Hernandonine-mediated autophagic cell death in hepatocellular carcinoma: Interplay of p53 and YAP signaling pathways.

Hepatocellular carcinoma (HCC), the primary form of liver cancer, is the third leading cause of cancer-related death globally. Hernandonine is a natural alkaloid derived from Hernandia nymphaeifolia that has been shown to exert various biological functions. In a previous study, hernandonine was shown to suppress the proliferation of several solid tumor cell lines without affecting normal human cell lines. However, little is known about the effect of hernandonine on HCC. Therefore, this study aimed to investigate the effect and mechanism of hernandonine on HCC in relation to autophagy. We found that hernandonine inhibited HCC cell growth in vitro and in vivo. In addition, hernandonine elicited autophagic cell death and DNA damage in HCC cells. RNA-seq analysis revealed that hernandonine upregulated p53 and Hippo signaling pathway-related genes in HCC cells. Small RNA interference of p53 resulted in hernandonine-induced autophagic cell death attenuation. However, inhibition of YAP sensitized HCC cells to hernandonine by increasing the autophagy induction. This is the first study to illustrate the complex involvement of p53 and YAP in the hernandonine-induced autophagic cell death in human HCC cells. Our findings provide novel evidence for the potential of hernandonine as a therapeutic agent for HCC treatment.

Structure and genome editing of type I-B CRISPR-Cas.

Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3+ T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.

FGF2 drives osteosarcoma metastasis through activating FGFR1-4 receptor pathway-mediated ICAM-1 expression.

Osteosarcoma is a malignant tumor with high metastatic potential, such that the overall 5-year survival rate of patients with metastatic osteosarcoma is only 20%. Therefore, it is necessary to unravel the mechanisms of osteosarcoma metastasis to identify predictors of metastasis by which to develop new therapies. Fibroblast growth factor 2 (FGF2) is a growth factor involved in embryonic development, cell migration, and proliferation. The overexpression of FGF2 and FGF receptors (FGFRs) has been shown to enhance cancer cell proliferation in lung, breast, gastric, and prostate cancers as well as melanoma. Nonetheless, the roles of FGF2 and FGFRs in human osteosarcoma cells remain unknown. In the present study, we found that FGF2 was overexpressed in human osteosarcoma sections and correlated with lung metastasis. Treatment of FGF2 induced migration activity, invasion activity, and intercellular adhesion molecule (ICAM)-1 expression in osteosarcoma cells. In particular, the downregulation or antagonism of FGFR1-4 suppressed FGF2-induced ICAM-1 expression and cancer cell migration. Furthermore, FGFR1, FGFR2, FGFR3, and FGFR4 were involved in FGF2-induced the phospholipase Cß/protein kinase Cα/proto-oncogene c-Src signaling pathway and triggered c-Jun nuclear translocation. Subsequent c-Jun upregulation of activator protein-1 transcription activity on the ICAM-1 promoter led to an increased migration of osteosarcoma cells. Moreover, the knockdown of endogenous FGF2 suppressed ICAM-1 expression and migration of osteosarcoma cells. These findings suggest that FGF2/FGFR1-4 signaling promotes metastasis via its direct downstream target gene ICAM-1, revealing a novel potential therapeutic target for osteosarcoma.

Critical role of heme oxygenase-1 in chaetoglobosin A by triggering reactive oxygen species mediated mitochondrial apoptosis in colorectal cancer.

The incidence rate of colorectal cancer (CRC) has been increasing and poses severe threats to human health worldwide and developing effective treatment strategies remains an urgent task. In this study, Chaetoglobosin A (ChA), an endophytic fungal metabolite from the medicinal herb-derived fungus Chaetomium globosum Km1126, was identified as a potent and selective antitumor agent in human CRC. ChA induced growth inhibition of CRC cells in a concentration-dependent manner but did not impair the viability of normal colon cells. ChA triggered mitochondrial intrinsic and caspase-dependent apoptotic cell death. In addition, apoptosis antibody array analysis revealed that expression of Heme oxygenase-1 (HO-1) was significantly increased by ChA. Inhibition of HO-1 increased the sensitivity of CRC cells to ChA, suggesting HO-1 may play a protective role in ChA-mediated cell death. ChA induced cell apoptosis via the induction of reactive oxygen species (ROS) and ROS scavenger (NAC) prevented ChA-induced cell death, mitochondrial dysfunction, and HO-1 activation. ChA promoted the activation of c-Jun N-terminal kinase (JNK), and co-administration of JNK inhibitor or siRNA markedly reversed ChA-mediated apoptosis. ChA significantly decreased the tumor growth without eliciting any organ toxicity or affecting the body weight of the CRC xenograft mice. This is the first study to demonstrate that ChA exhibits promising anti-cancer properties against human CRC both in vitro and in vivo. ChA is a potential therapeutic agent worthy of further development in clinical trials for cancer treatment.

Cryptocaryone induces apoptosis in human hepatocellular carcinoma cells by inhibiting aerobic glycolysis through Akt and c-Src signaling pathways.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with the second highest mortality rate in all cancer. Energy reprogramming is one of the hallmarks of cancer, and emerging evidence showed that targeting glycolysis is a promising strategy for HCC treatment. Cryptocaryone has been shown to display promising anti-cancer activity against numerous types of cancer. Previous study also indicated that cryptocaryone induces cytotoxicity by inhibiting glucose transport in cancer cells, but the detailed mechanism still needs to be elucidated. Therefore, this study aimed to investigate the relationship between the anti-cancer effect and glycolytic metabolism of cryptocaryone in human HCC cells. In this study, we found that cryptocaryone potently induced growth inhibition by apoptotic cell death in HCC cells. Cryptocaryone also suppressed the ATP synthesis, lactate production and glycolytic capacity of HCC cells. Mechanistic investigations showed that phosphorylation of Akt and c-Src, as well as the expression of HK1 were impeded by cryptocaryone. Moreover, cryptocaryone markedly increased the expression level of transcription factor FoxO1. Importantly, clinical database analysis confirmed the negative correlation between HK1 and FoxO1. High expression levels of HK-1 were positively correlated with poorer survival in patients with HCCs. These results suggest that cryptocaryone may promote cell apoptosis by inhibiting FoxO1-mediated aerobic glycolysis through Akt and c-Src signaling cascades in human HCC cells. This is the first study to indicate that cryptocaryone exerts anti-cancer property against human HCC cells. Cryptocaryone is a potential natural product worthy of further development into a promising candidate for HCC treatment.

Protodioscin Induces Mitochondrial Apoptosis of Human Hepatocellular Carcinoma Cells Through Eliciting ER Stress-Mediated IP3R Targeting Mfn1/Bak Expression.

Objective: Protodioscin (PD), a steroidal saponin, has a diverse pharmacological activity including neuroprotection, male fertility improvement, and cytotoxicity against various cancers cell lines of different origins. However, the effect of PD on hepatocellular carcinoma (HCC) is still unclear. Methods: Cell viability, colony formation and flow cytometry analysis for apoptosis profile, mitochondrial membrane potential endoplasmic reticulum (ER) expansion were employed to determine the effect of PD against HCC cells. Transient transfection of siRNA, immunofluorescent imaging and immunoprecipitation were used to elucidate the anti-cancer mechanism of PD. The in vivo toxicity and efficacy of PD were assessed by a xenograft mouse model. Results: PD induced apoptosis, loss of mitochondrial membrane potential and ER expansion in HCC cells. Either downregulation of Mfn1 or Bak reversed PD-induced apoptosis and loss of mitochondrial membrane potential. Further analysis revealed that Mfn1 and Bak will form a complex with IP3R to facilitate the transfer of Ca2+ from ER to mitochondria and apoptosis. In addition, our tumour xenograft model further verifies the in vivo anti-tumour effect of PD. Conclusion: Our study sheds light on the understanding of the anti-HCC effects of PD and may open new aspects for the development of novel treatment for human hepatocellular carcinoma.

Effect of phenylurea hydroxamic acids on histone deacetylase and VEGFR-2.

A series of phenylurea hydroxamic acids incorporating pharmacophores of inhibitors of HDAC inhibitors and VEGFR-2 has been designed. Most of the compounds show antiproliferative activity comparable to that of Vorinostat and Sorafenib, and better EPC inhibitory activity. Enzymatic assays and Western blotting results indicated that compound 14 not only inhibits HDAC but also has slight VEGFR-2 inhibitory activity. A docking study revealed that the polar hydroxamic acid retains the interaction with HDAC through a zinc ion and also interacts with some residues of the active site of VEGFR-2. Despite 14 displaying a weaker VEGFR-2 activity, a possible route to develop potent HDAC/VEGFR-2 inhibitors is suggested.

Overexpression of Lipocalin-2 Inhibits Proliferation and Invasiveness of Human Glioblastoma Multiforme Cells by Activating ERK Targeting Cathepsin D Expression.

Lipocalin-2 (LCN2) exhibits pro- and anti-carcinogenic effects in several cancers, but its role in the progression of glioblastoma multiforme (GBM) remains unclear. This study aims to elucidate the effect of LCN2 in human GBM cell, and the mechanism underlying its effects on GBM malignant progression. We observed that LCN2 expression was significantly lower in GBM than in normal tissues and was associated with poorer GBM patient survival. LCN2-overexpressing GBM cells showed significantly reduced proliferation and migration/invasion abilities. Human protease antibody array analysis showed that the expression of cathepsin D (CTSD) protein and mRNA was lower in LCN2-overexpressing GBM cells than in controls. Higher CTSD expression was observed in GBM tumors than in normal tissues, and higher CTSD expression was associated with poorer overall and disease-free survival. LCN2-overexpressing GBM cells exhibited increased ERK phosphorylation. Treatment of these cells with a MEK inhibitor (U0126) restored CTSD expression, cell migration, and cell invasiveness. In conclusion, LCN2 might be serving as a prognostic marker and promising anti-proliferative and anti-metastatic target for treating GBM.

Moscatilin Inhibits Metastatic Behavior of Human Hepatocellular Carcinoma Cells: A Crucial Role of uPA Suppression via Akt/NF-κB-Dependent Pathway.

Hepatocellular carcinoma (HCC) frequently shows early invasion into blood vessels as well as intrahepatic metastasis. Innovations of novel small-molecule agents to block HCC invasion and subsequent metastasis are urgently needed. Moscatilin is a bibenzyl derivative extracted from the stems of a traditional Chinese medicine, orchid Dendrobium loddigesii. Although moscatilin has been reported to suppress tumor angiogenesis and growth, the anti-metastatic property of moscatilin has not been elucidated. The present results revealed that moscatilin inhibited metastatic behavior of HCC cells without cytotoxic fashion in highly invasive human HCC cell lines. Furthermore, moscatilin significantly suppressed the activity of urokinase plasminogen activator (uPA), but not matrix metalloproteinase (MMP)-2 and MMP-9. Interestingly, moscatilin-suppressed uPA activity was through down-regulation the protein level of uPA, and did not impair the uPA receptor and uPA inhibitory molecule (PAI-1) expressions. Meanwhile, the mRNA expression of uPA was inhibited via moscatilin in a concentration-dependent manner. In addition, the expression of phosphorylated Akt, rather than ERK1/2, was inhibited by moscatilin treatment. The expression of phosphor-IκBα, and -p65, as well as κB-luciferase activity were also repressed after moscatilin treatment. Transfection of constitutively active Akt (Myr-Akt) obviously restored the moscatilin-inhibited the activation of NF-κB and uPA, and cancer invasion in HCC cells. Taken together, these results suggest that moscatilin impedes HCC invasion and uPA expression through the Akt/NF-κB signaling pathway. Moscatilin might serve as a potential anti-metastatic agent against the disease progression of human HCC.

Additional alkaloids from Zoanthus vietnamensis with neuroprotective and anti-angiogenic effects.

A chemical investigation of the zoantharian Zoanthus vietnamensis, collected off Taiwan, yielded eleven new alkaloids, 7α-hydroxykuroshine J (1), 18ß-hydroxykuroshine J (2), 5α-hydroxyzoanthenamine (3), 5ß-hydroxyzoanthenamine (4), 14α-hydroxyzoanthenamine (5), 30-hydroxyzoanthenamine (6), 11-dehydroxy-18-epi-kuroshine A (7), 5α-hydroxykuroshine A (8), 7ß-hydroxykuroshine A (9), 11-keto-oxyzoanthamine (10), and 30-hydroxyzoanthamine (11), along with eight known compounds (12-19). The structures of these compounds were identified by detailed spectroscopic data, including HRESIMS, IR, NMR, and UV spectra. All secondary metabolites isolated from Z. vietnamensis were investigated for the anti-angiogenic effect in human endothelial progenitor cells (EPCs). Compounds 6, 7, 11, and 13 exhibited mild anti-angiogenic effect by blocking cell growth and tube formation of EPCs. The neuroprotective potential of four major compounds 12, 14, 15, and 19 against paclitaxel-induced neurotoxicity was evaluated. Pretreatment of 14 and 15 protected paclitaxel-damaged neurite outgrowth of dorsal root ganglion (DRG) neurons, without interfering the cytotoxic activity of paclitaxel on cervical cancer SiHa cells.

Anti-lymphangiogenic diterpenes from the bark of Calocedrus macrolepis var. formosana.

Eight new diterpenes, 6α,7ß-dihydroxyferruginol (1), 6α,7α-dihydroxyferruginol (2), 6α-hydroxyhinokiol (3), 4α-hydroxy-7-oxo-18-norabieta-8,11,13-trien-4α-ol (4a), 15,16-dehydrosugiol (5), 7-methoxy-6,7-secoabieta-8,11,13-triene-6,12-diol (6), 7α-acetoxyabieta-8,12-diene-11,14-dione (7), 7α-butyloxyethyloxyabieta-8,12-diene-11,14-dione (8), along with four known compounds, 6,7-dehydroferruginol (9), 12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (10), 7α-11-dihydroxy-12-methoxy-8,11,13-abietatriene (11), and 11,14-dihydroxy-8,11,13-abietatrien-7-one (12) were successfully isolated from the bark of Calocedrus macrolepis var. formosana. The structures of all isolates were elucidated by physical data (appearance, UV, IR, optical rotation) and spectroscopic data (1D, 2D NMR, and HREIMS). Compounds 9, 10, 11, and 12 showed promising growth-inhibitory effect on human lymphatic endothelial cells (LECs). Among these compounds, compound 10 exerted the most potent anti-lymphangiogenesis property by suppressing cell growth and tube formation of LECs. In conclusion, the results revealed the anti-lymphangiogenic potentials of Formosan C. macrolepis var. formosana.

Praeruptorin A reduces metastasis of human hepatocellular carcinoma cells by targeting ERK/MMP1 signaling pathway.

Praeruptorin A (PA) is one of the active ingredients found in the dried root of Peucedanum praeruptorum Dunn, has been reported to possess anticancer effects against various types of cancer. However, the effect of PA on human hepatocellular carcinoma (HCC) remains uncleared. In this study, our results indicated that PA did not induce cytotoxicity or alter cell cycle distribution in human HCC cells (Huh-7, SK-Hep-1, and PLC/PRF/5 cells). Instead, PA inhibited the migration and invasion of human HCC cells while downregulating the expression of matrix metalloproteinase-1 (MMP1) and activating the extracellular signal-regulated kinase (ERK) signaling pathways. Furthermore, blocking the ERK signaling pathway through siERK restored the expression of MMP1 and the invasive ability of PA-treated HCC cells. In conclusion, our results demonstrate the antimetastatic activity of PA against human HCC cells, supporting its potential as a therapeutic agent of HCC treatments.

Endothelial Cell-Specific Molecule 1 Promotes Endothelial to Mesenchymal Transition in Renal Fibrosis.

The endothelial-to-mesenchymal transition (EndoMT) is involved in the complex pathogenesis of renal fibrosis. The soluble proteoglycan endothelial cell-specific molecule 1 (ESM1) is significantly upregulated in many tumor cells and cirrhosis-related disease. The role of ESM1 in renal fibrosis is unknown. This study investigates the role of ESM1 in renal fibrosis, using an in vivo unilateral ureteral obstruction (UUO) mouse model of renal fibrosis and in vitro mouse kidney MES 13 cells overexpressing ESM1. We observed that ESM1 overexpression significantly increased the motility and migration of MES 13 cells, independent of cell viability. In ESM1-overexpressing MES 13 cells, we also observed elevated expression of mesenchymal markers (N-cadherin, vimentin, matrix metallopeptidase 9 (MMP9)) and the fibrosis marker α-smooth muscle actin (α-SMA) and decreased expression of the endothelial marker vascular endothelial cadherin (VE-cadherin) and CD31. In a mouse model of fibrosis induced by unilateral ureter obstruction, we observed time-dependent increases in ESM1, α-SMA, and vimentin expression and renal interstitial collagen fibers in kidney tissue samples. These results suggest that ESM1 may serve as an EndoMT marker of renal fibrosis progression.

Inhibition of eIF2α dephosphorylation accelerates pterostilbene-induced cell death in human hepatocellular carcinoma cells in an ER stress and autophagy-dependent manner.

Hepatocellular carcinoma (HCC) is the one of the most common cancers worldwide. Because the side effects of current treatments are severe, new effective therapeutic strategies are urgently required. Pterostilbene (PT), a natural analogue of resveratrol, has diverse pharmacologic activities, including antioxidative, anti-inflammatory and antiproliferative activities. Here we demonstrated that PT inhibits HCC cell growth without the induction of apoptosis in an endoplasmic reticulum (ER) stress- and autophagy-dependent manner. Mechanistic studies indicated that the combination of salubrinal and PT modulates ER stress-related autophagy through the phospho-eukaryotic initiation factor 2α/activating transcription factor-4/LC3 pathway, leading to a further inhibition of eIF2α dephosphorylation and the potentiation of cell death. An in vivo xenograft analysis revealed that PT significantly reduced tumour growth in mice with a SK-Hep-1 tumour xenograft. Taken together, our results yield novel insights into the pivotal roles of PT in ER stress- and autophagy-dependent cell death in HCC cells.

Breeding and Rearing Naked Mole-Rats (Heterocephalus glaber) under Laboratory Conditions.

The unique biologic characteristics of naked mole-rats (NMR, Heterocephalus glaber) include longevity, cancer resistance, hypoxia tolerance, and pain insensitivity, making NMR an attractive model for biomedical research on aging, cancer, and neurobiology. However, breeding and rearing NMR in captivity is challenging. Here, we report a method for breeding NMR by using a closed-colony mating system. We selected sexually mature male and female NMR from different natal colonies and mated them 1:1. The 5 original colonies had an annual parity of 3.20 ± 0.84 (mean ± 1 SD), with 38.80 ± 9.50 pups born, 33.80 ± 8.32 pups weaned, and a survival rate of 87.19% ± 6.09% after weaning. The average annual parity of 22 N1 pairs (established from the progeny of the 5 original pairs) was 3.09 ± 0.81, with 34.86 ± 10.66 total pups born during the year, 30.14 ± 10.23 pups weaned, and a survival rate after weaning of 85.51% ± 6.60%. The average annual parity of 29 N2 pairs (that is, offspring of N1 pairs) was 3.04 ± 0.87, with 33.69 ± 11.42 pups born, 28.17 ± 10.43 pups weaned, and a survival rate after weaning of 83.66% ± 10.75%. None of these measures differed among the 3 generations, with average reproductive success exceeding 70% for each. In addition, the reproduction and growth of the N1 and N2 generations was similar to the original colonies. Our breeding method remarkably increases the production of NMR, thus representing a great potential to promote experimental NMR research and its applications.

Up-Regulated ATF4 Expression Increases Cell Sensitivity to Apoptosis in Response to Radiation.

BACKGROUND/AIMS: Activating transcription factor 4 (ATF4) is a member of the activating transcription factor family which regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses. ATF4 is also over-expressed in human solid tumors, although its effect on responsiveness to radiation is largely unexplored. METHODS: Real-time PCR was used to detect ATF4 mRNA levels in cells treated with different doses of 60Coγ radiation. Cell viability was assayed using a cell counting kit. The cell cycle was analyzed using flow cytometry, and cell apoptosis was assayed using Annexin V-PI double labeling. Small interfering RNA (siRNA) against ATF4 was transfected into ECV304 cells using Lipofectamine 2000. An ATF4 over-expression plasmid (p-ATF4-CGN) was transfected into HEK293 cells that endogenously expressed low levels of ATF4. The levels of intracellular reactive oxygen species (ROS) were measured using CM-H2DCFDA as a probe. RESULTS: ATF4 mRNA and protein expression levels were higher after radiation and increased in a dose- and time-dependent manner in AHH1 lymphoblast cells (P < 0.05). An increase in ATF4 levels was also observed after radiation in primary murine spleen cells, human endothelial ECV304 cells, human liver LO2 cells, breast cancer MCF7 cells, and human hepatocellular carcinoma HEPG2 cells. No change was observed in human embryonic kidney 293 (HEK293) cells. Over-expressing ATF4 in HEK293 cells inhibited cell proliferation, increased cell apoptosis and significantly increased the proportion of cells in G1 phase. Conversely, when ATF4 expression was knocked down using siRNA in ECV304 cells, it protected the cells from radiation-induced apoptosis. These findings suggest that ATF4 may play a role in radiation-induced cell killing by inhibiting cell proliferation and promoting cell apoptosis. CONCLUSIONS: In this study, we found that radiation up-regulated the expression of ATF4. We used ATF4 knockdown and over-expression systems to show that ATF4 may play a role in radiation-induced cellular apoptosis.

H2O2 treatment or serum deprivation induces autophagy and apoptosis in naked mole-rat skin fibroblasts by inhibiting the PI3K/Akt signaling pathway.

Naked mole-rats (NMR; Heterocephalus glaber) display extreme longevity and resistance to cancer. Here, we examined whether autophagy contributes to the longevity of NMRs by assessing the effects of the PI3K/Akt pathway inhibitor LY294002 and the autophagy inhibitor chloroquine (CQ) on autophagy and apoptosis in NMR skin fibroblasts. Serum starvation, H2O2 treatment, and LY294002 treatment all increased the LC3-II/LC3-I ratio and numbers of double-membraned autophagosomes and autophagic vacuoles, and decreased levels of p70S6K, p-AktSer473, and p-AktThr308. By contrast, CQ treatment decreased p70S6K, AktSer473, and AktThr308 levels. The Bax/Bcl-2 ratio increased after 12 h of exposure to LY294002 or CQ. These data show that inhibiting the Akt pathway promotes autophagy and apoptosis in NMR skin fibroblasts. Furthermore, LY294002 or CQ treatment decreased caspase-3, p53, and HIF1-α levels, suggesting that serum starvation or H2O2 treatment increase autophagy and apoptosis in NMR skin fibroblasts by inhibiting the PI3K/Akt pathway. CQ-induced inhibition of late autophagy stages also prevented Akt activation and induced apoptosis. Finally, the HIF-1α and p53 pathways were involved in serum starvation- or H2O2-induced autophagy in NMR skin fibroblasts.

Activation of the NLRP3 inflammasome in lipopolysaccharide-induced mouse fatigue and its relevance to chronic fatigue syndrome.

BACKGROUND: The NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3) is an intracellular protein complex that plays an important role in innate immune sensing. Its activation leads to the maturation of caspase-1 and regulates the cleavage of interleukin (IL)-1ß and IL-18. Various studies have shown that activation of the immune system plays a pivotal role in the development of fatigue. However, the mechanisms underlying the association between immune activation and fatigue remained elusive, and few reports have described the involvement of NLRP3 inflammasome activation in fatigue. METHODS: We established a mouse fatigue model with lipopolysaccharide (LPS, 3 mg/kg) challenge combined with swim stress. Both behavioural and biochemical parameters were measured to illustrate the characteristics of this model. We also assessed NLRP3 inflammasome activation in the mouse diencephalon, which is the brain region that has been suggested to be responsible for fatigue sensation. To further identify the role of NLRP3 inflammasome activation in the pathogenesis of chronic fatigue syndrome (CFS), NLRP3 KO mice were also subjected to LPS treatment and swim stress, and the same parameters were evaluated. RESULTS: Mice challenged with LPS and subjected to the swim stress test showed decreased locomotor activity, decreased fall-off time in a rota-rod test and increased serum levels of IL-1ß and IL-6 compared with untreated mice. Serum levels of lactic acid and malondialdehyde (MDA) were not significantly altered in the treated mice. We demonstrated increased NLRP3 expression, IL-1ß production and caspase-1 activation in the diencephalons of the treated mice. In NLRP3 KO mice, we found remarkably increased locomotor activity with longer fall-off times and decreased serum IL-1ß levels compared with those of wild-type (WT) mice after LPS challenge and the swim stress test. IL-1ß levels in the diencephalon were also significantly decreased in the NLRP3 KO mice. By contrast, IL-6 levels were not significantly altered. CONCLUSIONS: These findings suggest that LPS-induced fatigue is an IL-1ß-dependent process and that the NLRP3/caspase-1 pathway is involved in the mechanisms of LPS-induced fatigue behaviours. NLRP3/caspase-1 inhibition may be a promising therapy for fatigue treatment.

Toxicity of Smokeless Tobacco Extract after 184-Day Repeated Oral Administration in Rats.

The use of smokeless tobacco (ST) is growing rapidly and globally. The consumption of ST is associated with an increased risk for developing chronic diseases, such as diabetes, hypercholesterolemia, and myocardial infarction, and has led to many public health problems. It is very important to access the toxicity of ST. This experiment presents data from 184-day toxicology studies in Sprague-Dawley (SD) rats designed to characterize the chronic effects of a smokeless tobacco extract (STE). The control group and treatment groups were matched for a range of nicotine levels. Animals were given STE by oral gavage with doses of 3.75 (low-dose), 7.50 (mid-dose) and 15.00 (high-dose) mg · nicotine/kg body weight/day for 184 days, followed by 30 days for recovery. Variables evaluated included body weights, feed consumption, clinical observations, clinical and anatomic pathology (including organ weights), and histopathology. Decreased body weights and organ weights (heart, liver and kidney) were found in animals in the mid-dose and high-dose groups. STE also showed moderate and reversible toxicity in esophagus, stomach, liver, kidney and lung.