Avicularin alleviates acute liver failure by regulation of the TLR4/MyD88/NF-κB and Nrf2/HO-1/GPX4 pathways to reduce inflammation and ferroptosis

Acute liver failure (ALF) is an inflammation-mediated hepatocyte death process associated with ferroptosis. Avicularin (AL), a Chinese herbal medicine, exerts anti-inflammatory and antioxidative effects. However, the protective effect of AL and the mechanism on ALF have not been reported. Our in vivo results suggest that AL significantly alleviated lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced hepatic pathological injury, liver enzymes, inflammatory cytokines, reactive oxygen species and iron levels and increased the antioxidant enzyme activities (malondialdehyde and glutathione). Our further in vitro experiments demonstrated that AL suppressed inflammatory response in LPS-stimulated RAW 264.7 cells via blocking the toll-like receptor 4 (TLR4)/myeloid differentiation protein-88 (MyD88)/nuclear factor kappa B (NF-κB) pathway. Moreover, AL attenuated ferroptosis in D-GalN-induced HepG2 cells by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1)/glutathione peroxidase 4 (GPX4) pathway. Therefore, AL can alleviate inflammatory response and ferroptosis in LPS/D-GalN-induced ALF, and its protective effects are associated with blocking TLR4/MyD88/NF-κB pathway and activating Nrf2/HO-1/GPX4 pathway. Moreover, AL is a promising therapeutic option for ALF and should be clinically explored.     

吉粳88高效再生体系的建立

以吉粳88为材料,通过对不同培养基和激素的筛选,确定最佳的再生体系。结果表明:NMB培养基为吉粳88最适的愈伤组织诱导培养基。通过L₉(3³)正交实验研究最适激素浓度配比,筛选出吉粳88愈伤组织诱导的最佳激素配比为2.0 mg/L 2,4-D+0.2 mg/L 6-BA+0.10 mg/L NAA。在添加2.0 mg/L 6-BA+0.5 mg/L NAA的NMB基本培养基条件下,吉粳88的分化率最高,成培苗率也最高。     

TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

MyD88抑制乙型肝炎病毒复制依赖活化NF-κB信号通路

目的 研究髓样细胞分化蛋白(MyD88)抗乙型肝炎病毒(HBV)效应的作用机制。方法 构建MyD88的截短突变体,获得核因子kappa B(NF-κB)超抑制剂IkBa-SR或者NF-κB信号通路激活剂IKKα/IKKβ的表达质粒,分别与HBV复制型质粒瞬时转染Huh7细胞,检测细胞上清液中HBeAg,HBsAg的表达以及胞质中HBV复制中间体DNA的含量,并以NF-κB依赖的荧光素酶报道系统检测它们活化NF-κB的程度。结果 MyD88全长蛋白和2个截短突变体M(1-151)、M(151-296)活化NF-κB的程度与其抑制HBV蛋白以及复制中间体DNA合成的能力相一致。与空载相比,表达NF-κB信号通路激活剂IKKα/IKKβ的质粒共同瞬转细胞后,转染MyD88和HBV表达质粒的细胞中NF-κB的通路明显活化,同时HBV core蛋白的合成显著降低;而NF-κB的超抑制剂IκBα-SR共同瞬转的细胞中core蛋白的表达量显著增加,检测细胞培养上清液中HBeAg和HBsAg及胞质中HBV复制中间体DNA的合成,得到相似结果。结论 NF-κB信号通路的活化在MyD88抑制HBV复制中发挥了关键作用     

Effect of Glycosylation on the Catalytic and Conformational Stability of Homologous α-Amylases

summary. A thermostable -amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca²⁺. The stability of the native enzyme was found to increase upon modification as observed from the increase in t_1/2 in the absence of Ca²⁺ ions by about 1.5–13.7 times (at 85°C) in the case of BLA and 5.7–8.4 times (at 50°C) for BAA. The highest stability was observed for OSP400 modified enzyme with C_m and T_m values of 0.63 M and 7.92°C for BLA and 0.85 M and 5.3°C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.This article is dedicated to Dr. P.V. Sundaram.     

Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL‐1β/IL‐1R1/MyD88/NF‐κB pathway

Interleukin (IL)‐1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL‐1β is dependent upon caspase‐1‐containing multiprotein complexes called inflammasomes and IL‐1R1/MyD88/NF‐κB pathway. In this study, we explored whether a potential anti‐fibrotic agent fluorofenidone (FD) exerts its anti‐inflammatory and anti‐fibrotic effects through suppressing activation of NACHT, LRR and PYD domains‐containing protein 3 (NALP3) inflammasome and the IL‐1β/IL‐1R1/MyD88/NF‐κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α‐smooth muscle actin (α‐SMA), fibronectin, collagen I, caspase‐1, IL‐1R1, MyD88 were measured by Western blot and/or RT‐PCR. The human actue monocytic leukaemia cell line (THP‐1) were incubated with monosodium urate (MSU), with or without FD pre‐treatment. The expression of caspase‐1, IL‐1β, NALP3, apoptosis‐associated speck‐like protein containing (ASC) and pro‐caspase‐1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome‐associated molecules were measured by Co‐immunoprecipitation. RLE‐6TN (rat lung epithelial‐T‐antigen negative) cells were incubated with IL‐1β, with or without FD pre‐treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL‐1β, IL‐6, monocyte chemotactic protein‐1 (MCP‐1), myeloperoxidase (MPO), α‐SMA, fibronectin, collagen I, caspase‐1, IL‐1R1 and MyD88 in mice lung tissues. And FD inhibited MSU‐induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome‐associated molecules, decreased the level of caspase‐1 and IL‐1β in THP‐1 cells. Besides, FD inhibited IL‐1β‐induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM‐induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL‐1β/IL‐1R1/MyD88/ NF‐κB pathway.     

Melatonin,a toll-like receptor inhibitor: Current status and future perspectives

Toll-like receptors (TLRs) are crucial activators of inflammatory responses, they are considered immune receptors. TLRs are of fundamental importance in the pathophysiology of disorders related to inflammation including neurodegenerative diseases and cancer. Melatonin is a beneficial agent in the treatment of inflammatory and immune disorders. Melatonin is potent anti-inflammatory hormone that regulates various molecular pathways. Withal, limited studies have evaluated the inhibitory role of melatonin on TLRs. This review summarizes the current knowledge related to the effects of melatonin on TLRs in some common inflammatory and immunity disorders.     

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.     

Pod development of groundnut (Arachis hypogaea L.) in solution culture

Normal pods (containing seed) of groundnut (Arachis hypogaea L.) (cv. TMV-2) were successfully raised in darkened, aerated, nutrient solution, but not in the light. The onset of podding was evident 7 to 8 d after gynophores were submerged in the darkened nutrient solution. An examination of pods and submerged portions of gynophore surfaces by scanning electron microscopy showed the presence of two distinctly different protuberances: unicellular root-hair-like structures that first developed from epidermal cells of the gynophores and developing pods; and branched septate hairs that developed later from cells below the epidermal layer. The septate hairs became visible only after the epidermal and associated unicellular structures had been shed by the expanding gynophore and pods. Omission of Mn and Mg from the podding environment increased pod and seed weight, whilst omission of Zn reduced pod and seed weight.     

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology^1,2, behavior³, and memory and learning^4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access.