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Abstract

Mycosporine-like amino acids (MAAs) are secondary metabolites, produced by a large variety of microorganisms including algae, cyanobacteria, lichen and fungi. MAAs act as UV-absorbers and photo-protectants. MAAs are suggested to exert pharmaceutical relevant bioactivities in the human system. We particularly focused on their effect on defence and regulatory pathways that are active in inflamed environments. The MAAs shinorine and porphyra-334 were isolated and purified from the red algae Porphyra sp. using chromatographic methods. The effect of MAAs on central signaling cascades, such as transcription factor nuclear factor kappa b (NF-κB) activation, as well as tryptophan metabolism, was investigated in human myelomonocytic THP-1 and THP-1-Blue cells. Cells were exposed to the MAAs in the presence or absence of lipopolysaccharide (LPS). NF-κB activity and the activity of tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO-1) were used as readout. Compounds were tested in the concentration range from 12.5 to 200 µg/mL. Both MAAs were able to induce NF-κB activity in unstimulated THP-1-Blue cells, whereby the increase was dose-dependent and more pronounced with shinorine treatment. While shinorine also slightly superinduced NF-κB in LPS-stimulated cells, porphyra-334 reduced NF-κB activity in this inflammatory background. Modulation of tryptophan metabolism was moderate, suppressive in stimulated cells with the lower treatment concentration of both MAAs and with the unstimulated cells upon porphyra-334 treatment. Inflammatory pathways are affected by MAAs, but despite the structural similarity, diverse effects were observed.

2.1. Activation Degree of the Central Inflammation Marker NF-κB

Compared to control cells, treatment with shinorine in unstimulated cells activated NF-κB up to 33.63% at the highest treatment concentration (200 µg/mL). In addition, lower concentrations 12.5–100 µg/mL showed a dose-dependent and significant increase in NF-κB activity, starting from 4.78% upwards (p < 0.05). Treatment with shinorine in LPS-stimulated cells resulted in a slight but significant superinduction of NF-κB activity compared to LPS treatment alone, e.g., 100 and 200 µg/mL MAA treatment resulted in 6.01% and 10.16% activation compared to LPS-stimulated control cells (A).

2.3. Effects of Shinorine and Porphyra-334 on Tryptophan Metabolism

Treatment of LPS-stimulated cells with both MAAs at lower concentrations decreased Kyn/Trp levels slightly, but significantly. Shinorine decreased the Kyn/Trp at 12.5, 25 and 100 µg/mL treatment compared to LPS-stimulated control samples, with a maximum of 23.1% suppression. Shinorine treatment did not affect unstimulated cells (A).

Abstract

Many metabolomics studies focus on aboveground parts of the plant, while metabolism within roots and the chemical composition of the rhizosphere, as influenced by exudation, are not deeply investigated. In this study, we analysed exudate metabolic patterns of Arabidopsis thaliana and their variation in genetically diverse accessions. For this project, we used the 19 parental accessions of the Arabidopsis MAGIC collection. Plants were grown in a hydroponic system, their exudates were harvested before bolting and subjected to UPLC/ESI-QTOF-MS analysis. Metabolite profiles were analysed together with the genome sequence information. Our study uncovered distinct metabolite profiles for root exudates of the 19 accessions. Hierarchical clustering revealed similarities in the exudate metabolite profiles, which were partly reflected by the genetic distances. An association of metabolite absence with nonsense mutations was detected for the biosynthetic pathways of an indolic glucosinolate hydrolysis product, a hydroxycinnamic acid amine and a flavonoid triglycoside. Consequently, a direct link between metabolic phenotype and genotype was detected without using segregating populations. Moreover, genomics can help to identify biosynthetic enzymes in metabolomics experiments. Our study elucidates the chemical composition of the rhizosphere and its natural variation in A. thaliana, which is important for the attraction and shaping of microbial communities.

Matching metabolic and genetic patterns can indicate compound class

The hydroxylated fatty acid 9,12,13-trihydroxyoctadec-10-enoic acid (9,12,13-TriHOME, KEGG ) was not present in the exudates of Edi-0 and Zu-0 (). Its lack corresponds to a SNP pattern introducing a stop codon into a long-chain-alcohol O-fatty-acyltransferase gene (AT5G55360.1). The unsaturated variant 9,12,13-trihydroxyoctadec-10(E),15(Z)-enoic acid, however, could be detected in Edi-0 and Zu-0 exudates, but not in the Ct-1 accession, and accordingly, pointed to different polymorphism patterns. Besides, similar intensity distributions of both hydroxylated fatty acids were found across the exudates of the 19 accessions ().

Abstract

Taurine (TAU) has been shown to improve exercise time to exhaustion and 3-km running performance; however, no studies have considered the effect of acute TAU ingestion on short duration cycling time trial (TT) performance. The aim of this study was to determine the effects of a single oral acute dose of 1000 mg of TAU on a laboratory simulated 4-km cycling TT. Eleven trained male cyclists performed three, 4-km TTs. The first of the trials was a familiarisation, followed by two subsequent trials which were performed two hours after the consumption of either 1000 mg of TAU or placebo (P), using a double-blind randomised crossover design. Capillary blood samples were obtained prior to the start and immediately after each TT for the measurement of lactate, pH and HCO3 −. There was no effect of TAU (p = 0.731, d = 0.151) on performance (390 ± 27 and 388 ± 21 s for TAU and P, respectively), nor were there any condition main effects for VO2, lactate, pH, or HCO3 − (p > 0.05) despite post TT changes in lactate (7.3 ± 2.5 mmol l−1, p < 0.001, d = 2.86, 7.6 ± 2.0 mmol l−1 p < 0.001, d = 3.75); pH (−0.255 ± 0.1, p < 0.001, d = 2.62, −0.258 ± 0.09, p < 0.001, d = 2.87); HCO3 − (−13.58 ± 2.7 mmol l−1, p < 0.001, d = 5.04 vs. −13.36 ± 2.3, p < 0.001, d = 5.72 for TAU and P, respectively). The findings of this study suggest that a pre-exercise dose of 1000 mg TAU offers no performance advantage during 4-km TT nor does it alter the blood buffering responses in trained cyclists.

Method

Following ethical approval, health screening and informed consent, 11 male trained (De Pauw et al. ) cyclists (mean ± SD age 34.6 ± 11.5 years, height 178.1 ± 5.2 cm, weight 74.8 ± 6.2 kg and body fat 15.5 ± 4.6 %, training >3 h week−1 and at least 2 years’ racing experience) performed three, 4-km TTs on a CompuTrainer Pro cycle ergometer (RacerMate, Seattle, USA). The CompuTrainer was fitted with each participants own bicycle, which was then calibrated in accordance with the manufacturers guidelines. The cyclists were allowed to use either a road or TT bike, but they had to use the same machine and gear ratios for each trial. Participants were instructed to adopt a position on the handlebars which was most comfortable. Using this method, 4-km laboratory TTs have previously been shown to have a coefficient of variation of 1.6 % (Altareki et al. ) for performance times and produce reliable pacing strategies (Stone et al. ) in similar participant populations. Body composition data were measured using air-displacement plethysmography (BodPod V4.2.0, Life Measurement Inc., USA). Prior to each TT, participants were instructed to abstain from any food intake in the preceding 3 h and to adhere to their normal nutritional intake strategy. Nutritional intake was recorded for the 24-h period prior to attending the initial laboratory visit, and replicated on each subsequent occasion. For the preceding 24-h period, participants were also instructed to avoid performing any exercise. Pre-exercise urine samples were obtained to measure urine osmolality using a portable osmometer (Osmocheck, Vitech Scientific, West Sussex, UK) which has previously been shown to be both valid and reliable (Sparks and Close ). This was to ensure that this index of hydration status was consistent prior to each trial.

Experimental procedures

Participants performed a standardised warm-up which required them to cycle at a self-selected intensity for 5 min. This was replicated prior to each subsequent TT. Each laboratory session was performed at the same time of day to minimise the potential effects of circadian variations (Souissi et al. ). The first laboratory visit was a familiarisation TT as this has been shown to improve subsequent test–retest pacing strategy reliability (Ansley et al. ). This initial trial was followed by two experimental trials that took place between five and 7 days apart, which was chosen to limit the confounding effects of fatigue and potential changes in training status. All trials commenced 2 h after the consumption of either a 500 ml drink containing only water (Buxton still mineral water, UK), in the case of the familiarisation and the placebo trials, or after the consumption of 500 ml water containing 1000 mg of TAU (Acrōs Organics, Geel, Belgium). This pre-exercise consumption period was used to elicit peak plasma TAU concentrations at the start of the time trial (Galloway et al. ). The placebo and TAU TTs were conducted using a double-blind randomised crossover design. This dose was chosen because it has previously been shown to improve high intensity running performance in well-trained runners (Balshaw et al. ) and represents the typical dose in many commercially available energy and stimulant drinks. Data were downloaded from the ergometer software at a frequency of 37 Hz to determine each 500 m split time, speed and overall performance time.

Results

There were no main effects for experimental condition on blood lactate (mean difference = 0.19 mmol l−1, f = 1.06, CI = −0.22, 0.60, p = 0.33, ηp2 = 0.10), pO2 (mean difference = 0.10 kPa, f = 0.25, CI = −0.35, 0.56, p = 0.63, pη2 = 0.03), pCO2 (mean difference = 0.01 kPa, f = 0.01, CI = −0.26, 0.24, p = 0.93, ηp2 = 0.01), pH (mean difference = 0.003 pH units, f = 0.12, CI = −0.02, 0.01, p = 0.73, ηp2 = 0.010), nor HCO3 (mean difference = 0.17 mmol l−1, f = 0.50, CI = −0.37, 0.72, p = 0.50, ηp2 = 0.05), but there were significant main effects for time on all of the blood parameters (Table ). This was characterised by post exercise increases in blood lactate (mean difference = 7.41 mmol l−1, f = 112.72, CI = 5.85, 8.96, p < 0.001, ηp2 = 0.92) and pO2 (mean difference = 2.67 kPa, f = 148.39, CI = 2.17, 3.17, p < 0.001, ηp2 = 0.94). Conversely pCO2 (mean difference = −1.04 kPa, f = 155.88, CI = −1.23, −0.85, p < 0.001, ηp2 = 0.94) pH (mean difference = −0.26 pH units, f = 77.52, CI = −0.32, −0.19, p < 0.001, ηp2 = 0.89) and HCO3 (mean difference = −13.47 mmol l−1, f = 334.08, CI = −15.12, −11.83, p < 0.001, ηp2 = 0.97) were all significantly lowered (Table ) following the completion of the TTs. Base excess (Table ) was also consequently unaffected by taurine ingestion (mean difference = 0.18 mEq l−1, f = 0.51, CI = −0.74, 0.38, p = 0.49, ηp2 = 0.05), but significant decreases were observed following the 4-km TTs (mean difference = −16.04 mEq l−1, f = 222.51, CI = −18.44, −13.65, p < 0.001, ηp2 = 0.96).Table 1Mean (±SD) pre and post time trial blood parameter responses to the experimental conditionsParameterTaurinePlaceboLactate (mmol l−1) Pre0.9 ± 0.30.9 ± 0.5 Post8.2 ± 2.4*8.6 ± 2.0*pO2 (kPa) Pre9.6 ± 0.79.8 ± 1.1 Post12.3 ± 0.8*12.4 ± 0.6*pCO2 (kPa) Pre5.4 ± 0.35.3 ± 0.4 Post4.3 ± 0.3*4.3 ± 0.4*pH Pre7.42 ± 0.027.42 ± 0.01 Post7.16 ± 0.1*7.16 ± 0.08*HCO3 (mmol l−1) Pre25.5 ± 0.125.2 ± 1.6 Post11.9 ± 2.7*11.9 ± 2.3*Base Excess (mEq l−1) Pre1.29 ± 1.11.01 ± 1.6 Post−14.84 ± 3.9*−14.96 ± 3.2** Denotes significant post time trial changes (p < 0.001)

Abstract

The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest.

REA and NAD+ display different and coupled conformations

Our crystallographic data revealed that both REA and NAD+ bind to the enzyme active site with two different conformations that we observed in different monomers. In particular, in the monomers A and D, REA displays a closed conformation with its carboxyl group pointing toward the catalytic residue C314. In the same monomers, the nicotinic acid moiety of NAD+ is oriented in the direction of the active site. On the other hand, in monomers B and C, the REA adopts what we propose to be the product conformation, with its isoprenic chain moving away from the active site toward the protein surface, so that the entire REA molecule efficiently occupies a tunnel that provides substrate access to and product release from, the catalytic cysteine. In parallel, the NAD+ nicotinic ring also moves away from the active site pointing toward the protein surface (). In summary, we observed two different and linked conformations for the REA-NAD+ couple in two different monomers: the first showing the REA carboxylate pointing toward the C314 and the second showing the product conformation with the REA carboxylate moiety oriented toward the protein surface. The NAD+ and REA coupled conformations do not appear to be determined or influenced by contacts occurring between different monomers within the tetrameric assembly or from crystallographic symmetry mates. Therefore, we can surmise that in human ALDH1A3 structure, the different conformations adopted by the ligands are the consequence of specific interactions with ALDHs conserved residues.

Conclusion

Human ALDH1A3 has a key role in vertebrate development by participating in the synthesis of the differentiation factor retinoic acid. In the present study we report the crystal structure of human ALDH1A3 complexed with NAD+ and the physiological product all-trans retinoic acid. Our structural investigation provides the first description of the REA binding site in a member of the ALDH1A subfamily. Both the REA and NAD+ shows two different conformations that appear to be coupled. Indeed, in the closed-in conformation, both ligands point the reactive like moieties of the molecule toward the active site cysteine. On the other hand, in the product-like conformation both NAD+ and REA move away from the catalytic pocket to the protein surface and occupy a binding pocket located at the entrance of the catalytic tunnel. Remarkably, the conformational changes adopted by the ligands concern the catalytic reactive moieties on both molecules. Indeed, it is the nicotinamide ring of NAD+ and the isoprenic chain of REA that move in a concerted manner either close to the active site cysteine or toward the protein surface, while both the REA β-ionone ring and the ADP moiety of NAD+ maintain the same conformation. Structural superposition with human ALDH1A1 reveals the expected high level of conservation; however, few significantly different residues are observed in structurally equivalent positions in the catalytic pocket. In particular, in ALDH1A3 an asparagine at position 469 and a threonine at position 315, respectively replace a glycine and an isoleucine present in ALDH1A1. Remarkably, both residues contact REA in the closed conformation and therefore play a role in ligand recognition. The last observation suggests that the structure-based design of potent and, most importantly, specific inhibitors of potential medical interest, is achievable.

Results

This validated HPLC-LTQ-Orbitrap-HRMS method was used for the quantification analysis of myriocin in 7 batches of Cordyceps under the SRM mode. The negative ion spectra shows intense signal at m/z 400.2646 for myriocin. The main fragment in the low m/z spectral region derive from the threonine portion (formation of deprotonated serine, m/z 104.0333, C3H6NO3+), and fission of the bonds close to the C-14 carbonyl group yields a minor fragment at m/z 85.0226, C6H13+). The first-generation loss of the elements of formaldehyde (CH2O; −30 Da) and subsequent loss of two water molecules (H2O, H2O; −18–18 Da) were yielded from the precursor ion [M-H]− (m/z 400.2646) to derive the fragments at m/z 370.4453, m/z 352.2433 and m/z 334.2335, respectively, as shown in . The content of myriocin was calculated by its calibration curve, and the quantification results were presented in and . By comparison of the retention time and fragmentation pattern with a reference standard, myriocin was identified and confirmed, for the target peak showed the same retention time at 11.20 and the same fragmentation as the standard (). As presented in , myriocin was quantified in sample D, C and B at the levels of 189.36, 49.49, 9.80 μg/g, respectively. But it was very low and under the limit of determination in sample E, F, G and H, which were classified as Isaria cicadae.10.1080/21501203.2017.1383319-F0002Figure 2.Ion map(SIM)of vegetable cicada and its allies. (a): Myriocin (1: 11.20 min); (b): Cordyceps cicadae; (c): Fruiting body of Ophiocordyceps longissima; (d): Mycelium of O. longissima; (e): Fruiting body of vegetable cicada; (f): Substrate of vegetable cicada; (g): Wild vegetable cicada (Anhui); (h): Wild vegetable cicada (Henan).10.1080/21501203.2017.1383319-T0001Table 1.The content of myriocin in vegetable cicada and its allies.SampleOriginSourceContent(μg/g)BWild Cordyceps cicadaeYunnan9.80CFruiting body of O. longissimaAnhui Agricultural University49.49DMycelium of O. longissimaAnhui Agricultural University189.36EFruiting body of vegetable cicadaZhejiang Bioasia Institute of Life SciencesUDFSubstrate of vegetable cicadaZhejiang Bioasia Institute of Life SciencesUDGWild vegetable cicadaAnhuiUDHWild vegetable cicadaHenanUDUD: under the limit of determination.

Abstract

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are necessary for functional cell integrity. Preconditioning (PC), as we define it, is an acquired protection or resilience by a cell, tissue, or organ to a lethal stimulus enabled by a previous sublethal stressor or stimulus. In this study, we provide evidence that the omega-3 fatty acid docosahexaenoic acid (DHA) and its derivatives, the docosanoids 17-hydroxy docosahexaenoic acid (17-HDHA) and neuroprotectin D1 (NPD1), facilitate cell survival in both in vitro and in vivo models of retinal PC. We also demonstrate that PC requires the enzyme 15-lipoxygenase-1 (15-LOX-1), which synthesizes 17-HDHA and NPD1, and that this is specific to docosanoid signaling despite the concomitant release of the omega-6 arachidonic acid and eicosanoid synthesis. These findings advocate that DHA and docosanoids are protective enablers of PC in photoreceptor and retinal pigment epithelial cells.

Introduction

In this study, we demonstrate that the free pool size of the essential omega-3 fatty acid DHA is increased to form docosanoids, which play a critical role in survival signaling mechanisms during the protective actions of both in vitro and in vivo retinal PC. We show that, in human retinal pigment epithelial (RPE) cells and PRCs, this resilience is mitigated through 15-LOX-1. We also demonstrate that the neurotrophin pigment epithelium-derived factor (PEDF), which also stimulates docosanoid production, is involved. Moreover, we show that NPD1 prevents the loss of protection bestowed by 15-LOX-1 inhibition in vitro and protects PRCs from light damage in vivo, further signifying that DHA and its docosanoid metabolites are pivotal during RPE and photoreceptor PC.

Abstract

CD1 proteins present microbial lipids to T cells. Germline-encoded mycolyl lipid-reactive (GEM) T cells with conserved αβ T cell receptors (TCRs) recognize CD1b presenting mycobacterial mycolates. As the molecular basis underpinning TCR recognition of CD1b remains unknown, here we determine the structure of a GEM TCR bound to CD1b presenting glucose-6-O-monomycolate (GMM). The GEM TCR docks centrally above CD1b, whereby the conserved TCR α-chain extensively contacts CD1b and GMM. Through mutagenesis and study of T cells from tuberculosis patients, we identify a consensus CD1b footprint of TCRs present among GEM T cells. Using both the TCR α- and β-chains as tweezers to surround and grip the glucose moiety of GMM, GEM TCRs create a highly specific mechanism for recognizing this mycobacterial glycolipid.

A two-chain tweezers mechanism grips GMM

Whereas most GEM T cells recognize GMM, the extent of fine specificity for antigen structure remains unknown. One ‘atypical' GEM TCR expressed by clone 18 (GEM18 TCR) recognized free mycolic acid (MA). This antigen lacks the glucosyl moiety, pointing toward possible promiscuity. On the other hand, CD1b-reactive clones known as LDN5-like T cells, do show specificity for the glucosyl group of GMM,. We measured GEM clone 42 and clone 18 responses to molecules with altered head groups, including MA, which lacks glucose, as well as mannose-6-O-monomycolate (ManMM) and galactose-6-O-monomycolate (GalMM), which differ from GMM only in the orientation of one hydroxyl group in the hexosyl ring at position C2 or C4, respectively. In all cases alteration of the headgroup abolished the GEM clone 42 T cell response, demonstrating precise specificity for the glucosyl moiety ().

2.1. Dose-Response of Atorvastatin and GT3 on TM Expression

To measure the effects of atorvastatin and GT3 on TM expression, primary human umbilical vein endothelial cells (HUVECs) were treated with different doses of atorvastatin and GT3 for 24 h and TM expression was measured by flow cytometry. We observed dose-dependent increases in TM expression after atorvastatin treatment (A). Treatment with 1, 2.5, and 5 μM of atorvastatin resulted in a 1.36-, 2.45-, and 4.46-fold increase in TM expression, respectively, as compared to the vehicle treated group. GT3 treatment at 2.5 μM did not cause a significant change in TM expression, but 5 μM GT3 treatment for 24 h caused a 2.35-fold increase in TM expression (B).

2.2. Atorvastatin and GT3 Synergistically Up-Regulate TM Expression

Next, we investigated whether atorvastatin and GT3 has a synergistic effect on TM expression. To address the issue, we used low doses of atorvastatin and GT3. HUVECs were treated with 1 μM of atorvastatin, 1 μM of GT3, and 1 μM of atorvastatin plus GT3. They were also treated with 2.5 μM of atorvastatin, 2.5 μM of GT3, and 2.5 μM of atorvastatin plus 2.5 μM of GT3 for 24 h, and TM expression was measured in all groups by flow cytometry. For both the treatment strategies, we found that combined treatment resulted in significantly (p < 0.01) higher TM expression than singular treatment with atorvastatin or GT3 (A,B and ). TM expression was unaltered after 1 μM and 2.5 μM of GT3 treatment, but it was increased 1.36- and 2.45-fold after 1 μM and 2.5 μM, respectively, of atorvastatin treatment. Interestingly, TM expression increased 1.6-fold after treatment with 1 μM of atorvastatin plus 1 μM GT3, and it increased 3.38-fold after treatment with 2.5 μM atorvastatin plus 2.5 μM GT3. These findings show that atorvastatin and GT3 synergistically induce TM expression in this used dose range. However, when we treated HUVECs with 5 μM atorvastatin plus 5 μM GT3, the TM expression reduced to control level at 24 h ().

2.3. Atorvastatin and GT3 Synergistically Induce TM Functional Activity

Next, we determined whether atorvastatin and GT3 synergistically increased the functional activities of TM, which was measured by APC generation assay. HUVECs were treated with atorvastatin alone, GT3 alone, and atorvastatin plus GT3 for 24 h. We used three different doses of GT3 (1, 2.5, and 5 μM) and two doses of atorvastatin (1 and 2.5 μM). No significant change in APC generation was observed after the 1, 2.5 and 5 μM GT3 treatments. However, the 1 and 2.5 μM atorvastatin treatment for 24 h significantly (p < 0.001) induced APC generation when curve slopes were compared between vehicle-treated and atorvastatin-treated groups (). APC generation was maximally induced after combined treatment with 1 μM atorvastatin plus 1 μM GT3, 1 μM atorvastatin plus 2.5 μM GT3, 1 μM atorvastatin plus 5 μM GT3, and 2.5 μM atorvastatin plus 2.5 μM GT3 treatment compared to treatment with each drug alone (A–D), suggesting that atorvastatin and GT3 synergistically enhance the functional activity of TM as well as TM expression in this dose range.

2.4. Atorvastatin and GT3 Synergistically Up-Regulate KLF2 Expression

Since the transcription factor Kruppel-like factor 2 (KLF2) plays an important role in modulating TM expression, we hypothesized that atorvastatin and GT3 would also synergistically up-regulate KLF2 expression. HUVECs were treated with 2.5 μM of atorvastatin, 2.5 μM of GT3, and 2.5 μM of atorvastatin plus 2.5 μM of GT3. KLF2 mRNA expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) at three different time intervals (6, 12, and 24 h). GT3 treatment alone was not able to induce KLF2 expression at the 6 and 12 h time points; however, there was an approximately 2.5-fold increase (p = 0.0006) at the 24 h time point (A–C). On the other hand, treatment with atorvastatin alone significantly induced (p < 0.001) KLF2 expression at all three time points (A–C), but a maximum increase in KLF2 expression was observed after combined treatment with atorvastatin and GT3, suggesting a synergistic effect of these two compounds on KLF2 expression (A–C).

Abstract

A microporous hydrogel was developed using sodium alginate (alg) and 4-aminosalicylic acid (4-ASA). The synthesized hydrogel was characterized using various analytical techniques such as Fourier transform infrared spectroscopy (FTIR), Carbon-13 nuclear magnetic resonance (13C-NMR), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). Additonal carboxyl and hydroxyl functional groups of 4-ASA provided significant lubrication and stress-triggered sol-gel transition to the conjugated hydrogel. In addition, cytotoxicity analysis was undertaken on the conjugated hydrogel using human dermal fibroblast-adult (HDFa) cells, displaying non-toxic characteristics. Drug release profiles displaying 49.6% in the first 8 h and 97.5% within 72 h, similar to the native polymer (42.8% in first 8 h and 90.1% within 72 h). Under applied external stimuli, the modified hydrogel displayed significant gelling properties and structure deformation/recovery behaviour, confirmed using rheological evaluation (viscosity and thixotropic area of 8095.3 mPas and 26.23%, respectively). The modified hydrogel, thus, offers great possibility for designing smart synovial fluids as a biomimetic aqueous lubricant for joint-related injuries and arthritis-induced conditions. In addtion, the combination of thixotropy, non-toxicity, and drug release capabilities enables potential viscosupplementation for clinical application.

1. Introduction

In the current study, we developed a novel microporous hydrogel based on sodium alginate and 4-ASA via carbodiimide coupling chemistry. This is the first report on the synthesis of an alginate-4-aminosalicylic acid based amide conjugate to the best of our knowledge. Furthermore, the modified hydrogel was found to be nontoxic and possesed drug release profiles simmilar to sodium alginate. Gelling ability and gel structure deformation/recovery behaviour was confirmed using rheological studies to evaluate the hydrogel as a stress-triggered hydrogel system. Development and evaluation of biopolymer-based soft hydrogel systems provide great possibilities for designing smart synovial fluids and may act as a biomimetic aqueous lubricant for arthritis treatment. Moreover, the combination of thixotropy, nontoxicity and drug release capabilities of the hydrogel enables significant potential for clinical application such as viscosupplementation in arthritis intervention.