens epithelial cells in Bcl-2 and Bax expression in_2782

By Erin Hunter,2014-10-30 18:39
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Ginkgo biloba extract on H2O2-induced lens epithelial cells in Bcl-2 and Bax expression in_2782

    Ginkgo biloba extract on H2O2-induced lens epithelial cells in Bcl-2 and Bax expression in

     Authors: Li Shuang Yue, Qing-Li Luo, Li Zheng, the

    official Peng, Lu, Ai-Hua Chiang

     Abstract Objective: To observe the Ginkgo biloba extract

    (EGb761) on lens epithelial cell apoptosis-related gene

    protein Bcl-2 and Bax expression. Methods: The in vitro cultured lens epithelial cells in SD rats were divided into 3 groups: experimental group H2O2 EGb761 treatment, control group with H2O2 treatment, and control group without making any treatment. By immunohistochemistry method to detect epithelial cells, Bcl-2, Bax expression, analysis of the

    experimental group and control group and blank control group expression. Results: Bcl-2 protein expression positive cell

    rate: the experimental group over time to extend the rise, 24h when (20.7 ? 1.4)%, control group decline over time

    extension, 24h when (2.6 ? 0.6)% , blank control group (6.8

    ? 1.1)%; Bax protein expression positive cell rate: the

    extension of the experimental group over time no significant change, 24h when (10.7 ? 0.9)%; control group with the time of the rise in 24h when (33.4 ? 4.6)%, blank control group

    (4.6 ? 0.8)%. Conclusion: EGb761 by making the lens epithelial cells increased expression of Bcl-2 protein, Bcl-

    2/Bax to reduce and prevent oxidative damage induced lens epithelial cell apoptosis; apoptosis may be related to cataract formation, EGb 761 in the early formation of cataract may be a disincentive.

     Key Words Ginkgo biloba extract Bcl-2; Bax oxidative

    stress in lens epithelial cells

     0 Introduction

     Cataract incidence of injury and antioxidant status of relevant, age-related cataract patients with H2O2 in aqueous humor was higher than normal [1], oxidative stress may be

    related to age-related cataract-related. We simulate age-

    related cataract patients deal with aqueous H2O2 concentration in Sprague-Dawley (SD) rat lens epithelial cells by

    immunohistochemistry to observe the lens epithelial cell

    apoptosis-related genes Bcl-2 and Bax expression and by Ginkgo biloba extract material (EGb 761) on the role of H2O2 lens epithelial cells Bcl-2 and Bax expression in contrast to

    observations, to investigate the oxidative stress and anti-

    oxidants on apoptosis of lens epithelial cells the

    relationship between the biological behavior.

     1 Materials and methods

     1.1 Materials CO2 incubator (USA), Clean Bench (Suzhou), stereo microscope (Shanghai), Olympus optical microscope (Japan), a variety of flasks, and petri dishes, incubated with wet boxes. Phenol red-free M199 (Sigma), prepared according to the instructions into the culture medium by adding HEPES (Fluka) 25mmol / L, penicillin 100kU / L, streptomycin 100kU / L, filtration, sterilization, sub-loaded, -20 ? to save. With

    the pre-accession Ginaton (Germany Shu Pei major

    pharmaceutical companies, containing EGb761 17.5g / L), glucose oxidase (Sigma), 300mL / L H2O2 (Shanghai, AR), glucose (Shanghai, AR), and pH adjusted to 7.2 ~ 7.4. 0.01mol

    / L PBS pH7.6; 0.5mol / L Tris / HCL pH7.6; sheep serum working dilution (1:10); double-distilled water; 300ml / L

    H2O2; an anti-mouse anti-human Bcl -2 monoclonal antibody

    Santa Cruz Biotechnology products, 4 ? preservation, working

    dilution (1:100); an anti-goat anti-human Bax antibody Santa

    Cruz Biotechnology products, 4 ? preservation, working

    dilution (1:100 ); SP kit for ZYMED company's products, 4 ?

    for storage, the working dilution (1:100); DAB for the Sigma product.

     1.2 Methods SD rats age 4 weeks, quality, 110 ? 10g, of

    either sex, no diseases of the eye. Purchased from West China Medical University animal experiment center. CO2 inhalation death, removal of the eye, placed 500kU / L penicillin saline wash, in sterile conditions, take a transparent lens. Placed

    in a transparent lens with M199 1.5mL of the 24-well plate,

and placing temperature of 37 ?, 95% humidity, 50mL / L CO2

    incubator to cultivate 1 ~ 2h, spare. Simulated senile cataract aqueous H2O2 concentration on the in vitro rat lens

    epithelial cells in SD processing is divided into H2O2 added EGb761 processing experimental group (hereinafter referred to as the experimental group), H2O2 treatment control group (hereinafter referred to as the control group), only the M199 medium blank control group (hereinafter referred to as the blank group). In the M199 adding 0.95mmol / L glucose, glucose oxidase 1nkat, 90μmol / L H2O2. H2O2 concentration in the 8h period can be maintained at 90 ? 5μmol / L. It is reported

    in the literature [2,3]: glucose and glucose oxidase reaction to produce O2, because O2 pumping hydrogen generated H2O2. In order to maintain a more constant concentration of H2O2, the

    same conditions for every 8h to replace the medium. Take 3 of a group of transparent lens into the 10cm size, with M199 30mL dish, the set temperature of 37 ?, 95% humidity, 50mL / L CO2

    incubator. The following treatment, respectively, 3,6,18,24 h

    time taken to train the lens parameters were measured. The experimental group, in the above medium added to 0.95mmol / L glucose, glucose oxidase 1nkat, 100mg / L EGb761 and 90μmol /

    L H2O2. The control group, in the above-mentioned culture

    medium by adding 0.95mmol / L glucose, glucose oxidase 1nkat, 90μmol / LH2O2. Blank control group, only the M199 to cultivate. Will foster 3,6,18,24 h rat lens, full cut along the equatorial anterior capsule, in the next divided into two stereo microscope, the cell for the last tile on the glass slide, dry place will be stretched 4 ? acetone fixed 10min,

    dried out to be a natural place 4 ? refrigerator spare.

    Distilled water dipping acetone fixed slide, 0.01mol / L PBS immersion 3min × 3 times; home 30mL / L H2O2 aqueous solution

    at room temperature 10min, inactivation of endogenous peroxidase activity; 0.01mol / L PBS immersion 3min × 3

    times; 100mL / L normal goat serum closed, 37 ? 10min and

    dumped to the serum; dropping an anti-(1:100 working dilution)

    Rear 30min at room temperature 4 ? refrigerator overnight,

    0.01mol / L PBS immersion 3min × 3 times; dropping biotin-

    labeled secondary antibody (1:100), 37 ? 1h, 0.01mol / L PBS

    immersion 3min × 3 times; dropping horseradish peroxidase labeled streptavidin (1: 100), 37 ? 1h, 0.01mol / L PBS

    immersion 3min × 3 times; DAB color 1 ~ 5min; hematoxylin; dehydration, transparent, Feng Pian. Each laboratory experiment to provide Bcl-2, Bax-positive biopsy for positive

    control; to replace an anti-PBS as a negative control. Brown

granules within the cytoplasm appears as a Bcl-2, Bax positive

    cells, cytoplasm negative cells were not colored. Nuclei were stained blue. Each calculation of five cells, stretched, each stretched randomly selected five horizons under the microscope

    in the zoom 400 times, counting 1 000 cells, the number of positive cells and calculated the rate of positive cells.

     Statistical analysis: The SPSS 10.0 statistical software to do statistical analysis of results, using t test for each

    group were compared.

     2 Results

     2.1 Bcl-2 protein expression characteristics of the blank control group, Bcl-2 protein expression in the cytoplasm, the brownish yellow granules, the nucleus was blue. Negative cell cytoplasm is not colored, just see the nucleus was stained

    blue. In the blank control group, there are fewer lens epithelial cells expressed an average of (6.8 ? 1.1)%,

    (Figure 1). EGb761 role in the H2O2 early (3h) was not caused by the experimental group Bcl-2 protein expression increased

    significantly with time thereafter, the experimental group Bcl-2 protein expression gradually increased (Figure 1,2). H2O2 role of the control group showed an early expression of Bcl-2 protein in the increasing trend, and then decreased significantly (Figure 1,3).

     2.2 Bax protein expression characteristics of the blank control group, Bax protein expression in the cytoplasm, the brownish yellow granules, the nucleus was blue. Negative cell cytoplasm is not colored, just see the nucleus was stained

    blue. Blank control group, expressed in lens epithelial cells with little or no expression, the average (4.6 ? 0.8)%

    (Figure 2). EGb761 acting on H2O2 treated experimental group, in the 24h period, Bax protein expression was no significant change in the control group with the culture time extended, Bax protein expression increased significantly (Figure 4,5). Reposted elsewhere in the paper for free download http://

     3 Discussion

     Proto-oncogene bcl-2 is the inhibition of apoptosis

    (apoptosis) pathway is an important gene, bcl-2 by means of

    anti-oxidant to prevent cell apoptosis [2-6]. bcl-2 can be

    against H2O2-induced cell calcium concentration increase and subsequent reduction of glutathione system dysfunction caused by cell death [7-9]. Have found that antioxidants, such as the bad blood acid through increased expression of bcl-2 reduced

    the incidence of apoptosis, but the agent causing oxidative stress-induced cell proliferation or apoptosis and bcl-2

    function relationship has not yet fully understood [10]. Our

    experimental results show that: the control group, the role of H2O2 early increase in Bcl-2 protein expression and whether

    lens epithelial cells in their antioxidant defense mechanism of oxidative stress response, awaits further experimental

    confirmation. bax gene and bcl-2 gene has 21% homology, bax

    has a role in the promotion of cell apoptosis, oxidative stress-induced increased expression of Bax and Bcl-2 levels by

    making the drop, causing the cells took place followed by the

    withered death. Bax with Bcl-2 form homo-or heterodimers,

    intracellular Bax and Bcl-2 and Bcl-2/Bax determines the

    amount of Bax / Bax dimer ratio, and this in turn determines the percentage of cells susceptibility to apoptotic signals, regulating cell death and survival of the equilibrium state [11]. Experiments have confirmed that the body's antioxidant defense mechanisms, such as glutathione to restore the system capabilities can be reduced bax expression in apoptosis caused by the occurrence, we can see redox homeostasis in maintaining cell survival plays an essential the important role of [12]. In our experiments found that the role of the control group in the early H2O2 caused increased expression of Bcl-2 protein,

    followed by Bcl-2 expression decreased, Bax expression was

    significantly increased, as well as Bcl-2 / Bax ratio decrease

    may be related to the control group of lens epithelial Apoptosis rate was higher than the experimental group.

     Our experimental results show that, EGb761 acting on

    H2O2 treated lens epithelial cells, in the role of early (train 3h) did not cause significant increased expression of Bcl-2, whereby it can be assumed EGb761 lens epithelial cells to start or enhance their anti-oxidant defense mechanisms to

    protect cells from oxidative damage caused by Bcl-2 preceded

    increased expression. EGb761 could reduce the H2O2 on lens epithelial cells, the role of oxidative stress, maintaining the lens epithelial cells in high H2O2 redox environment, the dynamic equilibrium. While the whole culture period no

    significant changes in Bax expression, but as the culture time extended, the experimental group expression of Bcl-2 positive

cells increased gradually, Bcl-2 / Bax ratio increased

    gradually, that is, EGb761 by increasing the expression of

    Bcl-2 Non-reduced expression level of Bax, showing anti-

    oxidative damage induced apoptosis. Is consistent with the literature reported [11]. The hydrogen peroxide treatment the control group, the role of early H2O2 caused increased expression of Bcl-2, followed by Bcl-2 expression decreased,

    Bax expression was significantly increased, as well as Bcl-2 /

    Bax ratio decreased, and thus Erzhi apoptosis occurred. The Bax and oxidative stress in the cataract of the precise relationship remains to be further studied.

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