Epigallocatechin-3-Gallate Promotes Osteo-/Odontogenic Differentiation of Stem Cells from the Apical Papilla through Activating the BMP–Smad Signaling Pathway

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16–20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP–Smad signaling pathway.     

Anti-Hair Loss Effect of Adenosine Is Exerted by cAMP Mediated Wnt/β-Catenin Pathway Stimulation via Modulation of Gsk3β Activity in Cultured Human Dermal Papilla Cells

In the present study, we investigated the molecular mechanisms of adenosine for its hair growth promoting effect. Adenosine stimulated the Wnt/β-catenin pathway by modulating the activity of Gsk3β in cultured human dermal papilla cells. It also activated adenosine receptor signaling, increasing intracellular cAMP level, and subsequently stimulating the cAMP mediated cellular energy metabolism. The phosphorylation of CREB, mTOR, and GSK3β was increased. Furthermore, the expression of β-catenin target genes such as Axin2, Lef1, and growth factors (bFGF, FGF7, IGF-1) was also enhanced. The inhibitor study data conducted in Wnt reporter cells and in cultured human dermal papilla cells demonstrated that adenosine stimulates Wnt/β-catenin signaling through the activation of the adenosine receptor and Gsk3β plays a critical role in transmitting the signals from the adenosine receptor to β-catenin, possibly via the Gαs/cAMP/PKA/mTOR signaling cascade.     

Highly efficient detection of insulinotropic action of glucagon via GLP-1 receptor in mice pancreatic beta-cell with a novel perfusion microchip

Glucagon exhibits insulinotropic ability by activating cAMP through glucagon or glucagon-like peptide-1(GLP-1) receptors.To investigate the mechanism of endogenous and exogenous glucagon on insulin release,we studied the receptor selectivity on pancreatic islet beta-cells by switching the glucose concentration from 20 mmol/L to 0 mmol/L To measure the exact temporal relationship between glucagon and insulin release,we developed a quick,small volume,multi-channel polydimethylsiloxane(PDMS) microchip.At 0 mmol/L glucose,we observed an insulinotropic effect in both INS-1 cells and islets.Meanwhile,we observed a 63 ± 6.27 s delay of endogenous glucagon-induced insulin release.After treatment with glucagon and GLP-1 receptor antagonists,we found that endogenous glucagon utilized the glucagon receptor,whereas exogenous glucagon primarily utilized the GLP-1 receptor to promote insulin secretion.The microchip can also be used to describe the "glucagonocentric" vision of diabetes pathophysiology.Taken together,the insulinotropic mechanism of different receptors should be taken into account in clinical treatments.     

Antioxidant and Anti-Inflammatory Activities of Six Flavonoids from Smilax glabra Roxb

This study aimed to isolate, prepare and identify the main flavonoids from a standardized Smilax glabra flavonoids extract (SGF) using preparative HPLC, MS, ¹H NMR and ¹³C NMR, determine the contents of these flavonoids using UPLC, then compare their pharmacological activities in vitro. We obtained six flavonoids from SGF: astilbin (18.10%), neoastilbin (11.04%), isoastilbin (5.03%), neoisoastilbin (4.09%), engeletin (2.58%) and (−)-epicatechin (1.77%). The antioxidant activity of six flavonoids were evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS⁺) radical scavenging activity and ferric reducing antioxidant power (FRAP). In addition, the anti-inflammatory activity of six flavonoids were evaluated by determining the production of cytokines (IL-1β, IL-6), nitric oxide (NO) using enzyme linked immunosorbent assay and the NF-κB p65 expression using Western blotting in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that (−)-epicatechin, astilbin, neoastilbin, isoastilbin and neoisoastilbin had strong antioxidant activities, not only in DPPH and ABTS⁺ radicals scavenging capacities, but in FRAP system. Furthermore, all the six flavonoids could significantly inhibit the secretion of IL-1β, IL-6, NO (p < 0.01) and the protein expression of NF-κB p-p65 (p < 0.01) in LPS-stimulated RAW264.7 cells. This study preliminarily verified the antioxidant and anti-inflammatory activities of six flavonoids in S. glabra.     

Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [68Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in β-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R–avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic β-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [⁶⁸Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a ⁶⁸Ge/⁶⁸Ga Generator (GalliaPharma^®) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP^®). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 μg/mL) and [⁶⁸Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [⁶⁸Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5–0.75 μg/mL for both the analytes (NODAGA-exendin-4 and ⁶⁸Ga-NODAGA-exendin-4), with a correlation coefficient (R²) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [⁶⁸Ga]Ga-NODAGA-exendin-4 was linear with a R² of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 μg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [⁶⁸Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [⁶⁸Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [⁶⁸Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.     

Protective Effects of Gintonin on Reactive Oxygen Species-Induced HT22 Cell Damages: Involvement of LPA1 Receptor-BDNF-AKT Signaling Pathway

Gintonin is a kind of ginseng-derived glycolipoprotein that acts as an exogenous LPA receptor ligand. Gintonin has in vitro and in vivo neuroprotective effects; however, little is known about the cellular mechanisms underlying the neuroprotection. In the present study, we aimed to clarify how gintonin attenuates iodoacetic acid (IAA)-induced oxidative stress. The mouse hippocampal cell line HT22 was used. Gintonin treatment significantly attenuated IAA-induced reactive oxygen species (ROS) overproduction, ATP depletion, and cell death. However, treatment with Ki16425, an LPA1/3 receptor antagonist, suppressed the neuroprotective effects of gintonin. Gintonin elicited [Ca²⁺]_i transients in HT22 cells. Gintonin-mediated [Ca²⁺]_i transients through the LPA1 receptor-PLC-IP₃ signaling pathway were coupled to increase both the expression and release of BDNF. The released BDNF activated the TrkB receptor. Induction of TrkB phosphorylation was further linked to Akt activation. Phosphorylated Akt reduced IAA-induced oxidative stress and increased cell survival. Our results indicate that gintonin attenuated IAA-induced oxidative stress in neuronal cells by activating the LPA1 receptor-BDNF-TrkB-Akt signaling pathway. One of the gintonin-mediated neuroprotective effects may be achieved via anti-oxidative stress in nervous systems.     

Krüppel-like factor 4 mediates lysophosphatidic acid-stimulated migration and proliferation of PC3M prostate cancer cells

Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Accumulating evidence suggests that lysophosphatidic acid (LPA) serves as an autocrine/paracrine mediator to affect initiation, progression and metastasis of prostate cancer. In the current study, we demonstrate that LPA stimulates migration and proliferation of highly metastatic human prostate cancer, PC-3M-luc-C6 cells. LPA-induced migration of PC-3M-luc-C6 cells was abrogated by pretreatment of PC-3M-luc-C6 cells with the LPA receptor 1/3 inhibitor Ki16425 or small interfering RNA (siRNA)-mediated silencing of endogenous LPA receptor 1, implicating a key role of the LPA-LPA receptor 1 signaling axis in migration of PC-3M-luc-C6 cells. In addition, LPA treatment resulted in augmented expression levels of Krüppel-like factor 4 (KLF4), and siRNA or short-hairpin RNA (shRNA)-mediated silencing of KLF4 expression resulted in the abolishment of LPA-stimulated migration and proliferation of PC-3M-luc-C6 cells. shRNA-mediated silencing of KLF4 expression resulted in the inhibition of in vivo growth of PC-3M-luc-C6 cells in a xenograft transplantation animal model. Taken together, these results suggest a key role of LPA-induced KLF4 expression in cell migration and proliferation of prostate cancer cells in vitro and in vivo.     

Comparison of antioxidant capacities and antioxidant components of commercial bitter melon (Momordica charantia L.) products

In this study, the total phenolic contents and total antioxidant capacities of some commercial bitter melon products (powder, packaged powder, capsule, paste in olive oil), and of unripe and ripe fruits were determined by spectrophotometric and chromatographic methods. The total antioxidant capacities of unripe and ripe bitter melon samples, determined by using the CUPRAC (cupric reducing antioxidant capacity assay) and ABTS (2,2′-azino-bis(3-ethylbenzthiazolin-6-sulfonic acid))/HRP (horseradish peroxidase) methods, were 42.5 and 36.3 µmol TRE (Trolox equivalent) g–1, and 8.7 and 7.0 µmol TRE g–1, respectively. The TAC (total antioxidant capacity) order of the studied samples using the same 2 methods were determined as follows: capsule (CUPRAC value, 140.8; ABTS/HRP value, 143.6 µmol TRE g–1) > packaged powder (129.6; 126.1) > powder (52.3; 64.3) > unripe fruit (42.5; 36.3) > paste in olive oil (17.6; 14.4) > ripe fruit (8.7; 7.0). The order of phenolic content was found as follows: unripe fruit (193.2 µmol GAE (gallic acid equivalent) g-1) > capsule (162.0) > packaged powder (160.6) > powder (83.6) > paste in olive oil (38.3) > ripe fruit (14.6).     

Novel Phthalazin-1(2H)-One Derivatives Displaying a Dithiocarbamate Moiety as Potential Anticancer Agents

Nowadays, cancer disease seems to be the second most common cause of death worldwide. Molecular hybridization is a drug design strategy that has provided promising results against multifactorial diseases, including cancer. In this work, two series of phthalazinone-dithiocarbamate hybrids were described, compounds 6–8, which display the dithiocarbamate scaffold at N2, and compounds 9, in which this moiety was placed at C4. The proposed compounds were successfully synthesized via the corresponding aminoalkyl phthalazinone derivatives and using a one-pot reaction with carbon disulfide, anhydrous H₃PO₄, and different benzyl or propargyl bromides. The antiproliferative effects of the titled compounds were explored against three human cancer cell lines (A2780, NCI-H460, and MCF-7). The preliminary results revealed significant differences in activity and selectivity depending on the dithiocarbamate moiety location. Thus, in general terms, compounds 6–8 displayed better activity against the A-2780 and MCF-7 cell lines, while most of the analogues of the 9 group were selective toward the NCI-H460 cell line. Compounds 6e, 8e, 6g, 9a–b, 9d, and 9g with IC₅₀ values less than 10 µM were the most promising. The drug-likeness and toxicity properties of the novel phthalazinone-dithiocarbamate hybrids were predicted using Swiss-ADME and ProTox web servers, respectively.     

Development of Halogenated Pyrazolines as Selective Monoamine Oxidase-B Inhibitors: Deciphering via Molecular Dynamics Approach

Halogens have been reported to play a major role in the inhibition of monoamine oxidase (MAO), relating to diverse cognitive functions of the central nervous system. Pyrazoline/halogenated pyrazolines were investigated for their inhibitory activities against human monoamine oxidase-A and -B. Halogen substitutions on the phenyl ring located at the fifth position of pyrazoline showed potent MAO-B inhibition. Compound 3-(4-ethoxyphenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole (EH7) showed the highest potency against MAO-B with an IC₅₀ value of 0.063 µM. The potencies against MAO-B were increased in the order of –F (in EH7) > –Cl (EH6) > –Br (EH8) > –H (EH1). The residual activities of most compounds for MAO-A were > 50% at 10 µM, except for EH7 and EH8 (IC₅₀ = 8.38 and 4.31 µM, respectively). EH7 showed the highest selectivity index (SI) value of 133.0 for MAO-B, followed by EH6 at > 55.8. EH7 was a reversible and competitive inhibitor of MAO-B in kinetic and reversibility experiments with a Ki value of 0.034 ± 0.0067 µM. The molecular dynamics study documented that EH7 had a good binding affinity and motional movement within the active site with high stability. It was observed by MM-PBSA that the chirality had little effect on the overall binding of EH7 to MAO-B. Thus, EH7 can be employed for the development of lead molecules for the treatment of various neurodegenerative disorders.     

Preliminary Study of a 1,5-Benzodiazepine-Derivative Labelled with Indium-111 for CCK-2 Receptor Targeting

The cholecystokinin-2 receptor (CCK-2R) is overexpressed in several human cancers but displays limited expression in normal tissues. For this reason, it is a suitable target for developing specific radiotracers. In this study, a nastorazepide-based ligand functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator (IP-001) was synthesized and labelled with indium-111. The radiolabeling process yielded >95% with a molar activity of 10 MBq/nmol and a radiochemical purity of >98%. Stability studies have shown a remarkable resistance to degradation (>93%) within 120 h of incubation in human blood. The in vitro uptake of [¹¹¹In]In-IP-001 was assessed for up to 24 h on a high CCK-2R-expressing tumor cell line (A549) showing maximal accumulation after 4 h of incubation. Biodistribution and single photon emission tomography (SPECT)/CT imaging were evaluated on BALB/c nude mice bearing A549 xenograft tumors. Implanted tumors could be clearly visualized after only 4 h post injection (2.36 ± 0.26% ID/cc), although a high amount of radiotracer was also found in the liver, kidneys, and spleen (8.25 ± 2.21%, 6.99 ± 0.97%, and 3.88 ± 0.36% ID/cc, respectively). Clearance was slow by both hepatobiliary and renal excretion. Tumor retention persisted for up to 24 h, with the tumor to organs ratio increasing over-time and ending with a tumor uptake (1.52 ± 0.71% ID/cc) comparable to liver and kidneys.     

Preeclampsia serum-induced collagen I expression and intracellular calcium levels in arterial smooth muscle cells are mediated by the PLC-γ1 pathway

In women with preeclampsia (PE), endothelial cell (EC) dysfunction can lead to altered secretion of paracrine factors that induce peripheral vasoconstriction and proteinuria. This study examined the hypothesis that PE sera may directly or indirectly, through human umbilical vein ECs (HUVECs), stimulate phospholipase C-γ1-1,4,5-trisphosphate (PLC-γ1-IP₃) signaling, thereby increasing protein kinase C-α (PKC-α) activity, collagen I expression and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in human umbilical artery smooth muscle cells (HUASMCs). HUASMCs and HUVECs were cocultured with normal or PE sera before PLC-γ1 silencing. Increased PLC-γ1 and IP₃ receptor (IP₃R) phosphorylation was observed in cocultured HUASMCs stimulated with PE sera (P<0.05). In addition, PE serum significantly increased HUASMC viability and reduced their apoptosis (P<0.05); these effects were abrogated with PLC-γ1 silencing. Compared with normal sera, PE sera increased [Ca²⁺]_i in cocultured HUASMCs (P<0.05), which was inhibited by PLC-γ1 and IP₃R silencing. Finally, PE sera-induced PKC-α activity and collagen I expression was inhibited by PLC-γ1 small interfering RNA (siRNA) (P<0.05). These results suggest that vasoactive substances in the PE serum may induce deposition in the extracellular matrix through the activation of PLC-γ1, which may in turn result in thickening and hardening of the placental vascular wall, placental blood supply shortage, fetal hypoxia–ischemia and intrauterine growth retardation or intrauterine fetal death. PE sera increased [Ca²⁺]_i and induced PKC-α activation and collagen I expression in cocultured HUASMCs via the PLC-γ1 pathway.     

Process Optimization and Modeling of Phenol Adsorption onto Sludge-Based Activated Carbon Intercalated MgAlFe Ternary Layered Double Hydroxide Composite

A sewage sludge-based activated carbon (SBAC) intercalated MgAlFe ternary layered double hydroxide (SBAC-MgAlFe-LDH) composite was synthesized via the coprecipitation method. The adsorptive performance of the composite for phenol uptake from the aqueous phase was evaluated via the response surface methodology (RSM) modeling technique. The SBAC-MgAlFe-LDH phenol uptake capacity data were well-fitted to reduced RSM cubic model (R² = 0.995, R²-adjusted = 0.993, R²-predicted = 0.959 and p-values < 0.05). The optimum phenol adsorption onto the SBAC-MgAlFe-LDH was achieved at 35 °C, 125 mg/L phenol, and pH 6. Under the optimal phenol uptake conditions, pseudo-first-order and Avrami fractional-order models provided a better representation of the phenol uptake kinetic data, while the equilibrium data models’ fitting follows the order; Liu > Langmuir > Redlich–Peterson > Freundlich > Temkin. The phenol uptake mechanism was endothermic in nature and predominantly via a physisorption process (ΔG° = −5.33 to −5.77 kJ/mol) with the involvement of π–π interactions between the phenol molecules and the functionalities on the SBAC-LDH surface. The maximum uptake capacity (216.76 mg/g) of SBAC-MgAlFe-LDH was much higher than many other SBAC-based adsorbents. The improved uptake capacity of SBAC-LDH was attributed to the effective synergetic influence of SBAC-MgAlFe-LDH, which yielded abundant functionalized surface groups that favored higher aqueous phase uptake of phenol molecules. This study showcases the potential of SBAC-MgAlFe-LDH as an effective adsorbent material for remediation of phenolic wastewater     

Synthesis,DFT Calculations,Antiproliferative, Bactericidal Activity and Molecular Docking of Novel Mixed-Ligand Salen/8-Hydroxyquinoline Metal Complexes

Despite the common use of salens and hydroxyquinolines as therapeutic and bioactive agents, their metal complexes are still under development. Here, we report the synthesis of novel mixed-ligand metal complexes (MSQ) comprising salen (S), derived from (2,2′-{1,2-ethanediylbis[nitrilo(E) methylylidene]}diphenol, and 8-hydroxyquinoline (Q) with Co(II), Ni(II), Cd(II), Al(III), and La(III). The structures and properties of these MSQ metal complexes were investigated using molar conductivity, melting point, FTIR, ¹H NMR, ¹³C NMR, UV–VIS, mass spectra, and thermal analysis. Quantum calculation, analytical, and experimental measurements seem to suggest the proposed structure of the compounds and its uncommon monobasic tridentate binding mode of salen via phenolic oxygen, azomethine group, and the NH group. The general molecular formula of MSQ metal complexes is [M(S)(Q)(H₂O)] for M (II) = Co, Ni, and Cd or [M(S)(Q)(Cl)] and [M(S)(Q)(H₂O)]Cl for M(III) = La and Al, respectively. Importantly, all prepared metal complexes were evaluated for their antimicrobial and anticancer activities. The metal complexes exhibited high cytotoxic potency against human breast cancer (MDA-MB231) and liver cancer (Hep-G2) cell lines. Among all MSQ metal complexes, CoSQ and LaSQ produced IC₅₀ values (1.49 and 1.95 µM, respectively) that were comparable to that of cisplatin (1.55 µM) against Hep-G2 cells, whereas CdSQ and LaSQ had best potency against MDA-MB231 with IC₅₀ values of 1.95 and 1.43 µM, respectively. Furthermore, the metal complexes exhibited significant antimicrobial activities against a wide spectrum of both Gram-positive and -negative bacterial and fungal strains. The antibacterial and antifungal efficacies for the MSQ metal complexes, the free S and Q ligands, and the standard drugs gentamycin and ketoconazole decreased in the order AlSQ > LaSQ > CdSQ > gentamycin > NiSQ > CoSQ > Q > S for antibacterial activity, and for antifungal activity followed the trend of LaSQ > AlSQ > CdSQ > ketoconazole > NiSQ > CoSQ > Q > S. Molecular docking studies were performed to investigate the binding of the synthesized compounds with breast cancer oxidoreductase (PDB ID: 3HB5). According to the data obtained, the most probable coordination geometry is octahedral for all the metal complexes. The molecular and electronic structures of the metal complexes were optimized theoretically, and their quantum chemical parameters were calculated. PXRD results for the Cd(II) and La(III) metal complexes indicated that they were crystalline in nature.     

The Protective Effects of Neoastilbin on Monosodium Urate Stimulated THP-1-Derived Macrophages and Gouty Arthritis in Mice through NF-κB and NLRP3 Inflammasome Pathways

Gouty arthritis (GA) is a frequent inflammatory disease characterized by pain, swelling, and stiffness of joints. Neoastilbin is a flavonoid isolated from the rhizome of Smilax glabra, which possesses various anti-inflammatory effects. However, the mechanism of neoastilbin in treating GA has not yet been clarified. Thus, this study was to investigate the protective effects of neoastilbin in both monosodium urate (MSU) stimulated THP-1-derived macrophages and the animal model of GA by injecting MSU into the ankle joints of mice. The levels of key inflammatory cytokines in MSU stimulated THP-1-derived macrophages were detected by enzyme-linked immunosorbent assay (ELISA) kits. Protein expressions of nuclear factor kappa B (NF-κB) and NOD-like receptor protein 3 (NLRP3) inflammasome pathways were further detected by Western blotting. In addition, swelling degree of ankle joints, the levels of inflammatory factors, infiltration of inflammatory cells and the expressions of related proteins were determined. Swelling degree and histopathological injury in ankle joints of MSU-injected mice were significantly decreased after being treated with neoastilbin. Moreover, neoastilbin significantly diminished the secretion of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), suppressing the activation of NF-κB and NLRP3 inflammasome pathways in both MSU stimulated THP-1-derived macrophages and the mouse model of GA. In summary, neoastilbin could alleviate GA by inhibiting the NF-κB and NLRP3 inflammasome pathways, which provided some evidence for neoastilbin as a promising therapeutic agent for GA treatment.     

Contrasting Roles of Ang II and ACEA in the Regulation of IL10 and IL1β Gene Expression in Primary SHR Astroglial Cultures

Angiotensin (Ang) II is well-known to have potent pro-oxidant and pro-inflammatory effects in the brain. Extensive crosstalk between the primary Ang II receptor, Ang type 1 receptor (AT1R), and the cannabinoid type 1 receptor (CB1R) has been demonstrated by various groups in the last decade. Since activation of glial CB1R has been demonstrated to play a key role in the resolution of inflammatory states, we investigated the role of Ang II (100 nM) and/or ACEA (10 nM), a potent CB1R-specific agonist in the regulation of inflammatory markers in astrocytes from spontaneously hypertensive rats (SHR) and Wistar rats. Astrocytes were cultured from brainstems and cerebellums of SHR and Wistar rats and assayed for IL1β and IL10 gene expression and secreted fraction, in treated and non-treated cells, by employing qPCR and ELISA, respectively. mRNA expression of both IL10 and IL1β were significantly elevated in untreated brainstem and cerebellar astrocytes isolated from SHR when compared to Wistar astrocytes. No changes were observed in the secreted fraction. While ACEA-treatment resulted in a significant increase in IL10 gene expression in Wistar brainstem astrocytes (Log2FC ≥ 1, p < 0.05), its effect in SHR brainstem astrocytes was diminished. Ang II treatment resulted in a strong inhibitory effect on IL10 gene expression in astrocytes from both brain regions of SHR and Wistar rats (Log2FC ≤ −1, p < 0.05), and an increase in IL1β gene expression in brainstem astrocytes from both strains (Log2FC ≥ 1, p < 0.05). Co-treatment of Ang II and ACEA resulted in neutralization of Ang II-mediated effect in Wistar brainstem and cerebellar astrocytes, but not SHR astrocytes. Neither Ang II nor ACEA resulted in any significant changes in IL10 or IL1β secreted proteins. These data suggest that Ang II and ACEA have opposing roles in the regulation of inflammatory gene signature in astrocytes isolated from SHR and Wistar rats. This however does not translate into changes in their secreted fractions.     

Design,Synthesis, and Biological Evaluation of Pyridazinones Containing the (2-Fluorophenyl) Piperazine Moiety as Selective MAO-B Inhibitors

Twelve pyridazinones (T1–T12) containing the (2-fluorophenyl) piperazine moiety were designed, synthesized, and evaluated for monoamine oxidase (MAO) -A and -B inhibitory activities. T6 was found to be the most potent MAO-B inhibitor with an IC₅₀ value of 0.013 µM, followed by T3 (IC₅₀ = 0.039 µM). Inhibitory potency for MAO-B was more enhanced by meta bromo substitution (T6) than by para bromo substitution (T7). For para substitution, inhibitory potencies for MAO-B were as follows: -Cl (T3) > -N(CH₃)₂ (T12) > -OCH₃ (T9) > Br (T7) > F (T5) > -CH₃ (T11) > -H (T1). T6 and T3 efficiently inhibited MAO-A with IC₅₀ values of 1.57 and 4.19 µM and had the highest selectivity indices (SIs) for MAO-B (120.8 and 107.4, respectively). T3 and T6 were found to be reversible and competitive inhibitors of MAO-B with K_i values of 0.014 and 0.0071, respectively. Moreover, T6 was less toxic to healthy fibroblast cells (L929) than T3. Molecular docking simulations with MAO binding sites returned higher docking scores for T6 and T3 with MAO-B than with MAO-A. These results suggest that T3 and T6 are selective, reversible, and competitive inhibitors of MAO-B and should be considered lead candidates for the treatment of neurodegenerative disorders like Alzheimer’s disease.     

Induction of Apoptosis via Inactivating PI3K/AKT Pathway in Colorectal Cancer Cells Using Aged Chinese Hakka Stir-Fried Green Tea Extract

Food extract supplements, with high functional activity and low side effects, play a recognized role in the adjunctive therapy of human colorectal cancer. The present study reported a new functional beverage, which is a type of Chinese Hakka stir-fried green tea (HSGT) aged for several years. The extracts of the lyophilized powder of five HSGT samples with different aging periods were analyzed with high-performance liquid chromatography. The major components of the extract were found to include polyphenols, catechins, amino acids, catechins, gallic acid and caffeine. The tea extracts were also investigated for their therapeutic activity against human colorectal cancer cells, HT-29, an epithelial cell isolated from the primary tumor. The effect of different aging time of the tea on the anticancer potency was compared. Our results showed that, at the cellular level, all the extracts of the aged teas significantly inhibited the proliferation of HT-29 in a concentration-dependent manner. In particular, two samples prepared in 2015 (15Y, aged for 6 years) and 2019 (19Y, aged for 2 years) exhibited the highest inhibition rate for 48 h treatment (cell viability was 50% at 0.2 mg/mL). Further, all the aged tea extracts examined were able to enhance the apoptosis of HT-29 cells (apoptosis rate > 25%) and block the transition of G1/S phase (cell-cycle distribution (CSD) from <20% to >30%) population to G2/M phase (CSD from nearly 30% to nearly 10%) at 0.2 mg/mL for 24 h or 48 h. Western blotting results also showed that the tea extracts inhibited cyclin-dependent kinases 2/4 (CDK2, CDK4) and CylinB1 protein expression, as well as increased poly ADP-ribose polymerase (PRAP) expression and Bcl2-associated X (Bax)/B-cell lymphoma-2 (Bcl2) ratio. In addition, an upstream signal of one of the above proteins, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling, was found to be involved in the regulation, as evidenced by the inhibition of phosphorylated PI3K and AKT by the extracts of the aged tea. Therefore, our study reveals that traditional Chinese aged tea (HSGT) may inhibit colon cancer cell proliferation, cell-cycle progression and promoted apoptosis of colon cancer cells by inactivating PI3K/AKT signalling.     

Ultrasound Assisted Synthesis and In Silico Modelling of 1,2,4-Triazole Coupled Acetamide Derivatives of 2-(4-Isobutyl phenyl)propanoic acid as Potential Anticancer Agents

The development of an economical method for the synthesis of biologically active compounds was the major goal of this research. In the present study, we have reported the ultrasound-radiation-assisted synthesis of a series of novel N-substituted 1,2,4-triazole-2-thiol derivatives. The target compounds 6a–f were efficiently synthesized in significant yields (75–89%) by coupling 1,2,4-triazole of 2-(4-isobutylphenyl) propanoic acid 1 with different electrophiles using ultrasound radiation under different temperatures. The sonication process accelerated the rate of the reaction as well as yielded all derivatives compared to conventional methods. All derivatives were confirmed by spectroscopic (FTIR, ¹HNMR, ¹³CNMR, HRMS) and physiochemical methods. All derivatives were further screened for their anticancer effects against the HepG2 cell line. Compound 6d containing two electron-donating methyl moieties demonstrated the most significant anti-proliferative activity with an IC₅₀ value of 13.004 µg/mL, while compound 6e showed the lowest potency with an IC₅₀ value of 28.399 µg/mL. The order of anticancer activity was found to be: 6d > 6b > 6f > 6a > 6c > 6e, respectively. The in silico modelling of all derivatives was performed against five different protein targets and the results were consistent with the biological activities. Ligand 6d showed the best binding affinity with the Protein Kinase B (Akt) pocket with the lowest ∆G value of −176.152 kcal/mol. Compound 6d has been identified as a promising candidate for treatment of liver cancer.     

A comparative study on the monovalent and divalent cation separation of polymeric films and membranes from salt solutions under diffusion-dialysis

This study deals with selective separation of mono- and divalent cations from aqueous salt solutions using polymeric films based on polyethylene (PE) and polyamide6 (PA6), and two different commercial nanofiltration (NF) membranes. The diffusion rates (D) of ions (Na⁺ and Ca²⁺), separation factors (α) and ion rejections (R) of the films and NF membranes are examined comparatively as well as their surface morphology and hydrophilicity. It is observed that the diffusion rates of Na⁺ are in the range of 0.7–1.8 × 10⁻⁸cm² .s⁻¹ in the decreasing order of PE > NF90 > NF270 > PA6 while Ca²⁺ shows diffusion rates of 7.4–18.4 × 10⁻⁸ cm² .s⁻¹ in the increasing order of NF270 > NF90 ≈ PA6 > PE. Rejection values of the polymeric films and NF membranes against to Na⁺ and Ca²⁺ vary between 90% and 99.6%.The highest α(Ca²⁺/Na⁺) is found to be 20 for PA6 film. D, α, and R value of both polymeric films and NF membranes are strongly affected by the existence of osmosis during diffusion-dialysis and the sizes of hydrated sodiu and calcium ions. In conclusion, the film based on PA6 may be a good alternative for selective separation of mono- an divalent cations.