Gao Shi-yong

The Effect of Solanine on the Membrane Potential of Mitochondria in HepG2 Cells and [Ca²+]i in the Cells

To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca24]; in the cells, and to uncover the mechanism by which solanine induces apoptosis. HepG2 cells are double stained with TMRE and Fluo- 3/AM, and both the change in membrane potential of mitochondria and that of [Ca2+]; in the cells are observed using LCSM.The results of double staining with TMRE and Fluo-3/AM show that solanine can lower membrane potential and increase the concentration of Ca2+ in the cells Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca2+ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca2+ in the cell, turning on the mechanism for apoptosis.

The Effect of Solanine on the Membrane Potential of Mitochondria in HepG2 Cells and [Ca^2+]i in the Cells

To observe the effect of solanine on the membrane potential of mitochondria in HepG₂ cells and [Ca²⁺]_i in the cells, and to uncover the mechanism by which solanine induces apoptosis. HepG2 cells are double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca²⁺]_i in the cells are observed using LCSM. The results of double staining with TMRE and Fluo-3/AM show that solanine can lower membrane potential and increase the concentration of Ca²⁺ in the cells Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca²⁺ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca²⁺ in the cell, turning on the mechanism for apoptosis.

Calculation of the Effect of Solanine on the Michaelis Constant and the Maximum Reaction Rate of NAT

To explore how to calculate the effect of solanine on the Michaelis constant and the maximum reaction rate of NAT, high performance liquid chromatography (HPLC) was used, with 2-AF as substrate, and the rate at which 2-AF is acetylated into 2-AAF in intact HepG2 cells or in the cytoplasm of HepG2 cells as the reaction rate. The double reciprocal plot was made, with 1/S (reciprocal of the concentration of the substrate 2-AF) plotted against 1/V (reciprocal of the reaction rate), to yield a regression equation for calculating Km and Vmax. With intact HepG2 cells, the Km and Vmax for the negative control are 2.37times10-3plusmn8.37times10-5 mM and 9.16times10-4plusmn7.54times10-5 nmol/106 cells, respectively, and that the Km and Vmax for the solanine group are 2.22times10-3plusmn9.05times10-5 mM and 5.14times10-4plusmn3.72times10-5 nmol/106 cells, respectively. For the cytoplasm of HepG2 cells, the Km and Vmax for the negative control are 8.95times10-3plusmn2.61times10-4 mM and 2.55times10-6plusmn1.92times10-8 nmol/ minmiddotmg protein, respectively, and the Km and Vmax of the solanine group are 9.48times10-3plusmn3.63times10-4 Mm and 2.43times10-6plusmn1.32times10-8 nmol/minmiddotmg protein. Statistical analysis showed that, for both intact HepG2 cells and cytoplasm of HepG2 cells, Km does not differ significantly between the negative control and the solanine groups, but Vmax does differ significantly for these groups (P<0.05). Solanine is a non-competitive inhibitor for the 2-AF substrate of NAT.

Effects of solanine on the contents of Capspase-3 and Bcl-2 protern in HepG2 cells

To observe the effect of solanine on the contents of caspase-3 and Bcl-2 in HepG2 , and to explicate the mechanism by which solanine induces the apoptosis of tumor cells.Laser confocal scanning microscopy (LCSM) and Western blot were used to measure the contents of caspase-3 and Bcl-2 , and LCSM was used to determine their locations in the cell. Solanine markedly increased the content of caspase-3 while decreasing that of Bcl-2 in HepG2 , both in a dose-dependent way. Both caspase-3 and Bcl-2 are unevenly distributed in the cytoplasm , but are not found in the nucleus. Solanine does not affect the distribution of caspase-3 and Bcl-2. By suppressing the activity of Bcl-2 , solanine activates the caspase-3 protease family, thus inducing cell apoptosis.