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@#Objective To develop and verify a sulfosalicylic acid spectrophotometric method for the determination of trace iron ions in diphtheria toxin medium,and apply it preliminarily.Methods The maximum absorbance of the complex of iron and sulfosalicylic acid was scanned by full spectrum;A method for the determination of iron ion in culture medium was developed by linear regression between the absorbance of the complex and the content of iron ion,and the stability,accuracy and precision of the method were verified.The effects of Ca~(2+),Mg~(2+),K~+,Na~+ and reactants on the method were investigated.Spectrophotometric method with sulfosalicylic acid was used to determine trace iron in the self-made and commercial medium for diphtheria toxin production.Sulfosalicylic acid spectrophotometry,ferrizine colorimetry and o-phenanthroline spectrophotometry were used to detect iron content in two kinds of culture media(beef trypsin digestion liquid and 5% polypeptone),and the detection results of the three methods were compared.Results The complex of iron and sulfosalicylic acid showed the maximum absorbance at the wavelength of 425 nm;There was a good linear relationship between the absorbance and concentration of iron ion in the range of 1～0.05 μg/mL,the detection limit was 0.05 μg/mL,and the standard equation was:Y=0.027 9 X+0.046 1,R~2> 0.99;The coefficients of variation(CVs) of A_(425) value of each concentration of standards measured every 5 min were less than 5%;Low(0.05 μg/mL),medium(0.5 μg/mL)and high concentration(1 μg/mL) of Fe~(3+) standard solutions were continuously determined for 3 times.The CVs of 9groups of each concentration measured in parallel were all less than 5% and the recovery rates were higher than 95%;Ca~(2+),Mg~(2+),K~+,Na~+,15 μL of sulfo salicylic acid(20%) and 50 μL of ammonia hydroxide(1:1) showed no interference in the method;The results of toxin-producing medium were consistent with those of diphtheria bacteria;The results of the three detection methods were consistent.Conclusion The developed spectrophotometric method with sulfo salicylic acid can determine the content of trace iron ions in diphtheria toxin medium accurately and effectively.
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Background A large amount of iron deposition in the brain can cause neuronal damage by inducing oxidative stress, neuroinflammation, and abnormal mitochondrial function. In addition, iron deposition is also reported to be closely related to the pathogenesis of Alzheimer's disease (AD). The neurofibrillary tangles aggregated by tau hyperphosphorylation are one of the important pathological features of AD. Objective To investigate potential effect of exogenous trivalent iron ions on neuronal activity in human neuroblastoma (SH-SY5Y) cells and tau hyperphosphorylation and aggregation. Methods SH-SY5Y cells were treated with ferric chloride (FeCl3) at four concentrations (10, 100, 200, and 400 mg·L−1). Cell survival rate was then detected by CCK8 assay. Intracellular iron content was determined prussian blue (Perl's) by iron staining after 24 h exposure to FeCl3 at 10 or 200 mg·L−1. Transfection of tau-P301L plasmid was conducted to construct an AD-like cell model for tau overexpression. The differences in the expression of the phosphorylated tau (p-tau) protein in SH-SY5Y cells and SH-SY5Y cells with tau overexpression were detected by Western blotting after 24 h exposure to FeCl3 at 10 and 200 mg·L−1. After dilution with phosphate buffered saline (PBS), FeCl3, human tauR3, and FeCl3 + tauR3 were incubated at 37℃, and the fluorescence intensity reflecting tau aggregation level was measured by thioflavin T(ThT) method at 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively. Meanwhile, after 96 h coincubation of FeCl3 and tauR3, the fibers formed by tau aggregation were observed under a transmission electron microscope (TEM). Results After 24 h of FeCl3 exposure, the cell survival rate of SH-SY5Y cells among all groups was statistically different (F=8.63, P<0.01). The cell survival rates in the 200 and 400 mg·L−1 groups were 80.1% and 68.7% of the control group, respectively (P<0.05). Compared with the control group, the nuclei of the 200 mg·L−1 FeCl3 group were mainly yellowish-brown after iron staining and the positive cell rate was up-regulated by 12.9% (P<0.01). After 24 h of FeCl3 exposure , the p-tau (Ser396) protein expression was statistically different among all groups (F=11.6, P<0.01). Compared with the control group, the p-tau protein expression level of SH-SY5Y cells in the 200 mg·L−1 group was up-regulated by 72.7% (P<0.01). After FeCl3-treated SH-SY5Y cells with tau overexpression for 24 h, the p-tau (Ser396) protein expression was statistically different among all groups (F=27.8, P<0.01). Compared with the tau group, the p-tau (Ser396) protein expression level of SH-SY5Y cells in the tau + 200 mg·L−1 group was up-regulated by 44.6% (P<0.05). Compared with the tauR3 group, the fluorescence intensities in the 84 and 96 h tauR3 + FeCl3 groups were up-regulated by 49.9% and 53.7% (P<0.01) respectively. After 96 h of coincubation, compared with the tauR3 group, FeCl3 + tauR3 aggravated tau aggregation and formed fiber deposition under TEM. Conclusion Exogenous trivalent iron ions may inhibit SH-SY5Y cell viability, promote the phosphorylation of tau in SH-SY5Y cells transfected with tau-P301L plasmid, and aggravate tauR3 aggregation and fiber production.
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Objective@#To investigate the mechanism of Al (mal) 3-induced ferroptosis in rat adrenal pheochromocytoma cells (PC12), to explore the effect of deferoxamine (DFO) .@*Methods@#Taken PC12 cells growing at logarithmic phase and divided into 6 groups: control group, 200 μmol/L Al (mal) 3 group, 0.5% DMSO group, 200 μmol/L DFO group, Al (mal) 3+DMSO group, Al (mal) 3+DFO group. DMSO and DFO were added to the DMSO group and the Al (mal) 3+DMSO group, the DFO group and the Al (mal) 3+DFO group for 2 h, respectively, Al (mal) 3 was then added to the Al (mal) 3 group, Al (mal) 3+DMSO group, and the Al (mal) 3+DFO group to a final concentration of 200 μmol/L. The cell viability was detected by CCK8, the morphology and ROS levels of PC12 cells was observed by inverted microscope, the cell proliferation toxicity and intracellular iron ion content were detected by colorimetry, the GSH content and GSH-PX activity were detected by biochemical method.@*Results@#Al (mal) 3 exposure significantly inhibited the growth of PC12 cells and destroyed the cell morphological structure, resulting in increased LDH activity and intracellular iron ion content in PC12 cells, decreased GSH content and GSH-PX activity, increased ROS levels; the combined treatment of Al (mal) 3+DFO can significantly improve the cell viability of PC12 cells, improved cell morphology, decreased cell LDH activity and intracellular iron ion content (P>0.05), increased GSH content and GSH-PX activity, decreased ROS levels.@*Conclusion@#Al (mal) 3 can induce ferroptosis in PC12 cells, DFO may inhibit ferroptosis by reducing intracellular iron levels and reducing oxidative damage.
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Objective: To establish the in vitro screening method for determining what kinds of Chinese materia medica (CMM) and which main constituents in the medicine used for the treatment of Alzheimer's disease, Parkinson's disease, and osteoporosis can chelate with free iron ions (Fe3+) and evaluating the chelating strength. Methods: Based on the catalytic effect of Fe3+ on the spirolactam (nonfluorescence) to ring-open amide reaction (fluorescence), the catalytic spectrophotometric method was established to determine the free Fe3+ in the decoction of CMM, by adding a certain amount of iron ion and determinig the chelating level to evaluate the chelating strength of the constituents in CMM. Results: The chelating strength of kidney-tonifying CMM is stronger. The catalytic spectrophotometric method could be used to determine the free Fe3+ in the decoction of CMM and the linearity was good in the range of 1.68-22.4 mg/L, r = 0.999 0. The average recovery was between 90.45% and 104.11% with RSD < 5%. Conclusion: This method can be used to evaluate the chelating capacity of constituents in CMM with Fe3+.
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Intracerebral hemorrhage (ICH) is a common nervous system disease, its mortality and disability are very high. However, the mechanisms of brain injury after ICH have not yet been fully explained. "[he latest studies have suggested that iron overloading plays an important role in the brain injury after ICH. This article reviews the advances in research on the distribution and function of iron in the brain, the mechanisms of brain injury caused by iron overloading after ICH as well as the use of iron chelator.
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A series of pathophysiologic changes in brain tissues will occur after intracerebral hemorrhage, including the enlargement of hematoma, metabolism abnormality in perihematoma tissues, and formation of cerebral edema. Recent researches have demonstrated that iron ions play an important role in the secondary brain injury after intracerebral hemorrhage. Iron chelator can block iron-induced injury process by specifically binding iron ions. This article reviews the changes of iron metabolism, iron-related mechanisms of brain injury, and the neuroprotective effect of iron chelator.