Interactions between corn starch and lingonberry polyphenols and their effects on starch digestion and glucose transport.

This study investigated the interaction mechanism between corn starch (CS) and lingonberry polyphenols (LBP) during starch gelatinization, focusing on their effects on starch structure and physicochemical properties. Moreover, it explored the effect of this interaction on starch digestion and glucose transport. The results indicated that LBP interacted non-covalently with CS during starch gelatinization, disrupted the short-range ordered structure of starch, decreased gelatinization enthalpy of starch, and formed a dense network structure. Furthermore, the incorporation of LBP remarkably reduced the digestibility of CS. In particular, the addition of 10 % LBP decreased the terminal digestibility (C∞) from 77.87 % to 60.43 % and increased the amount of resistant starch (RS) by 21.63 %. LBP was found to inhibit α-amylase and α-glucosidase in a mixed manner. Additionally, LBP inhibited glucose transport in Caco-2 cells following starch digestion. When 10 % LBP was added, there was a 34.17 % decrease in glucose transport compared with starch digestion without LBP. This study helps establish the foundation for the development of LBP-containing starch or starch-based healthy foods and provides new insights into the mechanism by which LBP lowers blood glucose.

Ganoderic acid A mitigates dopaminergic neuron ferroptosis via inhibiting NCOA4-mediated ferritinophagy in Parkinson's disease mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum, a renowned tonic traditional Chinese medicine, is widely recognized for the exceptional activity in soothing nerves and nourishing the brain. It has been extensively employed to alleviate various neurological disorders, notably Parkinson's disease (PD). AIM OF THE STUDY: To appraise the antiparkinsonian effect of GAA, the main bioactive constituent of G. lucidum, and clarify the molecular mechanism through the perspective of ferritinophagy-mediated dopaminergic neuron ferroptosis. MATERIALS AND METHODS: PD mouse and cell models were established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+), respectively. Cell viability, behavioral tests and immunofluorescence analysis were performed to evaluate the neurotoxicity, motor dysfunction and dopaminergic loss, respectively. Biochemical assay kits were used to determine the levels of iron, lipid reactive oxygen species (ROS), malondialdehyde (MDA), total ROS and glutathione (GSH). Western blot and immunofluorescence were applied to detect the expressions of nuclear receptor co-activator 4 (NCOA4), ferritin heavy chain 1 (FTH1), p62 and LC3B. Additionally, NCOA4-overexpressing plasmid vector was constructed to verify the inhibitory effect of GAA on the neurotoxicity and ferroptosis-related parameters in PD models. RESULTS: GAA significantly mitigated MPP+/MPTP-induced neurotoxicity, motor dysfunction and dopaminergic neuron loss (p＜0.01 or p＜0.05). In contrast to MPP+/MPTP treatment, GAA treatment decreased the levels of iron, MDA, lipid and total ROS, while increasing the GSH level. GAA also reduced the levels of NCOA4 and LC3B, and enhanced the expressions of FTH1 and p62 in PD models (p＜0.01 or p＜0.05). However, the protective effect of GAA against the neurotoxicity, NCOA4-mediated ferritinophagy and ferroptosis in PD model was abolished by the overexpression of NCOA4 (p＜0.01). CONCLUSION: GAA exerted a protective effect on PD, and this effect was achieved by suppressing dopaminergic neuron ferroptosis through the inhibition of NCOA4-mediated ferritinophagy.

Rapid Discovery of Aß42 Fibril Disintegrators from Ganoderma lucidum via Ligand Fishing and Their Neuroprotective Effects on Alzheimer's Disease.

An amyloid-ß (Aß) fibril is a vital pathogenic factor of Alzheimer's disease (AD). Aß fibril disintegrators possess great potential to be developed into novel anti-AD agents. Here, a ligand fishing method was employed to rapidly discover Aß42 fibril disintegrators from Ganoderma lucidum using Aß42 fibril-immobilized magnetic beads, which led to the isolation of six Aß42 fibril disintegrators including ganodermanontriol, ganoderic acid DM, ganoderiol F, ganoderol B, ganodermenonol, and ergosterol. Neuroprotective evaluation in vitro exhibited that these Aß42 fibril disintegrators could significantly mitigate Aß42-induced neurotoxicity. Among these six disintegrators, ergosterol and ganoderic acid DM with stronger protecting activity were further selected to evaluate their neuroprotective effect on AD in vivo. Results showed that ergosterol and ganoderic acid DM could significantly alleviate Aß42-induced cognitive dysfunction and hippocampus neuron loss in vivo. Moreover, ergosterol and ganoderic acid DM could significantly inhibit Aß42-induced neuron apoptosis and Nrf2-mediated neuron oxidative stress in vitro and in vivo.

Transcriptional regulatory cascade of LcMYB71 and LcNAC73 affects low-temperature and drought stress response in Lonicera caerulea.

The development of stress tolerance is regulated via the transcriptional regulatory networks involving regulatory homeostasis mediated by protein-DNA interactions. LcNAC73 from Lonicera caerulea was characterized to understand the underlying mechanism of low-temperature and drought stress response in L. caerulea. To better understand the transcription pathway of LcNAC73, we cloned the promoter and screened proteins that could interact with the promoter. Using Yeast one-hybrid, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we found that the LcMYB71 protein specifically bound to the promoter of LcNAC73. The transient transformation and stable transgenic system were used to produce transgenic L. caerulea plants with overexpressed and silenced LcNAC73, elucidating the effect of LcNAC73 on low-temperature and drought stress tolerance. LcNAC73 positively regulated the proline content and enhanced the scavenging of reactive oxygen species, thus improving tolerance to low-temperature and drought stress. Further studies revealed that LcMYB71 and LcNAC73 had similar functions and could improve plant low-temperature and drought tolerance. It is necessary to identify the upstream regulators of a specific gene to characterize gene functions and the associated transcriptional pathways.

Human milk-derived MANF, as an immuno-nutritional factor, maintains the intestinal epithelial barrier and protects against necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of death in preterm infants. Compared to formula milk, breastfeeding protects against NEC. However, the composition of breast milk is quite complicated, and many immunological compositions remain unknown. In this study, we aimed to investigate the concentration of a secreted protein, Mesencephalic astrocyte-derived neurotrophic factor (MANF), in breastmilk and evaluate its immune-regulatory function in protecting the intestinal epithelial barrier. Our data indicated that MANF was secreted in human milk but could not be detected in infant formulas. More importantly, the amount of MANF in colostrum was higher than that in mature milk. We also clarified that MANF was mainly expressed in intestinal macrophages and was capable of inducing apoptosis and decreasing the inflammation of pro-inflammatory macrophages in both NEC intestinal tissues and BMDMs. Mechanismly, MANF protein significantly inhibited the apoptosis of intestinal epithelial cells and protected epithelial tight junctions through downregulation of the NF-κB pathway in pro-inflammatory macrophages. These results reveal the crucial function of human milk-derived MANF in intestinal macrophages, which contributes to downregulating the intestinal inflammatory response and protecting the homeostasis of intestinal epithelial cells. Our study not only demonstrates a potential mechanism underlying breastfeeding protective effects in NEC but also, more importantly, enables clinical translation, facilitating new strategies for the development of nutritional interventions in the prevention of NEC.

Role of Bmal1 in Type 2 Diabetes Mellitus-Related Glycolipid Metabolic Disorder and Neuropsychiatric Injury: Involved in the Regulation of Synaptic Plasticity and Circadian Rhythms.

Increasing data suggest a crucial role of circadian rhythm in regulating metabolic and neurological diseases, and Bmal1 is regarded as a key regulator of circadian transcription. The aim of this study is to investigate the role of Bmal1 in the disruption of circadian rhythm and neuropsychiatric injuries in type 2 diabetes mellitus (T2DM). A T2DM model was induced by the combination of high-fat-diet (HFD) and streptozotocin (STZ) in vivo or HT-22 cells challenged with palmitic-acid (PA) in vitro. The glucolipid metabolism indicators, behavioral performance, and expression of synaptic plasticity proteins and circadian rhythm-related proteins were detected. These changes were also observed after interference of Bmal1 expression via overexpressed plasmid or small interfering RNAs in vitro. The results showed that HFD/STZ could induce T2DM-like glycolipid metabolic turmoil and abnormal neuropsychiatric behaviors in mice, as indicated by the increased concentrations of fasting blood-glucose (FBG), HbA1c and lipids, the impaired glucose tolerance, and the decreased preference index of novel object or novel arm in the novel object recognition test (NOR) and Y-maze test (Y-maze). Consistently, the protein expression of synaptic plasticity proteins and circadian rhythm-related proteins and the positive fluorescence intensity of MT1B and Bmal1 were decreased in the hippocampus of HFD/STZ-induced mice or PA-challenged HT-22 cells. Furthermore, overexpression of Bmal1 could improve the PA-induced lipid metabolic dysfunction and increase the decreased expressions of synaptic plasticity proteins and circadian rhythm-related proteins, and vice versa. These results suggested a crucial role of Bmal1 in T2DM-related glycolipid metabolic disorder and neuropsychiatric injury, which mechanism might be involved in the regulation of synaptic plasticity and circadian rhythms.

Ultrasmall zirconium carbide nanodots for synergistic photothermal-radiotherapy of glioma.

Glioma is characterized by highly invasive, progressive, and lethal features. In addition, conventional treatments have been poorly effective in treating glioma. To overcome this challenge, synergistic therapies combining radiotherapy (RT) with photothermal therapy (PTT) have been proposed and extensively explored as a highly feasible cancer treatment strategy. Herein, ultrasmall zirconium carbide (ZrC) nanodots were successfully synthesized with high near-infrared absorption and strong photon attenuation for synergistic PTT-RT of glioma. ZrC-PVP nanodots with an average size of approximately 4.36 nm were prepared by the liquid exfoliation method and modified with the surfactant polyvinylpyrrolidone (PVP), with a satisfactory absorption and photothermal conversion efficiency (53.4%) in the near-infrared region. Furthermore, ZrC-PVP nanodots can also act as radiosensitizers to kill residual tumor cells after mild PTT due to their excellent photon attenuating ability, thus achieving a significant synergistic therapeutic effect by combining RT and PTT. Most importantly, both in vitro and in vivo experimental results further validate the high biosafety of ZrC-PVP NDs at the injected dose. This work systematically evaluates the feasibility of ZrC-PVP NDs for glioma treatment and provides evidence of the application of zirconium-based nanomaterials in photothermal radiotherapy.

ROS-mediated inflammatory response in liver damage via regulating the Nrf2/HO-1/NLRP3 pathway in mice with trichloroethylene hypersensitivity syndrome.

Trichloroethylene hypersensitivity syndrome (THS), mainly caused by occupational exposure to trichloroethylene (TCE), can give rise to serious and fatal hepatic damage. To date, the precise mechanisms of hepatic damage in THS remain unclear. Recent studies showed that reactive oxygen species (ROS) play a core role in cell death and inflammatory response. Therefore, the present study sought to explore whether ROS-mediated inflammatory responses contribute to the hepatic damage in TCE sensitization. To this end, a mouse model of TCE sensitization was established; in some cases, hosts were pretreated with tempol, an ROS scavenger. The results showed that TCE sensitization caused hepatic pathological/functional changes, ROS generation, and oxidative stress, alterations of the anti-oxidant defense Nrf2/HO-1/NLRP3 pathway, and pro-inflammatory cytokine formation in the liver. ROS scavenging via pretreatment with tempol was found not only to inhibit the hepatic oxidative stress, but also to regulate Nrf2/HO-1/NLRP3 pathway activity. In all cases, tempol was able to mitigate the pathologic changes induced by TCE sensitization. In summary, the results here demonstrated a novel molecular mechanism wherein ROS-mediated inflammatory responses play a central role in TCE-induced liver damage. Therapies targeting ROS scavenging could help to protect against hepatic damage by regulating Nrf2/HO-1/NLRP3 pathway activities in TCE-sensitized hosts.

Protective Effects of Three Flavonoids from Dendrobium huoshanense Flowers on Alcohol-Induced Hepatocyte Injury via Activating Nrf2 and Inhibiting NF-κB Pathways.

Dendrobium huoshanense flowers have been widely used for liver protection in China. This work was aimed to discover the natural products with activity of mitigating alcoholic hepatocyte injury from Dendrobium huoshanense flowers via bioactivity-guided isolation, and to clarify the underlying mechanisms of these natural products. As a result, three flavonoids, 3'-O-methylquercetin-3-O-ß-D-galactopyranoside (1), 3'-O-methylquercetin-3-O-ß-D-glucopyranoside (2) and quercetin-3-O-ß-D-glucopyranoside (3), were firstly isolated from D. huoshanense flowers. Results exhibited that flavonoids 1-3 could enhance the cell viability, decrease the expression of ALT and AST, inhibit the cell apoptosis, alleviate the oxidative stress, and mitigate the inflammatory response of alcohol-induced L02 cells. Mechanism study exhibited that flavonoids 1-3 could increase the expression of Nrf2 as well as its downstream antioxidation genes of alcohol-induced L02 cells, while ML-385 (Nrf2 inhibitor) could abolish the inhibitory effects of 1-3 on alcohol-induced hepatocyte injury. Flavonoids 1-3 could also reduce the phosphorylation levels of IκBα and NF-κB p65 of alcohol-induced L02 cells, while SC75741 (NF-κB inhibitor) could not enhance the inhibitory effects of 1-3 on alcohol-induced L02 cells injury. The data above indicated that flavonoids 1-3 could inhibit alcohol-induced hepatocyte injury, which might be attributed to alleviating oxidative stress and mitigating inflammatory response by activating Nrf2 and inhibiting NF-κB pathways.

Membrane Attack Complex C5b-9 Promotes Renal Tubular Epithelial Cell Pyroptosis in Trichloroethylene-Sensitized Mice.

Trichloroethylene (TCE), a commonly used organic solvent, is known to cause trichloroethylene hypersensitivity syndrome (THS), also called occupational medicamentosa-like dermatitis due to TCE (OMDT) in China. OMDT patients presented with severe inflammatory kidney damage, and we have previously shown that the renal damage is related to the terminal complement complex C5b-9. Here, we sought to determine whether C5b-9 participated in TCE-induced immune kidney injury by promoting pyroptosis, a new form of programed cell death linked to inflammatory response, with underlying molecular mechanisms involving the NLRP3 inflammasome. A BALB/c mouse-based model of OMDT was established by dermal TCE sensitization in the presence or absence of C5b-9 inhibitor (sCD59-Cys, 25µg/mouse) and NLRP3 antagonist (MCC950, 10 mg/kg). Kidney histopathology, renal function, expression of inflammatory mediators and the pyroptosis executive protein gasdermin D (GSDMD), and the activation of pyroptosis canonical NLRP3/caspase-1 pathway were examined in the mouse model. Renal tubular damage was observed in TCE-sensitized mice. GSDMD was mainly expressed on renal tubular epithelial cells (RTECs). The caspase-1-dependent canonical pathway of pyroptosis was activated in TCE-induced renal damage. Pharmacological inhibition of C5b-9 could restrain the caspase-1-dependent canonical pathway and rescued the renal tubular damage. Taken together, our results demonstrated that complement C5b-9 plays a central role in TCE-induced immune kidney damage, and the underlying mechanisms involve NLRP3-mediated pyroptosis.

Both high glucose and phosphate overload promote senescence-associated calcification of vascular muscle cells.

PURPOSE: The NAD+-dependent deacetylase, sirtuin 1 (SIRT1), plays an important role in vascular calcification induced by high glucose and/or high phosphate levels. However, the mechanism by which SIRT1 regulates this process is still not fully understood. Thus, this study aimed to determine the role of high glucose and phosphate in vascular calcification and the molecular mechanisms underlying SIRT1 regulation. METHODS: Vascular smooth muscle cells (VSMCs) were cultured under normal, high phosphate, and/or high-glucose conditions for 9 days. Alizarin red staining and calcification content analyses were used to determine calcium deposition. VSMC senescence was detected by ß-galactosidase (SA-ß-Gal) staining and p21 expression. RESULTS: Mouse VSMCs exposed to high phosphate and high glucose in vitro showed increased calcification, which was correlated with the induction of cell senescence, as confirmed by the increased SA-ß-galactosidase activity and p21 expression. SRT1720, an activator of SIRT1, inhibits p65 acetylation, the nuclear factor-κ-gene binding (NF-κB) pathway, and VSMC transdifferentiation, prevents senescence and reactive oxygen species (ROS) production, and reduces vascular calcification. In contrast, sirtinol, an inhibitor of SIRT1, increases p65 acetylation, activates the NF-κB pathway, induces vascular smooth muscle cell transdifferentiation and senescence, and promotes vascular calcification. CONCLUSIONS: High glucose and high phosphate levels induce senescence and vascular calcification in VSMCs, and the combined effect of high glucose and phosphate can inhibit SIRT1 expression. SIRT1 inhibits vascular smooth muscle cell senescence and osteogenic differentiation by inhibiting NF-κB activity, thereby inhibiting vascular calcification.

Tubular epithelial cell-to-macrophage communication forms a negative feedback loop via extracellular vesicle transfer to promote renal inflammation and apoptosis in diabetic nephropathy.

Background: Macrophage infiltration around lipotoxic tubular epithelial cells (TECs) is a hallmark of diabetic nephropathy (DN). However, how these two types of cells communicate remains obscure. We previously demonstrated that LRG1 was elevated in the process of kidney injury. Here, we demonstrated that macrophage-derived, LRG1-enriched extracellular vesicles (EVs) exacerbated DN. Methods: We induced an experimental T2DM mouse model with a HFD diet for four months. Renal primary epithelial cells and macrophage-derived EVs were isolated from T2D mice by differential ultracentrifugation. To investigate whether lipotoxic TEC-derived EV (EVe) activate macrophages, mouse bone marrow-derived macrophages (BMDMs) were incubated with EVe. To investigate whether activated macrophage-derived EVs (EVm) induce lipotoxic TEC apoptosis, EVm were cocultured with primary renal tubular epithelial cells. Subsequently, we evaluated the effect of LRG1 in EVe by investigating the apoptosis mechanism. Results: We demonstrated that incubation of primary TECs of DN or HK-2 mTECs with lysophosphatidyl choline (LPC) increased the release of EVe. Interestingly, TEC-derived EVe activated an inflammatory phenotype in macrophages and induced the release of macrophage-derived EVm. Furthermore, EVm could induce apoptosis in TECs injured by LPC. Importantly, we found that leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EVe activated macrophages via a TGFßR1-dependent process and that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-enriched EVm induced apoptosis in injured TECs via a death receptor 5 (DR5)-dependent process. Conclusion: Our findings indicated a novel cell communication mechanism between tubular epithelial cells and macrophages in DN, which could be a potential therapeutic target.

CD73 regulates hepatic stellate cells activation and proliferation through Wnt/ß-catenin signaling pathway.

Alcoholic liver fibrosis (ALF) is commonly associated with long-term alcohol consumption and the activation of hepatic stellate cells (HSCs). Inhibiting the activation and proliferation of HSCs is a critical step to alleviate liver fibrosis. Increasing evidence indicates that ecto-5'-nucleotidase (CD73) plays a vital role in liver disease as a critical component of extracellular adenosine pathway. However, the regulatory role of CD73 in ALF has not been elucidated. In this study, both ethanol plus CCl4-induced liver fibrosis mice model and acetaldehyde-activated HSC-T6 cell model were employed and the expression of CD73 was consistently elevated in vivo and in vitro. C57BL/6 J mice were intraperitoneally injected with CD73 inhibitor Adenosine 5'-(α, ß-methylene) diphosphate sodium salt (APCP) from 5th week to the 8th week in the development of ALF. The results showed APCP could inhibit the activation of HSCs, reduce fibrogenesis marker expression and thus alleviate ALF. Silencing of CD73 inhibited the activation of HSC-T6 cells and promoted apoptosis of activated HSC-T6 cells. What's more, the proliferation of HSC-T6 cells was inhibited, which was characterized by decreased cell viability and cycle arrest. Mechanistically, Wnt/ß-catenin pathway was activated in acetaldehyde-activated HSC-T6 cells and CD73 silencing or overexpression could regulate Wnt/ß-catenin signaling pathway. Collectively, our study unveils the role of CD73 in HSCs activation, and Wnt/ß-catenin signaling pathway might be involved in this progression.

Altered levels of complement components associated with non-immediate drug hypersensitivity reactions.

Nonimmediate drug hypersensitivity reactions (niDHRs) range from mild-type maculopapular exanthema (MPE) to severe type Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) with unentirely clarified pathogenesis. This study sought to explore whether complement components participated in niDHRs. The participants comprised of three groups as follows: MPE (n = 65), SJS/TEN (n = 13, contains 7 SJS, 2 SJS-TEN overlap and 4 TEN), and equal healthy controls (n = 78). Skin pathological changes were confirmed by hematoxylin and eosin staining. The mRNA and protein levels of complement components were assessed. In the MPE group, there were no alterations in complement components at the protein and mRNA levels found except for a decrease in factor H mRNA. In the SJS/TEN group, up-regulated levels of C3aR and C5aR mRNA and down-regulated factor H mRNA levels in blood were noted. A lower plasma protein level of C3, Factor H and a higher level of C3a, C5, C5a, C5b-9, Factor B (p < 0.05) were found in the SJS/TEN group compared with in the control (p < 0.05). In SJS/TEN skin lesions, indirect immunofluorescence assays showed positive specific staining for C5b-9, but not C3. Both C3aR and C5aR were positive staining in the SJS/TEN samples, while staining for C1q, mannose-binding lectin (MBL), Factor B, and Factor H were only weak or negative. The findings reported here are the first to define the expression profiles/extent of the presence of various complement components at the mRNA and protein levels in niDHRs, especially in SJS/TEN. These altered complement components might, at least in part, be integral to the mechanisms underlying the pathogeneses of SJS and TEN.

Arabidopsis heat shock transcription factor HSFA7b positively mediates salt stress tolerance by binding to an E-box-like motif to regulate gene expression.

Plant heat shock transcription factors (HSFs) are involved in heat and other abiotic stress responses. However, their functions in salt tolerance are little known. In this study, we characterized the function of a HSF from Arabidopsis, AtHSFA7b, in salt tolerance. AtHSFA7b is a nuclear protein with transactivation activity. ChIP-seq combined with an RNA-seq assay indicated that AtHSFA7b preferentially binds to a novel cis-acting element, termed the E-box-like motif, to regulate gene expression; it also binds to the heat shock element motif. Under salt conditions, AtHSFA7b regulates its target genes to mediate serial physiological changes, including maintaining cellular ion homeostasis, reducing water loss rate, decreasing reactive oxygen species accumulation, and adjusting osmotic potential, which ultimately leads to improved salt tolerance. Additionally, most cellulose synthase-like (CSL) and cellulose synthase (CESA) family genes were inhibited by AtHSFA7b; some of them were randomly selected for salt tolerance characterization, and they were mainly found to negatively modulate salt tolerance. By contrast, some transcription factors (TFs) were induced by AtHSFA7b; among them, we randomly identified six TFs that positively regulate salt tolerance. Thus, AtHSFA7b serves as a transactivator that positively mediates salinity tolerance mainly through binding to the E-box-like motif to regulate gene expression.

Transcription Factor-Centered Yeast One-Hybrid Assay.

The interaction between a protein and DNA is involved in almost all cellular functions, and is vitally important in transcriptional regulation. There are two complementary approaches used to detect the interactions between a transcription factor (TF) and DNA, i.e., the TF-centered or protein-DNA approach, and the gene-centered or DNA-protein approach. The yeast one-hybrid (Y1H) is a powerful and widely used gene-centered system to identify DNA-protein interactions. However, a powerful and simple TF-centered method to study protein-DNA interactions like Y1H is lacking. Here, we provide a TF-centered method based on the Y1H system to identify the motifs recognized by a defined TF, termed TF-centered Y1H. In this system, a random short DNA sequence insertion library is generated as the prey DNA sequences to interact with a defined TF as the bait. TF-centered Y1H could identify quickly the motifs bound by a defined TF, representing a reliable and efficient approach with the advantages of Y1H. Therefore, this TF-centered Y1H may have a wide application in protein-DNA interaction studies.

Role of selective blocking of bradykinin B1 receptor in attenuating immune liver injury in trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is able to induce trichloroethylene hypersensitivity syndrome (THS) with multi-system immune injuries. In our previous study, we found kallikrein-kinin system (KKS) activation, including the bradykinin B1 receptor (B1R), which contributed to immune organ injury in TCE sensitized mice. However, the mechanism of B1R mediating immune dysfunction is not clarified. The present study initiates to investigate the potential mechanism of B1R on liver injury. We establish a TCE sensitized BALB/c mouse model to explore the mechanism with or without a B1R inhibitor R715. We found B1R expression was increased in TCE sensitization-positive mice. As expect, hepatocyte intracellular organelles and mitochondria disappeared, glycogen particles reduced significantly as well in TCE sensitization-positive mice via the transmission electron microscopic examination, meanwhile, R715 alleviated the deteriorate above. The blockade of B1R resulted in a significant decreased p-ERK1/2 and increased p-AKT expression. The expression of CD68 kupffer cell and its relative cytokine, including IL-6 and TNF-α, increased in TCE sensitization-positive mice and decreased in R715 pretreatment TCE sensitization-positive mice. Together, the results demonstrate B1R plays a key role in ERK/MAPK and PI3K/AKT signal pathway activation and inflammation cytokine expression in immune liver injury induced by TCE. B1R exerts a pivotal role in the development of TCE induced liver injury.

Arabidopsis thaliana Trihelix Transcription Factor AST1 Mediates Salt and Osmotic Stress Tolerance by Binding to a Novel AGAG-Box and Some GT Motifs.

Trihelix transcription factors are characterized by containing a conserved trihelix (helix-loop-helix-loop-helix) domain that binds to GT elements required for light response, and they play roles in light stress and in abiotic stress responses. However, only a few of them have been functionally characterized. In the present study, we characterized the function of AST1 (Arabidopsis SIP1 clade Trihelix1) in response to salt and osmotic stress. AST1 shows transcriptional activation activity, and its expression is induced by osmotic and salt stress. A conserved sequence highly present in the promoters of genes regulated by AST1 was identified, which was bound by AST1, and termed the AGAG-box with the sequence [A/G][G/A][A/T]GAGAG. Additionally, AST1 also binds to some GT motifs including the sequence of GGTAATT, TACAGT, GGTAAAT and GGTAAA, but failed in binding to the sequence of GTTAC and GGTTAA. Chromatin immunoprecipitation combined with quantitative real-time reverse transcription-PCR analysis suggested that AST1 binds to the AGAG-box and/or some GT motifs to regulate the expression of stress tolerance genes, resulting in reduced reactive oxygen species, Na+ accumulation, stomatal apertures, lipid peroxidation, cell death and water loss rate, and increased proline content and reactive oxygen species scavenging capability. These physiological changes affected by AST1 finally improve salt and osmotic tolerance.

ThDof1.4 and ThZFP1 constitute a transcriptional regulatory cascade involved in salt or osmotic stress in Tamarix hispida.

Identification of the upstream regulators of a gene is important to characterize the transcriptional pathway and the function of the gene. Previously, we found that a zinc finger protein (ThZFP1) is involved in abiotic stress tolerance of Tamarix hispida. In the present study, we further investigated the transcriptional pathway of ThZFP1. Dof motifs are abundant in the ThZFP1 promoter; therefore, we used them to screen for transcriptional regulators of ThZFP1. A Dof protein, ThDof1.4, binds to the Dof motif specifically, and was hypothesized as the upstream regulator of ThZFP1. Further study showed that overexpression of ThDof1.4 in T. hispida activated the expression of GUS controlled by the ThZFP1 promoter. In T. hispida, transient overexpression of ThDof1.4 increased the transcripts of ThZFP1; conversely, transient RNAi-silencing of ThDof1.4 reduced the expression of ThZFP1. Chromatin immunoprecipitation indicated that ThDof1.4 binds to the ThZFP1 promoter. Additionally, ThDof1.4 and ThZFP1 share similar expression patterns in response to salt or drought stress. Furthermore, like ThZFP1, ThDof1.4 could increase the proline level and enhance ROS scavenging capability to improve salt and osmotic stress tolerance. Together, these results suggested that ThDof1.4 and ThZFP1 form a transcriptional regulatory cascade involved in abiotic stress resistance in T. hispida.