Figure 6. Cadmium-induced cytotoxicity in pancreatic beta-cell derived RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 1, 3, 5 and 10 μM) for 24h by MTT assay. Data are presented as means ± S.E. for four independent experiments with triplicate determinations. *P<0.05 as compared with control.
Figure 7. Cadmium on insulin secretion in pancreatic beta-cell derived RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 1, 3, 5 and 10 μM) for 24 h by insulin assay. Data are presented as means ± S.E. for four independent experiments with triplicate determinations. *P<0.05 as compared with control.
Figure 8.Flow cytometric analysis showing effects of cadmium on the cell cycle of RIN-m5F cells.
RIN-m5F Cells were treated with or without cadmium (5 and 10 μM) for 24 h in the presence or absence of JNK inhibitor(SP600125, 10 μM) Cells with genomic DNA fragmentation (sub-G1 DNA content). All data are presented as means ± SEM for three independent experiments with triplicate determinations. *P < 0.05 as compared with control. #P < 0.05 as compared with cadmium alone.
Figure 9. Effect of cadmium on the morphology of pancreatic beta-cell derived RIN-m5F cells.
RIN-m5F cells were exposed to different concentrations of cadmium for 24 h, (A and B) Control, (C and D) cadmium (10 μM), (E and F) JNK inhibitor(SP600125, 10μM), (G and F) RIN-m5F cells were exposed with cadmium (10 μM) in the presence of JNK inhibitor(SP600125, 10 μM). Apoptosis cells could be differentiated from necrotic cells by incubated with Ann Cy3 and 6-CFDA simultaneously. After labeled at room temperature, RIN-m5F cells were immediately observed by fluorescence microscopy (200×). Live cells would be labeled only with 6-CF (green fluorescence, A, C, E, G), while necrotics cells could be labeled only with Ann Cy3 (red
fluorescence, B, D, F, H). RIN-m5F cells in the early stage of apoptosis would be labeled with both Ann Cy3 (red fluorescence) and 6-CF (green fluorescence).
Figure 10. Induction of nuclear condensation after exposure to cadmium.
RIN-m5F cells were exposed with cadmium (0, 5 and 10 μM) in the presence or absence of JNK inhibitor(SP600125, 10 μM) by phase-contrast microscopy (left side) and Hoechst staining (right side)
Figure 11. Cadmium on reactive oxygen species (ROS) generation in RIN-m5f cells.
RIN-m5F cells were exposed with cadmium (5, and 10 μM) for 30, 60, and 120 min, and ROS were determined by flow cytometry. All data are presented as means ± SEM for three independent experiments with triplicate determinations.
Figure 12. JNK-inhibitor suppress cadmium-induced oxidative damage to membrane lipid by lipid peroxidation (LPO) assay in RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5 and 10 μM) in the presence or absence of JNK inhibitor(SP600125, 10 μM) by flow cytometry. Data are presented as means ± S.E. for four independent experiments with triplicate determinations.
*P<0.05 as compared with control. #P<0.05 as compared with CdCl2 alone.
Figure 13. Analysis of mitochondrial membrane potential (MMP) in cadmium-treated RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5, and 10 μM) in the presence or absence of JNK inhibitor(SP600125, 10 μM) for 8 h, and MMP was determined by flow cytometry. Data are presented as means ± S.E. for four independent experiments with triplicate determinations. *P<0.05 as compared with control. #P<0.05 as compared with CdCl2 alone.
Figure 14. Effects of cadmium-induced cytosolic cytochrome c release in RIN-m5F cells.
RIN-m5F cells were exposed with cadmium for various dose-depend (0, 5, 10 μM), and proteins expression of cytosolic cytochrome c was determined by western blot analysis. Data are representative of three independent experiments
Figure 15. JNK-inhibitor suppress cadmium-induced cytosolic cytochrome c release in RIN-m5F cells.
(A)Upper data was RIN-m5F cells were exposed with cadmium for various dose-depend (0, 5, 10 μM) in the presence or absence of the JNK inhibitor(SP600125, 10 μM) to investigate proteins expression of cytosolic cytochrome c was determined by western blot analysis. Data are representative of three independent experiments (B)Below data was Quantitative cytochrome c protein expression. Data are presented as means ± S.E. for four independent experiments with triplicate determinations.
*P<0.05 as compared with control. #P<0.05 as compared with CdCl2 alone.
Figure 16. JNK-inhibitor suppress Poly (ADP-ribose) polymerase (PARP) activation in cadmium-treated RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5, 10 μM) in the presence or absence of the JNK inhibitor(SP600125, 10 μM) for 8 h and 12 h to investigate proteins expression of PARP and cytochrome c release was analyzed by western blot. Data are representative of three independent experiments.
Figure 17. JNK-inhibitor suppress caspase-3 and caspase-7 activation in cadmium-treated RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5, 10 μM) in the presence or absence of the JNK inhibitor (SP600125, 10 μM) for 16 h anf 24 h to investigate proteins expression of caspase 3 and caspase-7 phosphorylation were determined by western blot analysis. Data are representative of three independent experiments.
Figure 18. Mitogen -activated protein (MAP) kinases are relationship to cadmium-induced cytotoxicity in pancreatic beta-cell derived RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5, 10 μM) in the presence or absence of JNK inhibitor (SP600125, 10 μM), P38 inhibitor (SB203580, 10 μM) and ERK inhibitor (PD98059, 10 μM)for 24h by MTT assay. Data are presented as means ± S.E.
for four independent experiments with triplicate determinations. #P<0.05 as compared with control.
Figure 19.JNK-inhibitor suppress the cadmium induced insulin secretion in pancreatic beta-cell derived RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5, 10 μM) in the presence or absence of JNK inhibitor(SP600125, 10 μM) for 24h by insulin assay. Data are presented as means ± S.E. for four independent experiments with triplicate determinations.
*P<0.05 as compared with control. #P<0.05 as compared with CdCl2 alone.
Figure 20. N-acetyl-L-cysteine (NAC ) suppress the cadmium-induced cytotoxicity in pancreatic beta-cell derived RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5, 10 μM) in the presence or absence of N-acetyl-L-cysteine (NAC, 1 mM) for 24h by MTT assay. Data are presented as means ± S.E. for four independent experiments with triplicate determinations.
*P<0.05 as compared with control. #P<0.05 as compared with CdCl2 alone.
Figure 21. N-acetyl-L-cysteine(NAC) suppress on the phosphorylation of mitogen -activated protein (MAP) kinases in cadmium-treated RIN-m5F cells.
RIN-m5F cells were treated with CdCl2 (0, 5, and 10 μM) in the presence or absence of N-acetyl-L-cysteine (NAC, 1 mM) for 30 min, and JNK, ERK1/2, p38
phosphorylation was analyzed by western blot. Data are representative of three independent experiments.
Figure 22. JNK-inhibitor suppress on the phosphorylation of JNK in cadmium-treated RIN-m5F cells.
RIN-m5F cells were treated with CdCl2 (0, 5, and 10 μM) in the presence or absence of the JNK inhibitor (SP600125, 10 μM) , and JNK phosphorylation was analyzed by western blot. Data are representative of three independent experiments.
Figure 23. Analysis of mitochondrial membrane potential (MMP) in JNK inhibitor compares with N-acetyl-L-cysteine (NAC) RIN-m5F cells.
RIN-m5F cells were exposed with cadmium (0, 5, and 10 μM) in the presence of the JNK inhibitor(SP600125, 10 μM) and N-acetyl-L-cysteine (NAC, 1 mM) for 1hr, and MMP was determined by flow cytometry. Data are presented as means ± S.E. for four independent experiments with triplicate determinations. *P<0.05 as compared with control. #P<0.05 as compared with pretreat NAC.
Figure 24. Cadmium induces related apoptosic mRNA expression in RIN-m5F
RIN-m5F cells were exposed with cadmium (0, 5, 10 μM) to investigate Bcl-2, Bax, p53 and Mdm2 mRNA were determined by real-time PCR. Data are presented as means ± S.E. for four independent experiments with triplicate determinations.
*P<0.05 as compared with control.
Figure 25. Effects of cadmium-induced plasma insulin secretion in ICR mice.
ICR mice were exposed with cadmium (10 mg/kg) for 1，2，4 and 6 weeks to investigate plasma insulin secretion by insulin assay. Data are presented as means ± S.E. for four independent experiments with triplicate determinations. *P<0.05 as compared with control.
Figure 26. Effects of cadmium-induced blood glucose in ICR mice.
ICR mice were treated with glucose (1 g/kg) at first, and ICR mice were exposed with cadmium (10 mg/kg) for 30，60，90 and 120 minute to investigate blood glucose level.
Data are presented as means ± S.E. for four independent experiments with triplicate determinations. *P<0.05 as compared with control.
Figure 27. Cadmium-induced the apoptotic effect of RIN-m5f involved in mitochondrial pathway.