[Selection of bak siRNA sequences and its influence on Al-induced apoptosis of SH-SY5Y cell line].

OBJECTIVE: To find the optimal design of small interfering RNA compounds, transfection concentration and transfection time to reduce the Al-induced apoptosis in SH-SY5Y cells. METHODS: Three siRNA sequences on bak gene were designed and transfected into SH-SY5Y cells, which were treated at various concentrations of aluminum. Cell viability was detected by CCK-8 kit on different siRNA sequences, various transfection concentrations, and diverse transfection courses. Transfection efficiency was determined by fluorescent staining of CY3, and interference efficiency was measured by QRT-PCR. Besides, immunohistochemical staining was used to express Bak protein content. Finally, apoptotic rate and necrotic rate in Al treated SH-SY5Y cells transfecting by the selected bak siRNA 1 were detected. RESULTS: Based on the viability of siRNA sequences, siRNA 1 was selected as the optimal siRNA sequences. The optimal transfection concentration was 10 nmol/L, and the optimal time course was 24 h after transfection. The transfection efficiency was above 90% and the interference efficiency with bak gene was 57.76%. Furthermore, there was significant transfection effect on Bak protein. The apoptotic rate in Al treated SH-SY5Y cells were significantly decreased by bak siRNA 1 transfection. CONCLUSION: Apoptosis is one of the major cell death pathways in SH-SY5Y cells induced by aluminum. When chemically synthesized siRNA is inducted to neural cells, it can significantly reduce bak gene level, decrease Bak protein expression and apoptotic rate, which may serve as the basis for preventing neural cells apoptosis and inhibiting the development of neurodegenerative diseases.

[Role of Bcl-2 and Bax protein contents and their gene expression in Al-induced neurons apoptosis].

OBJECTIVE: To study the role of Bcl-2 and Bax protein contents and their gene expression in Al-induced neurons apoptosis. METHODS: Neurons from 0 - 3 day rats were cultured and treated with different concentrations of AlCl(3 x 6) H2O. The cell apoptosis was observed by the TUNEL method and under the scan electron microscope. Bcl-2 and Bax protein contents were detected by the immunochemistry method while their gene expressions were measured by the RT-PCR method. RESULTS: (1) DNA fractions in the TUNEL method increased with the rising aluminum concentration. Blebbings and apoptosis bodies on the surface of the neurons were clearly observed under the scan electron microscope. (2) Bcl-2 protein contents and their gene expression decreased with the rising aluminum concentration (P < 0.01, r = -0.695; P < 0.05, r = -0.647), while Bax increased at the same time (P < 0.01, r = 0.676; P < 0.01, r = 0.794), the value of Bcl-2/Bax was related with the aluminum concentration (P < 0.01, r = -0.655; P < 0.01, r = -0.777). CONCLUSION: The aluminum may induce neurons apoptosis. Bcl-2 and Bax protein contents and their gene expression may play an important role in Al-induced apoptosis.

[Role of reactive oxygen species in sodium selenite induced DNA damage in HepG2 cells].

OBJECTIVE: To investigate the mechanisms of sodium selenite induced DNA damage in HepG2 cells. METHODS: HepG2 cells were treated with the designed concentrations of sodium selenite and the selenite (10 micromol/L) added simultaneously with GSH (10 mmol) and NAC (5 mmol). Then the cell viability was detected by MTT, and the flurescent intensity of reactive oxygen species (ROS) was determined by flow cytometry, and DNA damage was detected by commet assay. RESULTS: The level of ROS was increased after HepG2 was treated with 5, 10, 20 micromol/L sodium selenite for one hour, and the cell viability was decreased after 12 hours, and the DNA damage was enhanced. Compared with the control group, the difference was statistically significant (P < 0.05) . GSH and NAC effectively inhibited the ROS increased and cell viability decreased and DNA damage weakened. CONCLUSION: ROS may be the important reason that sodium selenite induced HepG2 cells DNA damage.

[Effects of aluminum on lipid peroxidation in rat's brain and its sex - related difference].

OBJECTIVE: To explore the mechanisms of aluminum (Al)-induced neurotoxicity by studying the effect of aluminum on lipid peroxidation in rat's brain and its sex related difference. METHODS: Forty SD rats, both male and female, were exposed to aluminum through intraperitoneal injection of AlCl3 solution for 60 days at different dose. After exposure, the step down test was performed to examine the learning and memory abilities of rats, and the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in rat's cerebrum were detected by chemical method. The changes of ultrastructure in cortex were observed by transmission electron microscopy. RESULTS: The differences between the changes of all indexes of female and male rats in the same dose-group wasn't statistically significant (P > 0.05). Compared with the rats in the control group, the learning and memory abilities of the Al-exposed rats were significantly decreased (P < 0.05). The content of MDA was increased (P < 0.01) while the activity of SOD was decreased (P < 0.05). The membrane structure of neurons in cerebrum cortex of the Al-exposed rats were broken, dissolved and gone. CONCLUSION: Aluminum can accelerate lipid peroxidation in rat's brain, which may be one of the important intoxication mechanisms of aluminum. However, the sex-related difference of this effect have not yet been observed.

[Effect of heat and BPDE on the expression of Hsp27 and Hsp70 in A549 cell line].

OBJECTIVE: To investigate the expression of the Hsp27 and Hsp70 in A549 cells treated with heat and BPDE. METHODS: A549 cells cultured in vitro were divided into heat stress group and BPDE group. Cells cultured at 37 degrees C used as control. The heat stress group was exposed to heat at different temperature (39 degrees C, 42 degrees C and 43 degrees C) for 2h. The BPDE group was treated with different concentrations of BPDE (0, 2, 4 and 8 micromol/L). Western-blot was used for the Hsp27 and Hsp70 expression analysis. RESULTS: In heat stress group, Hsp27 expression levels significantly increased compared to the control (P < 0.05) and reached its peak at 39 degrees C. Hsp70 expression level also increased after heat stress. It reached the peak at 42 degrees C and was higher than that of the control with significance (P < 0.05). In BPDE group, Hsp27 and Hsp70 expression levels were higher than those of the control. Hsp27 reached a maximum at 8 micromol/L, the highest concentration of BPDE in this experiment, and was significantly higher than that of the control (P < 0.05). But it was still on its elevatory phase. Hsp70 reached its peak at 4 micromol/L and was significantly higher than that of the control at 4 micromol/L and 8 micromol/L. CONCLUSION: Both heat and BPDE can induce the expressions of Hsp27 and Hsp70. In heat stress group, the Hsp27 and Hsp70 levels reached their peaks at 39 degrees C and 42 degrees C. In BPDE group, the expressional levels of Hsp27 and Hsp70 reached their peaks at 8 micromol/L and 4 micromol/L.

[Effect of aluminum on neuronal mitochondria of rats].

OBJECTIVE: To observe and explore the effect of aluminum on mitochondria in nerve cells of rats. METHODS: Nerve cells of new born rats (1-3 days) were cultured. Ultrastructure of mitochondria, cell death rate (CDR), reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and MTF were performed to investigate the alteration of mitochondrial structure and functions in cultured nerve cells. RESULTS: Aluminum can impair the ultrastructure of cultured nerve cells of rats. Increased CDR, enhanced ROS, decreased MMP, and decreased activity of enzyme in mitochondria were investigated in the group of Al3+ 100 micromol/L and Al3+ 500 micromol/L. CONCLUSION: The present study shows that the alteration of mitochondrial structure and functions have played important roles in neurotoxic mechanism induced by aluminum.