Regulation of NLGN3 and the Synaptic Rho-GEF Signaling Pathway by CDK5.

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes (NLGN1-3, 4X, and 4Y). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules.SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission.

DNA Markers, Pathogenicity Test, and Multilocus Sequence Analysis to Differentiate and Characterize Cereal-Specific Xanthomonas translucens Strains.

Xanthomonas translucens contains a group of bacterial pathogens that are closely related and have been divided into several pathovars based on their host range. X. translucens pv. undulosa (Xtu) and X. translucens pv. translucens (Xtt) are two important pathovars that cause bacterial leaf streak disease on wheat and barley, respectively. In this study, DNA markers were developed to differentiate Xtu and Xtt and were then used to characterize a collection of X. translucens strains with diverse origins, followed by confirmation and characterization with pathogenicity tests and multilocus sequence analysis/typing (MLSA/MLST). We first developed cleaved amplified polymorphic sequence markers based on the single-nucleotide polymorphisms within a cereal pathovar-specific DNA sequence. In addition, two Xtt-specific markers, designated Xtt-XopM and Xtt-SP1, were developed from comparative genomics among the sequenced Xtt/Xtu genomes. Using the developed markers, a collection of X. translucens strains were successfully identified as Xtu or Xtt. Pathogenicity tests on wheat and barley plants and MLSA of four housekeeping genes validated the pathovar assignation of those strains. Furthermore, MLSA revealed distinct subclades within both Xtu and Xtt groups. Seven and three sequence types were identified from MLST for Xtu and Xtt strains, respectively. The establishment of efficient Xtt/Xtu differentiation methods and characterization of those strains will be useful in studying disease epidemiology and host-pathogen interactions and breeding programs when screening for sources of resistance for these two important bacterial pathogens.

ERdj5 protects goblet cells from endoplasmic reticulum stress-mediated apoptosis under inflammatory conditions.

Endoplasmic reticulum stress is closely associated with the onset and progression of inflammatory bowel disease. ERdj5 is an endoplasmic reticulum-resident protein disulfide reductase that mediates the cleavage and degradation of misfolded proteins. Although ERdj5 expression is significantly higher in the colonic tissues of patients with inflammatory bowel disease than in healthy controls, its role in inflammatory bowel disease has not yet been reported. In the current study, we used ERdj5-knockout mice to investigate the potential roles of ERdj5 in inflammatory bowel disease. ERdj5 deficiency causes severe inflammation in mouse colitis models and weakens gut barrier function by increasing NF-κB-mediated inflammation. ERdj5 may not be indispensable for goblet cell function under steady-state conditions, but its deficiency induces goblet cell apoptosis under inflammatory conditions. Treatment of ERdj5-knockout mice with the chemical chaperone ursodeoxycholic acid ameliorated severe colitis by reducing endoplasmic reticulum stress. These findings highlight the important role of ERdj5 in preserving goblet cell viability and function by resolving endoplasmic reticulum stress.

Amelioration of DSS-Induced Acute Colitis in Mice by Recombinant Monomeric Human Interleukin-22.

IL-22, a pleiotropic cytokine, is known to have a profound effect on the regeneration of damaged intestinal barriers. The tissue-protective properties of IL-22 are expected to be potentially exploited in the attenuation and treatment of colitis. However, because of the disease-promoting role of IL-22 in chronic inflammation, a comprehensive evaluation is required to translate IL-22 into the clinical domain. Here, we present the effective production of soluble human IL-22 in bacteria to prove whether recombinant IL-22 has the ability to ameliorate colitis and inflammation. IL-22 was expressed in the form of a biologically active monomer and non-functional oligomers. Monomeric IL-22 (mIL-22) was highly purified through a series of 3 separate chromatographic methods and an enzymatic reaction. We reveal that the resulting mIL-22 is correctly folded and is able to phosphorylate STAT3 in HT-29 cells. Subsequently, we demonstrate that mIL-22 enables the attenuation of dextran sodium sulfate-induced acute colitis in mice, as well as the suppression of pro-inflammatory cytokine production. Collectively, our results suggest that the recombinant mIL-22 is suitable to study the biological roles of endogenous IL-22 in immune responses and can be developed as a biological agent associated with inflammatory disorders.

Differential expression pattern of novel MADS-box genes in early root formation and differentiation of sweet potato.

MADS-box genes are important transcription factors affecting overall development, but their role in sweet potato [Ipomoea batatas (L.) Lam.] has not been fully studied. This study isolated six novel MADS-box genes (IbSOC1, IbFUL1, IbAGL6, IbSVP1, IbSVP2, and IbSVP3) from sweet potato [Ipomoea batatas (L.) Lam. cv. Annouimo] during the early root differentiation stage using the de novo transcriptome assembly sequencing method. At the early root differentiation (between 0 and 3 days after transplanting), the IbSOC1, IbFUL1, and IbSVP2 genes decreased rapidly, whereas the IbSVP3 gene decreased gradually. In the early stages of root formation (0-30 days), the levels of IbSVP1 and IbSVP3 expression were steady, but the levels of IbSOC1 expression decreased gradually. The expression of six novel genes was also conducted in the tuberous root formation stage (30-90 days), and the IbSVP3 gene increased significantly according to the formation of the tuberous root. Six novel MADS-box genes that were believed to influence the entire root formation of sweet potato were isolated from the sweet potato. This study provides a genetic basis for further research on sweet potato root formation and development.

Drying Operation Effects on the Pigments and Phytochemical Properties of Rose Cultivars.

BACKGROUND: Roses are flowers which contain various phytochemicals and have been used for food and medicinal purposes in many countries. Materials, including flowers, are available for limited periods when plants can be cultivated. OBJECTIVE: To evaluate the quality of rose petals on the basis of cultivars and drying methods, chromaticity, phytochemical contents, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. METHOD: Petals of three rose cultivars were dried by hot air drying (HD) and freeze drying (FD). Subsequently, the chromaticity and the contents of pigment, total flavonoids, and ascorbic acid, and DPPH radical scavenging activity were analyzed. RESULTS: The total color difference (△E)values of rose red (RR, Calypso) and rose orange (RO, Lambada) were low in FD. In contrast, in rose yellow (RY, Ileos), there was no significant difference in chromaticity regulation regardless of the drying methods. The pigment contents were generally increased by drying. The carotenoid content in the RR and anthocyanin and carotenoid contents in RO were higher in FD than in HD. The drying operations increased the flavonoid and ascorbic acid content and antioxidant activity in roses, but the difference between the drying methods was not significant. Correlation coefficient analysis revealed ascorbic acid and anthocyanin to have a high positive correlation (r = 0.843), followed by flavonoid and DPPH radical scavenging activity. CONCLUSIONS: This study showed that the drying operations increased the flavonoid and ascorbic acid contents, and the chromaticity and pigment content of some cultivars was higher in FD. HIGHLIGHTS: Comparison of the color change, phytochemical content, and antioxidant activity by the drying methods (FD and HD treatment) in the three rose cultivars with red, orange, and yellow petals showed the increased phytochemical contents and antioxidant activity after drying, and chromaticity and pigment content were more stable and higher in FD.

Morin Hydrate Inhibits Influenza Virus entry into Host Cells and Has Anti-inflammatory Effect in Influenza-infected Mice.

Influenza virus is the major cause of seasonal and pandemic flu. Currently, oseltamivir, a potent and selective inhibitor of neuraminidase of influenza A and B viruses, is the drug of choice for treating patients with influenza virus infection. However, recent emergence of oseltamivir-resistant influenza viruses has limited its efficacy. Morin hydrate (3,5,7,2',4'-pentahydroxyflavone) is a flavonoid isolated from Morus alba L. It has antioxidant, anti-inflammatory, neuroprotective, and anticancer effects partly by the inhibition of the NF-кB signaling pathway. However, its effects on influenza virus have not been studied. We evaluated the antiviral activity of morin hydrate against influenza A/Puerto Rico/8/1934 (A/PR/8; H1N1) and oseltamivir-resistant A/PR/8 influenza viruses in vitro. To determine its mode of action, we carried out time course experiments, and time of addition, hemolysis inhibition, and hemagglutination assays. The effects of the co-administration of morin hydrate and oseltamivir were assessed using the murine model of A/PR/8 infection. We found that morin hydrate reduced hemagglutination by A/PR/8 in vitro. It alleviated the symptoms of A/PR/8-infection, and reduced the levels of pro-inflammatory cytokines and chemokines, such as TNF-α and CCL2, in infected mice. Co-administration of morin hydrate and oseltamivir phosphate reduced the virus titers and attenuated pulmonary inflammation. Our results suggest that morin hydrate exhibits antiviral activity by inhibiting the entry of the virus.

Plasmacytoid dendritic cells regulate colitis-associated tumorigenesis by controlling myeloid-derived suppressor cell infiltration.

Toll-like receptor (TLR)3 and TLR7 are important for stimulating plasmacytoid dendritic cells (pDCs), which secrete type I interferon. Mice deficient for TLR3 and TLR7 (TLR3-/-TLR7-/-) reportedly exhibit deteriorated colitis because of impaired pDCs. However, the role of pDCs in tumorigenesis-associated inflammation progression has not been studied. We treated wild-type or TLR3-/-TLR7-/- mice with dextran sulfate sodium (DSS) and/or azoxymethane (AOM) and examined colon mucosa, measured body weight and colon length of mice, and examined pDC and myeloid-derived suppressor cell (MDSC) accumulation. Further, we depleted pDCs in AOM/DSS-treated wild-type mice by treating them with anti-PDCA-1 antibodies. We found that MDSCs significantly increased, while pDCs decreased in TLR3-/-TLR7-/- mice. Moreover, TLR3-/-TLR7-/- mice developed colitis-associated colon cancer following AOM/DSS treatment. Additionally, we showed that a defect in TLR7 of pDCs is responsible for the aggravation of colitis-associated colon cancer. Further, we showed that TLR7 ligand mitigates colitis-associated colon cancer. Collectively, our results demonstrate that gut pDCs play a crucial role in reducing colorectal cancer development via the regulation of infiltrating MDSCs.

KRCA-0008 suppresses ALK-positive anaplastic large-cell lymphoma growth.

Anaplastic lymphoma kinase (ALK), which belongs to the insulin receptor tyrosine kinase superfamily, plays an important role in nervous system development. Due to chromosomal translocations, point mutations, and gene amplification, constitutively activated ALK has been implicated in a variety of human cancers, including anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer, and neuroblastoma. We evaluated the anti-cancer activity of the ALK inhibitor KRCA-0008 using ALCL cell lines that express NPM (nucleophosmin)-ALK. KRCA-0008 strongly suppressed the proliferation and survival of NPM-ALK-positive ALCL cells. Additionally, it induced G0/G1 cell cycle arrest and apoptosis by blocking downstream signals including STAT3, Akt, and ERK1/2. Tumor growth was strongly suppressed in mice inoculated with Karpas-299 tumor xenografts and orally treated with KRCA-0008 (50 mg/kg, BID) for 2 weeks. Our results suggest that KRCA-0008 will be useful in further investigations of ALK signaling, and may provide therapeutic opportunities for NPM-ALK-positive ALCL patients.

Esterase-sensitive cleavable histone deacetylase inhibitor-coupled hyaluronic acid nanoparticles for boosting anticancer activities against lung adenocarcinoma.

4-Phenylbutyric acid (PBA)-installed hyaluronic acid (HA)-based nanoparticles (NPs) were developed for amplifying the anticancer potential of curcumin (CUR) for lung cancer therapy. PBA was introduced to the HA backbone as a hydrophobic segment of a nanoassembled structure and as a histone deacetylase (HDAC) inhibitor for cancer therapy. PBA was released from the HA-PBA conjugate (HAPBA) via an esterase-responsive cleavage of ester bonds in cancer cells and may affect the dissociation of NP structure. CUR-entrapped HAPBA-based NPs, with 265 nm hydrodynamic size, unimodal size distribution, negative zeta potential, and sustained drug release, were fabricated. Co-treatment of A549 cells by PBA and CUR elevated the antiproliferation efficiency compared with CUR-treatment. CUR-loaded HAPBA NPs also exhibited a significantly lower IC50 value compared with the CUR and HAPBA10 + CUR groups (p < 0.05). Cy5.5-labeled HAPBA NPs containing CUR group displayed higher accumulation in tumor tissue and less distribution in liver and spleen after intravenous injection compared with the Cy5.5-injected group in A549 tumor-bearing mouse model. Multiple dosing of CUR-loaded HAPBA NPs in A549 tumor-bearing mouse model exhibited efficient tumor growth suppression and apoptosis-inducing effects. CD44 receptor targeting and HDAC inhibiting HAPBA NPs can be used to boost the anticancer potentials of drug cargo for the therapy of CD44 receptor-expressed cancers.

Manassantin B shows antiviral activity against coxsackievirus B3 infection by activation of the STING/TBK-1/IRF3 signalling pathway.

Coxsackievirus B3 (CVB3) is an important human pathogen associated with the development of acute pancreatitis, myocarditis, and type 1 diabetes. Currently, no vaccines or antiviral therapeutics are approved for the prevention and treatment of CVB3 infection. We found that Saururus chinensis Baill extract showed critical antiviral activity against CVB3 infection in vitro. Further, manassantin B inhibited replication of CVB3 and suppressed CVB3 VP1 protein expression in vitro. Additionally, oral administration of manassantin B in mice attenuated CVB3 infection-associated symptoms by reducing systemic production of inflammatory cytokines and chemokines including TNF-α, IL-6, IFN-γ, CCL2, and CXCL-1. We found that the antiviral activity of manassantin B is associated with increased levels of mitochondrial ROS (mROS). Inhibition of mROS generation attenuated the antiviral activity of manassantin B in vitro. Interestingly, we found that manassantin B also induced cytosolic release of mitochondrial DNA based on cytochrome C oxidase DNA levels. We further confirmed that STING and IRF-3 expression and STING and TBK-1 phosphorylation were increased by manassantin B treatment in CVB3-infected cells. Collectively, these results suggest that manassantin B exerts antiviral activity against CVB3 through activation of the STING/TKB-1/IRF3 antiviral pathway and increased production of mROS.

Deep convolutional neural network for the diagnosis of thyroid nodules on ultrasound.

BACKGROUND: We designed a deep convolutional neural network (CNN) to diagnose thyroid malignancy on ultrasound (US) and compared the diagnostic performance of CNN with that of experienced radiologists. METHODS: Between May 2012 and February 2015, 589 thyroid nodules in 519 patients were diagnosed as benign or malignant by surgical excision. Experienced radiologists retrospectively reviewed the US of the thyroid nodules in a test set. CNNs were trained and tested using retrospective data of 439 and 150 US images, respectively. Diagnostic performances were compared between the two groups. RESULTS: Of the 589 thyroid nodules, 396 were malignant and 193 were benign. The area under the curve (AUC) for diagnosing thyroid malignancy was 0.805-0.860 for radiologists. The AUCs for diagnosing thyroid malignancy for the three CNNs were 0.845, 0.835, and 0.850. There was no significant difference in AUC between radiologists and CNNs. CONCLUSIONS: CNNs showed comparable diagnostic performance compared to experienced radiologists in differentiating thyroid malignancy on US.

BIG2-ARF1-RhoA-mDia1 Signaling Regulates Dendritic Golgi Polarization in Hippocampal Neurons.

Proper dendrite development is essential for establishing neural circuitry, and Rho GTPases play key regulatory roles in this process. From mouse brain lysates, we identified Brefeldin A-inhibited guanine exchange factor 2 (BIG2) as a novel Rho GTPase regulatory protein involved in dendrite growth and maintenance. BIG2 was highly expressed during early development, and knockdown of the ARFGEF2 gene encoding BIG2 significantly reduced total dendrite length and the number of branches. Expression of the constitutively active ADP-ribosylation factor 1 ARF1 Q71L rescued the defective dendrite morphogenesis of ARFGEF2-null neurons, indicating that BIG2 controls dendrite growth and maintenance by activating ARF1. Moreover, BIG2 co-localizes with the Golgi apparatus and is required for Golgi deployment into major dendrites in cultured hippocampal neurons. Simultaneous overexpression of BIG2 and ARF1 activated RhoA, and treatment with the RhoA activator lysophosphatidic acid in neurons lacking BIG2 or ARF1 increased the number of cells with dendritic Golgi, suggesting that BIG2 and ARF1 activate RhoA to promote dendritic Golgi polarization. mDia1 was identified as a downstream effector of BIG2-ARF1-RhoA axis, mediating Golgi polarization and dendritic morphogenesis. Furthermore, in utero electroporation of ARFGEF2 shRNA into the embryonic mouse brain confirmed an in vivo role of BIG2 for Golgi deployment into the apical dendrite. Taken together, our results suggest that BIG2-ARF1-RhoA-mDia1 signaling regulates dendritic Golgi polarization and dendrite growth and maintenance in hippocampal neurons.

Trans-scirpusin A showed antitumor effects via autophagy activation and apoptosis induction of colorectal cancer cells.

Trans-Scirpusin A (TSA) is a resveratrol oligomer found in Borassus flabellifer L. We found that TSA inhibited the growth of colorectal cancer Her2/CT26 cells in vivo in mice. Although some cytotoxic T lymphocytes (CTLs) were induced against the tumor-associated antigen Her2, TSA treatment did not significantly increase the level of Her2-specific CTL response compared to that with vehicle treatment. However, there was a significant increase in the level of TNF-α mRNA in tumor tissue and Her2-specific Ab (antibody) production. More importantly, we found that TSA overcomes the tumor-associated immunosuppressive microenvironment by reducing the number of CD25+FoxP3+ regulatory T cells and myeloid-derived suppressor cells (MDSCs). We detected the induction of autophagy in TSA-treated Her2/CT26 cells, based on the increased level of the mammalian autophagy protein LC3 puncta, and increased conversion of LC3-I to LC3-II. Further, TSA induced 5' AMP-activated protein kinase (p-AMPK) (T172) and inhibited mammalian target of rapamycin complex 1 (mTORC1) activity as estimated by phosphorylated ribosomal protein S6 kinase beta-1 (p-p70S6K) levels, thereby suggesting that TSA-mediated AMPK activation and inhibition of mTORC1 pathway might be associated with autophagy induction. TSA also induced apoptosis of Her2/CT26 cells, as inferred by the increased sub-G1 mitotic phases in these cells, Annexin V/PI-double positive results, and TUNEL-positive cells. Finally, we found that the combined treatment of mice with docetaxel and TSA successfully inhibited tumor growth to a greater extent than docetaxel alone. Therefore, we propose the use of TSA for supplementary anticancer therapy to support anti-neoplastic drugs, such as docetaxel, by inducing apoptosis in cancer cells and resulting in the induction of neighborhood anti-cancer immunity.

Boronic acid-tethered amphiphilic hyaluronic acid derivative-based nanoassemblies for tumor targeting and penetration.

(3-Aminomethylphenyl)boronic acid (AMPB)-installed hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated for tumor-targeted delivery. The amine group of AMPB was conjugated to the carboxylic acid group of hyaluronic acid (HA) via amide bond formation, and synthesis was confirmed by spectroscopic methods. HACE-AMPB/MB NPs with a 239-nm mean diameter, narrow size distribution, negative zeta potential, and >90% drug encapsulation efficiency were fabricated. Exposed AMPB in the outer surface of HACE-AMPB NPs (in the aqueous environment) may react with sialic acid of cancer cells. The improved cellular accumulation efficiency, in vitro antitumor efficacy, and tumor penetration efficiency of HACE-AMPB/MB NPs, compared with HACE/MB NPs, in MDA-MB-231 cells (CD44 receptor-positive human breast adenocarcinoma cells) may be based on the CD44 receptor-mediated endocytosis and phenylboronic acid-sialic acid interaction. Enhanced in vivo tumor targetability, infiltration efficiency, and antitumor efficacies of HACE-AMPB NPs, compared with HACE NPs, were observed in a MDA-MB-231 tumor-xenografted mouse model. In addition to passive tumor targeting (based on an enhanced permeability and retention effect) and active tumor targeting (interaction between HA and CD44 receptor), the phenylboronic acid-sialic acid interaction can play important roles in augmented tumor targeting and penetration of HACE-AMPB NPs. STATEMENT OF SIGNIFICANCE: (3-Aminomethylphenyl)boronic acid (AMPB)-tethered hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated and their tumor targeting and penetration efficiencies were assessed in MDA-MB-231 (CD44 receptor-positive human adenocarcinoma) tumor models. MB, which exhibited antitumor efficacies via the inhibition of angiogenesis and hypoxia inducible factor (HIF)-1, was entrapped in HACE-AMPB NPs in this study. Phenylboronic acid located in the outer surface of HACE-AMPB/MB NPs (in the aqueous milieu) may react with the sialic acid over-expressed in cancer cells and intramolecular B‒O bond can be formed. This phenylboronic acid-sialic acid interaction may provide additional tumor targeting and penetration potentials together with an enhanced permeability and retention (EPR) effect (passive tumor targeting) and HA-CD44 receptor interaction (active tumor targeting). Developed HACE-AMPB NP may be one of promising nanocarriers for the imaging and therapy of CD44 receptor-expressed cancers.

Interleukin-10 attenuates tumour growth by inhibiting interleukin-6/signal transducer and activator of transcription 3 signalling in myeloid-derived suppressor cells.

Interleukin-10 (IL-10) is a well-characterized anti-inflammatory cytokine, but its role in anti-cancer immunity is controversial. After injection with TC-1 cancer cells, we observed more rapid tumour growth and significantly higher interleukin-6 (IL-6) production in IL-10 knockout (IL-10(-/-)) mice than wild-type (WT) mice. Blocking IL-6 with an anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) inhibited tumour growth and myeloid-derived suppressor cell (MDSC) generation, which were significantly increased in IL-10-deficient mice. MDSCs and tumour cells from IL-10(-/-) mice had increased phosphorylated signal transducer and activator of transcription 3 (p-STAT3) levels. Treatment with a STAT3 inhibitor, S3I, reduced tumour growth, inhibited MDSC expansion, reduced IL-6 in tumours, and relieved T cell suppression. The combination of anti-IL-6R mAb and S3I further inhibited tumour growth compared to S3I treatment alone. These results suggested that the inhibition of the IL-6/STAT3 signalling axis is a candidate anti-cancer strategy, especially under systemic inflammatory conditions with high IL-6.

Steamed and Fermented Ethanolic Extract from Codonopsis lanceolata Attenuates Amyloid-ß-Induced Memory Impairment in Mice.

Codonopsis lanceolata (C. lanceolata) is a traditional medicinal plant used for the treatment of certain inflammatory diseases such as asthma, tonsillitis, and pharyngitis. We evaluated whether steamed and fermented C. lanceolata (SFC) extract improves amyloid-ß- (Aß-) induced learning and memory impairment in mice. The Morris water maze and passive avoidance tests were used to evaluate the effect of SFC extract. Moreover, we investigated acetylcholinesterase (AChE) activity and brain-derived neurotrophic factor (BDNF), cyclic AMP response element-binding protein (CREB), and extracellular signal-regulated kinase (ERK) signaling in the hippocampus of mice to determine a possible mechanism for the cognitive-enhancing effect. Saponin compounds in SFC were identified by Ultra Performance Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS). SFC extract ameliorated amyloid-ß-induced memory impairment in the Morris water maze and passive avoidance tests. SFC extract inhibited AChE activity and also significantly increased the level of CREB phosphorylation, BDNF expression, and ERK activation in hippocampal tissue of amyloid-ß-treated mice. Lancemasides A, B, C, D, E, and G and foetidissimoside A compounds present in SFC were determined by UPLC-Q-TOF-MS. These results indicate that SFC extract improves Aß-induced memory deficits and that AChE inhibition and CREB/BDNF/ERK expression is important for the effect of the SFC extract. In addition, lancemaside A specifically may be responsible for efficacious effect of SFC.

Antiviral Activity of Oroxylin A against Coxsackievirus B3 Alleviates Virus-Induced Acute Pancreatic Damage in Mice.

The flavonoids mosloflavone, oroxylin A, and norwogonin, which were purified from Scutellaria baicalensis Georgi, significantly protected Vero cells against Coxsackievirus B3 (CVB3)-induced cell death. To investigate the in vivo antiviral activity of oroxylin A, we intraperitoneally inoculated CVB3 into 4-week-old BALB/c mice. Body weights and blood glucose levels of the mice were decreased after CVB3 infection, and these changes were attenuated by the administration of oroxylin A. Importantly, treatment of mice with oroxylin A reduced viral titers in the pancreas and decreased the serum levels of the inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α. Additionally, the administration of oroxylin A mitigated the histological pancreatic lesions and apoptotic cell death induced by CVB3 infection and increased the levels of phospho-eIF2α in infected pancreata. The results suggest that oroxylin A may represent a potent antiviral agent against CVB3 infection.

Anti-Influenza Activity of Betulinic Acid from Zizyphus jujuba on Influenza A/PR/8 Virus.

Betulinic acid, a pentacyclic triterpene isolated from Jujube tree (Zizyphus jujuba Mill), has been known for a wide range of biological and medicinal properties such as antibacterial, antimalarial, anti-inflammatory, antihelmintic, antinociceptive, and anticancer activities. In the study, we investigated the antiviral activity on influenza A/PR/8 virus infected A549 human lung adenocarcinoma epithelial cell line and C57BL/6 mice. Betulinic acid showed the anti-influenza viral activity at a concentration of 50 µM without a significant cytotoxicity in influenza A/PR/8 virus infected A549 cells. Also, betulinic acid significantly attenuated pulmonary pathology including increased necrosis, numbers of inflammatory cells and pulmonary edema induced by influenza A/PR/8 virus infection compared with vehicle- or oseltamivir-treated mice in vivo model. The down-regulation of IFN-γ level, which is critical for innate and adaptive immunity in viral infection, after treating of betulinic acid in mouse lung. Based on the obtained results, it is suggested that betulinic acid can be the potential therapeutic agent for virus infection via anti-inflammatory activity.

Coadministration of Hedera helix L. Extract Enabled Mice to Overcome Insufficient Protection against Influenza A/PR/8 Virus Infection under Suboptimal Treatment with Oseltamivir.

Several anti-influenza drugs that reduce disease manifestation exist, and although these drugs provide clinical benefits in infected patients, their efficacy is limited by the emergence of drug-resistant influenza viruses. In the current study, we assessed the therapeutic strategy of enhancing the antiviral efficacy of an existing neuraminidase inhibitor, oseltamivir, by coadministering with the leaf extract from Hedera helix L, commonly known as ivy. Ivy extract has anti-inflammatory, antibacterial, antifungal, and antihelminthic properties. In the present study, we investigated its potential antiviral properties against influenza A/PR/8 (PR8) virus in a mouse model with suboptimal oseltamivir that mimics a poor clinical response to antiviral drug treatment. Suboptimal oseltamivir resulted in insufficient protection against PR8 infection. Oral administration of ivy extract with suboptimal oseltamivir increased the antiviral activity of oseltamivir. Ivy extract and its compounds, particularly hedrasaponin F, significantly reduced the cytopathic effect in PR8-infected A549 cells in the presence of oseltamivir. Compared with oseltamivir treatment alone, coadministration of the fraction of ivy extract that contained the highest proportion of hedrasaponin F with oseltamivir decreased pulmonary inflammation in PR8-infected mice. Inflammatory cytokines and chemokines, including tumor necrosis factor-alpha and chemokine (C-C motif) ligand 2, were reduced by treatment with oseltamivir and the fraction of ivy extract. Analysis of inflammatory cell infiltration in the bronchial alveolar of PR8-infected mice revealed that CD11b+Ly6G+ and CD11b+Ly6Cint cells were recruited after virus infection; coadministration of the ivy extract fraction with oseltamivir reduced infiltration of these inflammatory cells. In a model of suboptimal oseltamivir treatment, coadministration of ivy extract fraction that includes hedrasaponin F increased protection against PR8 infection that could be explained by its antiviral and anti-inflammatory activities.