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IL-10+ regulatory B (Breg) cells play a vital role in regulating the immune responses in experimental autoimmune encephalomyelitis, colitis, and contact hypersensitivity (CHS). Several stimulants such as lipopolysaccharide (LPS), CD40 ligand, and IL-21 spur the activation and maturation of IL-10+ Breg cells, while the epigenetic mechanism for the IL-10 expression remains largely unknown. It is well accepted that the histone acetylation/ deacetylation is an important mechanism that regulates the expression of IL-10. We found that entinostat, an HDAC inhibitor, stimulated the induction of IL-10+ Breg cells by LPS in vitro and the formation of IL-10+ Breg cells to suppress CHS in vivo. We further demonstrated that entinostat inhibited HDAC1 from binding to the proximal region of the IL-10 expression promoter in splenic B cells, followed by an increase in the binding of NF-κB p65, eventually enhancing the expression of IL-10 in Breg cells. [BMB Reports 2021; 54(10): 534-539].

Conjugation of carbohydrate antigens with a carrier protein is a clinically proven strategy to overcome the poor immunogenicity of bacterial polysaccharide. In addition to its primary role, which is to help generate a T cell-mediate long-lasting immune response directed against the carbohydrate antigen, the carrier protein in a glycoconjugate vaccine can also play an important role as a protective antigen. Among carrier proteins currently used in licensed conjugate vaccines, non-typeable Haemophilus influenzae protein D has been used as an antigenically active carrier protein. Our previous studies also indicate that some carrier proteins provide B cell epitopes, along with T cell helper epitopes. Herein we investigated the dual role of truncated rotavirus spike protein ΔVP8* as a carrier and a protective antigen. Capsular polysaccharide lipoarabinomannan (LAM), purified from Mycobacterium tuberculosis (M.tb), was chemically conjugated with ΔVP8*. Mouse immunization experiments showed that the resultant conjugates elicited strong and specific immune responses against the polysaccharide antigen, and the responses were comparable to those induced by Diphtheria toxoid (DT)-based conjugates. The conjugate vaccine induced enhanced antibody titers and functional antibodies against ΔVP8* when compared to immunization with the unconjugated ΔVP8*. Thus, these results indicate that ΔVP8* can be a relevant carrier protein for glycoconjugate vaccine and the glycoconjugates consisting of ΔVP8* with LAM are effective bivalent vaccine candidates against rotavirus and tuberculosis.

Introduction Endoplasmic reticulum (ER) stress condition is characterized as the accumulation of misfolded or unfolded proteins in lumen of ER. This condition has been implicated in various diseases and pathologies including β-cell apoptosis, Alzheimer’s disease and atherosclerosis. We have reported that hydroxynaphthoic acids (HNA), naphthalene analogues of salicylic acid (SA), reduced ER stress. In this study, we explored structural modification to bi-aryl analogues of SA. Methods Palladium-catalyzed cross-coupling was applied to synthesize bi-aryl analogues of SA. Anti-ER stress activity was monitored by using our cell-based assay system where ER stress is induced by tunicamycin. To monitor ER stress markers, ER stress was induced physiologically relevant palmitate system. Results Many analogues decreased ER stress signal induced by tunicamycin. Compounds creating dihedral angle between Ar group and SA moiety generally increased the activity but gave some cytotoxicity to indicate the crucial role of flat conformation of aromatic region. The best compound (16e) showed up to almost 6-fold and 90-fold better activity than 3-HNA and tauro-ursodeoxycholic acid, positive controls, respectively. ER stress markers such as p-PERK and p-JNK were accordingly decreased in Western blotting upon treatment of 16e under palmitate-induced condition. Conclusion Anti-ER stress activity and toxicity profile of bi-aryl analogues of SA could provide a novel platform for potential therapy for protein misfolding diseases., Graphical Abstract

Background Korean Red ginseng extract (KRGE) has beneficial effects on the cardiovascular system by improving endothelial cell function. However, its pharmacological effect on endothelial cell senescence has not been clearly elucidated. Therefore, we examined the effect and molecular mechanism of KRGE on the senescence of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were grown in normal or KRGE-supplemented medium. Furthermore, they were transfected with heme oxygenase-1 (HO-1) gene or treated with its inhibitor, a NF-κB inhibitor, and a miR-155-5p mimic or inhibitor. Senescence-associated characteristics of endothelial cells were determined by biochemical and immunohistochemical analyses. Results Treatment of HUVECs with KRGE resulted in delayed onset and progression of senescence-associated characteristics, such as increased lysosomal acidic β-galactosidase and decreased telomerase activity, angiogenic dysfunction, and abnormal cell morphology. KRGE preserved the levels of anti-senescent factors, such as eNOS-derived NO, MnSOD, and cyclins D and A: however, it decreased the levels of senescence-promoting factors, such as ROS, activated NF-κB, endothelial cell inflammation, and p21 expression. The beneficial effects of KRGE were due to the induction of HO-1 and the inhibition of NF-κB-dependent biogenesis of miR-155-5p that led to the downregulation of eNOS. Moreover, treatment with inhibitors of HO-1, NF-κB, and miR-155-5p abolished the anti-senescence effects of KRGE. Conclusion KRGE delayed or prevented HUVEC senescence through a signaling cascade involving the induction of HO-1, the inhibition of NF-κB-dependent miR-155-5p biogenesis, and the maintenance of the eNOS/NO axis activity, suggesting that it may protect against vascular diseases associated with endothelial senescence.

Phytochemical investigation of the methanol extract of the aerial parts of Salvia plebeia aided by a proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression screening assay in HepG2 cells led to the identification of 19 compounds including one new norsesquiterpene (1), six new eudesmane sesquiterpenoids (2-5, 8, and 11), and 12 known compounds. The structures of all compounds were elucidated by interpretation of their 1D and 2D NMR spectroscopic and MS data. Furthermore, computational prediction of ECD or chemical shifts was used to propose the absolute configurations of the new structures. All isolates were assessed for their inhibitory activities against PCSK9 mRNA expression and PCSK9-low-density lipoprotein receptor (LDLR) interactions. None of the isolated compounds inhibited PCSK9 and LDLR interactions. However, compounds 1, 9, and 10 downregulated PCSK9 mRNA expression.

Mast cells (MCs) are systemically distributed and secrete several allergic mediators such as histamine and leukotrienes to cause type I hypersensitivity. Dasatinib is a type of anti-cancer agent and it has also been reported to inhibit human basophils. However, dasatinib has not been reported for its inhibitory effects on MCs or type I hypersensitivity in mice. In this study, we examined the inhibitory effect of dasatinib on MCs and MC-mediated allergic response in vitro and in vivo. In vitro, dasatinib inhibited the degranulation of MCs by antigen stimulation in a dose-dependent manner (IC50, ~34 nM for RBL-2H3 cells; ~52 nM for BMMCs) without any cytotoxicity. It also suppressed the secretion of inflammatory cytokines IL-4 and TNF-α by antigen stimulation. Furthermore, dasatinib inhibited MC-mediated passive cutaneous anaphylaxis (PCA) in mice (ED50, ~29 mg/kg). Notably, dasatinib significantly suppressed the degranulation of MCs in the ear tissue. As the mechanism of its effect, dasatinib inhibited the activation of Syk and Syk-mediated downstream signaling proteins, LAT, PLCγ1, and three typical MAP kinases (Erk1/2, JNK, and p38), which are essential for the activation of MCs. Interestingly, in vitro tyrosine kinase assay, dasatinib directly inhibited the activities of Lyn and Fyn, the upstream tyrosine kinases of Syk in MCs. Taken together, dasatinib suppresses MCs and PCA in vitro and in vivo through the inhibition of Lyn and Fyn Src-family kinases. Therefore, we suggest the possibility of repositioning the anti-cancer drug dasatinib as a treatment for various MC-mediated type I hypersensitive diseases.

Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in cells, which induces sunburn cell formation, melanoma, photoaging, and skin cancer. This study examines the anti-photodamage effects of kudzu root vinegar and adenosine in UVB-exposed human keratinocytes (HaCaT cells). UVB significantly decreased HaCaT cell viability, whereas kudzu root vinegar and adenosine did not exhibit cytotoxic effects and increased the viability of HaCaT cells. To investigate the protective effects of kudzu root vinegar and adenosine on UVB-induced oxidative stress in HaCaT cells, ROS, matrix metalloproteinases (MMPs), and mitogen-activated protein kinase (MAPK) were analyzed. UVB-induced treatment reduced the activity of antioxidant enzymes; however, kudzu root vinegar and adenosine increased their activity. These results indicated that kudzu root vinegar and adenosine exert cytoprotective activity against UVB-induced oxidative stress in HaCaT cells. Moreover, they suppressed the UVB-induced downregulation of MMPs and inhibited the phosphorylation of MAPK induced by UVB-irradiation. Therefore, kudzu root vinegar and adenosine offer anti-oxidative effects, via lowering ROS production, suppressing JNK activation, and downregulating expression of MMPs. Our findings suggest that kudzu root vinegar and adenosine have potential application in preventing skin damage owing to UVB exposure. Keywords: reactive oxygen species (ROS), HaCaT cell, UVB, skin damage, anti-aging

Panax notoginseng (PN) is a traditional herbal medicine containing several active compounds such as saponins and ginsenosides with many therapeutic applications including anti-obesity activity. Fermentation by lactic acid bacteria has the potential to metabolize ginsenosides to more active forms. This study examined whether fermentation has any benefits on the protective effects of a PN extract against obesity using a high-fat diet (HFD)-fed mouse model. PN was fermented with Lactobacillus plantarum which exhibited high β-glucosidase activity. Upon fermentation, the PN extract exhibited an altered ginsenoside profile, a dramatic increase in the lactate level. Treatment of the HFD group with fermented PN (FPN), but not PN, decreased both the food and calorie intake significantly, which was consistent with the more potent suppressing effects of FPN than PN on the signaling pathways involved in appetite and energy intake. The PN treatment also modulated the gut microbial composition. The PN and FPN treatment groups showed clear differences in the population of gut microbiota. The relative abundance of Bacteroidetes, Erysipelotrichaceae, Coprococus, and Dehalobacterium were significantly higher in the FPN group then the normal, HFD, and XEN groups. Furthermore, the relative abundances of Akkermansia, Dehalobacterium, Erysipeliotrichaceae and parpabacteroides were significantly higher in the FPN group than the PN group, but the relative abundances of Allobaculum, Erysipelotrichi and Erysipelotrichale were significantly lower. The relative abundance of Bacteroides and Lactococcus was significantly higher and lower, respectively in the PN and FPN groups than the HFD group. In conclusion, the altered ginsenoside and organic acid’s profile, and altered gut microbial composition are believed to be the major factors contributing to the anti-obesity properties of FPN.

The aim of this study was to examine the estrogen-like effects of gentiopicroside, macelignan, γ-mangostin, and three lignans (schisandrol A, schisandrol B, and schisandrin C), and their possible mechanism of action. Their effects on the proliferation of the estrogen receptor (ER)-positive breast cancer cell line (MCF-7) were evaluated using Ez-Cytox reagents. The expression of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), AKT, and estrogen receptor α (ERα) was measured by performing Western blot analysis. 17β-estradiol (E2), also known as estradiol, is an estrogen steroid and was used as a positive control. ICI 182,780 (ICI), an ER antagonist, was used to block the ER function. Our results showed that, except for gentiopicroside, all the compounds promoted proliferation of MCF-7 cells, with schisandrol A being the most effective; this effect was better than that of E2 and was mitigated by ICI. Consistently, the expression of ERK, PI3K, AKT, and ERα increased following treatment with schisandrol A; this effect was slightly better than that of E2 and was mitigated by ICI. Taken together, the ERα induction via the PI3K/AKT and ERK signaling pathways may be a potential mechanism underlying the estrogen-like effects of schisandrol A. This study provides an experimental basis for the application of schisandrol A as a phytoestrogen for the prevention of menopausal symptoms.

Phytochemical investigation on the n-BuOH-soluble fraction of the aerial parts of Epimedium koreanum using the PCSK9 mRNA monitoring assay led to the identification of four previously undescribed acylated flavonoid glycosides and 18 known compounds. The structures of new compounds were elucidated by NMR, MS, and other chemical methods. All isolated compounds were tested for their inhibitory activity against PCSK9 mRNA expression in HepG2 cells. Of the isolates, compounds 6, 7, 10, 15, and 17–22 were found to significantly inhibit PCSK9 mRNA expression. In particular, compound 7 was shown to increase LDLR mRNA expression. Thus, compound 7 may potentially increase LDL uptake and lower cholesterol levels in the blood.

Modifications of polyvinylidene fluoride (PVDF) membranes were carried out to improve both hydrophobicity and stability through four steps: pore expansion by a plasma treatment, hydroxylation of the membrane by the Fenton reaction, generation and growth of microparticles (MPs) on the hydroxylated functional groups in pores, and hydrophobic modification. The membranes modified by the methods proposed in this study did not lose their hydrophobicity and maintained the flux over a significantly longer period. The PVDF membrane modified by hydrophobic MPs attached inside enlarged pores exhibited a minimized flux reduction and significantly higher antiwetting stability.

The fouling propensity of organic matter categorized by their origins has been investigated during the reuse of tertiary wastewater effluent by a ceramic ultrafiltration membrane with molecular weight cut-off (MWCO) around 1 kDa. Selective removal of organic matter was carried out with three carbon-based adsorbents including granular activated carbon (GAC), expanded graphite (EG), and multi-walled carbon nanotubes (MWNTs). Analysis on the residual organic matter after the adsorption revealed that EG and MWNTs mainly adsorbed microbial-oriented organic matters (MOOMs) such as the soluble microbial products (SMP)-like and aromatic protein (AP)-like organic compounds, while GAC showed non-selective organic removals. At the identical organic loading, feed solution treated by EG and MWNTs exhibited significantly lower fouling propensity than that of the GAC-treated sample. Consequently, the selective removal of MOOMs from tertiary effluents can provide an effective strategy to reduce organic fouling on ceramic membranes.

In this study, new type of thin film nanocomposite (TFN) membrane with polysulfone (PSf) support layer having highly permeable interlayer using halloysite nanotubes (HNT) and hydrophilic polydopamine (pDA) was fabricated to improve the surface wettability of the PSf support layer, and to play a positive role in the development of a PA active layer. ATR-FTIR and XPS analysis confirmed the presence of the pDA/HNTx interlayer on the PSf support layer. Contact angle measurements confirmed that the surface wettability of support layer for TFN FO membrane was enhanced with increasing HNT loading. It was revealed that a pDA/HNT nanocomposite interlayer has considerable potential for enhancing the performance of TFN FO membranes. A TFN membrane with a pDA/HNT interlayer showed outstanding FO performance, due to its superior water flux of 26.9 L/m2 h (FO mode, 1 M NaCl draw solution), without a significant increase in reverse salt flux. This was attributed to the superior hydrophilicity and permeability of the interlayer, as well as the formation of a selective PA layer on the interlayer.

We report polyethylene glycol (PEG)-grafting antifouling surfaces using a plasma copolymerized (PcP) technique to monitor protease activity in complex media. By varying the mixing ratio of the PEG and ethylenediamine (EDA) precursors, the PcP-PEG-EDA (PcP-PE) film was able to easily control surface amine density with good preservation of the internal PEG structure. We found that nonspecific protein adsorption was dramatically reduced in serum-containing media on the PcP-PE films, as opposed to that on plasma polymerized-EDA (PP-E) films without PEG. When SPR sensor chips coated with PcP-PE film were employed to detect protease activity, biotinylated luciferase probes (luciferase-peptide-biotin) on streptavidin-conjugated SPR chips enabled real-time and label-free measurement of matrix metalloproteinase activity in cell culture media. Owing to its excellent antifouling ability, this newly developed method boasts minimal nonspecific binding and can serve as a biochip platform to promote a wide range of applications in the biological field.

Seven new prenylated flavonoids (1-7) and one new prenylated phenylpropiophenone (8) were isolated from roots and rhizomes of Sophora tonkinensis, along with nine known compounds (9-17). The structures 1-8 were elucidated by spectroscopic data analysis and comparison with reported values. Compounds 8 and 12 (7-methoxyebenosin) showed inhibitory activities against nitric oxide production in lipopolysaccharide-induced RAW264.7 cells, with IC50 values of 8.1 and 6.2 μM, respectively. They also significantly lowered expression of CSF2, TNF, and IL-1β. Lonchocarpol A (10) and erybraedin D (16) at concentrations of 20 μM downregulated proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression in HepG2 cells. Moreover, erybraedin D (16) inhibited PCSK9 protein synthesis (IC50 7.8 μM), while simultaneously activating AMP-activated protein kinase and acetyl-CoA carboxylase.

Hepatic fibrosis, characterized by persistent deposition of extracellular matrix (ECM) proteins, occurs in most types of chronic liver disease. The prevention of liver damage using extract of Dendropanax morbifera has been widely studied, but its molecular mechanism on the therapeutic efficacy of hepatic fibrosis is unclear. The aim of this study was to assess whether aquatic extract (DM) of D. morbifera ameliorates thioacetamide (TAA)-induced hepatic fibrosis. Hepatic fibrosis was induced by an intraperitoneal (i.p.) injection (150 mg/kg, twice per week) of TAA for 6 weeks. DM (50 mg/kg/day) or silymarin (50 mg/kg/day) was administered daily for 6 weeks. DM markedly reduced serum AST, ALT, ALP, and r-GTP in TAA-treated rats. DM significantly ameliorated the total glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) activity in TAA-treated rats. In particular, DM significantly reduced expression of α-SMA, type I collagen, vimentin, TGF-β1 and p-Smad2/3 in hepatic fibrosis rats. The protective effects of DM on progression of hepatic fibrosis were clearly shown by detecting 4-hydroxyproline concentration and histopathological examination in the liver. Therefore, our data suggest that DM dramatically prevented hepatic fibrosis by inhibiting oxidative stress and the TGF-β1/Smads signaling pathways.

Three acylated saponins and three flavonoid glycosides, along with nine known flavonoids, were isolated from the fruits of Stewartia koreana Nakai ex Rehder (Theaceae) using relative mass defect filtering analysis. The structures of these compounds were determined by performing spectroscopic analyses and using chemical methods. Furthermore, all the isolates were evaluated for their effects on the mRNA expression of the genes for proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) as well as their inhibitory activities on PCSK9 and LDLR binding. None of the isolates was deemed to be active in PCSK9-LDLR binding inhibition. However, (+)-catechin was found to inhibit PCSK9 expression and increase LDLR expression, suggesting the potential of (+)-catechin to lower cholesterol level via the downregulation of PCSK9 expression.

Ethnopharmacological relevance Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, jaundice and to treat liver problems. Recent evidences provided the potential for the clinical use of P. chinense in the treatment of metabolic disease. Aim of the study: Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism. Materials and methods The isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape. Results Two new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 μM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay. Conclusion (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one.

[Image: see text] This study evaluated the potential use of senescence-inducing small molecules in the treatment of melanoma. We screened commercially available small-molecule libraries with high-throughput screening and high-content screening image-based technology. Our findings showed an initial hit with the embedded N-arylpiperidine-3-carboxamide scaffold-induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. A focused library containing diversely modified analogues were constructed and examined to evaluate the structure–activity relationship of N-arylpiperidine-3-carboxamide derivatives starting from hit 1. This work identified a novel compound with remarkable antiproliferative activity in vitro and demonstrated the key structural moieties within.

Natural products have been fundamental materials in drug discovery. Traditional strategies for observing natural products with novel structure and/or biological activity are challenging due to large cost and time consumption. Implementation of the MS/MS-based molecular networking strategy with the in silico annotation tool is expected to expedite the dereplication of secondary metabolites. In this study, using this tool, two new dilignans with a 2-phenyl-3-chromanol motif, obovatolins A (1) and B (2), were discovered from the stem barks of Magnolia obovata Thunb. along with six known compounds (3–8), expanding chemical diversity of lignan skeletons in this natural source. Their structures and configurations were elucidated using spectroscopic data. All isolates were evaluated for their PCSK9 mRNA expression inhibitory activity. Obovatolins A (1) and B (2), and magnolol (3) showed potent lipid controlling activities. To identify transcriptionally controlled genes by 1 along with downregulation of PCSK9, using small set of genes (42 genes) related to lipid metabolism selected from the database, focused bioinformatic analysis was carried out. As a result, it showed the correlations between gene expression under presence of 1, which led to detailed insight of the lipid metabolism caused by 1.

The aim of the present study was to examine the protective effect of &gamma, mangostin, a component of the mangosteen shell, against oxidative damage to nerve cells induced by excessive glutamate, a known excitatory neurotransmitter. To investigate the effect of &gamma, mangostin on apoptosis, 5 mM of glutamate was used to induce apoptotic cell death in mouse hippocampal HT22 cells. In this study, &gamma, mangostin was found to exert a stronger protection than N-acetyl cysteine against glutamate-induced cell damage. &gamma, Mangostin showed prevented glutamate-induced apoptosis in HT22 cells by reducing the production of reactive oxygen species and stimulating the expression of heme oxygenase-1 protein. In addition, glutamate significantly induced the accumulation of intracellular calcium ions, whereas treatment with &gamma, mangostin markedly reduced it. Hoechst 33342 staining showed an improvement in glutamate-induced nuclear condensation following &gamma, mangostin treatment. Furthermore, the number of annexin V-positive cells was significantly reduced following treatment with &gamma, mangostin. Western blot analysis showed the inhibition of glutamate-induced mitogen-activated protein kinase phosphorylation by &gamma, mangostin. &gamma, mangostin also inhibited the regulation of the intrinsic mitochondrial apoptotic pathway. Thus, the results of this study suggest that &gamma, mangostin is an active ingredient of mangosteen and exerts neuroprotective activities in HT22 cells.

Following our report that A(3) adenosine receptor (AR) antagonist 1 exhibited a polypharmacological profile as a dual modulator of peroxisome proliferator-activated receptor (PPAR)γ/δ, we discovered a new template, 1′-homologated adenosine analogues 4a–4t, as dual PPARγ/δ modulators without AR binding. Removal of binding affinity to A(3)AR was achieved by 1′-homologation, and PPARγ/δ dual modulation was derived from the structural similarity between the target nucleosides and PPAR modulator drug, rosiglitazone. All the final nucleosides were devoid of AR-binding affinity and exhibited high binding affinities to PPARγ/δ but lacked PPARα binding. 2-Cl derivatives exhibited dual receptor-binding affinity to PPARγ/δ, which was absent for the corresponding 2-H derivatives. 2-Propynyl substitution prevented PPARδ-binding affinity but preserved PPARγ affinity, indicating that the C2 position defines a pharmacophore for selective PPARγ ligand designs. PPARγ/δ dual modulators functioning as both PPARγ partial agonists and PPARδ antagonists promoted adiponectin production, suggesting their therapeutic potential against hypoadiponectinemia-associated cancer and metabolic diseases.

Background: Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, and jaundice. So far there is no report regarding proprotein convertase subtilisin/kexin type 9 inhibitory constituents from this plant. The aim of the present study was to discover new active constituents with PCSK9 expression inhibitory activities from P. chinenseMethods: All structures were established by interpreting NMR spectroscopic data and MS data. Further experimental and calculated ECD data were used to determine the absolute configuration of the two new neolignans. To monitor the inhibitory activity on proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression and PCSK9-low density lipoprotein receptor (LDLR) interaction, quantitative real time-PCR, Western blot analysis, and an enzyme-linked immunosorbent assay (ELISA) method by a PCSK9-biotinylated-LDLR binding assay were performed.Results: 39 compounds were isolated and identified including two new oxepine-type neolignans, penthorinols A (1) and B (2) and a naturally occurring chalcone, 6'-hydroxy-2'-methoxychalcone-4'-O-β-D-glucopyranoside (20). Of all tested compounds, penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 15.56 and 11.66 µM, respectively. Furthermore, penthorin A (4) and methyl gallate (25) downregulated PCSK9 protein expression. However, all compounds seemed to be inactive in PCSK9-LDLR interaction.Conclusion: In the present study, two new compounds was discovered from this plant and active constituents with PCSK9 expression inhibitory activities were suggested.

In order to identify anti-tubercular agents with a novel scaffold, commercial libraries of small organic compounds were screened against a fluorescent strain of Mycobacterium tuberculosis H37Rv, using a dual phenotypic assay. Compounds were assessed against bacteria replicating in broth medium, as well as inside macrophages, and thienothiazolocarboxamide (TTCA) scaffold was identified as hit in both assays, with submicromolar inhibitory concentrations. Derivatives of TTCA were further synthesized and evaluated for their inhibitory effects on M. tuberculosis H37Rv. In the present study we report the structure–activity relationship of these TTCA derivatives. Compounds 28, 32 and 42 displayed good anti-tubercular activities, as well as favorable ADME and PK properties. Compound 42 exhibited excellent oral bioavailability in mice with high distribution to lungs, within 1 h. It was found to be efficacious in a dose dependent manner in a murine model of M. tuberculosis infection. Hence, compound 42 is now under evaluation as a potential lead candidate for treatment of tuberculosis.

Aim of the study The aim of this study was to test the anti-rheumatic effects of A. jaluense tubers in acute and chronic arthritis rats, and to assign its ingredients through UPLC-TOF/MS. Materials and methods Subcutaneous injection of carrageenan for acute arthritis and complete Freund's adjuvant (CFA) for chronic arthritis was carried out in the hind paw of SD rats. The paw volume was measured by a plethysmometer; thermal hyperalgesia was tested using a thermal plantar tester, and mechanical hyperalgesia was evaluated by ankle flexion evoked vocalizations. The expression of c-Fos in the brain hippocampus was measured with the avidin-biotin-peroxidase technique. The ingredients were assigned by UPLC-TOF/MS, chromatography was performed by UHPLC system with DAD detector and BEH C18 column, and spectroscopy was conducted by ESI-MS system. Results and discussion The 80% ethanoic extract of A. jaluense tubers showed an acute anti-inflammatory effect by suppressing the edema volume in the hind paw of carrageenan-stimulated rats. In addition, A. jaluense tubers exerted an anti-rheumatic activity by reducing the secondary swelling volume from an immunological reaction in the left hind paw of CFA-induced chronic arthritis rats. Additionally, oral treatment with the 80% ethanoic extract -showed potent analgesic effects in the arthritis rats by recovering the paw withdrawal latency stimulated by the thermal hyperalgesia and by reducing the vocalization scores evoked by ankle flexion on both hind paws. Moreover, its treatment also indicated an anti-psychiatric effect by controlling the c-Fos protein expression of the brain hippocampus in CFA-stimulated arthritis rats. These results suggested that these therapeutic effects were exhibited by less toxic monoester diterpenoid alkaloids (MDAs), and nontoxic non ester diterpenoid alkaloids (NDAs). Conclusion A. jaluense tubers may act as viable therapeutic or preventive candidates for acute and chronic arthritis, particularly, for immune-inflammatory rheumatoid arthritis to suppress the pain and psychiatric condition.

The beneficial effects of light-emitting diode (LED) irradiation have been reported in various pathologies, including cancer. However, its effect in pancreatic cancer cells remains unclear. Herein, we demonstrated that blue LED of 460 nm regulated pancreatic cancer cell proliferation and apoptosis by suppressing the expression of apoptosis-related factors, such as mutant p53 and B-cell lymphoma 2 (Bcl-2), and decreasing the expression of RAC-β serine/threonine kinase 2 (AKT2), the phosphorylation of protein kinase B (AKT), and mammalian target of rapamycin (mTOR). Blue LED irradiation also increased the levels of cleaved poly-(ADP-ribose) polymerase (PARP) and caspase-3 in pancreatic cancer cells, while it suppressed AKT2 expression and inhibited tumor growth in xenograft tumor tissues. In conclusion, blue LED irradiation suppressed pancreatic cancer cell and tumor growth by regulating AKT/mTOR signaling. Our findings indicated that blue LEDs could be used as a nonpharmacological treatment for pancreatic cancer.

Mast cells are critical effector cells in IgE-mediated allergic responses. The aim of this study was to investigate the anti-allergic effects of 3-[(aminocarbonyl)amino]-5-(3-fluorophenyl)-N-(3S)-3-piperidinyl-2-thiophenecarboxamide (AZD7762) in vitro and in vivo. AZD7762 inhibited the antigen-stimulated degranulation from RBL-2H3 (IC50, ∼27.9 nM) and BMMCs (IC50, ∼99.3 nM) in a dose-dependent manner. AZD7762 also inhibited the production of TNF-α and IL-4. As the mechanism of its action, AZD7762 inhibited the activation of Syk and its downstream signaling proteins, such as Linker of activated T cells (LAT), phospholipase (PL) Cγ1, Akt, and mitogen-activated protein (MAP) kinases (Erk1/2, p38, and JNK) in mast cells. In in vitro protein kinase assay, AZD7762 inhibited the activity of Lyn and Fyn kinases, which are important for the activation of Syk in mast cells. Furthermore, AZD7762 also suppressed the degranulation of LAD2 human mast cells (IC50, ∼49.9 nM) and activation of Syk in a dose-dependent manner. As observed in experiments with mast cells in vitro, AZD7762 inhibited antigen-mediated passive cutaneous anaphylaxis in mice (ED50, ∼35.8 mg/kg). Altogether, these results suggest that AZD7762 could be used as a new therapeutic agent for mast cell-mediated allergic diseases.

High mobility group box 1 (HMGB1) has been proposed as crucial in the pathogenesis of many diseases including sepsis. Acetylation of HMGB1 prevents its entry into the nucleus and leads to its secretion from the cell where it can trigger inflammation. We hypothesized that histone deacetylase 4 (HDAC4) controls the acetylation of HMGB1 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells via the janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. The results showed that LPS treatment promoted the degradation of HDAC4 in a proteasome-dependent manner, which led to HMGB1 acetylation. In LPS-activated RAW264.7 cells, treatment with TAK-242 (a toll like receptor 4 inhibitor) and pyridone 6 (a JAK inhibitor) significantly inhibited HDAC4 degradation and acetylation of HMGB1, and thus prevented secretion of HMGB1. Decreased phosphorylation of STAT1 was also observed. Interestingly, HDAC4 overexpression significantly prevented the acetylation and secretion of HMGB1 in both RAW264.7 cells and isolated murine peritoneal macrophages. We conclude that HDAC4 might be a useful target for the treatment of sepsis.

Alterations in sulfur amino acid metabolism are associated with an increased risk of a number of common late-life diseases, which raises the possibility that metabolism of sulfur amino acids may change with age. The present study was conducted to understand the age-related changes in hepatic metabolism of sulfur amino acids in 2-, 6-, 18- and 30-month-old male C57BL/6 mice. For this purpose, metabolite profiling of sulfur amino acids from methionine to taurine or glutathione (GSH) was performed. The levels of sulfur amino acids and their metabolites were not significantly different among 2-, 6- and 18-month-old mice, except for plasma GSH and hepatic homocysteine. Plasma total GSH and hepatic total homocysteine levels were significantly higher in 2-month-old mice than those in the other age groups. In contrast, 30-month-old mice exhibited increased hepatic methionine and cysteine, compared with all other groups, but decreased hepatic S-adenosylmethionine (SAM), S-adenosylhomocysteine and homocysteine, relative to 2-month-old mice. No differences in hepatic reduced GSH, GSH disulfide, or taurine were observed. The hepatic changes in homocysteine and cysteine may be attributed to upregulation of cystathionine β-synthase and down-regulation of γ-glutamylcysteine ligase in the aged mice. The elevation of hepatic cysteine levels may be involved in the maintenance of hepatic GSH levels. The opposite changes of methionine and SAM suggest that the regulatory role of SAM in hepatic sulfur amino acid metabolism may be impaired in 30-month-old mice.

Background/Aims: Airborne particulate matter with a diameter of < 10 µm (PM10) causes oxidative damage, inflammation, and premature skin aging. In this study, we evaluated whether polyphenolic antioxidants attenuate the inflammatory responses of PM10-exposed keratinocytes. Methods: Primary human epidermal keratinocytes were exposed in vitro to PM10 in the absence or presence of punicalagin and (–)-epigallocatechin-3-gallate (EGCG), which are the major polyphenolic antioxidants found in pomegranate and green tea, respectively. Assays were performed to determine cell viability, production of reactive oxygen species (ROS), and expression of NADPH oxidases (NOX), proinflammatory cytokines, and matrix metalloproteinase (MMP)-1. Results: PM10 decreased cell viability and increased ROS production in a dose-dependent manner. It also increased the expression levels of NOX-1, NOX-2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and MMP-1. Punicalagin was not cytotoxic up to 300 μM, and (–)-EGCG was cytotoxic above 30 μM, respectively. Further, punicalagin (3–30 μM) and EGCG (3–10 μM) rescued the viability of PM10-exposed cells. They also attenuated ROS production and the expression of NOX-1, NOX-2, TNF-α, IL-1β, IL-6, IL-8, and MMP-1 stimulated by PM10. Conclusions: This study demonstrates that polyphenolic antioxidants, such as punicalagin and (–)-EGCG, rescue keratinocyte viability and attenuate the inflammatory responses of these cells due to airborne particles.

The Nardostachys jatamansi DC (NJ) root has been used as a sedative or analgesic to treat neurological symptoms and pain in traditional Korean medicine. Here, we investigate the potential effects of NJ on Alzheimer's disease (AD) and reveal the molecular mechanism through which NJ exerts its effects. The neuroprotective effect of the NJ root ethanol extract against β amyloid (Aβ) toxicity was examined in vitro using a cell culture system and in vivo using a Drosophila AD model. The NJ extract and chlorogenic acid, a major component of NJ, inhibited Aβ-induced cell death in SH-SY5Y cells. Moreover, the NJ extract rescued the neurological phenotypes of the Aβ42-expressing flies (decreased survival and pupariation rate and a locomotor defect) and suppressed Aβ42-induced cell death in the brain. We also found that NJ extract intake reduced glial cell number, reactive oxygen species level, extracellular-signal-regulated kinase (ERK) phosphorylation, and nitric oxide level in Aβ42-expressing flies, without affecting Aβ accumulation. These data suggest that the neuroprotective activity of NJ might be associated with its antioxidant and anti-inflammatory properties, as well as its inhibitory action against ERK signaling; thus, NJ is a promising medicinal plant for the development of AD treatment.

Background and Purpose The aim of this study is to evaluate the prevalence of hypothyroidism and the change of thyroid hormone level in the children with epilepsy on a ketogenic diet (KD). Methods The levels of serum free thyroxine (fT4) and thyroid-stimulation hormone (TSH) were measured at the start of the KD and at 6- to 12-month intervals in children with intractable epilepsy. Hypothyroidism was defined as fT4 level < 0.8 ng/dL and TSH level > 6.0 μIU/mL. Results A total of 28 children (17 boys and 11 girls) were enrolled in the study. The mean age of onset of seizure was 1.4 ± 1.6 years, the mean age of the start of the KD was 3.2 ± 2.4 years, and the mean duration of KD was 1.9 ± 1.5 years. Overall, there was no significant longitudinal change in the mean fT4 (0.99 ± 0.25 vs. 0.94 ± 0.71 ng/dL, p = 0.28) and TSH (2.94 ± 1.32 vs. 3.18 ± 1.21 μIU/mL, p = 0.44) levels from the start of the KD to last follow-up. The patients with a younger age of seizure onset, earlier initiation of KD, and higher serum levels of cholesterol and triglyceride had a significant decrease in fT4 levels and increase in TSH levels during the KD. Sex, duration of the seizure or KD therapy, seizure types, seizure frequency, seizure outcomes, brain lesion, ratio of KD, and being overweight did not affect the longitudinal change of fT4 and TSH levels during KD. Conclusion Thyroid function had no significant longitudinal decrease in pediatric epilepsy during KD therapy. However, careful monitoring of the serum levels of fT4/TSH should be recommended in children on KDs, especially in those with earlier seizure onset, earlier start of KD, and higher levels of lipid profiles.

Regulatory B cells, also well-known as IL-10-producing B cells, play a role in the suppression of inflammatory responses. However, the epigenetic modulation of regulatory B cells is largely unknown. Recent studies showed that the bromodomain and extra-terminal domain (BET) protein inhibitor JQ1 controls the expression of various genes involving cell proliferation and cell cycle. However, the role of BET proteins on development of regulatory B cells is not reported. In this study, JQ1 potently suppressed IL-10 expression and secretion in murine splenic and peritoneal B cells. While bromodomaincontaining protein 4 (BRD4) was associated with NF-κB on IL-10 promoter region by LPS stimulation, JQ1 interfered the interaction of BRD4 with NF-κB on IL-10 promoter. In summary, BRD4 is essential for toll like receptor 4 (TLR4)-mediated IL-10 expression, suggesting JQ1 could be a potential candidate in regulating IL-10-producing regulatory B cells in cancer. [BMB Reports 2017; 50(12): 640-646].

The aim of our study was to investigate the effect of three lignans (schisandrol A, schisandrol B, and schisandrin C) on insulin secretion in rat INS-1 pancreatic β-cells and glucose uptake in mouse C2C12 skeletal muscle cells. Schisandrol A and schisandrin C enhanced insulin secretion in response to high glucose levels with no toxic effects on INS-1 cells. The effect of schisandrin C was superior to that of gliclazide (positive control), a drug commonly used to treat type 2 diabetes (T2D). In addition, western blot analysis showed that the expression of associated proteins, including peroxisome proliferator-activated receptor γ (PPARγ), pancreatic and duodenal homeobox 1 (PDX-1), phosphatidylinositol 3-kinase (PI3K), Akt, and insulin receptor substrate-2 (IRS-2), was increased in INS-1 cells after treatment with schisandrin C. In addition, insulin secretion effect of schisandrin C were enhanced by the Bay K 8644 (L-type Ca2+ channel agonist) and glibenclamide (K+ channel blocker), were abolished by the nifedipine (L-type Ca2+ channel blocker) and diazoxide (K+ channel activator). Moreover, schisandrin C enhanced glucose uptake with no toxic effects on C2C12 cells. Western blot analysis showed that the expression of associated proteins, including insulin receptor substrate-1 (IRS-1), AMP-activated protein kinase (AMPK), PI3K, Akt, glucose transporter type 4 (GLUT-4), was increased in C2C12 cells after treatment with schisandrin C. Schisandrin C may improve hyperglycemia by enhancing insulin secretion in pancreatic β-cells and improving glucose uptake into skeletal muscle cells. Our findings may provide evidence that schisandrin C may be beneficial in devising novel anti-T2D strategies.

Receptor activator of NF-kB ligand (RANKL) generates intracellular reactive oxygen species (ROS), which increase RANKL-mediated signaling in osteoclast (OC) precursor bone marrow macrophages (BMMs). Here we show that a ROS scavenging protein DJ-1 negatively regulates RANKL-driven OC differentiation, also called osteoclastogenesis. DJ-1 ablation in mice leads to a decreased bone volume and an increase in OC numbers. In vitro, the activation of RANK-dependent signals is enhanced in DJ-1-deficient BMMs as compared to wild-type BMMs. DJ-1 suppresses the activation of both RANK-TRAF6 and RANK-FcRγ/Syk signaling pathways because of activation of Src homology region 2 domain-containing phosphatase-1, which is inhibited by ROS. Ablation of DJ-1 in mouse models of arthritis and RANKL-induced bone disease leads to an increase in the number of OCs, and exacerbation of bone damage. Overall, our results suggest that DJ-1 plays a role in bone homeostasis in normal physiology and in bone-associated pathology by negatively regulating osteoclastogenesis., Osteoclasts are involved in arthritis, and their differentiation depends on RANKL signaling. The author show that the ROS-scavenging protein DJ-1 negatively regulates RANKL signaling and that its ablation increases osteoclast numbers and exacerbates bone damage in mouse models of arthritis.

Mast cells trigger IgE-mediated allergic reactions by releasing various allergic mediators. 8-Formyl-7-hydroxy-4-methylcoumarin, also called 4μ8C, was originally known as an inositol-requiring enzyme 1 (IRE1) suppressant, but no study has examined its relationship with mast cells and allergic diseases. Therefore, the purpose of this study was to determine whether 4μ8C is effective in suppressing allergic reactions in mast cells and in IgE-mediated allergic animal model. 4μ8C suppressed the degranulation of IgE-mediated mast cells (IC50=3.2μM) and the production of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in a dose-dependent manner. 4μ8C also suppressed passive cutaneous anaphylaxis (PCA) in mice (ED50=25.1mg/kg). In an experiment on mast cell signaling pathways stimulated by antigen, the phosphorylation and activation of Syk was decreased by 4μ8C, and phosphorylation of downstream signaling molecules, such as linker for activated T cells (LAT), Akt, and the three MAP kinases, ERK, p38, and JNK, were suppressed. Mechanistic studies showed that 4μ8C inhibited the activity of Lyn and Fyn in vitro. Based on the results of those experiments, the suppressor mechanism of allergic reaction by 4μ8C involved reduced activity of Lyn and Fyn, which is pivotal in an IgE-mediated signaling pathway. In summary, for the first time, this study shows that 4μ8C inhibits Lyn and Fyn, thus suppressing allergic reaction by reducing the degranulation and the production of inflammatory cytokines. This suggests that 4μ8C can be used as a new medicinal candidate to control allergic diseases such as seasonal allergies and atopic dermatitis.

Biofouling, a critical issue in membrane filtration, is influenced by several factors such as membrane characteristics and feed water quality. The organic matter (OM) composition is known to significantly influence biofilm formation, but few studies on this subject have been reported. Optical coherence tomography (OCT) allows direct monitoring of biofilm development on the membrane surface without the need for membrane autopsy. The purposes of the present study are 1) to quantify biofouling formation on a membrane surface using OCT; 2) to monitor the temporal variation of OM composition during membrane formation; and 3) to investigate the variation of OM composition. Substantial variations in thickness was observed from OCT images. It demonstrates that biovolume quantified from a single 2D cross-sectional image could result in inaccurate quantification. Here, we quantified foulants on the membrane surface using 3D images and validated the results using confocal laser scanning microscopy (CLSM). As well, variations in the OM composition driven by bacterial activity were observed by fluorescence excitation–emission matrix analysis and size exclusion chromatography. The biovolume estimated by the OCT system was 103.7 μm 3 /μm 2 . CLSM demonstrated that 10% of the foulants on the membrane surface was composed of live and dead bacteria.

Sulfonated poly(arylene ether sulfone) (SPAES) copolymers possessing three different dihydroxynaphthalene isomers and a biphenol (BP) moiety were synthesized as membranes for energy generation. Membrane samples of three different weight-based ion exchange capacities (IECw, 1.42, 1.82 and 2.2 meq g−1) were evaluated through a thermal, mechanical and electrochemical analysis. In order to investigate the isomeric effects of naphthalene and BP units in a polymer matrix, their ion transport properties were studied for potential energy generation applications, exemplified by reverse electrodialysis (RED) and polymer electrolyte membrane fuel cell (PEMFC) applications. Interestingly, the prepared polymeric membranes comprised of three different naphthalene isomers or BP exhibited considerably disparate ion transport properties in both RED and PEMFC applications. In RED applications, 2,6-dihydroxynaphthalene containing SPAES showed high Na+ ion permselectivity and low membrane resistance due to the dense membrane structure and linear conformation of the resulting membrane. In addition, despite having the same IECw, a large difference in the electrochemical performance of the membranes was observed due to isomeric effects in the naphthalene containing polymer backbone. Moreover, in PEMFC single cell tests at 80 °C under RH 100% and H2/air feed conditions, the membrane composed of 1,5-dihydroxynaphthalene (IECw=2.2 meq g−1), which had a helical conformation, achieved a promising current density (614 mA cm−2 at 0.6 V) among the tested membranes.

In this paper, we present a novel pressure-cascaded stripping configuration for membrane contactor biogas upgrading processes. The work clearly illustrates that optimizing the stripping unit for the biogas upgrading process is crucial to maximize the plant productivity. Employing polypropylene (PP) hollow fiber membrane modules and water as physical absorbent, the effects of absorption pressure, gas-liquid flowrate, and vacuum mode were systematically studied. In addition, exploiting the solubility difference between CO 2 and CH 4 , as well as the preferential absorption of CO 2 in water, a pressure-cascaded stripping tank was implemented to recover and recycle the absorbed CH 4 in high purity. The proposed configuration simultaneously enhanced the biomethane yield (90%) and purity (97%), high enough to be linked to the natural gas pipeline. A preliminary analysis showed that the proposed configuration offers cost benefits for large-scale biogas upgrading plants, and a significant reduction of global warming potential (GWP) compared to conventional configuration. The process developed in this work will be implemented in a biogas pilot-plant site to assess its overall feasibility.

Cassia obtusifolia L. (Leguminosae) seeds are a well-known medicinal food in East Asia and are used to clear liver heat, sharpen vision, lubricate the intestines, and promote bowel movement. The aims of the present study were to identify the hepatoprotective components of C. obtusifolia seeds by bioactivity-guided isolation and to elucidate their mechanisms of action. Ten phenolic glycosides were isolated from the most active ethyl acetate fraction, and their chemical structures were elucidated by spectroscopic analyses. Among the isolated compounds, toralactone 9-O-gentiobioside (5) had the highest hepatoprotective efficacy against tert-butylhydroperoxide-induced cell death in HepG2 cells. Immunoblotting and real-time polymerase chain reaction analyses revealed that the hepatoprotective effects were exerted through nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent antioxidative signaling. Together, these results provide insights into the effects of this medicinal plant as well as a basis for developing hepatoprotective agents as pharmaceuticals and/or nutraceuticals.

Here, we demonstrate the improved electrochemical performance and stability of end-group cross-linked anion exchange membranes (AEM) for the first time via the introduction of imidazolium groups in poly(arylene ether sulfone) (Imd-PAES). A novel feature of the cross-linking reaction is that basic additives are not required to prevent gelation with the cationic functional groups. In this work, the sodium salt of 3-hydroxyphenylacetylene acted directly as the end-group cross-linker, and it was cross-linked by thermal treatment at 180 °C. The gel fraction and hydroxide conductivity of the cross-linked membranes (XE-Imds) depended on the cross-linking temperature and time. The prepared XE-Imd70 (70 refers to the degree of functionalization) membranes with an ion exchange capacity (IEC) of 2.2 meq g−1 achieved a high hydroxide conductivity (107 mS cm−1). This material also showed good single cell performance (XE-Imd70: 202 mA cm−2 at 0.6 V and a maximum power density of 196.1 mW cm−2) at 80 °C, 100% relative humidity (RH), and improved durability and alkaline stability. The excellent hydroxide conductivity and electrochemical performance of XE-Imd70 was due to the fact that the ion cluster size of XE-Imd membranes was larger (12.1–14 nm) than that of E-Imd (5.5–8.14 nm), indicating that XE-Imd membranes have a closely associated ion-clustered morphology, which was confirmed by transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) measurements.