Biogenic selenium nanoparticles alleviate intestinal epithelial barrier injury by regulating mitochondria-lysosome crosstalk.

The intestinal epithelial barrier plays a fundamental role in human and animal health. Mitochondrial dysfunction can lead to intestinal epithelial barrier damage. The interaction between mitochondria and lysosomes has been proved to regulate each other's dynamics. Our previous studies have demonstrated that biogenic selenium nanoparticles (SeNPs) can alleviate intestinal epithelial barrier injury through regulating mitochondrial autophagy. In this study, we hypothesize that the protective effects of SeNPs against intestinal epithelial barrier dysfunction are associated with mitochondrial-lysosomal crosstalk. The results showed that lipopolysaccharide (LPS) and TBC1D15 siRNA transfection both caused the increase of intestinal epithelial permeability, activation of mitophagy, and mitochondrial and lysosomal dysfunction in porcine jejunal epithelial cells (IPEC-J2). SeNP pretreatment significantly up-regulated the expression levels of TBC1D15 and Fis1, down-regulated Rab7, caspase-3, MCOLN2 and cathepsin B expression levels, reduced cytoplasmic Ca2+ concentration, effectively alleviated mitochondrial and lysosomal dysfunction, and maintained the integrity of the intestinal epithelial barrier in IPEC-J2 cells exposed to LPS. Furthermore, SeNPs obviously reduced cytoplasmic Ca2+ concentration and activated the TBC1D15/Fis/Rab7-mediated signaling pathway, shortened the contact time between mitochondria and lysosomes, inhibited mitophagy, maintained mitochondrial and lysosomal homeostasis, and effectively attenuated intestinal epithelial barrier injury in IPEC-J2 cells transfected with TBC1D15 siRNA. These results indicated that the protective effect of SeNPs on intestinal epithelial barrier injury is closely associated with the TBC1D15/Rab7-mediated mitochondria-lysosome crosstalk signaling pathway.

GAP positions catalytic H-Ras residue Q61 for GTP hydrolysis in molecular dynamics simulations, complicating chemical rescue of Ras deactivation.

Functional interaction of Ras signaling proteins with upstream, negative regulatory GTPase activating proteins (GAPs) represents a crucial step in cellular decision making related to growth and survival. Key components of the catalytic transition state for Ras deactivation by GAP-accelerated hydrolysis of Ras-bound guanosine triphosphate (GTP) are thought to include an arginine residue from the GAP (the arginine finger), a glutamine residue from Ras (Q61), and a water molecule that is likely coordinated by Q61 to engage in nucleophilic attack on GTP. Here, we use in-vitro fluorescence experiments to show that 0.1-100 mM concentrations of free arginine, imidazole, and other small nitrogenous molecule fail to accelerate GTP hydrolysis, even in the presence of the catalytic domain of a mutant GAP lacking its arginine finger (R1276A NF1). This result is surprising given that imidazole can chemically rescue enzyme activity in arginine-to-alanine mutant protein tyrosine kinases (PTKs) that share many active site components with Ras/GAP complexes. Complementary all-atom molecular dynamics (MD) simulations reveal that an arginine finger GAP mutant still functions to enhance Ras Q61-GTP interaction, though less extensively than wild-type GAP. This increased Q61-GTP proximity may promote more frequent fluctuations into configurations that enable GTP hydrolysis as a component of the mechanism by which GAPs accelerate Ras deactivation in the face of arginine finger mutations. The failure of small molecule analogs of arginine to chemically rescue catalytic deactivation of Ras is consistent with the idea that the influence of the GAP goes beyond the simple provision of its arginine finger. However, the failure of chemical rescue in the presence of R1276A NF1 suggests that the GAPs arginine finger is either unsusceptible to rescue due to exquisite positioning or that it is involved in complex multivalent interactions. Therefore, in the context of oncogenic Ras proteins with mutations at codons 12 or 13 that inhibit arginine finger penetration toward GTP, drug-based chemical rescue of GTP hydrolysis may have bifunctional chemical/geometric requirements that are more difficult to satisfy than those that result from arginine-to-alanine mutations in other enzymes for which chemical rescue has been demonstrated.

Yin and yang regulation of stress granules by Caprin-1.

Stress granules (SGs) are cytoplasmic biomolecular condensates containing proteins and RNAs in response to stress. Ras-GTPase-activating protein binding protein 1 (G3BP1) is a core SG protein. Caprin-1 and ubiquitin specific peptidase 10 (USP10) interact with G3BP1, facilitating and suppressing SG formation, respectively. The crystal structures of the nuclear transport factor 2-like (NTF2L) domain of G3BP1 in complex with the G3BP1-interacting motif (GIM) of Caprin-1 and USP10 show that both GIMs bind to the same hydrophobic pocket of G3BP1. Moreover, both GIMs suppressed the liquid-liquid phase separation (LLPS) of G3BP1, suggesting that Caprin-1 likely facilitates SG formation via other mechanisms. Thus, we dissected various domains of Caprin-1 and investigated their role in LLPS in vitro and SG formation in cells. The C-terminal domain of Caprin-1 underwent spontaneous LLPS, whereas the N-terminal domain and GIM of Caprin-1 suppressed LLPS of G3BP1. The opposing effect of the N- and C-terminal domains of Caprin-1 on SG formation were demonstrated in cells with or without the endogenous Caprin-1. We propose that the N- and C-terminal domains of Caprin-1 regulate SG formation in a "yin and yang" fashion, mediating the dynamic and reversible assembly of SGs.

Liquiritigenin alleviates doxorubicin-induced chronic heart failure via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway.

Chronic heart failure (CHF) is one of the most common chronic diseases with increasing incidence and mortality. Liquiritigenin (LQG) is shown to protect mice from cardiotoxicity. However, its underlying mechanism remains unclear. Our study aimed to reveal the role of ARHGAP18 in LQG-mediated cardioprotective effects in CHF. In the current study, CHF cell model and rat model were established by the application of doxorubicin (DOX). The reactive oxygen species (ROS) level and cell apoptosis were determined by flow cytometry. The cardiac function of rats was evaluated by measuring left ventricular systolic pressure, left ventricular end diastolic pressure, and serum level of lactate dehydrogenase and brain natriuretic peptide. The expression of active RhoA was elevated and that of ARHGAP18 was decreased in DOX-induced CHF cell model. ARHGAP18 could reduce DOX-induced RhoA activation, ROS elevation, and cell apoptosis. Meanwhile, the knockdown of ARHGAP18 could promote the activation of RhoA, the level of ROS, and the rate of cell apoptosis, which could be reversed by the application of RhoA inhibitor. LQG promoted the expression of ARHGAP18 and exerted similar effects of ARHGAP18 in CHF cell model. The application of LQG could also reverse the effects mediated by ARHGAP18 knockdown. Moreover, LQG significantly improved cardiac function and ameliorated DOX-induced cardiotoxicity of CHF rats. In conclusion, LQG could alleviate DOX-induced CHF via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway. LQG was a potential agent for CHF treatment.

Coeloglossum viride var. bracteatum extract attenuates Aß-induced toxicity by inhibiting RIP1-driven inflammation and necroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Tibetan ginseng named Wangla (tuber of Coeloglossum viride var. bracteatum) is a traditional tonic that has Yang-strengthening and qi-enhancing, tranquilizing, intelligence-enhancing and longevity-enhancing properties. It has been used to treat impotence, spermatorrhea, anemia and insomnia. Therefore, its characteristic components and neuronal modulating effects were studied. AIM OF THE STUDY: To investigate the elimination of Aß-induced toxicity by CE and to elucidate the molecular mechanisms involving BDNF, FGF2, and their related signaling axis, and the RIP1-driven inflammatory pathway. MATERIALS AND METHODS: We established Aß-induced toxicity models in cultured neurons and ICR mice, respectively. MWM and fear conditioning tests were performed for behavioral analysis of cognitive functions in mice. Western blot was used to investigate the levels of BDNF, FGF2, and their downstream effector TrkB/Akt/Bcl-2, as well as the RIP1-driven RIP1/RIP3/MLKL pathway. Immunofluorescence assay is used to examine the status of glial cells. RESULTS: CE abrogated Aß toxicity and inhibited apoptosis in cultured neurons, mainly by regulating the BDNF, FGF2, and TrkB/Akt signaling pathways as well as RIP1-driven inflammation and necroptosis. Similarly, mice injected intracerebrally with Aß exhibited cognitive deficits and had elevated oxidative stress and inflammatory factors detected in their serum and brain. However, CE-treated mice showed recovery of cognitive abilities and quelled levels of oxidative stress and inflammatory factors. Moreover, Aß toxicity led to a reduction in BDNF, FGF2, and related signaling regulators in the hippocampus and prefrontal cortex, accompanied by activation of RIP1-driven inflammatory signaling pathways, and a reduction in TBK1 and Bcl-2. However, CE restored the levels of BDNF, FGF2, and TrkB/Akt signaling pathway, while inhibiting RIP1-induced RIP1/RIP3/MLKL pathway, thereby antagonizing apoptosis and maintaining neuronal activity. CONCLUSIONS: CE effectively eliminated the toxicity of Aß in cultured neurons and mouse models, which holds promise for drug development.

Members of the ELMOD protein family specify formation of distinct aperture domains on the Arabidopsis pollen surface.

Pollen apertures, the characteristic gaps in pollen wall exine, have emerged as a model for studying the formation of distinct plasma membrane domains. In each species, aperture number, position, and morphology are typically fixed; across species they vary widely. During pollen development, certain plasma membrane domains attract specific proteins and lipids and become protected from exine deposition, developing into apertures. However, how these aperture domains are selected is unknown. Here, we demonstrate that patterns of aperture domains in Arabidopsis are controlled by the members of the ancient ELMOD protein family, which, although important in animals, has not been studied in plants. We show that two members of this family, MACARON (MCR) and ELMOD_A, act upstream of the previously discovered aperture proteins and that their expression levels influence the number of aperture domains that form on the surface of developing pollen grains. We also show that a third ELMOD family member, ELMOD_E, can interfere with MCR and ELMOD_A activities, changing aperture morphology and producing new aperture patterns. Our findings reveal key players controlling early steps in aperture domain formation, identify residues important for their function, and open new avenues for investigating how diversity of aperture patterns in nature is achieved.

Zooming in on cells reveals patterns on their outer surfaces. These patterns are actually a collection of distinct areas of the cell surface, each containing specific combinations of molecules. The outer layers of pollen grains consist of a cell wall, and a softer cell membrane that sits underneath. As a pollen grain develops, it recruits certain fats and proteins to specific areas of the cell membrane, known as 'aperture domains'. The composition of these domains blocks the cell wall from forming over them, leading to gaps in the wall called 'pollen apertures'. Pollen apertures can open and close, aiding reproduction and protecting pollen grains from dehydration. The number, location, and shape of pollen apertures vary between different plant species, but are consistent within the same species. In the plant species Arabidopsis thaliana, pollen normally develops three long and narrow, equally spaced apertures, but it remains unclear how pollen grains control the number and location of aperture domains. Zhou et al. found that mutations in two closely related A. thaliana proteins ­ ELMOD_A and MCR ­ alter the number and positions of pollen apertures. When A. thaliana plants were genetically modified so that they would produce different levels of ELMOD_A and MCR, Zhou et al. observed that when more of these proteins were present in a pollen grain, more apertures were generated on the pollen surface. This finding suggests that the levels of these proteins must be tightly regulated to control pollen aperture numbers. Further tests revealed that another related protein, called ELMOD_E, also has a role in domain formation. When artificially produced in developing pollen grains, it interfered with the activity of ELMOD_A and MCR, changing pollen aperture shape, number, and location. Zhou et al. identified a group of proteins that help control the formation of domains in the cell membranes of A. thaliana pollen grains. Further research will be required to determine what exactly these proteins do to promote formation of aperture domains and whether similar proteins control domain development in other organisms.

14-Deoxy-11,12-Didehydroandrographolide Ameliorates Glucose Intolerance Enhancing the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 Signaling Pathway and Inducing GLUT4 Expression in Myotubes and Skeletal Muscle of Obese Mice.

14-Deoxy-11,12-didehydroandrographolide (deAND), a bioactive component of Andrographis paniculata, has antidiabetic activity. AMP-activated protein kinase (AMPK) regulates glucose transport and ameliorates insulin resistance. The aim of the present study was to investigate whether activation of AMPK is involved in the mechanism by which deAND ameliorates insulin resistance in muscles. deAND amounts up to 40 [Formula: see text]M dose-dependently activated phosphorylation of AMPK[Formula: see text] and TBC1D1 in C2C12 myotubes. In addition, deAND significantly activated phosphorylation of LKB1 at 6 h after treatment, and this activation was maintained up to 48 h. deAND increased glucose uptake at 18 h after treatment, and this increase was time dependent up to 72 h. Compound C, an inhibitor of AMPK, suppressed deAND-induced phosphorylation of AMPK[Formula: see text] and TBC1D1 and reversed the effect on glucose uptake. In addition, the expression of GLUT4 mRNA and protein in C2C12 myotubes was up-regulated by deAND in a time-dependent manner. Promotion of GLUT4 gene transcription was verified by a pGL3-GLUT4 (837 bp) reporter assay. deAND also increased the nuclear translocation of MEF-2A and PPAR[Formula: see text]. After 16 weeks of feeding, the high-fat diet (HFD) inhibited phosphorylation of AMPK[Formula: see text] and TBC1D1 in skeletal muscle of obese C57BL/6JNarl mice, and deactivation of AMPK[Formula: see text] and TBC1D1 by the HFD was abolished by deAND supplementation. Supplementation with deAND significantly promoted membrane translocation of GLUT4 compared with the HFD group. Supplementation also significantly increased GLUT4 mRNA and protein expression in skeletal muscle compared with the HFD group. The hypoglycemic effects of deAND are likely associated with activation of the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 signaling pathway and stimulation of MEF-2A- and PPAR[Formula: see text]-dependent GLUT4 gene expression, which account for the glucose uptake into skeletal muscle and lower blood glucose levels.

Rosemary extract increases neuronal cell glucose uptake and activates AMPK.

Glucose is the primary metabolic substrate of neurons and is responsible for supporting many vital functions including neuronal signalling. Decreases in glucose uptake and utilization are common characteristics of dementia, particularly Alzheimer's disease, and thus agents that can restore neuronal glucose availability may be especially valuable to the field. Diets rich in antioxidants and polyphenols have been associated with reductions in the risk of chronic disease that are associated with aging. In previous studies, rosemary extract (RE) has been reported to have antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. The purpose of the present study was to explore the effects of RE on neuronal glucose uptake. Human SH-SY5Y neuroblastoma cells exposed to varied concentrations of RE showed a dose-dependent increase in glucose uptake, with a significant increase observed following treatment with 5 µg/mL RE for 2 h (159% ± 20.81% of control) that was comparable to maximum insulin stimulation (135.6% ± 3.2% of control). This increase in glucose uptake was paralleled by increases in AMP-activated protein kinase (AMPK), but not Akt, phosphorylation/activation. The present study is the first to report that treatment with rosemary extract can stimulate glucose uptake in a neuronal cell line. These results demonstrate the potential of RE to be used as an agent to regulate neuronal glucose homeostasis. Novelty: RE increases neuronal glucose uptake. RE activates AMPK in neurons. RE increases neuronal glucose uptake independently of insulin signalling.

Limonene, a food additive, and its active metabolite perillyl alcohol improve regeneration and attenuate neuropathic pain after peripheral nerve injury: Evidence for IL-1ß, TNF-α, GAP, NGF and ERK involvement.

BACKGROUND: Limonene (LIM) and its main metabolite perillyl alcohol (POH) are ingredients found in food with promising chemical entities due to their pharmacological profile. In this study, we hypothesized that LIM and POH are two molecules capable of accelerating the regenerative process and alleviating neuropathic pain. METHODS: Animals were treated daily (LIM, POH and saline) for 28 days and during this period evaluated for mechanical hyperalgesia, astrocyte participation by immunofluorescence for GFAP, and ELISA was used to quantify IL-1ß and TNF-α in the spinal cord. Western blot analysis of the following proteins was also performed: GFAP, GAP-43, NGF and ERK. For motor deficit analysis, tests were performed to assess hind paw muscle strength and footprints through gait (SFI). RESULTS: Both POH and LIM accelerated the regenerative process and improved motor deficits comparing to positive control; however, POH was more effective, particularly between the 2nd and 3rd week after the nerve injury, increasing GAP-43, NGF and the phosphorylated ERK immunocontent. Moreover, POH and LIM were able to reduce hyperalgesia and astrocytosis. CONCLUSIONS: Both substances, LIM and POH, improved the regeneration process and sensory and motor function recovery in the PNI model in mice by mitigating the inflammatory reactions and up-regulating the neurotrophic process.

Protective role of Astragaloside IV in chronic glomerulonephritis by activating autophagy through PI3K/AKT/AS160 pathway.

Astragaloside IV(AS-IV), a saponin purified from Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao, has been widely used in traditional Chinese medicine. However, the underlying mechanisms in treating chronic glomerular nephritis (CGN) have not been fully understood. The aim of the present study was to evaluate the potential mechanism of AS-IV on CGN. CGN rats were administrated with AS-IV at 10 mg·kg-1 ·d-1 (ASL) and 20 mg·kg-1 ·d-1 (ASH). Twenty four hour proteinuria, blood urea nitrogen (BUN), and serum creatinine (SCr) were detected. Hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining were performed to evaluate the kidney lesion. Transmission electron microscope and GFP-RFP-LC3 transfection assay were used to monitor the effect of AS-IV on autophagy. IL-6 and IL-1ß were detected. The expression of CyclinD1, PI3K/AKT/AS160 pathway and autophagy related proteins were detected by Western Blot. The results demonstrated that AS-IV improved kidney function, ameliorated kidney lesion, and diminished inflammatory in CGN rats. Further, both in vivo and vitro study demonstrated that AS-IV inhibited the proliferation of mesangial cells. AS-IV further displayed a remarkable effect on inhibiting the activation of PI3K/AKT/AS160 pathway and improved the activation of autophagy in vivo and vitro. These results suggested that AS-IV is a potential therapeutic agent for CGN and merits further investigation.

Quantitative proteomic analysis reveals that the Rap1/MAPK/ERK pathway is inhibited through selenomethionine strengthening antioxidant activity.

To investigate the influence on the proteome of chicken skeletal muscles of Selenomethionine (SeMet) use, 36 chicks were fed with SeMet feeding for 35 days. A total of 72 1-day old broiler chicks were randomly allocated into two groups (n = 36/group): the control group (C group), the SeMet supplemented group (SeMet group). The Selenium (Se) concentrations of skeletal muscles from the chicks with basal diet (negative control group) and SeMet feeding were found to be 0.01 mg/kg and 0.40 mg/kg, respectively. The skeletal muscles from the two groups were investigated using isobaric Tags for Relative and Absolute Quantitation (iTRAQ), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This proteomic analysis identified proteins that were differentially expressed between the two groups. A total of 3564 proteins from the SeMet and the control (C) groups at 35 days were analyzed. 86 proteins were found by iTRAQ to be differentially expressed in the SeMet group, including 38 up-regulated proteins and 48 down-regulated proteins. These differential proteins were later identified as being mainly involved in antioxidant and enzyme-regulating activities. Fluorescent quantitative PCR(qPCR) and Western blot analyse proved to be consistent with the results of iTRAQ identification. The differentially expressed proteins (DEPs) identified in our work could be specific biomarkers related to SeMet intake in chicks. SeMet intake may strengthen antioxidant activity through Rap1/mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) signal pathways.

Danhong Injection Alleviates Cardiac Fibrosis via Preventing the Hypermethylation of Rasal1 and Rassf1 in TAC Mice.

BACKGROUND/AIM: Danhong injection (DHI) is a Chinese patent drug used for relieving cardiovascular diseases. Recent studies have suggested that DNA methylation plays a pivotal role in the maintenance of cardiac fibrosis (CF) in cardiovascular diseases. This study was aimed at identifying the effect and the underlying mechanism of DHI on CF, especially the DNA methylation. METHODS: A CF murine model was established by thoracic aortic constriction (TAC). A 28-day daily treatment with or without DHI via intraperitoneal injection was carried out immediately following TAC surgery. The changes in cardiac function, pathology, and fibrosis following TAC were measured by echocardiography and immunostaining. We used methyl-seq analysis to assess the DNA methylation changes in whole genes and identified the methylation changes of two Ras signaling-related genes in TAC mice, including Ras protein activator like-1 (Rasal1) and Ras-association domain family 1 (Rassf1). Next, the methylation status and expression levels of Rasal1 and Rassf1 genes were consolidated by bisulfite sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting, respectively. To determine the underlying molecular mechanism, the expressions of DNA methyltransferases (DNMTs), Tet methylcytosine dioxygenase 3 (TET3), fibrosis-related genes, and the activity of Ras/ERK were measured by RT-qPCR and Western blotting. RESULTS: DHI treatment alleviated CF and significantly improved cardiac function on day 28 of TAC. The methyl-seq analysis identified 42,606 differential methylated sites (DMSs), including 19,618 hypermethylated DMSs and 22,988 hypomethylated DMSs between TAC and sham-operated mice. The enrichment analysis of these DMSs suggested that the methylated regulation of Ras signal transduction and focal adhesion-related genes would be involved in the TAC-induced CF development. The results of bisulfite sequencing revealed that the TAC-induced methylation affected the CpG site in both of Rasal1 and Rassf1 genes, and DHI treatment remarkably downregulated the promoter methylation of Rasal1 and Rassf1 in CF hearts. Furthermore, DHI treatment upregulated the expressions of Rasal1 and Rassf1, inhibited the hyperactivity of Ras/ERK, and decreased the expressions of fibrosis-related genes. Notably, we found that DHI treatment markedly downregulated the expression of DNMT3B in CF hearts, while it did not affect the expressions of DNMT1, DNMT3A, and TET3. CONCLUSION: Aberrant DNA methylation of Rasal1 and Rassf1 genes was involved in the CF development. DHI treatment alleviated CF, prevented the hypermethylation of Rasal1 and Rassf1, and downregulated DNMT3B expression in CF hearts.

Weichang'an suppressed migration and invasion of HCT116 cells by inhibiting Wnt/ß-catenin pathway while upregulating ARHGAP25.

Evidence suggests that Weichang'an (WCA) inhibited the metastasis of colorectal cancer (CRC) in vitro and downregulates oncogenic ß-catenin; more intriguingly, we also found an upregulation of ARHGAP25 in this process. This study aimed to investigate the mechanisms by which WCA regulated CRC metastasis in vitro. Here, HCT116 cells were transfected with siRNAs to interfere ARHGAP25 expression. WCA decoction, XAV939 (a specific Wnt/ß-catenin pathway inhibitor), and LiCl (an activator for Wnt/ß-catenin pathway) were used for treatment. Cell migratory and invasive capacities were determined using Transwell chamber. The activation of Wnt/ß-catenin pathway was assessed by determining the expression of MMP7, MMP9, ZEB1, and ß-catenin. The study suggests that WCA inhibited the migration and invasion of HCT116 cells and suppressed the activation of Wnt/ß-catenin pathway, as evidenced by retarding MMP7, MMP9, ZEB1, and ß-catenin. However, siRNA-ARHGAP25 resulted in the opposite. In siRNA-ARHGAP25-transfected HCT116 cells, WCA (0.4 mg/mL) induced the antimetastatic effects and the inactivation of Wnt/ß-catenin pathway was remarkably reversed with additional LiCl treatment. Our study concludes that inhibiting Wnt/ß-catenin pathway while promoting ARHGAP25 was the mechanism, whereby WCA retarded migration and invasion of CRC in vitro.

Herbs-partitioned moxibustion alleviates aberrant intestinal epithelial cell apoptosis by upregulating A20 expression in a mouse model of Crohn's disease.

BACKGROUND: A20 inhibits intestinal epithelial cell apoptosis in Crohn's disease, and herbs-partitioned moxibustion (HPM) has been demonstrated to be an effective treatment for Crohn's disease. However, the mechanism by which HPM reduces intestinal epithelial cell apoptosis in Crohn's disease has not been thoroughly elucidated to date. AIM: To elucidate whether HPM exerts its effects by upregulating A20 to affect intestinal epithelial cell apoptosis in a Crohn's disease mouse model. METHODS: In this study, mice with A20 deletion in intestinal epithelial cells (A20IEC-KO) were utilized to establish a Crohn's disease mouse model with 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration, as well as wild-type mice. Mice were randomly divided into normal control (NC), model control (MC), mesalazine (MESA), and HPM groups. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and serum endotoxin and apoptosis of epithelial cells were evaluated by enzyme-linked immunosorbent assay and terminal dUTP nick-end labeling assay accordingly. The protein expression levels of A20 and tumor necrosis factor receptor 1 (TNFR1)-related signaling molecules were evaluated by Western blot, and co-expression of A20 and TNFR1-associated death domain (TRADD) and co-expression of A20 and receptor-interacting protein 1 (RIP1) were observed by double immunofluorescence staining. RESULTS: The intestinal epithelial barrier was noted to have an improvement in the HPM group of wild-type (WT) mice compared with that in A20IEC-KO mice. Compared with A20 IEC-KO HPM mice, serum endotoxin levels and apoptosis percentages were decreased (P < 0.01), A20 expression levels were increased (P < 0.01), and expression of TNFR1, TRADDD, and RIP1 was decreased in the HPM group of WT mice (P TNFR1 < 0.05, P TRADD < 0.01, P RIP1 < 0.01). Both of the co-expression of A20/TRADD and A20/RIP1 showed a predominantly yellow fluorescence in the HPM group of WT mice, while a predominantly red fluorescence was noted in the HPM group of A20IEC-KO mice. CONCLUSION: Our findings suggest that HPM in treating Crohn's disease functions possibly via upregulation of the A20 expression level, resulting in downregulation of TNFR1, TRADD, and RIP1 to alleviate increased cell apoptosis in the intestinal epithelial barrier in Crohn's disease.

Evaluation of the Liver Toxicity of Pterocephalus hookeri Extract via Triggering Necrosis.

Pterocephalus hookeri (C. B. Clarke) Höeck, recorded in the Chinese Pharmacopoeia (2015 version) as a Tibetan medicine for the treatment of various diseases, especially rheumatoid arthritis, was believed to possess a slight toxicity. However, hardly any research has been carried out about it. The present study aimed to evaluate the toxicity in vivo and in vitro. Toxicity was observed by the evaluation of mice weight loss and histopathological changes in the liver. Then, the comparison research between ethyl acetate extract (EAE) and n-butanol extract (BUE) suggested that liver toxicity was mainly induced by BUE. The mechanical study suggested that BUE-induced liver toxicity was closely associated with necrosis detected by MTT and propidium iodide (PI) staining, via releasing lactate dehydrogenase (LDH), reducing the fluidity, and increasing the permeability of the cell membrane. Western blot analysis confirmed that the necrosis occurred molecularly by the up-regulation of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3), as well as the activation of the nuclear factor-kappa-gene binding (NF-κB) signaling pathway in vivo and in vitro. This finding indicated that the liver toxicity induced by BUE from P. hookeri was mainly caused by necrosis, which provides an important theoretical support for further evaluation of the safety of this folk medicine.

p53 Up-regulated Modulator of Apoptosis Induction Mediates Acetaminophen-Induced Necrosis and Liver Injury in Mice.

Acetaminophen (APAP) overdose is one of the leading causes of hepatotoxicity and acute liver failure in the United States. Accumulating evidence suggests that hepatocyte necrosis plays a critical role in APAP-induced liver injury (AILI). However, the mechanisms of APAP-induced necrosis and liver injury are not fully understood. In this study, we found that p53 up-regulated modulator of apoptosis (PUMA), a B-cell lymphoma-2 (Bcl-2) homology domain 3 (BH3)-only Bcl-2 family member, was markedly induced by APAP in mouse livers and in isolated human and mouse hepatocytes. PUMA deficiency suppressed APAP-induced mitochondrial dysfunction and release of cell death factors from mitochondria, and protected against APAP-induced hepatocyte necrosis and liver injury in mice. PUMA induction by APAP was p53 independent, and required receptor-interacting protein kinase 1 (RIP1) and c-Jun N-terminal kinase (JNK) by transcriptional activation. Furthermore, a small-molecule PUMA inhibitor, administered after APAP treatment, mitigated APAP-induced hepatocyte necrosis and liver injury. Conclusion: Our results demonstrate that RIP1/JNK-dependent PUMA induction mediates AILI by promoting hepatocyte mitochondrial dysfunction and necrosis, and suggest that PUMA inhibition is useful for alleviating acute hepatotoxicity attributed to APAP overdose.

Cancer growth regulation by 4-hydroxynonenal.

While reactive oxygen species (ROS) gain their carcinogenic effects by DNA mutations, if generated in the vicinity of genome, lipid peroxidation products, notably 4-hydroxynonenal (HNE), have much more complex modes of activities. Namely, while ROS are short living and have short efficiency distance range (in nm or µm) HNE has strong binding affinity for proteins, thus forming relatively stable adducts. Hence, HNE can diffuse from the site or origin changing structure and function of respective proteins. Consequently HNE can influence proliferation, differentiation and apoptosis of cancer cells on one hand, while on the other it can affect genome functionality, too. Although HNE is considered to be important factor of carcinogenesis due to its ability to covalently bind to DNA, it might also be cytotoxic for cancer cells, as well as it can modulate their growth. In addition to direct cytotoxicity, HNE is also involved in activity mechanisms by which several cytostatic drugs and radiotherapy exhibit their anticancer effects. Complementary to that, the metabolic pathway for HNE detoxification through RLIP76, which is enhanced in cancer, may be a target for anti-cancer treatments. In addition, some cancer cells can undergo apoptosis or necrosis, if exposed to supraphysiological HNE levels in the cancer microenvironment, especially if challenged additionally by pro-oxidative cytostatics and/or inflammation. These findings could explain previously observed disappearance of HNE from invading cancer cells, which is associated with the increase of HNE in non-malignant cells close to invading cancer utilizing cardiolipin as the source of cancer-inhibiting HNE.

Benefits of L-alanine or L-arginine supplementation against adiposity and glucose intolerance in monosodium glutamate-induced obesity.

PURPOSE: L-alanine (Ala) and L-arginine (Arg) have been reported to regulate pancreatic ß-cell physiology and to prevent body fat accumulation in diet-induced obesity. Here, we assessed growth and adiposity parameters, glucose tolerance, insulin secretion and the expression of insulin and nutrient-regulated proteins in monosodium glutamate (MSG)-obese mice supplemented with either Ala or Arg. METHODS: Male newborn C57Bl/6 mice received a daily subcutaneous injection of MSG or saline solution (CTL group), during the first 6 days of life. From 30 to 90 days of age, MSG and CTL mice received or not 2.55 % Ala (CAla or MArg groups) or 1.51 % Arg-HCl (CArg or MArg groups) in their drinking water. RESULTS: Adult MSG mice displayed higher adiposity associated with lower phosphorylation of the adipogenic enzyme, ACC, in adipose tissue. Glucose intolerance in MSG mice was linked to lower insulin secretion and to lower expression of IRß in adipose tissue, as well as AS160 phosphorylation in skeletal muscle. Perigonadal fat depots were smaller in Ala and Arg mice, while retroperitoneal fat pads were decreased by Ala supplementation only. Both Ala and Arg improved fed-state glycemia as well as IRß and pAS160 content, but only Ala led to improved glucose tolerance and insulin secretion. Adipostatic signals were increased in MAla mice, as indicated by enhanced AMPK phosphorylation and pACC content in fat depots. CONCLUSIONS: Ala supplementation led to more pronounced metabolic improvements compared to Arg, possibly due to suppression of lipogenesis through activation of the AMPK/ACC pathway.

TSC2 Deficiency Unmasks a Novel Necrosis Pathway That Is Suppressed by the RIP1/RIP3/MLKL Signaling Cascade.

Tuberous sclerosis complex (TSC) is a genetic multiorgan disorder characterized by the development of neoplastic lesions in kidney, lung, brain, heart, and skin. It is caused by an inactivating mutation in tumor suppressor genes coding the TSC1/TSC2 complex, resulting in the hyperactivation of mTOR- and Raf/MEK/MAPK-dependent signaling that stimulates tumor cell proliferation and metastasis. Despite its oncogenic effect, cells with TSC deficiency were more sensitive to oxidative stress and dependent on mitochondrial metabolism, providing a rationale for a new therapeutic approach. The current study shows that simultaneous inhibition of two major pathways regulating redox homeostasis using l-buthionine-sulfoximine (BSO, glutathione synthesis inhibitor) and auranofin (thioredoxin reductase inhibitor) induces oxidative burst, mitochondrial damage, and necrotic cell death in TSC-deficient cells in a highly synergistic and cell context-specific manner. Furthermore, blocking RIP1/RIP3/MLKL-dependent signaling using chemical inhibitors necrostatin-1 (Nec-1) and necrosulfonamide (NSA) synergizes with BSO and auranofin in killing TSC-deficient cells. Expression analysis demonstrated that RIP1, RIP3, and MLKL protein levels are elevated in cells with TSC2 deficiency, and their inactivation enhances mitochondrial dysfunction in a glutaminolysis-dependent and autophagy-independent manner. Finally, supplementation with the mitochondrial metabolite α-ketoglutarate, whose synthesis is regulated by RIP1/RIP3/MLKL, rescues cells from the sensitizing effect of Nec-1 and NSA. Together, this study identifies a previously unrecognized novel regulated necrotic death pathway that involves mitochondrial homeostasis, is suppressed by the RIP1/RIP3/MLKL signaling in TSC-deficient cells, and could be a promising therapeutic target for TSC-associated tumors. Cancer Res; 76(24); 7130-9. ©2016 AACR.

Upregulation of miR-124 by physcion 8-O-ß-glucopyranoside inhibits proliferation and invasion of malignant melanoma cells via repressing RLIP76.

Melanoma is the most malignant type of skin cancer. In recent years, mounting studies have evidenced the involvement of miRNAs in melanoma. One of these miRNAs, miR-124 has been found aberrantly downregulated in a variety of human malignancies. In this study, our results showed that the expression of miR-124 was significantly lower in malignant melanoma tissues and cell lines and miR-124 functioned as a tumor suppressor in melanoma. Moreover, our findings showed that miR-124 exerted anti-tumor effect by directly targeting RLIP76, a stress-inducible non-ABC transporter that plays a crucial role in the development of melanoma. Furthermore, our study also showed that physcion 8-O-ß-glucopyranoside, a natural compound from medicinal plant, could inhibit the proliferation and invasion of melanoma cells by targeting miR-124/RLIP76 signaling.