Sr-bi Suppresses Cytotoxicity-induced By Trivalent Iron

OBJECTIVETo evaluate the histological changes and iron deposition in the Red Pulp of spleens of SR-BI-deficient mice, effects of Fe-NTA on SR-BI expression in HepG2(-) cells and effects of SR-BI on suppression of cytotoxicity-induced by trivalent iron, such as Fe-NTA, hemoglobin or hemin. Furthermore, explore the underlying mechanism of the cell damage and death induced by Fe-NTA, so to provide a theoretical and experimental evidence of SR-BI suppresses cytotoxicity-induced by trivalent iron and a new focus on the function of SR-BI research.METHODS1. Heterozygous SR-BI+/- mice on a mixed C57BL/6 x129-background were obtained from the Jackson Laboratory. SR-BI-/- and SR-BI+/+ littermates were generated by mating SR-BI+/- mice.8-12 week-old mice were sacrificed by anesthesia. Spleens were isolated and removed for the conventional tissue sections and HE and Perls Prussian Blue staining. Evaluate the histological changes and hemosiderin deposition in the Red Pulp of spleens of SR-BI+/+ and SR-BI-/- mice under microscope.2. Using Western blot to detect the expression levels of SR-BI in HepG2 (-) cells that treated by various concentrations of Fe-NTA; transfect the human SR-BI-cDNA into CHO cells to generate CHO-Vector cells and CHO-SR-BI cells, then 2- to 3-generations used in experiments were treated with different concentrations of Fe-NTA (0,0.2,0.4,0.8 and 1.0mM), NTA (0,0.8,1.0 and 2.0mM) and H2O2 (0,0.2,0.4 0.8 and 1.0mM). Assess the cells ability by microscope, detect the LDH levels in the cultured cells medium and the effects of SR-BI on the cytotoxicity induced by Fe-NTA.3.2- to 3-generations of CHO-SR-BI cells and CHO-Vector cells were treated with0.2 mM Fe-NTA for 24 hours and 0.05mM Fe-NTA for 48 hours. Assess the cells ability using the microscope, extract cells DNA and identificate DNA ladder of the cell apoptosis by using DNA agarose gel electrophoresis to explore the underlying mechanism of cytotoxicity induced by Fe-NTA.2- to 3-generations of cultured CHO-SR-BI cells and CHO-Vector cells were treated with different concentrations of Fe-NTA-H2O2 complexes (0,0.2,0.4,0.8 and 1.0mM), hemoglobin-H202 complexes (0,6.25,12.5 and 25.0uM) and hemin-H2O2 complexes (0,6.25,12.5 and 25.0uM). Assess the cells ability using the microscope, detect the LDH levels in the two cultured cells medium and the effects of the expression of SR-BI on cytotoxicities induced by complexes used in our experiments.RESULTS1. HE and Perls Prussian Blue staining:Compared to SR-BI+/+ littermates, the histological structure of SR-BI-/- mice was changed, the distribution of red pulp and the various size of white pulp is irregular, and the amount of hemosiderin and the content of iron in the red pulp region of spleens of SR-BI-/- mice were significantly increased.2. In our experimental conditions, the results indicated that Fe-NTA could result in the non-apoptotic cell damage and death. Western blot detection and assay:There was no obviously up-regulation expression of SR-BI of the human HepG2 (-) cells under the various concentrations of Fe-NTA (p>0.05)3. Cytotoxicity-induced by Fe-NTA was markly enhanced by H2O2 (p<0.05); compared to control groups (CHO-Vector cells), the LDH release percents were obviously lower in the experiment groups (CHO-SR-BI) in the different concentrations of Fe-NTA, Fe-NTA and H2O2 complexes, hemoglobin and H2O2 complexes or hemin and H2O2 complexes, there is significant difference of the percent between control groups and experiment groups (p<0.05).CONCLUSION1. Compared to SR-BI+/+ littermates, the histological structure of SR-BI-/- mice was changed, the distribution of red pulp and the various size of white pulp is irregular, and the amount of hemosiderin and the content of iron in the red pulp region of spleens of SR-BI-/-mice were significantly increased.2. Cytotoxicity induced by Fe-NTA is in a dose-dependent way to result in the non-apoptotic cell damage and death in our experimental conditions. There were no significant changes of SR-BI expression levels in HepG2 (-) cells treated by various concentrations of Fe-NTA.3. Cytotoxicity induced by Fe-NTA was markly enhanced by H2O2 SR-BI suppresses cytotoxicity-induced by trivalent iron, such as Fe-NTA, Fe-NTA-H2O2 complexes, hemoglobin-H2O2 complexes or hemin-H2O2 complexes.