Heme and Cu2+-induced vasoactive intestinal peptide (VIP) tyrosine nitration: A possible molecular mechanism for the attenuated anti-inflammatory effect of VIP in inflammatory diseases.

Vasoactive intestinal peptide (VIP) is a neuropeptide that play an important role in immunoregulation and anti-inflammation. Numerous inflammatory/autoimmune disorders are associated with decreased VIP binding ability to receptors and diminished VIP activation of cAMP generation in immune cells. However, the mechanisms linking oxidative/nitrative stress to VIP immune dysfunction remain unknown. It has been reported that the elevated heme or Cu2+ in inflammatory diseases can cause oxidative and nitrative damage to nearby biological targets under high oxidative stress conditions, which affects the structure and activity of linked peptides or proteins. Thus, the VIP down-regulated immune response may be interfered by redox metal catalyzed VIP tyrosine nitration. To explore this, we systematically investigated the possibility of heme or Cu2+ to catalyze VIP tyrosine nitration. The results showed that Tyr10 and Tyr22 of VIP can both be nitrated in heme/H2O2/NO2- system as well as in Cu2+/H2O2/NO2- system. Then, we used synthetic mutant VIPs with tyrosine residues substituted by 3-nitrotyrosine to study the impact of tyrosine nitration on VIP activity in SHSY-5Y cells. Our findings demonstrated that VIP nitration dramatically decreased the content of its α-helix and random coil, suggesting that VIP nitration might reduce its affinity to the receptor. This was further confirmed in the cAMP assay. The results showed that 10 nM of these tyrosine nitrated VIPs could significantly (p < 0.01) decrease cAMP secretion compared to the wild type VIP. Our data reveal that the attenuation of the neuroprotective effect of VIP in inflammation-related diseases might be attributed to metal-catalyzed VIP tyrosine nitration.

Inhibitory activities of flavonoids from Scutellaria baicalensis Georgi on amyloid aggregation related to type 2 diabetes and the possible structural requirements for polyphenol in inhibiting the nucleation phase of hIAPP aggregation.

Human islet amyloid polypeptide (hIAPP)-mediated cytotoxicity is identified as a potential target for developing new anti-diabetic molecules. Herein, we investigated the effect of the major bioactive compounds of Scutellaria baicalensis Georgi (S. baicalensis), including baicalein, baicalin, wogonin and oroxylin A, on hIAPP aggregation. We found that all of these compounds inhibited hIAPP fibril formation in a dose-dependent manner. But baicalein and baicalin, especially baicalein are more effective than wogonin and oroxylin A in stabilizing hIAPP monomers and eliminating toxic hIAPP assembly, suggesting that flavonoids with ortho-hydroxyl group on the A-ring exhibited higher anti-hIAPP nucleation potential than those without this structure. This stimulated our interest in further studying the possible structure-activity relationship between polyphenol and hIAPP aggregation inhibition. Our results demonstrated that flavonoids with ortho-hydroxyl group on the B-ring are also more effective against hIAPP nucleation than those without this structure. These results suggest that the ortho-hydroxybenzene structure is a key structural feature required for polyphenols to effectively inhibit hIAPP nucleation. This was further confirmed by the effects of polyphenol and phenols in inhibiting hIAPP nucleation. The conclusion that pyrogallol-type polyphenols are potential lead inhibitors may provide a valuable structural template for the further development of polyphenol-based inhibitor of amyloid peptides.

Poly(tannic acid) nanocoating based surface modification for construction of multifunctional composite CeO2NZs to enhance cell proliferation and antioxidative viability of preosteoblasts.

Ceria (CeO2) based materials possess many antioxidant enzyme-like activities and unique properties for bone repair, but their free radical scavenging function is still insufficient. In order to deal with the complex oxidative stress environment in bone repair, multifunctional composite CeO2 nanozymes (CeO2NZs), featuring multiple antioxidative properties, were constructed via surface modification on CeO2NZs with nanoscale poly(tannic acid) (PTA) coatings. Moreover, we adjusted pH conditions (ranging from 4 to 9) to effectively control the formation and antioxidative properties of PTA coatings on CeO2NZ surfaces. Here, the physical properties of this novel inorganic and organic composite antioxidant, such as surface morphology, particle size, crystal structure, surface charge and element composition, were thoroughly characterized. The PTA/CeO2NZs showed obvious coating morphology under weak acid conditions (pH = 5-6), and the PTA layer at pH = 5 is about 1 nm in thickness. Compared with untreated CeO2NZs, the PTA/CeO2NZs showed stronger SOD-like activity and obviously higher free radical scavenging rate (for both ABTS+˙ and DPPH˙).Notably, this composite antioxidative nanozyme not only exhibited favorable cell proliferation of preosteoblasts (MC3T3-E1) but also provided strong antioxidative property to maintain cell vitality against H2O2 induced oxidative damage. In particular, this study provides new insights into the designing of surface polyphenolic coatings at the nanoscale, and these multiple antioxidative properties shown by PTA coated CeO2NZs make them suitable for protecting cells under the oxidative stress environment.

Structure relationship of metalloporphyrins in inhibiting the aggregation of hIAPP.

Metalloporphyrins (FeTBAP, MnTBAP, FeTMPyP and MnTMPyP) have been proposed as effective therapeutic agents in ONOO--related disease including type 2 diabetes (T2D). As these metalloporphyrins share the structural similarities of the planar aromatic conjugation with a valuable class of inhibitors against amyloids fibrillation, they might be effective inhibitors via aromatic π-π stacking interactions with amyloid peptides. Here, we found that the anionic metalloporphyrins (FeTBAP and MnTBAP) are effective inhibitors against hIAPP fibrillation, while, the cationic metalloporphyrins (FeTMPyP and MnTMPyP) only have limited inhibitory effects. Besides, the porphyrin with iron center is more effective than the one with manganese center. Our results favor the electrostatic attraction contributes the main reason to the inhibitory effect between the anionic porphyrins and hIAPP, followed by the π-π stacking interactions between aromatic ring of porphyrins and hIAPP and the stronger coordination ability of iron center to hIAPP. Additionally, by comparison with FeTBAP, which can completely inhibit cytotoxicity induced by hIAPP via stabilizing hIAPP monomers, MnTBAP fails to reverse the cytotoxicity due to that it can only delay the transition of hIAPP from α-helix to ß-sheet rich oligomers. Our results provide theoretical significance for further designing or screening of metalloporphyrins as bifunctional antidiabetic drugs.

Y12 nitration of human calcitonin (hCT): A promising strategy to produce non-aggregation bioactive hCT.

Irreversible aggregation can extremely limit the bioavailability and therapeutic activity of peptide-based drugs. There is therefore an urgent demand of effective strategy to control peptide aggregation. Recently, we found that tyrosine nitration at certain sites of peptide can effectively inhibit its aggregation. This minor modification may be an ideal strategy to the rational design of peptide-based drugs with low aggregation propensity yet without loss of bioactivity. Human calcitonin (hCT) is such a peptide hormone known for its hypocalcaemic effect but has limited pharmaceutical potential due to a high tendency to aggregate. In this study, by using multiple techniques including Fluorescence, TEM, Nu-PAGE and CD, we demonstrated that Y12 nitration of hCT would significantly inhibit its self-assembles, and we also found that this modification would not only reduce the cytotoxicity induced by peptide aggregation, but also had little effect on its potency. This finding may provide a novel strategy for clinically application of hCT instead of sCT.

Peroxynitrite scavenger FeTPPS effectively inhibits hIAPP aggregation and protects against amyloid induced cytotoxicity.

Type 2 diabetes (T2D) is associated with pancreatic ß-cell dysfunction, which can be induced by oxidative stress or/and the aggregation of human islet amyloid polypeptide (hIAPP). Therefore, ONOO- and hIAPP become the crucial targets of T2D treatment. Previously, we found heme could be an effective inhibitor of hIAPP aggregation. However, heme causes serious toxic effects on cells, tissues and organs through oxidative stress, which block it as a potential drug candidate for T2D treatment. 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato iron(III) chloride (FeTPPS), a water-soluble derivative of heme, is recognized as a high-efficient ONOO- decomposition catalyst, which is reported to have a great therapeutic potential in ONOO- -related diseases, including T2D. Here, we explored the potentiality of FeTPPS to be an inhibitor of hIAPP aggregation and the protective effects on cytotoxicity of hIAPP aggregation. It was found that the interaction between FeTPPS and hIAPP remarkably affected hIAPP fibrillation by both stabilizing hIAPP monomers and disaggregating the long fibrils into small oligomeric species. Furthermore, unlike heme, the addition of FeTPPS completely reversed the cytotoxicity and ROS level induced by hIAPP, which was consistent with its strong inhibitory activity. These results implied that FeTPPS could be a promising agent for the treatment of T2D.

Structure effect of water-soluble iron porphyrins on catalyzing protein tyrosine nitration in the presence of nitrite and hydrogen peroxide.

Water-soluble iron porphyrins, such as FeTPPS (5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III)), FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphyrinato iron (III) chloride) and FeTBAP (5,10,15,20-tetrakis (4-benzoic acid) porphyrinato iron (III)), are highly active catalysts for peroxynitrite decomposition and thereby have been suggested as therapeutic agent for inflammatory diseases that implicate the involvement of nitrotyrosine formation. Here, we systemically investigated catalytic properties of FeTPPS, FeTMPyP and FeTBAP on protein nitration in the presence of hydrogen peroxide and nitrite. We showed that FeTPPS, FeTBAP and FeTMPyP all exhibited higher peroxidase activity in compared with hemin. As to protein nitration, the catalytic effect of FeTPPS and FeTBAP are effective in the presence of hydrogen peroxide and nitrite, while negligible BSA nitration was observed in the case of FeTMPyP. Moreover, the underlying mechanism of the oxidation of FeTPPS, FeTBAP and FeTMPyP was further studied. Collectively, our results suggest that, compound I and II species are involved in as the key intermediates in FeTMPyP/H2O2 system as similar as those in FeTPPS/H2O2 and FeTBAP/H2O2 system. As compared to weak antioxidants, TPPS and TBAP, however, TMPyP scavenges oxo-Fe (IV) intermediates of FeTMPyP at a faster rate by significant self-degradation; results in the shortest lifetimes of OFeIV-TMPyP and the lowest catalytic activity on oxidizing tyrosine and nitrite; and therefore, attributes to inactivation of FeTMPyP in protein nitration. In addition, association of FeTMPyP to BSA was found weak, while strong binding of FeTPPS and FeTBAP were observed. The weak binding keeps away of target residue of BSA from the center of FeTMPyP where the RNS is generated, which might be attributed as additional factors to the inactivation of FeTMPyP in protein nitration.

Nitration of hIAPP promotes its toxic oligomer formation and exacerbates its toxicity towards INS-1 cells.

Amyloid formation of human islet amyloid polypeptide (hIAPP) is one of the most common pathological features of type 2 diabetes (T2D). Increasing evidences have shown that the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an important role in the development of the T2D. Interestingly, our previous studies indicated that heme could bind to hIAPP, and the complex might induce the nitration of tyrosine residue (Y37) of hIAPP in the presence of hydrogen peroxide and nitrite. However, it remains unclear about effect of the nitration on the implicated function of hIAPP in the development of T2D. In this study, fluorescent assays, transmission electron microscopy (TEM), atomic force microscope (AFM) were used to demonstrate that nitration of hIAPP significantly decreased its fibril formation. But the decreased fibril formation was not through the diminished aggregation of hIAPP monomer as suggested by the results of circular dichroism spectroscopy (CD) and gel electrophoresis assay. Surface-enhanced raman spectroscopy (SERS) indicated that nitration of hIAPP impaired the intermolecular hydrogen bonding. On the basis of these results, we hypothesize that nitration of hIAPP may block the intermolecular hydrogen bonding, leading to the inhibition of its fibril formation. In addition, cytotoxicity study of native and modified hIAPP was also performed on INS-1 cells, which revealed exacerbated toxicity of hIAPP by its nitration. The findings in this study that nitration of hIAPP promotes its oligomer formation and thus exacerbates its cytotoxicity suggests a possible link between the nitrite (or the sum of nitrite and nitrate) levels and T2D, and ameliorated nitration of hIAPP by diminishing nitrative stress might be a promising therapeutic strategy for T2D.

Study on the detoxification mechanisms to 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) chloride (FeTPPS), an efficient pro-oxidant of heme water-soluble analogue.

5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato iron(III) chloride (FeTPPS) is a water-soluble analog of heme and widely employed as peroxynitrite scavenger in vivo. However, previous studies have showed that like heme, FeTPPS could also act as an effective pro-oxidant towards appreciable substrates in vitro in the presence of oxidant. The reason that FeTPPS did not show any pro-oxidative damage in previous studies when it was used as peroxynitrite decomposition catalyst in vivo, has not been studied. Herein, the effects of two main detoxification mechanisms of heme, i.e., serum albumin (SA) binding and heme oxygenase-1 (HO-1) induction, were examined on FeTPPS in vitro. Fluorescence quenching studies showed bovine serum albumin (BSA) could bind to FeTPPS with high affinity (Kb ~ 109 M-1). Molecular docking studies presented us the details of the binding site that is not a heme pocket. Furthermore, the intrinsic pro-oxidative activity of FeTPPS was found effectively inhibited by forming BSA-FeTPPS complex of low reactivity, which could be thought to protect against the potentially toxic effects of FeTPPS on blood components. In addition, this binding could protect FeTPPS against oxidative degradation. In albumin-free cell system, cell viability results indicated FeTPPS was innoxious to living cells and could protect cells against the oxidative impairment of H2O2 effectively rather than promoting damage. Using western blot, we illustrated that HO-1 expression could not be induced by FeTPPS, which suggested that HO-1 was not related to the protective capacity of FeTPPS. Our results provide a better understanding of FeTPPS and lead to a new guidance to its application.

5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS), a peroxynitrite decomposition catalyst, catalyzes protein tyrosine nitration in the presence of hydrogen peroxide and nitrite.

5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS) is a water-soluble heme analog, which has been used as a scavenger of peroxynitrite in many studies. Similar to heme, it may also possess pseudo-peroxidase activity that could cause protein tyrosine nitration through the peroxidase-H2O2-NO2- pathway. In this paper, we used western blotting and spectrophotometry analysis to study the capability of FeTPPS in catalyzing protein tyrosine nitration. Furthermore, the capability of FeTPPS in catalyzing protein nitration in tissue homogenate and cultured cells was also investigated. Our results showed that FeTPPS induced bovine serum albumin (BSA) nitration in the presence of H2O2 and NaNO2, and the reaction was dose-, time- and pH-dependent. In acidic condition, more protein was nitrated by FeTPPS than heme, which corresponded to their peroxidase activities. Meanwhile, our results also confirmed the catalytic effect of FeTPPS on protein tyrosine nitration in rat brain homogenate and human hepatocellular carcinoma (HepG2) cells. At the end of this study, we used liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to investigate differences of site selectivity between heme and FeTPPS catalyzed protein tyrosine nitration. The result indicated that FeTPPS tended to catalyze tyrosine residues locating in more hydrophilic sites, whereas heme was more likely to induce nitration of tyrosine residues locating in relatively hydrophobic environment. Taken together, this is the first report that FeTPPS is an effective and convenient nitration catalyzer in vitro, and this study confirms that the hydrophilicity of the nitrating agents would play an important role in nitration site selection.

Strong Inhibitory Effect of Heme on hIAPP Fibrillation.

The deposition of human islet amyloid polypeptide (hIAPP) within ß-cells is implicated in the etiology of type 2 diabetes mellitus (T2Dm). It was reported that heme could bind to hIAPP. We speculate that binding may affect the aggregation of hIAPP. In this study, UV-vis spectroscopy was used to detect the interaction pattern between the heme and hIAPP. ThT and Bis-ANS fluorescence assay, circular dichroism spectroscopy, gel electrophoresis assay, and transmission electron microscopy were employed to study the effect of heme on the aggregation of hIAPP. We found that heme dramatically inhibited hIAPP aggregation, even partially dismantled hIAPP aggregates by preventing its conformational changes. Moreover, a similar inhibitory effect was also observed on mutant hIAPP. In the compared group, the inhibitory effects of protoporphyrin on hIAPP and its mutants aggregation were weaker. Similarly, its effect on the dismantlement of the aggregates was also weaker. On the basis of these results, we revealed that the heme iron center was not required for the inhibitory effect on hIAPP but affected the binding affinity of heme to hIAPP. Besides Arg11 and His18, other hydrophobic residues of hIAPP may also play important roles in heme binding. Our results may help to develop an in-depth understanding of the interaction between heme and hIAPP, which would be helpful in designing new therapeutic strategies against T2Dm.

Interaction of glyceraldehyde-3-phosphate dehydrogenase and heme: The relevance of its biological function.

GAPDH was speculated to function as a transient trap to reduce the potential toxicity of free heme by a specific and reversible binding with heme. Up to now, there has been lack of studies focused on this effect. In this paper, the efficiency of GAPDH-heme complex on catalyzing protein carbonylation and nitration, the cross-linking of heme to protein formation, and cytotoxicity of GAPDH-heme were studied. It was found that the binding of GAPDH could inhibit H2O2-mediated degradation of heme. Peroxidase activity of GAPDH-heme complex was higher than that of free heme, but significantly lower than that of HSA-heme. Catalytic activity of heme corresponded complex toward tyrosine oxidation/nitration was decreased in the order of HSA-heme, heme and GAPDH-heme. GAPDH also inhibited heme-H2O2-NO2- induced protein carbonylation. No covalent bond was formed between heme and GAPDH after treated with H2O2. GAPDH was more effective than HSA on protecting cells against heme-NO2--H2O2 induced cytotoxicity. These results indicate that binding of GAPDH inhibits the activity of heme in catalyzing tyrosine nitration and protects the coexistent protein against oxidative damage, and the mechanism is different from that of HSA. This study may help clarifying the protective role of GAPDH acting as a chaperone in heme transfer to downstream areas.

Nitration of Tyrosine Residue Y10 of Aß1-42 Significantly Inhibits Its Aggregation and Cytotoxicity.

Amyloid-ß plaques and oxidative stress are the major hallmarks of Alzheimer's disease. Our previous study found that the heme-Aß complex enhanced the catalytic effect of free heme on protein tyrosine nitration in the presence of hydrogen peroxide (H2O2) and nitrite (NO2-). Y10 in Aß could be the first target to be nitrated. We also found that nitration of Aß1-40 significantly decreased its aggregation. However, a contrary report showed that nitration of Aß1-42 by peroxynitrite enhanced its aggregation. To rule out the interference of peroxynitrite caused Aß oxidation, we used synthetic Y10 nitrated Aß1-42 to study the influence of Y10 nitration on Aß1-42's aggregation and cytotoxicity in this study. We confirmed that Aß1-42 could be nitrated in the presence of H2O2, NO2-, and heme by dot blotting. CD spectroscopy showed an increase of ß-sheet structure of Aß1-42 and its mutants. The thioflavin T (ThT) flourescence assay revealed that both nitration and chlorination significantly inhibited Aß1-42 fibril formation. TEM and AFM observations of Aß peptide aggregates further confirmed that Y10 modification inhibited Aß1-42 fibril formation. The cytotoxicity study of native and modified Aß peptides on SH-SY5Y cells revealed that nitration of Aß1-42 remarkably decreased the neurotoxicity of Aß1-42. On the basis of these results, we hypothesized that nitration of Y10 may block the π-π stacking interactions of Aß1-42 so that it inhibit its aggregation and neurotoxicity. More importantly, considerable evidence suggested that the levels of nitrite plus nitrate significantly decreased in the brain of AD patients. Thus, we believe that these findings would be helpful for further understanding the function of Aß in AD.

Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation.

It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction.

Association of cardiac injury with iron-increased oxidative and nitrative modifications of the SERCA2a isoform of sarcoplasmic reticulum Ca(2+)-ATPase in diabetic rats.

The role of iron in the etiology of diabetes complications is not well established. Thus, this study was performed to test whether the iron-induced increase of oxidative/nitrative damage is involved in SERCA2a-related diabetic heart complication. Four randomly divided groups of rats were used: normal control group; iron overload group; diabetes group, and diabetic plus iron overload group. Iron supplementation stimulated cardiomyocyte hypertrophy and led to an increase in cardiac protein carbonyls, nitrotyrosine (3-NT) formation, and iNOS protein expression, thus resulting in abnormal myocardium calcium homeostasis of diabetic rats. The levels of SECA2a oxidation/nitration were significantly increased in the iron overload diabetic rats, along with a decrease in SECA2a expression and activity. In order to elucidate the possible role of iron in SERCA2a dysfunction, the effects of iron (Fe(3+) or hemin) on peroxynitrite (ONOO(-)) induced SERCA2a oxidation and nitration were further investigated in vitro. It was found that tyrosine nitration played more important role in SERCA2a inactivation than thiol oxidation. These results present a potential mechanism in which iron exacerbates the diabetes-induced oxidative/nitrative modification of SERCA2a, which may cause functional deficits in the myocyte associated with diabetic cardiac dysfunction. Our findings may help to further understand the role of iron in the pathogenesis of diabetic complications.

Nitration of Y10 in Aß1-40: is it a compensatory reaction against oxidative/nitrative stress and Aß aggregation?

Amyloid ß peptide (Aß) aggregation is considered to be a crucial pathological biomarker of Alzheimer's disease (AD). It was found that Aß and heme can form an Aß-heme complex, which results in increased heme pseudoperoxidase activity. Recently, we found that increasing pseudoperoxidase activity induces elevated tyrosine nitration on Aß in the presence of nitrite and hydrogen peroxide. However, the nature of tyrosine nitration of Aß and its physiologic significance are still unknown. In this study, we revealed that Aß1-40 can be nitrated in vitro by binding to heme in the presence of nitrite and hydrogen peroxide. Moreover, we found that tyrosine nitration had little effect on Aß1-40's binding activity with heme. A TMB assay also revealed that the peroxidase activity of the heme-Aß1-40Y10(3N)T (tyrosine 10 was replaced with 3-nitrtotyrosine in Aß1-40) complex was moderately increased compared with that of the heme-Aß1-40 complex. Furthermore, Thioflavin T fluorescence and transmission electron microscopic characterization indicated that tyrosine nitration significantly decreased the aggregation of Aß1-40. In addition, a cytotoxicity test verified that wild-type Aß1-40 was more cytotoxic than that of Aß1-40Y10(3N)T. These results suggest that nitration of Aß1-40 might be an Aß detoxicant process and a compensatory reaction to nitrative stress. Our findings may lead to a detailed understanding of the function of Aß1-40 and may be helpful in preventing and curing AD.

Iron increases diabetes-induced kidney injury and oxidative stress in rats.

Diabetic nephropathy is both a common and a severe complication of diabetes mellitus. Iron is an essential trace element. However, excess iron is toxic, playing a role in the pathogenesis of diabetic nephropathy. The present study aimed to determine the extent of the interaction between iron and type 2 diabetes in the kidney. Male rats were randomly assigned into four groups: control, iron (300-mg/kg iron dextran), diabetes (a single dose of intraperitoneal streptozotocin), and iron + diabetes group. Iron supplementation resulted in a higher liver iron content, and diabetic rats showed higher serum glucose compared with control rats, which confirmed the model as iron overload and diabetic. It was found that iron + diabetes group showed a greater degree of kidney pathological changes, a remarkable reduction in body weight, and a significant increase in relative kidney weight and iron accumulation in rat kidneys compared with iron or diabetes group. Moreover, malondialdehyde values in the kidney were higher in iron + diabetes group than in iron or diabetes group, sulfhydryl concentration and glutathione peroxidase activity were decreased by the diabetes and iron + diabetes groups, and protein oxidation and nitration levels were higher in the kidney of iron + diabetes group as compared to iron or diabetes group. However, iron supplementation did not elevate the glucose level of a diabetic further. These results suggested that iron increased the diabetic renal injury probably through increased oxidative/nitrative stress and reduced antioxidant capacity instead of promoting a rise in blood sugar levels; iron might be a potential cofactor of diabetic nephropathy, and strict control of iron would be important under diabetic state.

Aß interacts with both the iron center and the porphyrin ring of heme: mechanism of heme's action on Aß aggregation and disaggregation.

Amyloid ß peptide (Aß) aggregation is a pathological hallmark of Alzheimer's disease (AD). Modulation of the self-assembly processes, therefore, is thought to be an attractive strategy for the prevention and treatment of AD. Interestingly, heme has been found to inhibit Aß aggregation and even dismantle Aß aggregates. However, the mechanism remains unresolved. Recent research has shown that heme binds preferentially to the His(13) residue of Aß with the iron center, while the hydrophobic domain of Aß is also able to bind to heme. Herein, absorption spectrometric, Thioflavin T fluorescence, and circular dichroism spectroscopic and transmission electron microscopic measurements revealed that the iron center is not required for the inhibition of Aß aggregation but do influence the binding affinity of heme toward Aß and the dismantlement rate and degree of the Aß aggregates. By studying the interaction of different truncated or mutated Aß peptides with heme or protoporphyrin, we further found that the porphyrin ring of heme is implicated to interact preferentially with the Phe(19) residue, facilitating the binding of heme to Aß and disturbing the interstrand aromatic interaction between the Phe residues, which is crucial for Aß fibrillation. These findings open new avenues in the understanding of the interaction between the heme and Aß and the pathways for modulation of Aß aggregation and disaggregation, which would be helpful in designing therapeutic strategies against AD.

Iron increases liver injury through oxidative/nitrative stress in diabetic rats: Involvement of nitrotyrosination of glucokinase.

Excessive tissue iron levels are associated with the increase of oxidative/nitrative stress which contributes to tissue damage that may elevate the risk of diabetes. Therefore, we investigated the effects of iron on diabetes-associated liver injury and whether iron-related tyrosine nitration participated in this process. Rats were randomly divided into four groups: control, iron overload (300 mg/kg iron dextran, i.p.), diabetic (35 mg/kg of streptozotocin i.p. after administration of a high-fat diet) and diabetic simultaneously treated with iron. Iron supplement markedly increased diabetes-mediated liver damage and hepatic dysfunction by increasing liver/body weight ratio, serum levels of aspartate and alanine aminotransferase, and histological examination, which were correlated with elevated levels of lipid peroxidation, protein carbonyls and tyrosine nitration, oxidative metabolism of nitric oxide, and reduced antioxidant capacity. Consequently, the extent of oxidized/nitrated glucokinase was markedly increased in the iron-treated diabetic rats that contribute to a decrease in its expression and activity. Further studies revealed a significant contribution of iron-induced specific glucokinase nitration sites to its inactivation. In conclusion, iron facilitates diabetes-mediated elevation of oxidative/nitrative stress, simultaneously impairs liver GK, and can be a link between enzymatic changes and hepatic dysfunction. These findings may provide new insight on the role of iron in the pathogenesis of diabetes mellitus.

Ferric citrate CYP2E1-independently promotes alcohol-induced apoptosis in HepG2 cells via oxidative/nitrative stress which is attenuated by pretreatment with baicalin.

In the case of alcoholic liver injury, an iron overload is always present. Both alcohol and iron can individually induce oxidative stress in liver. However, the combined effect of physiological concentrations of alcohol and iron on oxidative stress in hepatocytes remains unknown. Baicalin has been demonstrated to be an antioxidant or iron chelator in animal experiments. In this study, we investigated the injury to hepatocytes CYP2E1-independently induced by the combination of alcohol and iron and the protective effect of baicalin. Compared with cells treated with ethanol alone, ferric citrate enhanced the accumulation of reactive oxygen and nitrogen species, increased the occurrence of protein carbonylation/nitration and the levels of 4-hydroxy-2-nonenal, changed the distribution of iNOS, and eventually resulted in apoptosis. However, pretreatment with baicalin inhibited the oxidative stress induced by the combination of alcohol and iron, mainly by chelating iron. Our findings therefore suggest that iron could CPY2E1-independently enhance the oxidative stress induced by alcohol, which probably contributes to the pathogenesis of alcoholic liver disease. Baicalin is a promising phytomedicine for preventing alcoholic liver disease.