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As2O3 caused autophagosome accumulated in livers

To investigate whether autophagy was involved in As2O3-induced toxicity, we utilized transmission electron microscopy to observe the ultrastructure in As2O3-treatedlivers. Morphological hallmark of autophagy was the presence of autophagosomes (). Quantification of the autophagosomes numbers per cell demonstrated that As2O3 increased autophagosomes number significantly and in a dose-dependent manner. The number of autophagosomes in As2O3-treated rats was obviously decreased by pretreatment with Tau ().

As2O3 activated autophagy in offsprings’ livers

In this study, we evaluated the expression of autophagy biomarkers, LC3-II and P62, by Western blot analysis (). The level of LC3-II was increased dramatically and the level of P62 was decreased in As2O3-treated livers as shown in Western blot assay. After pretreatment with Tau, the expression of LC3-II was decreased and the expression of P62 was increased dramatically in As2O3-treated cells (), this gave us a clue to investigate the correspondence of autophagy and cytotoxicity at different As2O3 concentration and whether Tau could protect against the autophagy induced by As2O3.

As2O3 reduced the expression of PPARγ in offsprings’ livers

PPARγ was a metabolism related protein in livers. The expression of PPARγ was decreased in As2O3-treated offsprings’ livers (). Tau could protect against the reduction of PPARγ induced by As2O3 in offsprings’ livers ().

As2O3 accelerated Malonic Dialdehyde (MDA) level in offsprings’ livers

As2O3 could induce autophagy through ROS generation in vitro. To investigate the level of oxidative stress, we used MDA test kit to measure the level of MDA. We found that the level of MDA was increased significantly after treatment with As2O3. After pretreatment with Tau, the level of MDA was decreased in the livers of As2O3-treated rats (). It suggested that treatment with As2O3 could accelerate the generation of ROS, and pretreatment with Tau could withstand the oxidative stress induced by As2O3.

Tau reduced cell death of As2O3-treated HepG2 cells

We found that the autophagy in livers was decreased significantly after pretreatment of Tau. To investigate the action of Tau in vitro, the As2O3-treated HepG2 cells were pretreated with Tau at a concentration of 20 μM. The cell viability was elevated significantly (). It suggested that Tau reduced the cell death in As2O3-treated HepG2 cells.

RGS and Tau reduced autophagy in As2O3-treated HepG2 cells

In the study, the level of LC3-II was increased significantly and the expression of P62 was decreased in As2O3-treated HepG2 cells (). After pretreatment with RGS, we found that autophagy was inhibited in As2O3-treated cells (), suggesting that inhibition of PPARγ contributed to As2O3-induced autophagy. The level of autophagy was decreased after pretreatment with Tau in As2O3-treated cells (), suggesting that Tau could protect hepatocytes against As2O3 through modulating PPARγ–autophagy pathway.

Abstract

We previously isolated the transaminase from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, β-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, . Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via . In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of as a β-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions for Pseudomonas sp. strain AAC.

INTRODUCTION

Previously, we characterized a number of transaminases from Pseudomonas sp. strain AAC (NCBI accession number ), some of which were able to metabolize 12-aminododecanoic acid, a nonnatural compound with potential as a building block for industrial nylon-12 synthesis (). Hazards associated with the synthesis of nylon-12 (), as well as global shortages of the material in recent years (), have afforded opportunities for the application of biocatalysis within the materials field (), and a number of examples have been reported to date (, ). However, while most organisms are unable to metabolize 12-aminododecanoic acid, the Pseudomonas sp. strain AAC bacterium contains three enzymes with activity against the compound. Catalytic promiscuity is often thought to be the result of an enzymatic activity that was acquired coincidentally and evolved under selective pressure. In this case, the substrate is neither toxic nor abundant in the environment, and it is unlikely that there was a strong selective pressure to evolve with a metabolic activity against the compound. Examples of secondary reactions which provide no apparent benefit and are only encountered under laboratory conditions, such as the dechlorination of chloropurines by adenosine deaminase (), have been reported, and without further evidence it is more likely that this is the case for Pseudomonas sp. strain AAC. However, the discovery of three genes encoding proteins with this activity within the same organism suggests that the compound has a broader metabolic context, or that the genes coevolved from a single parent gene ().

INTRODUCTION

As stated above, among Pseudomonas sp. strain AAC proteins, three transaminases were identified with 12-aminododecanoic acid activity: , , and . The most promising candidate biocatalyst in terms of ease of protein production, solubility, and activity was , and here we report the subsequent characterization of that protein.

Abstract

S-Alk(en)yl-L-cysteine sulfoxides are pharmaceutically important secondary metabolites produced by plants that belong to the genus Allium. Biosynthesis of S-alk(en)yl-L-cysteine sulfoxides is initiated by S-alk(en)ylation of glutathione, which is followed by the removal of glycyl and γ-glutamyl groups and S-oxygenation. However, most of the enzymes involved in the biosynthesis of S-alk(en)yl-L-cysteine sulfoxides in Allium plants have not been identified. In this study, we identified three genes, AsGGT1, AsGGT2, and AsGGT3, from garlic (Allium sativum) that encode γ-glutamyl transpeptidases (GGTs) catalyzing the removal of the γ-glutamyl moiety from a putative biosynthetic intermediate of S-allyl-L-cysteine sulfoxide (alliin). The recombinant proteins of AsGGT1, AsGGT2, and AsGGT3 exhibited considerable deglutamylation activity toward a putative alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine, whereas these proteins showed very low deglutamylation activity toward another possible alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine sulfoxide. The deglutamylation activities of AsGGT1, AsGGT2, and AsGGT3 toward γ-glutamyl-S-allyl-L-cysteine were elevated in the presence of the dipeptide glycylglycine as a γ-glutamyl acceptor substrate, although these proteins can act as hydrolases in the absence of a proper acceptor substrate, except water. The apparent Km values of AsGGT1, AsGGT2, and AsGGT3 for γ-glutamyl-S-allyl-L-cysteine were 86 μM, 1.1 mM, and 9.4 mM, respectively. Subcellular distribution of GFP-fusion proteins transiently expressed in onion cells suggested that AsGGT2 localizes in the vacuole, whereas AsGGT1 and AsGGT3 possess no apparent transit peptide for localization to intracellular organelles. The different kinetic properties and subcellular localizations of AsGGT1, AsGGT2, and AsGGT3 suggest that these three GGTs may contribute differently to the biosynthesis of alliin in garlic.

ASSAYS OF GGT ENZYME ACTIVITIES

For the analysis of deglutamylation activities toward γ-glutamyl-S-allyl-L-cysteine and γ-glutamyl-S-allyl-L-cysteine sulfoxide, the enzyme assay reaction mixture consisted of 0.67 μg μl-1 yeast crude protein, 50 mM Tris-HCl (pH 8.0), 10 mM glycylglycine, and 1 mM γ-glutamyl-S-allyl-L-cysteine or γ-glutamyl-S-allyl-L-cysteine sulfoxide was incubated for 6 h at 37°C. For the determination of the enzyme activity in a pH range of 6.0–7.0, the reaction mixture containing 50 mM 2-(N-morpholino)ethanesulfonic acid buffer, instead of 50 mM Tris-HCl buffer, was used. For the determination of the enzyme activity in a pH range of 7.0–9.0, 50 mM Tris-HCl buffer at pH values from 7.0 to 9.0, instead of 50 mM Tris-HCl (pH 8.0), was used. For the analysis of the effects of glycylglycine as a γ-glutamyl acceptor, deglutamylation activity was determined in the reaction mixture with or without 10 mM glycylglycine. The reaction was initiated by the addition of yeast crude proteins. After incubation at 37°C, proteins in the reaction mixture were removed using a centrifugal ultrafiltration device (molecular weight cut-off, 10 kD; Kurabo, Osaka, Japan). S-Allyl-L-cysteine and alliin in the ultrafiltrated solution were quantified using high-performance liquid chromatography (HPLC). For the kinetic analysis, assays were carried out with γ-glutamyl-S-allyl-L-cysteine concentrations ranging from 12.5 to 1000 μM for AsGGT1, 0.5–8 mM for AsGGT2, and 1–25 mM for AsGGT3. Km values were calculated from triplicate date sets according to the Michaelis-Menten equation.

Par‐C10 culture and treatment

A quantity of 100 μl of growth factor‐reduced Matrigel (GFR‐MG) (8 mg/mL; 2:1 GFR‐MG: DMEM‐Ham's F12 [1:1] medium; Becton Dickinson, Franklin Lakes, NJ) was allowed to solidify in a 37°C incubator for 1 h in eight‐well chambers mounted on No. 1.5 German borosilicate coverglass (Nalge Nunc International, Naperville, IL). Then, Par‐C10 cells (15,000 cells/well; passages 30–60) were plated on the GFR‐MG in DMEM‐Ham's F12 (1:1) medium with supplements and treated with TNF‐α (100 ng/mL; Becton Dickinson), then incubated for 1 h or 6 h at 37°C. Cells were then treated with AT‐RvD1 (100 ng/mL; Cayman Chemical, Ann‐Arbor, MI), and varying doses of DEX (25–100 ng/mL). Three‐dimensional Par‐C10 cell clusters were used for assays after incubation at 37°C with 95% air and 5% CO2 for 72 h.

Treatment with AT‐RvD1 and DEX alone and in combination enhances Par‐C10 cell cluster growth

Par‐C10 cells were plated on GFR‐MG and preincubated with TNF‐α followed by treatment with AT‐RvD1 and DEX alone and in combination as described in . Then, salivary cell clusters were quantified using light microscopy as described in . As shown in Figure , untreated control cells as well as cells subjected to single treatments with AT‐RvD1 (100 ng/mL) or DEX (100 ng/mL) for 72 h showed increased salivary cell cluster growth as compared to cells treated with TNF‐α alone (100 ng/mL). Additionally, cells that were preincubated with TNF‐α (100 ng/mL) for 1 h followed by treatment with AT‐RvD1 (100 ng/mL) and DEX (25–100 ng/mL) alone and in combination recovered the number of salivary cell clusters after 72 h. Surprisingly, cells preincubated with TNF‐α for 1 h and then treated with AT‐RvD1 (100 ng/mL) and ¼ dose of DEX (25 ng/mL) for 72 h displayed a significant increase in the number of cell clusters grown as compared to cells treated with TNF‐α alone. Note that only cells preincubated with TNF‐α without any subsequent treatment showed a statistically significant cell cluster reduction from the control group.

Treatment with AT‐RvD1 and DEX alone and in combination mitigates TNF‐α mediated cellular apoptosis

To assess the effects of AT‐RvD1 and DEX on cell death following preincubation with TNF‐α, we performed a TUNEL assay on the Par‐C10 cell clusters grown on GFR‐MG. As shown in Figure A and B, a basal level of TUNEL‐positive cells (indicating percentage of apoptotic cells) was determined using untreated (control) cells. Both single treatments with DEX (100 ng/mL) and TNF‐α (100 ng/mL) resulted in a significant increase in the degree of apoptosis from the untreated cells, although AT‐RvD1 alone (100 ng/mL) did not increase the percentage of apoptotic cells. After preincubation with TNF‐α for 1 h, AT‐RvD1 alone and in combination with DEX caused a significant reduction in apoptotic cells as compared to cells treated with TNF‐α alone (see asterisks). Interestingly, the treatment consisting of AT‐RvD1 (100 ng/mL) and a reduced dose of DEX (25 ng/mL) was the most effective in reducing apoptosis, with values similar to untreated cells. Figure B shows representative images of percentages of TUNEL‐positive cells in each treatment group.

17-oxo-DHA displays anti-inflammatory actions and additive effects with FP in COPD patients and healthy individuals

Freshly isolated PBMCs from moderate to severe COPD patients (N = 10) and healthy individuals (N = 11, control group) were treated for 1 h with 17-oxo-DHA, FP or 17-oxo-DHA + FP, then stimulated with LPS for 18 hours. Afterwards, TNFα and IL-1β concentrations were measured in the supernatants. The induction of TNFα and IL-1β release in response to LPS stimulation was significantly higher in PBMCs from COPD patients (median (range): 17.88 ng/ml (9.003; 23.91) for TNFα and 1.528 ng/ml (1.173; 3.901) for IL-1β) compared to PBMCs from healthy individuals (median (range): 8.92 ng/ml (4.96; 11.5) for TNFα and 1.096 ng/ml (0.8440; 1.528) for IL-1β). 17-oxo-DHA and FP, when administered alone, displayed a similar effect in suppressing TNFα release, while 17-oxo-DHA was more potent than FP in inhibiting IL-1β release (). The combination of 17-oxo-DHA and FP resulted in even greater suppression of TNFα and IL-1β release, showing additive effects in both study groups. To further investigate the impact of 17-oxo-DHA on FP actions, dose-response experiments were performed and the effect of 17-oxo-DHA on half maximal inhibitory concentration (IC50) and maximum inhibition (Imax) of FP was evaluated (, ). No differences of IC50 and Imax were observed between the two study groups. When administered in combination with FP, 17-oxo-DHA significantly increased FP-Imax for both cytokines and in both study groups, without modifying FP-IC50.

17-oxo-DHA, but not FP, suppresses IL-1β release by inhibiting the NLRP3 inflammasome

To evaluate the effect of 17-oxo-DHA on NLRP3 inflammasome activation, LPS-primed PBMCs from healthy volunteers were stimulated with the NLRP3 inflammasome activator nigericin. The effect of 17-oxo-DHA on inflammasome activation was specifically evaluated by adding 17-oxo-DHA after LPS priming and before nigericin as previously reported. The effect of FP on NLRP3 inflammasome activation was also evaluated. 17-oxo-DHA effectively inhibited nigericin-induced IL-1β release while FP had no effect (). The actions of 17-oxo-DHA and FP on inflammasome activation were further assessed by evaluating the formation of cleaved caspase-1 and IL-1β as well as the expression of NLRP3, pro-IL-1β and procaspase-1. 17-oxo-DHA, but not FP, completely blocked the cleavage of pro-IL-1β and procaspase-1 (). In the presence of nigericin, the strong effect on the extracellular release of IL-1β was accompanied with reduced intracellular levels of pro-IL-1β (). The expression of the procaspase-1 did not show significant variations among different conditions while NLRP3 was induced by LPS, which is consistent with previous reports. The inhibitory effect of 17-oxo-DHA on caspase-1 activity was further confirmed by flow cytometry using a fluorescent probe specific for active caspase-1. Double staining with fluorescent anti-human CD14 antibody was performed on LPS-primed and nigericin-activated PBMCs to evaluate the specific contribution of CD14+ monocytes to caspase-1 activation. Caspase-1 activation was inhibited by 17-oxo-DHA, but not by FP ().

17-oxo-DHA inhibits the activation of multiple inflammasomes as well as nigericin-induced pyroptosome formation and mitochondrial ROS generation

To further investigate the molecular mechanisms of inflammasome inhibition by 17-oxo-DHA and consistent with the activation of the inflammasome in peripheral blood monocytes, our analysis was expanded using the human monocyte-like cell line THP-1. The strong inhibitory effect of 17-oxo-DHA, but not FP, on nigericin-induced release of IL-1β, and cleavage of procaspase-1 and pro-IL-1β was confirmed (). Similar to PBMCs, the expression of procaspase-1 did not vary among conditions while pro-IL-1β was induced by the LPS treatment. The action of 17-oxo-DHA on NLRP3 inflammasome activation was confirmed also in response to ATP and MSU crystals (). Stimulation of LPS-primed THP-1 cells with nigericin induces the formation of the pyroptosome, a large supramolecular assembly of ASC that is believed to mediate caspase-1 activation. Immunostaining for endogenous ASC in THP-1 cells revealed that 17-oxo-DHA, but not FP, strongly inhibited nigericin-triggered ASC pyroptosome formation (). Since the activation of the NLRP3 inflammasome is mediated by mitochondrial reactive oxygen species (ROS), we investigated the effect of 17-oxo-DHA on mitochondrial ROS generation. As reported in , 17-oxo-DHA, unlike FP, inhibited nigericin-induced mitochondrial ROS generation. We also tested the effects of 17-oxo-DHA on other inflammasome complexes. We found that 17-oxo-DHA significantly inhibited the activation of AIM2 and NAIP-NLRC4 inflammasomes induced by dA-dT and flagellin from S. typhimurium, respectively (). In addition, we performed a colorimetric assay using human recombinant caspase-1 to test whether the effects of 17-oxo-DHA were mediated by inhibition of caspase-1. As reported in , 17-oxo-DHA was able to inhibit caspase-1 activity.

Abstract

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disease characterized by diminished secretory function of the exocrine glands. Treatments for hyposalivation are limited to the use of saliva substitutes and medications that provide only temporary relief. In light of the high degree of need and the limitations of current therapies, development of alternative treatments to restore functioning is essential. Resolvins (Rv), which are highly potent lipid mediators, offer a viable alternative for better treating inflammatory diseases such as SS. The goal of this study was to determine whether systemic preventive treatment with Aspirin-triggered RvD1 (AT-RvD1) reduces inflammation and preserves secretory functioning in NOD/ShiLtJ SS-like mice. Our results indicate that systemic treatment with AT-RvD1 diminishes the progression of the disease in salivary epithelium from female mice as follows: (a) improves secretory function, (b) reduces pro-inflammatory molecule gene expression, (c) increases anti-inflammatory molecule gene expression and (d) induces M2 macrophage polarization. Finally, AT-RvD1 decreases lymphocytic infiltration into the salivary glands when used with small doses of the steroid, dexamethasone, and promotes the tissue healing process.

Experimental animals

Female and male NOD/ShiLtJ mice were treated twice a wk starting at 4 wk for 16 wk via tail vein injections with a vehicle control (EtOH, 8.9%) and AT-RvD1 (Cayman Chemical, Ann Arbor, MI) at 0.1 mg/kg. We used DEX (Sigma Aldrich, St. Louis, MO) at 8.25 mg/kg as a positive control and for a combination of AT-RvD1 and DEX (4.25 mg/kg). The doses of AT-RvD1 and EDX used in this study were chosen based on previous studies indicating that 0.1 mg/kg produces a significant downregulation of systemic inflammatory genes as compared to 0.05 mg/kg (data not shown). At 12 wk and 20 wk of age, the mice were anesthetized with 80–100 mg/kg Ketamine +5 mg/kg Xylazine and subsequently euthanized by abdominal exsanguination. 20 wk old untreated C57BL/6, NOD/ShiLtJ and ALX/FPR2−/− mice were used for [Ca2+]i measurements. The animal protocol (14-006007) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at University of Utah, according to the animal welfare act of the United States (7 U.S.C. 2131 et. seq.). All mouse experiments were carried out at the animal care facility of the University of Utah, Colorow 383 building, in accordance with approved guidelines.

Treatment with AT-RvD1 before disease onset maintains healthy saliva flow rates in female NOD/ShiLtJ mice

In order to verify whether AT-RvD1 was able to stimulate its receptor, ALX/FPR2, in SMG from NOD/ShiLtJ mice, we measured [Ca+2]i mobilization in response to this agonist in mouse SMG cells. As shown in , SMG cells from 20 wk NOD/ShiLtJ mice were able to mobilize [Ca2+]i in response to AT-RvD1. In contrast, SMG cells from 20 wk ALX/FPR2−/− mice (used as a negative control) did not show such increase. However, SMG cells from both NOD/ShiltJ and ALX/FPR2−/− mice responded to carbachol (Cch; acetylcholine analog; used as a positive control). Finally, SMG cells from C57BL/6 control mice showed responses to both AT-RvD1 and Cch ( and ). In order to determine whether AT-RvD1 was able to preserve salivary secretory function in NOD/ShiLtJ mice, we treated them with AT-RvD1 before disease onset as described in the Materials and Methods, then measured saliva flow rate at 20 wk. As shown in , female NOD/ShiLtJ mice treated with AT-RvD1 displayed saliva flow rates comparable to healthy controls (i.e., 4 wk old mice) and significantly higher flow rates when compared to vehicle controls. In contrast, male NOD/ShiLtJ mice subjected to the same treatment displayed saliva flow rates lower than healthy controls and comparable to vehicle controls ().

Treatment with AT-RvD1 prior to disease onset reduces pro-inflammatory and increases anti-inflammatory molecule gene expression in SMG

Upregulation of pro-inflammatory cytokines has previously been observed in the NOD mouse. To determine whether AT-RvD1 altered the inflammatory cytokine profiles, we performed a quantitative PCR analysis in SMG from female NOD/ShiLtJ mice. As shown in , SMG from 12 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed significant downregulation of expression of the pro-inflammatory cytokines, TNFα, IL-1β and IFNγ, as compared to vehicle-treated controls. In addition, SMG from 20 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant downregulation of the pro-inflammatory cytokines, TNFα, IL-1β, IFNγ and IL-17 as compared to vehicle-treated controls (). In contrast, SMG from 20 wk male NOD/ShiLtJ mice treated with AT-RvD1 did not display these changes ().

Treatment with AT-RvD1 prior to disease onset reduces pro-inflammatory and increases anti-inflammatory molecule gene expression in SMG

Anti-inflammatory molecules were also investigated in this study. As shown in , SMG from 12 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant upregulation of Del-1 (endogenous leukocyte-endothelial adhesion inhibitor), TGFβ and IL-10 as compared to vehicle-treated controls. Moreover, SMG from 20 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant upregulation of Annexin A1 (ANXA1), Del-1, TGFβ and IL-10 expression (). However, SMG from 20 wk male NOD/ShiLtJ mice treated with AT-RvD1 did not show significant changes as compared to vehicle-treated controls ().

Treatment with AT-RvD1 prior to disease onset triggers macrophage (Mϕ) M2 polarization in SMG

Previous studies have shown that treatment with AT-RvD1 polarizes pro-inflammatory M1-type macrophages (Mϕ) toward a pro-resolution M2-type in mouse and human cells. Therefore, we aimed to characterize the Mϕ phenotype in SMG from different treatment groups. Specifically, gene signatures of either M1 (IL-23 and iNOS) or M2 (IL-1ra and Arg-1) type Mϕ were analyzed in this study. Our results showed that SMG from 12 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant downregulation of M1-type Mϕ genes (IL-23 and iNOS, ) and upregulation of pro-resolution M2-type Mϕ genes (IL-1ra and Arg-1, ) as compared to vehicle-treated controls (). Furthermore, SMG from 20 wk female NOD/ShiLtJ mice treated with AT-RvD1 demonstrated similar effects (i.e. M1-type downregulation/M2-type upregulation) as compared to vehicle-treated controls ().

2.3. Experimental Design

After the animals had acclimated for one week, sub-chronic exposure was performed. Fifty male SD rats (80 ± 20 g, 4-weeks-old) were divided randomly into five groups (with 10 rats in each groups), including control, Mn-treated, and three doses of PAS-Na (100, 200, and 300 mg/kg) treatment (Mn + 100, Mn + 200 and Mn + 300 PAS) groups. As in , the Mn-treated, Mn + 100, Mn + 200, and Mn + 300 PAS groups received intraperitoneal (i.p.) injections of MnCl2 (as 6.55 mg/kg Mn) once a day, five days per week, for 12 weeks, before subsequent treatment with physiological saline (for the Mn-treated group) and different doses of PAS-Na (100, 200, and 300 mg/kg, subcutaneous (s.c.) injections) in the back area of rats once a day, five days per week, for an additional six weeks. The control group received sterile physiological saline in the meantime. All of the i.p. and s.c. injections were given at volumes of 0.2 mL per 100 g rat weight. MnCl2 and PAS-Na were dissolved in sterile physiological saline each day before the injections.

3.1. PAS-Na Treatment Recovered Sub-Chronic Mn Exposure Induced Impairments of Spatial Learning and Memory Abilities

As shown in the spatial navigation test for assessing learning abilities, Mn-treated rats showed learning impairments that were characterized by an increased escape latency and swimming distance, when compared with those of the control group (repeated measures ANOVA F(4, 45) = 3.382, p = 0.017 for escaping latency; and F(4, 45) = 3.505, p = 0.014 for swimming distance (A,B). In contrast, PAS-Na treatments (100, 200, or 300 mg/kg) significantly improved the Mn-induced learning impairments, by decreasing the escape latency and the swimming distance (Tukey’s post-hoc test, p < 0.05 and p < 0.01 respectively, A,B). On the other hand, in the spatial probe trial for assessing memory, the number of platform crosses of the Mn-treated group was less than those of the control group (3.50 ± 1.96 vs. 6.20 ± 2.28 s, one-way ANOVA, F = 3.278, p = 0.021, C), suggesting memory deficits. As expected, PAS-Na treatment (300 mg/kg) also significantly attenuated the memory deficits induced by sub-chronic Mn exposure, restoring the number of platform crosses to control levels (multiple comparisons, p = 0.023, D). Furthermore, there was no difference observed in the swimming speed between different groups (one-way ANOVA, F = 0.538, p = 0.677, C), indicating that the aforementioned impairments by Mn did not result from possible muscular side effects of the Mn injections.

3.2. PAS-Na Treatment Failed to Restore Mn-Induced Decrease in GABA Levels in the Basal Ganglia

Next, we measured the GABA level in the basal ganglia to determine the possible involvement of GABA metabolism. Sub-chronic Mn exposure for 12 weeks and a six-week exposure cessation led to decreased GABA levels compared with the control [F(4, 45) = 4.337, p = 0.003, multiple comparisons, p = 0.02, ]. In contrast to the protective effects of PAS-Na in Mn-induced neurotoxicity, all doses of PAS-Na treatment for six weeks failed to affect the Mn-induced changes in the GABA levels (multiple comparisons, p > 0.05).

3.3. PAS-Na Treatment Restored Mn-Induced Changes of GABA Enzymes

To further investigate the effects of Mn on GABA metabolism, the enzymatic activities of GAD (a GABA-synthesizing enzyme) and GABA-T (a GABA-degrading enzyme) in the basal ganglia were analyzed. After Mn exposure for 12 weeks and a six-week exposure cessation, the GAD activity in the Mn-treated group was significantly increased compared with the control [F(4, 45) = 5.061, p = 0.003, and multiple comparisons, p = 0.0005, ]. In a similar pattern, the GABA-T activity of the Mn-treated group was significantly increased compared with the control [153% of control, F(4, 45) = 4.261, p = 0.004, and multiple comparisons, p = 0.0002, B]. In contrast, treatment with either 200 or 300 mg/kg PAS-Na for six weeks caused significant reductions in GAD activity, to 41.06% and 41.31% of the Mn-treated group, respectively(multiple comparisons, p = 0.005 or 0.009 respectively, A). PAS-Na treatment at the highest dose (300 mg/kg, but not 100 or 200 mg/kg) for six weeks also significantly reduced GABA-T activity by 24.0%, compared with the Mn-treated group (multiple comparisons, p = 0.005, B).

3.4. PAS-Na Prevented Mn-Induced Transcriptional Changes in GABAA and GAT-1 Expression

To investigate whether Mn affected GABA metabolism through the deregulation of its receptor or transporter, the mRNA and protein expressions of GABAA and GAT-1 in the basal ganglia were measured. Sub-chronic Mn exposure decreased GABAA mRNA expression in the basal ganglia [F(4, 20) = 12.182, p = 0.0008, multiple comparisons, p = 0.007, left panel of ], but significantly increased GAT-1 mRNA levels [F(4, 20) = 12.182, p = 0.003, multiple comparisons, p = 0.0002, right panel of ], when compared with the control. Treatment with 300 mg/kg PAS-Na (but not 100 and 200 mg/kg) for six weeks restored the above-mentioned changes by increasing GABAA mRNA expression (multiple comparisons, p = 0.033) and decreasing GAT-1 mRNA expression (multiple comparisons, p = 0.006). Although it seemed that sub-chronic Mn exposure increased GAT-1 protein expression and PAS-Na treatment could recover this, all of the changes failed to reach statistical significance (p > 0.05, ).

Methods

Gene expression of 2 resolvin receptors (ALX and ChemR23) was quantified in synovium and medial tibial plateau specimens obtained from patients with OA at the time of joint replacement surgery. Two models of OA joint pain were used for the mechanistic studies. Gene expression in the joint and central nervous system was quantified. The effects of exogenous administration of the D series resolvin precursor 17(R)‐hydroxy‐docosahexaenoic acid (17[R]‐HDoHE) on pain behavior, joint pathology, spinal microglia, and astroglyosis were quantified. Plasma levels of relevant lipids, resolvin D2, 17(R)‐HDoHE, and arachidonic acid, were determined in rats, using liquid chromatography tandem mass spectrometry.

Results

There was a positive correlation between resolvin receptor and interleukin‐6 (IL‐6) expression in human OA synovial and medial tibial plateau tissue. In rats, synovial expression of ALX was positively correlated with expression of IL‐1β, tumor necrosis factor, and cyclooxygenase 2. Treatment with 17(R)‐HDoHE reversed established pain behavior (but not joint pathology) in 2 models of OA pain. This was associated with a significant elevation in the plasma levels of resolvin D2 and a significant reduction in astrogliosis in the spinal cord in the monosodium iodoacetate–induced OA rat model.

Animals and model induction

Animal experiments were approved by the Nottingham University ethics committee, and all procedures were approved by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 and conform to the guidelines of the International Association for the Study of Pain. Adult male Sprague‐Dawley rats (n = 166) were used (Charles River). All procedures and testing were performed in a blinded manner. The model of monosodium iodoacetate (MIA)–induced OA pain was generated as previously described . The medial meniscal transection (MNX) induction model of OA pain was based on previously described methods (see also Supplementary Methods, available on the Arthritis & Rheumatology web site at ).

Pharmacologic interventions and assessment of pain behavior

The 17(R)‐HDoHE (stock solution 100 μg/ml in ethanol) was diluted in normal sterile saline to provide a concentration of 1 ng/μl. The vehicle solution consisted of 1% ethanol solution in 99% saline. A series of different pharmacologic studies were performed using 300 ng of 17(R)‐HDoHE in 300 μl saline administered intraperitoneally. Study 1 determined the effects of a single injection of 17(R)‐HDoHE on pain behavior on day 14 after induction of the model. Study 2 determined the effects of repeated administration of 17(R)‐HDoHE (300 ng in 300 μl saline, every other day from day 14 until day 28 after induction of the model) on pain behavior in the MIA‐induced OA pain model and the MNX induction model of OA pain. Study 3 determined the effects of discontinuous administration of 17(R)‐HDoHE (300 ng in 300 μl saline, from day 14 to day 22 after induction of the model) on pain behavior, quantified until day 35 after induction of the model. All of the drug intervention studies were conducted in a blinded manner.

Reversal of MIA‐ and MNX‐induced OA pain by the D series precursor 17(R)‐HDoHE

To further investigate the potential mechanisms underlying the effects of17(R)‐HDoHE on OA‐induced pain behavior, the effects of repeated treatment with 17(R)‐HDoHE on joint pathology were determined (Figures A–C). Intraarticular injection of MIA was associated with a significant increase in chondropathy, synovitis, and chondrocyte appearance and increased numbers of subchondral osteoclasts (Figures D–G). Following repeated administration of 17(R)‐HDoHE (300 ng in 300 μl every other day from day 14 to day 28) there were no significant changes in any of these features of OA joint pathology (Figures D–G). Similarly, repeated treatment with 17(R)‐HDoHE did not alter MNX‐induced joint pathology (data not shown).

Spinal effects of 17(R)‐HDoHE correlated with behavioral analgesia

We previously reported a significant increase in GFAP immunofluorescence, a marker for astrogliosis, in the spinal cord at later time points in the MIA‐induced model of OA . Consistent with previous findings, GFAP immunofluorescence was significantly increased in the ipsilateral dorsal horn (Figures E and F) but not the contralateral dorsal horn (data not shown) of MIA‐treated rats that received vehicle, compared with saline‐treated controls. Repeated systemic administration of 17(R)‐HDoHE (300 ng in 300 μl every other day from day 14 to day 28) significantly inhibited GFAP immunofluorescence in the ipsilateral dorsal horn of the spinal cord in MIA‐treated rats, compared with vehicle‐treated rats with MIA‐induced OA pain (Figures E and F). Correlation analysis revealed that spinal GFAP expression was positively correlated with weight‐bearing asymmetry (Figure G) and negatively correlated with ipsilateral paw withdrawal thresholds (Figure H).

Methods

Gene expression of 2 resolvin receptors (ALX and ChemR23) was quantified in synovium and medial tibial plateau specimens obtained from patients with OA at the time of joint replacement surgery. Two models of OA joint pain were used for the mechanistic studies. Gene expression in the joint and central nervous system was quantified. The effects of exogenous administration of the D series resolvin precursor 17(R)‐hydroxy‐docosahexaenoic acid (17[R]‐HDoHE) on pain behavior, joint pathology, spinal microglia, and astroglyosis were quantified. Plasma levels of relevant lipids, resolvin D2, 17(R)‐HDoHE, and arachidonic acid, were determined in rats, using liquid chromatography tandem mass spectrometry.

Results

There was a positive correlation between resolvin receptor and interleukin‐6 (IL‐6) expression in human OA synovial and medial tibial plateau tissue. In rats, synovial expression of ALX was positively correlated with expression of IL‐1β, tumor necrosis factor, and cyclooxygenase 2. Treatment with 17(R)‐HDoHE reversed established pain behavior (but not joint pathology) in 2 models of OA pain. This was associated with a significant elevation in the plasma levels of resolvin D2 and a significant reduction in astrogliosis in the spinal cord in the monosodium iodoacetate–induced OA rat model.

Animals and model induction

Animal experiments were approved by the Nottingham University ethics committee, and all procedures were approved by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 and conform to the guidelines of the International Association for the Study of Pain. Adult male Sprague‐Dawley rats (n = 166) were used (Charles River). All procedures and testing were performed in a blinded manner. The model of monosodium iodoacetate (MIA)–induced OA pain was generated as previously described . The medial meniscal transection (MNX) induction model of OA pain was based on previously described methods (see also Supplementary Methods, available on the Arthritis & Rheumatology web site at ).

Abstract

Many researchers suggest an increased risk of atherosclerosis in women with polycystic ovary syndrome. In the available literature, there are no studies on the mediators of inflammation in women with PCOS, especially after dietary intervention. Eicosanoids (HETE and HODE) were compared between the biochemical phenotypes of women with PCOS (normal and high androgens) and after the 3-month reduction diet. Eicosanoid profiles (9(S)-HODE, 13(S)-HODE, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE, 5(S)-oxoETE, 16(R)-HETE, 16(S)-HETE and 5(S), 6(R)-lipoxin A4, 5(S), 6(R), 15(R)-lipoxin A4) were extracted from 0.5 ml of plasma using solid-phase extraction RP-18 SPE columns. The HPLC separations were performed on a 1260 liquid chromatograph. No significant differences were found in the concentration of analysed eicosanoids in phenotypes of women with PCOS. These women, however, have significantly lower concentration of inflammatory mediators than potentially healthy women from the control group. Dietary intervention leads to a significant (p < 0.01) increase in the synthesis of proinflammatory mediators, reaching similar levels as in the control group. The development of inflammatory reaction in both phenotypes of women with PCOS is similar. The pathways for synthesis of proinflammatory mediators in women with PCOS are dormant, but can be stimulated through a reduction diet. Three-month period of lifestyle change may be too short to stimulate the pathways inhibiting inflammatory process.

2.5.2. Sample Preparation

Venous blood was collected from the patients after an overnight fasting. Whole blood was collected and placed in tubes containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant. The blood was immediately placed on ice or in a refrigerator, and next the samples were centrifuged at 3500 rpm for 10 min at 4°C within max 2 h of collection. Plasma was stored at −80°C. Standard blood biochemical analyses were performed in the University Hospital Laboratory. 5(S), 6(R)-lipoxin A4, 5(S), 6(R), 15(R)-lipoxin A4, 5(S)-HETE, 5(S)-oxoETE, 12(S)-HETE, 15(S)-HETE, 16(R)/16(S)-HETE, 9(S)-HODE, and 13(S)-HODE were extracted from 0.5 ml of plasma using a solid-phase extraction RP-18 SPE columns (Agilent Technologies, Cheadle, UK) []. Recovery and sensitivity of method were described previously []. The total recovery for all sample extraction and processing steps (mean ± SD) was 46 ± 8%.

3. Results

When comparing the concentrations of analysed inflammatory state mediators between the group of women with PCOS (PCOS-I) and the control group (CG), it was observed that statistically significant differences were present in levels of almost all eicosanoids except lipoxins A4 (, ). Similar relation with respect to the control group (CG) was observed when the patients were divided into two phenotypes: with high (HT) and normal (NT) levels of androgens. When we compared two phenotypes of women with PCOS (HT and NT) to each other, we did not observe any statistically significant differences. However, an increasing tendency was noted in the group of women with normal level of androgens (NT) (). After the three-month reduction diet with lower glycemic index, a significant increase in the majority of inflammatory factors was observed, except for 6(R), 15(R)-lipoxin A4 (). Women with PCOS (PCOS-II) after being three months on the diet did not differ significantly from the control group (CG), however, the levels of 12(S)-HETE were much higher ().

2.2. Reagents

Chloramphenicol (Mw = 323.13 g·mol−1) was purchased from Sigma (Diegem, Belgium). Tris buffer containing 100 × 10−3 mol·L−1 NaCl, 20 × 10−3 mol·L−1 Tris HCl, 2 × 10−3 mol·L−1 MgCl2, 5 × 10−3 mol·L−1 KCl, and 1 × 10−3 mol·L−1 CaCl2 (pH = 7.6) was obtained from VWR (Leuven, Belgium) and used as a binding buffer solution. CAP binding aptamer sequence (5'-SH-(CH2)6-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-3') selected and designed at the SPHERE laboratory of the University of Antwerp [,] was purchased from Eurogentec (Seraing, Belgium). Type B gelatine (GelB, IEP = 5, Bloom strength = 257) isolated from bovine skin by the alkaline process and Type A gelatin (GelA, IEP = 8.8, Bloom strength = 202) isolated from porcine skin by the acid treatment were provided by Tessenderlo Chemie (Brussels, Belgium).

2.3. Procedure

In first step of procedure, the electrochemical pretreatment and biomodification of SPE were done. Prior to immobilization of thiolated DNA aptamer, a multiple-pulse amperometric pretreatment on the gold surface is carried out in a stirred 0.5 mol·L−1 H2SO4, 10 mmol·L−1 KCl solution with following triple-potential pulse sequence: −0.3 V for 3.0 s; 0.0 V for 3.0 s and +1.0 V for 1.5 s (15 cycles) [,]. The gold working electrode surface of SPE was then exposed to the mixture of aptamer (5 µM) containing GelB solution (5 w/v%; solved in Tris buffer at 40 °C). The percentage of the incorporation was 70:30 v/v% for aptamer:Gel solution in Tris buffer (pH 7.6). Their interaction was allowed to proceed for around 4 h, while the electrodes were stored in a wet chamber for protection from evaporation. Four hours was the best time for the immobilization of aptamer; because the minimum time for formation of a self-assembled monolayer of thiolated groups on the gold surface was 3 h [,,] and the drop of gelatin was completely liquid in the wet chamber (≈40 °C) during this period. After that, the wet chamber was switched off and cooled down gradually to reach room temperature. At room temperature, the drop of apt/GelB became rigid.

3. Results and Discussion

depicts the diagnostic performance of the aptasensor. To study the role of gelB as matrix in function of the biosensor, the CAP reduction signal was investigated on the aptamer/GelB-modified SPE. It can be seen that the peak current increases with increasing CAP concentration (0.10 to 12.0 nmol·L−1) with two different slopes [,,]. The peak current shows a linear relationship with the concentration of CAP in the range from 0.30 to 2.0 nmol·L−1 (inset). The calculated detection limit is 1.83 × 10−10 mol·L−1 based on three times standard deviation of the blank divided by the slope of the calibration curve (3σ/m).