ABSTRACT
Objective To investigate the effect and mechanism of advanced oxidation protein products (AOPPs) on the apoptosis of HaCaT cells. Methods AOPPs were prepared by incubation of human serum albumin (HSA) with sodium hypochlorite solution. MTT assay was used to determine the effects of different concentrations (0, 50, 100, 200 and 400µg/ml) of AOPPs on HaCaT cell activity by incubating the cells for 24h for selecting optimum working concentration of AOPPs. HaCaT cells were treated based on time effect or concentration effect. The incidence of cell apoptosis was measured by flow cytometry. Intracellular reactive oxygen species (ROS) production was determined with dichlorofluorescein diacetate (DCFH-DA). And the protein expressions of NOX4, Bax and Bcl-2 were examined by Western blotting. Results Viability of HaCaT cells treated with 400µg/ml AOPPs was significantly lowered (P<0.05). AOPPs, in a time- and concentration-dependent manner, induced the apoptosis of HaCaT cells (P<0.05), increased the expression of NOX4 and Bax (P<0.05) and down-regulated the expression of Bcl-2 (P<0.05); prolonged exposure to AOPPs and increase of AOPPs concentration both led to an increase of intracellular ROS production (P<0.05). Conclusion AOPPs may induce apoptosis of HaCaT cells through NADPH oxidase pathway.
ABSTRACT
Objective To study whether the uremic toxins accumulated long-term in uremia patients may be involved in oxidation of protein by forming advanced oxidative protein products (AOPPs). Methods Malonylaldehyde (MDA), hippuric acid (HA) and p-cresol were used as the representatives of uremic toxins. Human albumin serum (HSA), plasma specimens from normal or uremia patients were incubated respectively with MDA (10 retool/L), HA (20 mmol/L) and p-cresol (10 retool/L) or PBS (20 retool/L, pH 7.4, as control groups) at 37℃ for 30 minutes or 24 hours, respectively. Those indices such as AOPPs, protein thiol groups (Pt-SH) and dityrosine were used as biomarkers of protein injury. High performance liquid chromatography (HPLC) was employed to identify the aggregation and cross-links of modified proteins. Results AOPPs levels in all groups containing poison compounds were significantly increased by 121.5%(P<0.05) compared to that in control groups. Uremic toxins also resulted in over 14.7% loss in Pt-SH (P< 0.05) and 119.2% increment in dityrosine, respectively (P<0.05). Meanwhile, the formation of HMW-AOPPs in a time-dependent manner was observed by HPLC and cross-linked protein levels were significantly increased by 148.45%～333.3% in comparison with control groups. Conclusion Uremic toxins can directly mediate the damage of proteins by inducing the formation of HMW- AOPPs in a time-dependent manner, which is also one of the mechanism of AOPPs production in vivo besides the activation of the myeloperoxidase-H2O2-Cl pathway.
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Objective To study the effect of oxidative modification of hydrochlorous acid (HOCl) on human serum albumin (HSA) and the relationship between the AOPPs and HOCl-treated HSA. Methods Purified HSA (60 mg/ml) was treated with HOCl (0, 1, 5, 10, 20, 30, 40, 50 and 60 mmol/L). Size-exclusion chromatography was applied to estimate molecular weights of oxidized products of HSA by HOCl and spectrum scan from 190 nm -400 nm was performed to observe the spectrum characteristics of all variants of HSA. Results Major products of HSA after exposure to HOC1 were dimer and hexmer of HSA. The first-order process could be employed to describe the oxidative dynamics of monomer and dimer of HSA oxidized by HOCl. To AOPPs formation mediated by oxidant was identified as pseudo first-order reaction. However, formation hexmer was much in accordance with second-order reaction. Hexmer was also a major contributor to AOPPs in all types of modified HSA. Spectral analysis showed that red shift of absorbance maximum of polymers of HSA occurred, suggesting that a possibility that polymers of HSA were cross linked by tyrosine residues in protein. Conclusions Protein aggregation is primary consequence of HSA after its exposure to HOCl. Hexmer of HSA is the major contributor to AOPPs.
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Objective To study the effects of oxidants on the structure of albumin. Methods Using both AOPPs and protein carbonyl content as indices. The oxidative stress level in normal controls and uremia patients was evaluated. Albumin in plasma was purified by HPLC and then was subjected to amino acids composition assay. Results Both AOPPs level and protein carbonyl content in uremic patients were significantly higher than those in controls (P