Avian pathogenic Escherichia coli (APEC) causes inflammation in multiple organs of chickens called avian colibacillosis, and results in serious economic loss to the chicken industry. Polyphenolic compounds possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases. In this study, the curative effect and mechanism of action of the polyphenolic extracts from Punica granatum L. and Terminalia chebula Retz. in chickens challenged with APEC were studied. Specific-pathogen-free white Leghorn chickens (males, 21-d old) were challenged with APEC and then given oral administration of extracts of P. granatum and T. chebula. The extracts decreased the morbidity and inflammation induced by APEC. Data from quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay showed that the extracts of P. granatum and T. chebula polyphenols (GCP) reversed the over-expression genes of the Toll-like receptor (TLR) 2, 4, and 5, down-regulated the activation of nuclear factor-kappa B signal transduction pathways, and inhibited the production of pro-inflammatory cytokines. Naturally occurring GCP may be a potential alternative medicine for the prevention or treatment of avian colibacillosis.

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Ginsenosides are major bioactive constituents that are responsible for the diverse pharmacological activities of ginseng. This work aimed to assess the skin anti-photoaging activities of the two stereoisomeric forms of ginsenoside Rg3, 20(S)-Rg3 and 20(R)-Rg3. When the two Rg3 stereoisomers were added to cultured human keratinocyte HaCaT cells prior to irradiation with 70 mJ/cm² UV-B, 20(S)-Rg3, but not 20(R)-Rg3, decreased the UV-B-induced intracellular reactive oxygen species (ROS) levels in a concentration-dependent manner, as detected by both fluorometric and confocal microscopic analyses. Likewise, 20(S)-Rg3, but not 20(R)-Rg3, decreased the UV-B-induced ROS levels in human dermal fibroblast cells. Both stereoisomers were unable to modulate the nitric oxide levels in HaCaT cells under UV-B irradiation, and induced no cytotoxicity in cultured keratinocytes and fibroblasts. 20(S)-Rg3 suppressed the UV-B-induced matrix metalloproteinase (MMP)-2 activities in HaCaT cells. Taken together, these results indicate that 20(S)-Rg3 possesses both ROS-scavenging and MMP-2 inhibitory activities, while 20(R)-Rg3 possesses neither activity. These findings imply that ginsenoside Rg3 stereoselectively demonstrates skin anti-photoaging activities.

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Our previous studies found that hydroxysafflor yellow A (HSYA), an active ingredient in Carthamus tinctorius L., has anti-inflammatory and anti-fibrosis properties. In this study, we investigated the effect of HSYA on small airway remodeling (SAR) in a chronic obstructive pulmonary disease (COPD) rat model induced by cigarette smoke and lipopolysaccharide (LPS). SAR is a common lesion in COPD characterized by thickening of the airway wall, mainly by subepithelial fibrosis. In this study the thickness of the small airway was determined by total wall area/basement membrane perimeter (WAt/Pbm). Collagen deposition of the small airway was assessed by Masson’s trichrome staining. HSYA significantly attenuated the thickening and collagen deposition of the small airway and inhibited transforming growth factor β1 (TGF-β1) mRNA and protein expression in COPD rat. In addition, HSYA inhibited the phosphorylation of p38 mitogen-activated protein kinases (MAPK) in the lung tissue of rat. HSYA can attenuate experimentally induced airway remodeling and this attenuation may be attributed to suppression of TGF-β1 expression.

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Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury via inflammation and cell apoptosis. Volatile anesthetics have been shown to exert organ-protective effects against kidney damage in vivo and in vitro. In the present study, we investigated the effects of isoflurane, a commonly used volatile anesthetic, on renal I/R injury and the underlying mechanisms. Rats subjected to renal I/R displayed higher serum creatinine and blood urea nitrogen levels than sham rats as well as severe histopathological damage. Renal I/R also resulted in a nuclear factor-κB (NF-κB)-mediated inflammatory response and dysfunction of the p53-Bax-caspase-3 apoptotic pathway. Rats preconditioned with 1.5% isoflurane for 2 h had better renal function and less tubular apoptosis 24 h after I/R injury than control rats. Pretreatment with isoflurane suppressed renal NF-κB activation, leading to a reduction in proinflammatory molecules (high-mobility group box 1, interleukin-1β, and tumor necrosis factor-α) both in the kidneys and circulation. In addition, rats subjected to isoflurane preconditioning had a higher Bcl-2/Bax ratio and less cleaved caspase-3. Our findings suggest that preconditioning with a clinically relevant concentration of isoflurane attenuates renal I/R injury, based at least in part on its ability to modulate renal inflammation and apoptosis.

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Inflammatory pain and neuropathic pain are major health issues that represent considerable social and economic burden worldwide. In this study we investigated the potential of obtusifolin and gluco-obtusifolin, two anthraquinones found in the seeds of the widely used traditional Chinese medical botanical Cassia obtusifolia, to reduce neuropathic and inflammatory pain. The potential analgesic effects of obtusifolin and gluco-obtusifolin were evaluated by mice formalin test and complete Freund’s adjuvant (CFA)-induced nociceptive behaviors in rats. Chronic constriction injury (CCI), L5 spinal nerve ligation (L5 SNL), diabetes, and chemotherapeutics inducing allodynia were used to test whether repeated treatment with obtusifolin and gluco-obtusifolin ameliorated neuropathic pain. Finally, we explored whether obtusifolin and gluco-obtusifolin altered the degree of neuroinflammation in rat spinal cord after CFA administration and CCI induction. Obtusifolin and gluco-obtusifolin (0.25, 0.5, 1, and 2 mg/kg) reduced licking/biting time in dose-dependent manner in phase 2 of formalin-induced behavior in mice. Furthermore, repeated administration of obtusifolin and gluco-obtusifolin (0.25, 0.5, 1, and 2 mg/kg) reversed mechanical allodynia induced by CFA, CCI, L5 SNL, diabetes, and oxaliplatin in a dose-dependent manner in rats. Levels of activated nuclear factor-kappa B (NF-κB) and proinflammatory cytokines (interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α)) in lumbar spinal cord were elevated in rats following CFA treatment and CCI induction, and obtusifolin and gluco-obtusifolin significantly inhibited these effects. Our results demonstrate that obtusifolin and gluco-obtusifolin produce significant antinociceptive action in rodent behavioral models of inflammatory/neuropathic pain, and that this activity is associated with modulation of neuroinflammation in spinal cord.

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An environmental isolate of Salmonella enterica serovar Enteritidis (SE) clone, SE Cl#15-1, loses its culturability during 72-h culture in M9 minimal medium containing 0.8% glucose, a concentration twice higher than that in normal M9 medium, whereas the bacterium retains its culturability in normal M9 medium. Live/dead analysis using the 5-cyano-2,3-di(p-tolyl) tetrazolium chloride (CTC)-reduction assay revealed that SE cells cultured in M9 medium containing 0.8% glucose died with time when in the “viable but non-culturable” (VBNC) state. Assay of the culturability of SE cells in the used supernatant (0.4 spent M9 or 0.8 spent M9) also indicated that 0.8 spent M9 soon showed a lethal effect on intact SE cells. These results suggest that large amounts of glucose metabolites might have been responsible for the toxicity. Analysis of the 0.8 spent M9 revealed that formate rapidly accumulated in the medium. The pH of the medium rapidly dropped to 4.7, leading to conversion of formate to formic acid, which might have damaged the bacterial cell membrane. These results suggest that the excessive amount of glucose in the M9 medium might have injured SE cells in the VBNC state by being metabolized to formic acid and other acidic compounds.

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Recently, Banhabackchulchunmatang (HMC05) has been implicated as a preventive and/or therapeutic candidate for cardiovascular diseases due to its inhibition of atherosclerosis lesions and its reduction of neointima formation. Knowledge of the mechanism of HMC05 in smooth muscle cells (SMC) is limited. However, SMC may be a potential target for HMC05 therapy because they are supported by the HMC05-mediated preservation of medial smooth muscle cell layers in pathogenic progression. Therefore, in the present study, we hypothesized that the effect of HMC05 is associated with reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H):quinone oxidoreductase-1 (NQO-1) gene regulation, which precipitates an antioxidant effect in SMC. HMC05 significantly increased NQO-1 gene expression in a dose- and time-dependent manner. The reactive oxygen species-mediated toxicity that was generated by xanthine/xanthine oxidase was suppressed by HMC05. The knockdown of the NQO-1 gene abrogated the HMC05-mediated cytoprotection. Interestingly, pretreatment with a chemical inhibitor of geranylgeranyl transferase 1 or farnesyl transferase abolished the NQO-1 gene induction and cytoprotection by HMC05. The transfection of dominant negative RhoA or Ras suppressed HMC05-induced gene expression. Berberine and hesperidin, which are found in large quantities in HMC05, also induced NQO-1 gene expression. Taken together, this is the first study to demonstrate that HMC05 is efficacious in protection against oxidative stress through NOQ-1 gene induction via the regulation of RhoA and/or Ras, and that berberine and hesperidin are major components of NQO-1 gene induction. This study provides mechanistic targets of HMC05 in reducing atherosclerotic lesions in atherosclerosis.

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Tryptanthrin [6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline], originally isolated from Isatidis radix, has been characterized as having anti-microbial and anti-tumor activities. It is well-known that excess oxidative stress is one of the major factors causing cell damage in the liver. This study investigated the cytoprotective effects and molecular mechanism of tryptanthrin against tert-butyl hydroperoxide (tBHP)-induced oxidative stress in human hepatocyte-derived HepG2 cells. Tryptanthrin pre-treatment blocked the reactive oxygen species production, mitochondrial dysfunction, and cell death induced by tBHP. Moreover, tryptanthrin reversed tBHP-induced GSH reduction. This study also confirmed the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by tryptanthrin as a plausible molecular mechanism for its cytoprotective effects. Specifically, tryptanthrin treatment induced nuclear translocation and transactivation of Nrf2 as well as phosphorylation of extracellular signal-regulated kinase (ERK), a potential upstream kinase of Nrf2. Tryptanthrin also up-regulated the expression of the heme oxygenase 1 and glutamate–cysteine ligase catalytic subunits, which are representative target genes of Nrf2. Moreover, inhibitor of ERK was used to verify the important role of the ERK-Nrf2 pathway in the hepatoprotective effects of tryptanthrin. In conclusion, this study demonstrated that tryptanthrin protects hepatocytes against oxidative stress through the activation of the ERK/Nrf2 pathway in HepG2 cells.

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Chondroitin sulfate–glycyl-prednisolone conjugate (CS–GP) was previously demonstrated to exhibit superior anti-arthritic effects compared to prednisolone (PD) alone. In this study, CS–GP was examined for its pharmacokinetic features and tropism for inflammatory joints using rats with adjuvant-induced arthritis in order to identify the mechanism of the potential enhancement. After intravenous injection (2.5 mg PD eq./kg), CS–GP yielded an area under the curve (AUC) of the total (free+conjugated) drug much higher than that of PD alone. After intravenous administration at the same dose, the drug distribution to the hind paw inflammatory joints was investigated. For PD alone, the PD concentration was 1.2–1.7 µg/g at 1 h and fell to 0.12–0.14 µg/g at 24 h. In contrast, CS–GP maintained the total concentration in the range of 0.55–0.97 µg/g for 1–24 h, and maintained the free PD concentration at 0.06–0.16 µg/g for 1–24 h. Furthermore, at 24 h after intravenous administration (2.5 mg PD eq./kg), CS–GP exhibited a higher total drug concentration in arthritic rats than in healthy rats. These findings suggested that CS–GP may have the ability to target inflammatory joints. As the apparent molecular weight of CS–GP became greater in plasma, it might interact with blood components and cause high plasma retention and good tropism to the inflammatory sites. Enhancement of the anti-inflammatory potential of CS–GP was found to be due to good maintenance of drug levels in the inflamed area.

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We purified ORI2 [3-(3,4-dihydroxyphenyl)acrylic acid 1-(3,4-dihydroxyphenyl)-2-methoxycarbonylethyl ester] from an extract of the plant Isodon excisus. We tested the antiviral effect of ORI2 in a coxsackievirus-induced myocarditis model. Coxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. Activation of extracellular signal-regulated kinase (ERK) and Akt signaling in virus-infected cells is essential for CVB3 replication. Antiviral compounds were screened by HeLa cell survival assay. Several purified natural compounds were added to HeLa cells cultured in 96-well plates for 30 min after 1 multiplicity of infection (m.o.i) CVB3 infection. ORI2 significantly improved HeLa cell survival in a dose-dependent manner. For in vivo studies, BALB/c mice (n=20) were infected with CVB3, then 10 of the mice were treated by daily intraperitoneal injections of ORI2 (100 mM) for 3 consecutive days. ORI2 treatment significantly improved early survival in the treated mice compared to untreated mice (85% vs. 50%, respectively). Organ virus titers and myocardial damage were significantly lower in the ORI2-treated mice than in untreated mice. These results demonstrate that ORI2, delivered by intraperitoneal injection after CVB3 infection, has a significant antiviral effect by markedly inhibiting virus replication, resulting in a decrease in organ virus titer and myocardial damage. ORI2 may be developed as a potential therapeutic agent for the treatment of CVB3 infections.

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Diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step in triacylglycerol (TG) synthesis, is a key enzyme associated with hepatic steatosis and insulin resistance. Here, using an in vitro screen of 20000 molecules, we identified a class of compounds with a substituted 1H-pyrrolo[2,3-b]pyridine core which proved to be potent and selective inhibitors of human DGAT2. Of these compounds, H2-003 and -005 exhibited a considerable reduction in TG biosynthesis in HepG2 hepatic cells and 3T3-L1 preadipose cells. These compounds exert DGAT2-specific-inhibitory activity, which was further confirmed in DGAT2- or DGAT1-overexpressing HEK293 cells. In addition, these compounds almost completely abolished lipid droplet formation in 3T3-L1 cells when co-treated with a DGAT1 inhibitor, which was not attained using either a DGAT2 or DGAT1 inhibitor alone. Collectively, we identified two DGAT2 inhibitors, H2-003 and -005. These compounds will aid in DGAT2-related lipid metabolism research as well as in therapeutic development for the treatment of metabolic diseases associated with excessive TG.

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A transdermal oil-in-water type emulsified formulation containing propiverine hydrochloride, used for treatment of an overactive bladder (OAB), was evaluated for in vitro skin permeation under finite conditions and in vivo transdermal absorption. Propiverine hydrochloride solubility was determined using 1,3-butyleneglycol, polyoxyethylene (2) oleylether, isostearyl alcohol, and lauryl alcohol. The solubility increased as the solubility parameter value increased. In vitro skin permeation in hairless mouse skin and in vivo transdermal absorption in rats were measured using propiverine hydrochloride dissolved in a simple solution containing these solvents. Dependent on the increase in in vitro flux, the in vivo area under the curve up to 72 h (AUC_0–72) was increased. Therefore, the emulsified formulation was prepared containing these ingredients using polyoxyethylene (20) stearylether for optimization. The emulsified formulation was used to conduct in vivo single- and repeated-dose absorption studies in rats. After single-dose transdermal administration of the emulsified formulation, the AUC_0–72 was equivalent to that of the simple solution. Furthermore, results using the emulsified formulation indicated an increase in AUC_0–72 and significant extension of the elimination half-life, in comparison with oral administration. After repeated-dose administration, a significant minimum plasma concentration was observed compared with oral administration. These results demonstrate that the emulsified formulation is a good option for transdermal delivery of propiverine hydrochloride.

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Histochemical visualization of phosphatase is exclusively required for Western immunoblotting and antigen-positive cell staining using an alkaline phosphatase (AP)-labeled secondary antibody. This detection has been performed by several reagents including 5-bromo-4-chloro-3-indolyl-phosphate (X-Phos), nitro blue tetrazolium (NBT), 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane and 2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4-[3H]-quinazolinone (ELF® 97 Phosphate). We previously reported that 2-(benzothiazol-2-yl)-4-bromophenol bonded with N-acetylneuraminic acid (BTP3-Neu5Ac), enabled fluorescent histochemical visualization of sialidase activity. 2-(Benzothiazol-2-yl)-4-bromophenol (BTP3), which is formed from BTP3-Neu5Ac by sialidase reaction, is a crystalline, insoluble and stable fluorogenic compound, deposited at the site of enzyme activity. We developed a BTP3 phosphate ester (BTP3-Phos) for the purpose of fluorescent histochemical visualization of phosphatase activity. BTP3-Phos emitted fluorescence in a manner dependent on the concentration of the AP-labeled antibody. BTP3-Phos also enabled fluorescent histochemical visualization of AP-blotted dots in a manner dependent on the concentration of the AP-labeled antibody. The detection sensitivity of BTP3-Phos was estimated to be greater than that of the conventional method using X-Phos and NBT. Influenza A virus-infected cells were fixed and reacted with anti-influenza A virus antibodies and incubated continuously with an AP-labeled secondary antibody. BTP3-Phos stained the infected cells with distinct green fluorescence. These results indicate that BTP3-Phos can enable fluorescent immunohistochemical staining analysis using an AP-labeled antibody. BTP3-Phos would be beneficial for histochemical staining of AP activity, and may be applicable for multi-color staining or a cell sorter.

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To develop an external vehicle for skin hydration and enhanced dermal drug delivery, a hydrogel-based ultra-moisturizing cream (HUMC) was successfully formulated with carbopol 934P, urea, Tinocare GL, grape seed oil, and other excipients. The HUMC showed plastic flow behavior due to a gel structure with a cream base. Different types of drug-free vehicles such as a hydrogel, conventional cream (CC), and three HUMCs were prepared and subjected to an in vivo skin hydration test on a hairless mouse using a corneometer. Hydration effect (∆AU) was in the order of HUMC2>HUMC1 ≥ CC>HUMC3>hydrogel. Using nile red (NR) and 5-carboxyfluorescein (5-CF) as lipophilic and hydrophilic fluorescent probes, respectively, in vitro skin permeation and accumulation studies were conducted using Franz diffusion cells. The values of steady-state flux (J_ss, ng/h/cm²) were obtained: 74.8 (CC), 145.6 (HUMC1), and 161.9 (HUMC2) for NR delivery; 6.8 (CC), 8.3 (HUMC1), and 10.9 (HUMC2) for 5-CF delivery. The amounts retained in the skin at 12 h (Q_r, ng/cm²) were determined: 86.4 (CC) and 102.0 (HUMC2) for NR; and 70.1 (CC) and 195.6 (HUMC2) for 5-CF. Confocal microscopy was used to visualize the distribution of the fluorescent probes. NR tended to be localized into the deeper part of the skin with adipose tissue whereas 5-CF localized in the upper layer of the skin. Thus we propose that HUMC2 is an efficacious vehicle for skin hydration and enhances dermal delivery of lipophilic and hydrophilic drugs.

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We previously reported the lipase inhibitory activity of the n-BuOH fraction of Dioscorea opposita (DOB) and its isolates. This study sought to evaluate their anti-adipogenic activity in terms of their effects on the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) as well as phosphorylated AMP-activated protein kinase (p-AMPK) and carnitine palmitoyl transferase-1 (CPT-1). DOB apparently attenuated 3T3-L1 adipocyte differentiation (33.6% decrease at 20 µg/mL). In addition, a marked decrease (90.4%) in the expression of PPARγ was observed in the DOB-treated 3T3-L1 cells. Four isolates from DOB: (4E,6E)-1,7-bis(4-hydroxyphenyl)-4,6-heptadien-3-one (1), (3R,5R)-1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanediol (2), batatasin I (3), and (1E,4E,6E)-1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (4), suppressed adipocyte differentiation by inhibiting PPARγ at 20 µM (85.9%, 68.6%, 76.2%, and 90.2% decrease, respectively) and C/EBPα (51.7%, 3.1%, 20.9%, and 59.8% decrease, respectively). Batatasin I was found to increase p-AMPK and CPT-1 at a concentration of 20 µM in 3T3-L1 adipocytes, resulting in inhibiting adipogenesis. Taken together, batatasin I might be responsible for the anti-adipogenic effect of DOB via inhibition of PPARγ and C/EBPα and activation of p-AMPK and CPT-1.

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We screened 2400 compounds to find novel inhibitors of the adenylyl cyclase (AC)–protein kinase A (PKA)–cAMP response-element-binding protein (CREB) signaling pathway (AC/PKA/CREB pathway). Using a multistep cell-based screening system employing split luciferase technique, we narrowed down the candidates effectively from 2400 chemical compounds and identified a novel AC inhibitor (compound 1). Since dysregulation of the AC/PKA/CREB pathway is known to cause diseases not only in the nervous system but also in other organs, compound 1 is expected to be developed as a medicine for these diseases.

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Although pregabalin has been shown to have preclinical and clinical efficacy in neuropathic pain, the mechanism of its antinociceptive action is still unknown in other pain states. This study aimed to evaluate the antinociceptive effect of pregabalin and its underlying spinal mechanisms related to mitogen activated protein kinases (MAPKs) in neuron and microglia following intraplantar injection of zymosan model. Zymosan evoked thermal hyperalgesia, mechanical hyperalgesia, and mechanical allodynia starting from 1 h and persistent until 5 h post-injection, which were dose-dependently reversed by oral pretreatment of pregabalin (3, 10, and 30 mg/kg). Pregabalin dramatically inhibited zymosan-induced Fos expression (a marker for neuronal activation) and microglia activation (using markers CD11b and ED1) in the spinal dorsal horn. Moreover, zymosan significantly increased phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2 (double labeling with neuron), ERK5 (double labelling with neuron and microglia) and p38 MAPK (double labeling with microglia) in the spinal dorsal horn, which overall elevations were reversed by pregabalin. These findings suggest that blockage of MAPKs activation in neuron and microglia might be closely related to the antinociceptive effect of pregabalin on zymosan-induced peripheral inflammatory pain.

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Caspases are well-known enzymes that work as initiators and effectors of apoptosis. To elucidate the role of caspases in neurodevelopment, we sought to determine if caspases are involved in the proliferation of neural stem/progenitor cells (NPCs) in the developing mouse brain. Labeling with 5-bromo-2′-deoxyuridine (BrdU) from days 14 to 18 of pregnant mice revealed that the 18-d old fetus had many BudU-positive cells in its brain. Double-labeling revealed that active caspase-3 was co-localized with these BrdU-positive cells in the neocortex, hippocampus, and subventricular zone of the fetal brain. Active caspase-3 was detected in cultures of NPCs derived from the neocortex of 15-d old fetuses during culture periods. Importantly, the pan-caspase inhibitor z-VAD-FMK was effective at completely inhibiting neurosphere formation by the NPCs. These results suggest the possibility that the caspase cascade is essential for the proliferation of neocortical NPCs in the developing mouse brain.

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