Dioscin Activates Endoplasmic Reticulum Unfolded Protein Response for Defense Against Pathogenic Bacteria in Caenorhabditis elegans via IRE-1/XBP-1 Pathway.

The unfolded protein response (UPR) is an evolutionarily conserved pathway that senses and responds to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen during bacterial infection. The IRE-1/XBP-1 pathway is a major branch of the UPRER that has been conserved from yeast to human. Dioscin, a steroidal saponin exhibits a broad spectrum of properties. However, whether dioscin influences the immune response and the underlying molecular mechanisms remain obscure. We find that dioscin increases resistance to Gram-negative pathogen Pseudomonas aeruginosa. Furthermore, dioscin also inhibits the growth of pathogenic bacteria. Meanwhile, dioscin enhances the resistance to pathogens by reducing bacterial burden in the intestine. Through genetic screening, we find that dioscin activates the UPRER to promote innate immunity via IRE-1/XBP-1 pathway. Intriguingly, dioscin requires the neural XBP-1 for immune response. Our findings suggest that dioscin may be a viable candidate for the treatment of infectious diseases.

Caffeic acid activates mitochondrial UPR to resist pathogen infection in Caenorhabditis elegans via the transcription factor ATFS-1.

Mitochondria play roles in the resistance of Caenorhabditis elegans against pathogenic bacteria by regulating mitochondrial unfolded protein response (UPRmt). Caffeic acid (CA) (3,4-dihydroxy cinnamic acid) is a major phenolic compound present in several plant species, which exhibits biological activities such as antioxidant, anti-fibrosis, anti-inflammatory, and anti-tumor properties. However, whether caffeic acid influences the innate immune response and the underlying molecular mechanisms remains unknown. In this study, we find that 20 µM caffeic acid enhances innate immunity to resist the Gram-negative pathogen Pseudomonas aeruginosa infection in C. elegans. Meanwhile, caffeic acid also inhibits the growth of pathogenic bacteria. Furthermore, caffeic acid promotes host immune response by reducing the bacterial burden in the intestine. Through genetic screening in C. elegans, we find that caffeic acid promotes innate immunity via the transcription factor ATFS-1. In addition, caffeic acid activates the UPRmt and immune response genes for innate immune response through ATFS-1. Our work suggests that caffeic acid has the potential to protect patients from pathogen infection.

The homeodomain transcription factor CEH-37 regulates PMK-1/p38 MAPK pathway to protect against intestinal infection via the phosphatase VHP-1.

Increasing evidence indicate that the expression of defense genes at the right place and the right time are regulated by host-defense transcription factors. However, the precise mechanisms of this regulation are not well understood. Homeodomain transcription factors, encoded by homeobox genes, play crucial role for the development of multicellular eukaryotes. In this study, we demonstrated that homeodomain transcription factor CEH-37 (known as OTX2 in mammals) was a key transcription factor for host defense in Caenorhabditis elegans. Meanwhile, CEH-37 acted in the intestine to protect C. elegans against pathogen infection. We further showed that the homeodomain transcription factor CEH-37 positively regulated PMK-1/ p38 MAPK activity to promote the intestinal immunity via suppression phosphatase VHP-1. Furthermore, we demonstrated that this function was conserved, because the human homeodomain transcription factor OTX2 also exhibited protective function in lung epithelial cells during Pseudomonas aeruginosa infection. Thus, our work reveal that CEH-37/OTX2 is a evolutionarily conserved transcription factor for defense against pathogen infection. The finding provides a model in which CEH-37 decreases VHP-1 phosphatase activity, allowing increased stimulation of PMK-1/p38 MAPK phosphorylation cascade in the intestine for pathogen resistance.

Evaluation of volatile components from the tuber, fibrous roots, bud, stem and leaf tissues of Bletilla striata for its anti-colon cancer activity.

Bletilla striata (Thunb.) Rchb.f., a medicinal plant in the Orchidaceae family, is mainly found in East Asia and has extensive pharmacological activities. Plant's volatile components are important active ingredients with a wide range of physiological activities, and B. striata has a special odor and unique volatile components. Yet it has received little attention, hindering a full understanding of its phytochemical components. Employing the ultrasonic-assisted extraction method, the volatile components of B. striata's fibrous root, bud, aerial part and tuber were extracted, resulting in yields of 0.06%, 0.64%, 3.38% and 4.47%, respectively. A total of 78 compounds were identified from their chemical profiles using gas chromatography-mass spectrometry (GC-MS), including 45 components with the main compounds of linoleic acid (content accounting for 31.23%), n-hexadecanoic acid (13.53%), and octadecanoic acid (9.5%) from the tuber, 34 components with the main compounds of eicosane, 2-methyl- (28.42%), linoelaidic acid (10.43%), linoleic acid (4.53%), and n-hexadecanoic acid (6.91%) from the fibrous root, 38 components with the main compounds of pentadeca-6,9-dien-1-ol (9.29%), n-hexadecanoic acid (11%), eicosane,2-methyl- (23.43%), and linoleic acid (23.53%) from the bud, and 27 components with the main compounds of linoelaidic acid (5.97%), n-hexadecanoic acid (15.99%), and linolenic acid ethyl ester (18.9%) from the aerial part. Additionally, the growth inhibition activity against colon cancer HCT116 cells was evaluated using sulforhodamine B (SRB) assay and the thiazolyl blue tetrazolium bromide (MTT) assay, and the accumulation of reactive oxygen species (ROS) was determined using dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining and fluorescence intensity analysis. The volatile extracts exhibited significant growth inhibitory efficacy against HCT116 cells, with half-maximal inhibitory concentration (IC50) values of 3.65, 2.32, 2.42 and 3.89 mg/mL in the SRB assay, and 3.55, 2.58, 3.12 and 4.80 mg/mL in the MTT assay for the root, bud, aerial part, and tuber, respectively. Notably, treatment with the aerial part extract caused morphological changes in the cells and significantly raised the intracellular ROS level. In summary, the chemical profiles of the volatile components of B. striata were revealed for the first time, demonstrating a certain tissue specificity. Additionally, it demonstrated for the first time that these volatile extracts possess potent anti-colon cancer activity, highlighting the importance of these volatile components in B. striata's medicinal properties.

The combination of Biochanin A and SB590885 potentiates the inhibition of tumour progression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most aggressive and frequently diagnosed malignancy of the liver. Despite aggressive therapy, life expectancy of many patients in these cases is extended by only a few months. Hepatocellular carcinoma (HCC) has a particularly poor prognosis and would greatly benefit from more effective therapies. METHODS: The CCK-8 assay and colony formation assays were used to test the cell proliferation and viability. The effects of combination Biochanin A and SB590885 on apoptosis and cell cycle arrest of HCC cells were analysed by flow cytometry. The expression of ERK MAPK and PI3K/AKT/mTOR signalling as well as apoptosis and cell cycle-related proteins in HCC cells were tested by western blotting. The HCC cell xenograft model was established to test the tumor proliferation. Serum and plasma were tested for liver and kidney safety markers (ALP, ALT, AST, total bilirubin, creatinine, urea nitrogen) by using SpectraMax i3X. RESULTS: The combination of natural product Biochanin A with the BRAF inhibitor SB590885 synergistically suppressed proliferation, and promoted cell cycle arrest and apoptosis in vitro. Furthermore, we demonstrated that the combination of Biochanin A and SB590885 led to increased impairment of proliferation and HCC tumour inhibition through disrupting of the ERK MAPK and the PI3K/AKT pathways in vitro. The volumes tumors and the weights of tumours were significantly reduced by the combination treatment compared to the control or single treatments in vivo. In addition, we found that there was no significant hepatorenal toxicity with the drug combination, as indicated by the hepatorenal toxicity test. CONCLUSION: The results identify an effective combination therapy for the most aggressive form of HCC and provide the possibility of therapeutic improvement for patients with advanced HCC.

Genome analysis and gene nblA identification of Microcystis aeruginosa myovirus (MaMV-DC) reveal the evidence for horizontal gene transfer events between cyanomyovirus and host.

The genome sequence, genetic characterization and nblA gene function of Microcystis aeruginosa myovirus isolated from Lake Dianchi in China (MaMV-DC) have been analysed. The genome DNA is 169 223 bp long, with 170 predicted protein-coding genes (001L­170L) and a tRNA gene. About one-sixth of these genes have homologues in the host cyanobacteria M. aeruginosa. The genome carries a gene homologous to host nblA, which encodes a protein involved in the degradation of cyanobacterial phycobilisome. Its expression during MaMV-DC infection was confirmed by reverse transcriptase PCR and Western blot detection and abundant expression was companied by the significant decline of phycocyanin content and massive release of progeny MaMV-DC. In addition, expressing MaMV-DC nblA reduced the phycocyanin peak and the phycocyanin to chlorophyll ratio in model cyanobacteria. These results confirm that horizontal gene transfer events have occurred between cyanobacterial host and cyanomyovirus and suggest that MaMV-DC carrying host-derived genes (such as 005L, that codes for NblA) is responsible for more efficient expression of cyanophage genes and release of progeny cyanophage. This study provides novel insight into the horizontal gene transfer in cyanophage and the interactions between cyanophage and their host.

Gamabufotalin inhibits colitis-associated colorectal cancer by suppressing transcription factor STAT3.

Constitutive activation of STAT3 plays important role in the pathogenesis of colorectal cancer (CRC). Inhibition of STAT3 has been proposed as a reasonable strategy to suppress CRC. Gamabufotalin (Gam), an effective bioactive compound of ChanChu, has been used for cancer therapy due to its desirable metabolic stability and less adverse effect. However, its effect on CRC is still unclear. In this study, we found that Gam significantly inhibited the CRC in vitro and vivo. Furthermore, Gam induced apoptosis to inhibit the viability of HCT-116 and HT-29 cell lines in dose-dependent manner by suppressing the transcription factor STAT3. In addition, Gam was also found to inhibit carcinogenesis of colitis-associated cancer (CAC) in AOM/DSS mice model by inhibiting STAT3. Our findings suggest that Gam may be an effective way to prevent occurrence and development of CRC and CAC.

Cynaroside extends lifespan and improves the neurondegeneration diseases via insulin/IGF-1 signaling pathway in Caenorhabditis elegans.

The evolutionarily conserved insulin/IGF-1 signaling pathway plays a central role in aging and aging related diseases such as neurodegeneration diseases. Inhibition of insulin/IGF-1 signaling pathway has been proposed as an effective way to extend lifespan and delay neurodegeneration diseases in different organisms. Cynaroside (Cyn), a flavonoid contained in many medical plants and in vegetables, had been shown to exhibit pharmacological properties such as anti-inflammatory, anti-tumor, and anti-oxidant effects. The study demonstrated that lifespan extension and neurodegeneration diseases improving could be achieved by targeting evolutionarily conserved insulin/IGF-1 pathway through using pharmacological interventions. Via using this approach in tractable model Caenorhabditis elegans, we found that 10 µM Cynaroside significantly promoted the healthy lifespan in wild-type animals. Furthermore, via genetic screen, we showed that Cynaroside acted on IGF-1-R /DAF-2, which was followed by the activation of transcription factor DAF-16/FOXO to extend the healthy lifespan. Intriguingly, Cynaroside also improved neurodegeneration diseases such as Alzheimer's and polyglutamine disease by suppressing insulin/IGF-1 signaling pathway. Our work suggests that Cynaroside may be a promising candidate for the prevention and treatment of aging and neurodegeneration diseases.

Schisandrin A enhances pathogens resistance by targeting a conserved p38 MAPK pathway.

Schizandrin A (SA), also known as deoxyschizandrin, is one of the most biologically active lignans isolated from the traditional Chinese medicine Fructus schisandrae chinensis. Schisandrin A has proven benefits for anti-cancer, anti-inflammation, hepatoprotection, anti-oxidation, neuroprotection, anti-diabetes. But the influence of Schisandrin A to the innate immune response and its molecular mechanisms remain obscure. In this study, we found that Schisandrin A increased resistance to not only the Gram-negative pathogens Pseudomonas aeruginosa and Salmonella enterica but also the Gram-positive pathogen Listeria monocytogenes. Meanwhile, Schisandrin A protected the animals from the infection by enhancing the tolerance to the pathogens infection rather than by reducing the bacterial burden. Through the screening of the conserved immune pathways in Caenorhabditis elegans, we found that Schisandrin A enhanced innate immunity via p38 MAPK pathway. Furthermore, Schisandrin A increased the expression of antibacterial peptide genes, such as K08D8.5, lys-2, F35E12.5, T24B8.5, and C32H11.12 by activation PMK-1/p38 MAPK. Importantly, Schisandrin A-treated mice also enhanced resistance to P. aeruginosa PA14 infection and significantly increased the levels of active PMK-1. Thus, promoted PMK-1/p38 MAPK-mediated innate immunity by Schisandrin A is conserved from worms to mammals. Our work provides a conserved mechanism by which Schisandrin A enhances innate immune response and boosts its therapeutic application in the treatment of infectious diseases.

S-Adenosylmethionine (SAM) diet promotes innate immunity via histone H3K4me3 complex.

S-adenosylmethionine (SAM) was a methyl donor for modifying histones, which had crucial roles in lipid accumulation, tissue injury, and immune responses. SAM fluctuation might be linked to variations in histone methylation. However, the underlying molecular mechanisms of whether the SAM diet influenced the immune response via histone modification remained obscure. In this study, we utilized the Caenorhabditis elegans as a model to investigate the role of SAM diet in innate immunity. We found that 50 µM SAM increased resistance to Gram-negative pathogen Pseudomonas aeruginosa PA14 by reducing the bacterial burden in the intestine. Furthermore, through the genetic screening in C. elegans, we found that SAM functioned in germline to enhance innate immunity via an H3K4 methyltransferase complex to upregulate the immune response genes, including irg-1 and T24B8.5. Intriguingly, SAM also protected mice from P. aeruginosa PA14 infection by reducing the bacterial burden in lung. These findings provided insight into the mechanisms of molecular connections among SAM diet, histone modifications and innate immunity.

Luteolin promotes pathogen resistance in Caenorhabditis elegans via DAF-2/DAF-16 insulin-like signaling pathway.

The DAF-2/DAF-16 insulin-like signaling pathway was an evolutionarily conserved pathway, which regulated many aspects of organismal physiology, such as pathogen resistance, metabolism, stress response, longevity. Luteolin, a flavone contained in many medical plants and in vegetables, had been shown to exhibit activities such as anti-tumor, anti-oxidant and neuroprotective effects. However, whether the Luteolin influenced the immune response and the underlying molecular mechanisms remained obscure. We found that Luteolin increased resistance to not only the Gram-negative pathogens Pseudomonas aeruginosa and Salmonella enterica but also the Gram-positive pathogens Enterococcus faecalis and Staphylococcus aureus in dose dependent manner. Meanwhile, Luteolin promoted host immune response via inhibiting the growth of pathogenic bacteria. Through the genetic screening in C. elegans, we found that Luteolin promoted innate immunity via DAF-2/DAF-16 insulin-like signaling pathway rather than p38 MAPK pathway and SKN-1. Furthermore, Luteolin activated the DAF-16/FOXO transcription factor for innate immune response. Our work suggested that Luteolin had the potential of improving the patients with pathogen infection.

Dioscin integrates regulation of monosaturated fatty acid metabolism to extend the life span through XBP-1/SBP-1 dependent manner.

Delay aging, especially in healthy life extension, brought the most interest to the medical field. Searching for anti-aging drugs with relative safety profiles bring natural products in hotspot. In this study, we find that dioscin promotes the health span extension in wild-type Caenorhabditis elegans. Through the genetic screening in C. elegans, we further reveal that dioscin activates the transcription factor SBP-1/SREBP by the UPRER transcription factor XBP-1 to upregulate transcription of the Δ9 desaturase FAT-5 and FAT-7, resulting in increased monounsaturated fatty acid content which requires for healthy life span extension. Intriguingly, through tissue-specific knockdown, we find that dioscin modulates the health span by activating SBP-1 in the intestine. Unexpectedly, dietary supplementation of POA and OA rescues XBP-1, SBP-1 mutants-induced shortened life span phenotype. Considering the conservation of MUFAs metabolism, dioscin may promote health span in other species, including mammals. Our work suggests that dioscin might be a promising candidate for developing anti-aging agent.

A time-resolved fluorescence microsphere-lateral flow immunochromatographic strip for quantitative detection of Pregnanediol-3-glucuronide in urine samples.

Introduction: Pregnanediol-3-glucuronide (PdG), as the main metabolite of progesterone in urine, plays a significant role in the prediction of ovulation, threatened abortion, and menstrual cycle maintenance. Methods: To achieve a rapid and sensitive assay, we have designed a competitive model-based time-resolved fluorescence microsphere-lateral flow immunochromatography (TRFM-LFIA) strip. Results: The optimized TRFM-LFIA strip exhibited a wonderful response to PdG over the range of 30-2,000 ng/mL, the corresponding limit of detection (LOD) was calculated as low as 8.39 ng/mL. More importantly, the TRFM-LFIA strip was innovatively used for the quantitative detection of PdG in urine sample, and excellent recovery results were also obtained, ranging from 97.39% to 112.64%. Discussion: The TRFMLFIA strip possessed robust sensitivity and selectivity in the determination of PdG, indicating the great potential of being powerful tools in the biomedical and diagnosis region.

[Influence of temperature on spermatogenesis-related genes in spermatogonia cultured in vitro].

OBJECTIVE: To investigate the influence of temperature on the expressions of c-kit and PI3K in spermatogonia cultured in vitro at 32 degrees C and 37 degrees C, and provide basic scientific data for the mechanism of spermatogenic impairment due to body temperature (37 degrees C). METHODS: Isolated spermatogenic cells were cultured in vitro at 32 degrees C and 37 degrees C, and their adherence, proliferation and morphologic changes were observed and recorded under the inverted phase contrast microscope. At 8 days, the spermatogonia were separated by Percoll density gradient centrifugation and the differential adhesion method. The expressions of c-kit and PI3K mRNA and proteins in the separated cells were detected by real time polymerase chain reaction and Western blot, respectively. The c-kit gene was sequenced to identify the occurrence of mutations. RESULTS: Adherence, division and proliferation of the cells were observed in both the 32 degrees C and 37 degrees C groups. The expressions of c-kit and PI3K mRNA and proteins in the spermatogonia were significantly higher in the 32 degrees C group than in the 37 degrees C group (P < 0.05). The 32 degrees C group showed no mutation of c-kit in exon 9, 11 and 13; the 37 degrees C group exhibited no mutation in exon 11 and 13, but possible insertion or deletion mutations in exon 9. CONCLUSION: Culturing in vitro at 37 degrees C could inhibit the expression of proliferation- and differentiation-related genes in spermatogenic cells and lead to the mutation of the c-kit gene.

Metformin induces S-adenosylmethionine restriction to extend the Caenorhabditis elegans healthspan through H3K4me3 modifiers.

Metformin, a widely prescribed first-line drug for the treatment of type II diabetes mellitus, has been shown to extend lifespan and delay the onset of age-related diseases. The precisely mechanisms by which these effects are realized remain elusive. We find that metformin exposure is restricted to adults, which is sufficient to extend lifespan. However, limiting metformin exposure to the larvae has no significant effect on Caenorhabditis elegans longevity. Here, we show that after metformin treatment, the level of S-adenosylmethionine (SAM) is reduced in adults but not in the larvae. Potential mechanisms by which reduced SAM might increase lifespan include altering the histone methylation. However, the molecular connections between metformin, SAM limitation, methyltransferases, and healthspan-associated phenotypes are unclear. Through genetic screening of C. elegans, we find that metformin promotes the healthspan through an H3K4 methyltransferase/demethylase complex to downregulate the targets, including mTOR and S6 kinase. Thus, our studies provide molecular links between meformin, SAM limitation, histone methylation, and healthspan and elucidate the mode action of metformin-regulated healthspan extension will boost its therapeutic application in the treatment of human aging and age-related diseases.

Bufogarlides A-C, three new bufadienolides with Δ14,15 double bond from the skins of Bufo bufo gargarizans.

Three new bufadienolides with a Δ14,15 double bond, named bufogarlides A-C (1-3), together with three known analogs (4-6), were isolated from the skins of Bufo bufo gargarizans. Their structures were identified by analyses of spectroscopic data (1 D and 2 D NMR, HR-ESIMS), and comparison with the literature data. All the isolates were evaluated for their cytotoxic activities against ovarian carcinoma cell lines A2780 and SKOV3. Among them, compound 5 showed the highest potential for the growth inhibition of cancerous cells A2780 and SKOV3 with the IC50 values of 21.09 and 67.08 nM, respectively.

Brevilin A enhances innate immunity and the resistance of oxidative stress in Caenorhabditis elegans via p38 MAPK pathway.

The conserved p38/PMK-1 pathway that is an evolutionarily conserved module used by mammals and nematodes in immune response against bacterial infections. Brevilin A (BA), a sesquiterpene lactone compound of Centipeda minima has been shown to exhibit activities such as anti-tumor, anti-bacterial and anti-protozoal. However, whether the Brevilin A influences the immune response and the underlying molecular mechanisms remain obscure. We find that 10 µM Brevilin A increases resistance to not only the Gram-negative pathogens Pseudomonas aeruginosa and Salmonella enterica but also the Gram-positive pathogens Enterococcusfaecalis and Staphylococcus aureus. Meanwhile, Brevilin A enhances the resistance to pathogens by reducing the bacterial burden in the intestine. Through the genetic screening in C. elegans, we find that Brevilin A promotes innate immunity via p38 MAPK pathway. Furthermore, Brevilin A activates the p38/PMK-1 in the intestine for innate immune response. In addition, we also find that Brevilin A increases the resistance of oxidative stress and extends lifespan through p38 MAPK pathway. Our work suggests that Brevilin A may be a viable candidate for the treatment of infectious diseases.

Usnic Acid extends healthspan and improves the neurodegeneration diseases via mTOR/PHA-4 signaling pathway in Caenorhabditis elegans.

The Mammalian/mechanistic target of rapamycin (mTOR) played a central role in cellular survival and aging. Inhibition of mTOR had been proposed as a reasonable strategy to promote lifespan and delay age-related diseases in evolutionarily diverse organisms. The study showed that lifespan extension and age-related diseases improvement could be achieved by targeting evolutionarily conserved mTOR pathways and mechanisms using pharmacological interventions. Using this approach in Caenorhabditis elegans, We found that 2 µM Usnic Acid significantly extended the healthy lifespan in wild-type animals. Furthermore, via genetic screen, we showed that Usnic Acid acted on mTOR, which was followed by the activation of PHA-4/Foxa to extend the healthy lifespan. Intriguingly, Usnic Acid also delayed neurodegeneration diseases such as Alzheimer's and polyglutamine disease through mTOR-dependent manner. Our work suggested that Usnic Acid might be a viable candidate for the prevention and treatment of aging and age-related diseases.

Sanguinarine promotes healthspan and innate immunity through a conserved mechanism of ROS-mediated PMK-1/SKN-1 activation.

The longevity of an organism is influenced by both genetic and environmental factors. With respect to genetic factors, a significant effort is being made to identify pharmacological agents that extend lifespan by targeting pathways with a defined role in the aging process. Sanguinarine (San) is a benzophenanthridine alkaloid that exerts a broad spectrum of properties. In this study, we utilized Caenorhabditis elegans to examine the mechanisms by which sanguinarine influences aging and innate immunity. We find that 0.2 µM sanguinarine extends healthspan in C. elegans. We further show that sanguinarine generates reactive oxygen species (ROS), which is followed by the activation of PMK-1/SKN-1pathway to extend healthspan. Intriguingly, sanguinarine increases resistance to pathogens by reducing the bacterial burden in the intestine. In addition, we also find that sanguinarine enhances innate immunity through PMK-1/SKN-1 pathway. Our data suggest that sanguinarine may be a viable candidate for the treatment of age-related disorders.

Two new 19-hydroxy bufadienolides with cytotoxic activity from the skins of Bufo melanosticus.

Two new 19-hydroxy bufadienolides, named bufohydrolide A (1) and bufohydrolide B (2), and four known analogs (3-6) were isolated from the aqueous extracts of the skins of Bufo melanosticus. Their structures were established by spectral data analyses, such as UV, IR, 1 D/2D NMR and mass spectra. Compounds 1-6 were evaluated for their cytotoxic activity against SMMC-7721, HT-29 and A549 cells. Noteworthily, all six isolates exhibited various levels of anti-proliferative activities with IC50 values ranging from 0.02 ± 0.0002 to 25 ± 0.5 µM.