Liang-Ge-San: a classic traditional Chinese medicine formula, attenuates acute inflammation via targeting GSK3ß.

Sepsis is a serious life-threatening health disorder with high morbidity and mortality rates that burden the world, but there is still a lack of more effective and reliable drug treatment. Liang-Ge-San (LGS) has been shown to have anti-inflammatory effects and is a promising candidate for the treatment of sepsis. However, the anti-sepsis mechanism of LGS has still not been elucidated. In this study, a set of genes related to inflammatory chemotaxis pathways was downloaded from Encyclopedia of Genes and Genomes (KEGG) and integrated with sepsis patient information from the Gene Expression Omnibus (GEO) database to perform differential gene expression analysis. Glycogen synthase kinase-3ß (GSK-3ß) was found to be the feature gene after these important genes were examined using the three algorithms Random Forest, support vector machine recursive feature elimination (SVM-REF), and least absolute shrinkage and selection operator (LASSO), and then intersected with possible treatment targets of LGS found through the search. Upon evaluation, the receiver operating characteristic (ROC) curve of GSK-3ß indicated an important role in the pathogenesis of sepsis. Immune cell infiltration analysis suggested that GSK-3ß expression was associated with a variety of immune cells, including neutrophils and monocytes. Next, lipopolysaccharide (LPS)-induced zebrafish inflammation model and macrophage inflammation model was used to validate the mechanism of LGS. We found that LGS could protect zebrafish against a lethal challenge with LPS by down-regulating GSK-3ß mRNA expression in a dose-dependent manner, as indicated by a decreased neutrophils infiltration and reduction of inflammatory damage. The upregulated mRNA expression of GSK-3ß in LPS-induced stimulated RAW 264.7 cells also showed the same tendency of depression by LGS. Critically, LGS could induce M1 macrophage polarization to M2 through promoting GSK-3ß inactivation of phosphorylation. Taken together, we initially showed that anti-septic effects of LGS is related to the inhibition on GSK-3ß, both in vitro and in vivo.

Functional biochar fabricated from red mud and walnut shell for phosphorus wastewater treatment: Role of minerals.

A novel functional biochar (BC) was prepared from industrial waste red mud (RM) and low-cost walnut shell by one facile-step pyrolysis method to adsorb phosphorus (P) in wastewater. The preparation conditions for RM-BC were optimized using Response Surface Methodology. The adsorption characteristics of P were investigated in batch mode experiments, while a variety of techniques were used to characterize RM-BC composites. The impact of key minerals (hematite, quartz, and calcite) in RM on the P removal efficiency of the RM-BC composite was studied. The results showed that RM-BC composite produced at 320 °C for 58 min, with a 1:1 mass ratio of walnut shell and RM, had a maximum P sorption capacity of 15.48 mg g-1, which was more than double that of the raw BC. The removal of P from water was found to be facilitated significantly by hematite, which forms Fe-O-P bonds, undergoes surface precipitation, and exchanges ligands. This research provides evidence for the effectiveness of RM-BC in treating P in water, laying the foundation for future scaling-up trials.

Dihydroquercetin protects against renal fibrosis by activating the Nrf2 pathway.

BACKGROUND: Dihydroquercetin (DHQ) is an antifibrotic agent. However, whether DHQ can prevent renal fibrosis remains unknown. PURPOSE: This study aimed to investigate the effects of DHQ on tubulointerstitial fibrosis and its underlying mechanisms in unilateral ureteral obstruction (UUO) mice in vivo and NRK-49F cells in vitro. METHODS: In vivo, UUO mice received vehicle or DHQ treatment. In vitro, NRK-49F cells were pretreated with DHQ and exposed to transforming growth factor-ß1 (TGF-ß1). Changes in fibroblast activation, collagen synthesis, oxidative stress, and related signaling pathways were assessed by immunohistochemical staining, Western blot analysis, real-time reverse transcription-PCR, and fluorescence microscopy. RESULTS: UUO induced tubular atrophy, inflammation, fibroblast differentiation into myofibroblast, and collagen deposition, whereas DHQ ameliorated these effects. UUO also resulted in decreased levels of nuclear factor-erythroid-2-related factor 2 (Nrf2), catalase, and heme oxygenase-1, but increased H2O2 and malondialdehyde levels. DHQ treatment corrected these changes. In vitro, the intracellular Nrf2 level of NRK-49F exposed to TGF-ß1 decreased. However, DHQ rescued intracellular Nrf2 level and promoted nuclear translocation of Nrf2. DHQ scavenged TGF-ß1-induced accumulation of reactive oxygen species, inhibited TGF-ß1-induced Smad3 phosphorylation, and prevented TGF-ß1-induced fibroblast activation and collagen synthesis in NRK-49F. Nrf2 knockdown could suppress the DHQ-mediated inhibitory effects on oxidative stress, Smad3 phosphorylation, fibroblast activation, and collagen deposition. Furthermore, DHQ ameliorated established renal fibrosis in UUO mice. CONCLUSIONS: DHQ posed remarkable preventive and therapeutic effects on UUO-induced renal fibrosis and suppressed fibroblast activation by reducing oxidative stress and Smad3 phosphorylation via Nrf2 signaling. This study provided a mechanistic basis for the clinical application of DHQ in renal fibrosis treatment.

Elucidation of the Mechanisms of Long-Distance mRNA Movement in a Nicotiana benthamiana/Tomato Heterograft System.

Recent heterograft analyses showed that large-scale messenger RNA (mRNA) movement takes place in the phloem, but the number of mobile transcripts reported varies widely. However, our knowledge of the mechanisms underlying large-scale mRNA movement remains limited. In this study, using a Nicotiana benthamiana/tomato (Solanum lycopersicum) heterograft system and a transgenic approach involving potato (Solanum tuberosum), we found that: (1) the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root; (2) increasing the expression levels of nonmobile mRNAs in the companion cells does not promote their mobility; (3) mobile mRNAs undergo degradation during their movement; and (4) some mRNAs arriving in roots move back to shoots. These results indicate that mRNA movement has both regulated and unregulated components. The cellular origins of mobile mRNAs may differ between herbaceous and woody species. Taken together, these findings suggest that the long-distance movement of mRNAs is a complex process and that elucidating the physiological roles associated with this movement is challenging but remains an important task for future research.

Protective Effect of Phillyrin on Lethal LPS-Induced Neutrophil Inflammation in Zebrafish.

BACKGROUND/AIMS: Forsythia suspensa Vahl. (Oleaceae) fruits are widely used in traditional Chinese medicine to treat pneumonia, typhoid, dysentery, ulcers and oedema. Antibacterial and anti-inflammatory activities have been reported for phillyrin (PHN), the main ingredient in Forsythia suspensa Vahl fruits, in vitro. However, the underlying mechanisms in vivo remain poorly defined. In this study, we discovered that PHN exerted potent anti-inflammatory effects in lethal LPS-induced neutrophil inflammation by suppressing the MyD88-dependent signalling pathway in zebrafish. METHODS: LPS-yolk microinjection was used to induce a lethal LPS-infected zebrafish model. The effect of PHN on the survival of zebrafish challenged with lethal LPS was evaluated using survival analysis. The effect of PHN on neutrophil inflammation grading in vivo was assessed by tracking neutrophils with a transgenic line. The effects of PHN on neutrophil production and migration were analysed by SB+ cell counts during consecutive hours after modelling. Additionally, key cytokines and members of the MyD88 signalling pathway that are involved in inflammatory response were detected using quantitative RT-PCR. To assess gene expression changes during consecutive hours after modelling, the IL-1ß, IL-6, TNF-α, MyD88, TRIF, ERK1/2, JNK, IκBa and NF-κB expression levels were measured. RESULTS: PHN could protect zebrafish against a lethal LPS challenge in a dose-dependent manner, as indicated by decreased neutrophil infltration, reduced tissue necrosis and increased survival rates. Up-regulated IL-1ß, IL-6 and TNF-α expression also showed the same tendencies of depression by PHN. Critically, PHN significantly inhibited the LPS-induced activation of MyD88, IκBa, and NF-κB but did not affect the expression of ERK1/2 MAPKs or JNK MAPKs in LPS-stimulated zebrafish. Additionally, PHN regulated the MyD88/IκBα/NF-κB signalling pathway by controlling IκBα, IL-1ß, IL-6, and TNF-α expression. CONCLUSION: This study provides a rationale for the clinical application of PHN as an anti-inflammatory agent.

Puerarin attenuates renal fibrosis by reducing oxidative stress induced-epithelial cell apoptosis via MAPK signal pathways in vivo and in vitro.

Puerarin (PR) is an isoflavonoid isolated from the root of the plant Pueraria lobata and has been widely used in traditional Chinese herbal medicine for the treatment of various diseases. Oxidative stress and epithelial cell apoptosis play important roles in the renal fibrotic process. The present study aimed to determine whether or not PR inhibits renal fibrosis by reducing oxidative stress induced-epithelial cell apoptosis. In vivo, unilateral ureteral obstruction (UUO) induced renal fibrosis, and epithelial cell apoptosis. A total of 24 mice were randomly assigned to four experimental groups: sham, UUO alone, UUO +50 mg/kg PR, and UUO +100 mg/kg PR. In vitro, 200 µM hydrogen peroxide (H2O2) induced epithelial cell apoptosis. The experiments were dived into four groups: control, H2O2 alone, H2O2+50 µM PR, and H2O2+100 µM PR. Tubular injury was measured in the renal cortex of the mice through periodic acid-Schiff (PAS) staining, and the extracellular matrix (ECM) was measured through Sirius red (SR), immunohistochemistry (IHC) staining, and Western blot. Renal epithelial cell apoptosis was measured through terminal deoxynucleotidyl transferase-mediated dUTP Nick-End labeling (TUNEL), flow cytometry (FCM), and Hoechst assays. The protein expression of NOX4, caspase3, ERK, P38, and JNK was assessed through Western blot. PAS staining showed that PR decreased renal tubular injury in UUO mice. SR and IHC staining demonstrated that PR decreased the accumulation of ECM. PR treatment significantly inhibited epithelial cell apoptosis according to the results of TUNEL, FCM, Hoechst, and Western blot. Furthermore, NOX4 increased in UUO mice and decreased with PR treatment. H2O2-derived oxidative stress activated epithelial apoptosis and mitogen-activated protein kinases (MAPK), and PR treatment significantly reversed it. These results suggest that PR treatment ameliorates renal fibrosis by inhibiting oxidative stress induced-epithelial cell apoptosis through MAPK signaling.

StMYB44 negatively regulates phosphate transport by suppressing expression of PHOSPHATE1 in potato.

Phosphorus is an important macronutrient for plant growth, but often deficient in soil. To understand the molecular basis of the complex responses of potato (Solanum tuberosum L.) to phosphate (Pi) deficiency stress, the RNA-Seq approach was taken to identify genes responding to Pi starvation in potato roots. A total of 359 differentially expressed genes were identified, among which the Solanum tuberosum transcription factor gene MYB44 (StMYB44) was found to be down-regulated by Pi starvation. StMYB44 was ubiquitously expressed in potato tissues and organs, and StMYB44 protein was exclusively localized in the nucleus. Overexpression of StMYB44 in potato resulted in lower accumulation of Pi in shoots. Transcriptomic analysis indicated that the abundance of S. tuberosum PHOSPHATE1 (StPHO1), a Pi transport-related gene, was reduced in StMYB44 overexpression lines. In contrast, knock-out of StMYB44 by a CRISPR/Cas9 system failed to increase transcription of StPHO1. Moreover, StMYB44 was found to interact in the nucleus with AtWRKY6, a known Arabidopsis transcription factor directly regulating PHO1 expression, and StWRKY6, indicating that StMYB44 could be a member of the regulatory complex controlling transcription of StPHO1. Taken together, our study demonstrates that StMYB44 negatively regulates Pi transport in potato by suppressing StPHO1 expression.

Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-кB in vivo and in vitro.

Renal fibrosis is characterized by infiltration of inflammatory cells, activation and proliferation of fibroblasts, and accumulation of extracellular matrix (ECM). Astragalus membranaceus (AM) is traditional Chinese medicine and has a range of pharmacological effects. Astragaloside IV (As IV) is the main compound of AM and has anti-inflammation activities. Whether As IV ameliorates renal interstitial fibrosis by inhibiting inflammation remains unknown. Accordingly, this study investigated the ameliorating effect of As IV on renal fibrosis. Renal fibrosis was induced in vivo using the unilateral ureteral obstruction (UUO) model. UUO mice were administered intragastrically with As IV (20 and 40mg/kg/day). After a week, ECM including fibronectin and collagen I was examined by Immunohistochemistry and Western blot, inflammatory cells (CD68 and CD3) were detected by Immunohistochemistry, the release of inflammatory cytokines (tumor necrosis factor-α and interleukin-1ß) was inspected by polymerase chain reaction, and signaling pathway was determined by Western blot. In vitro, 100ng/ml lipopolysaccharide (LPS) stimulated epithelial cells to construct the inflammatory model; these cells were treated by As IV (10 and 20µM) with or without TAK-242 (1µM) for 48h. The released inflammatory cytokines were assayed by enzyme-linked immunosorbent assay, and signaling pathway was evaluated by Western blot. As IV decreased accumulation of ECM and infiltration of inflammatory cells in UUO-induced renal fibrosis. Furthermore, As IV markedly attenuated pro-inflammatory cytokines in UUO mouse and LPS-induced epithelial cells. As IV also inhibited the TLR4 and nuclear factor (NF)-кB signaling pathway in vivo and vitro. These results demonstrate that As IV protects against the progression of renal fibrosis by inhibiting inflammation via the TLR4/NF-кB signaling pathway.

Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting tubular epithelial-mesenchymal transition in vivo and in vitro.

Epithelial-mesenchymal transition (EMT) induces the progression of renal tubulointerstitial fibrosis. Astragalus membranaceus (AM) is a traditional Chinese herbal medicine that has been demonstrated to exert anti-inflammatory and anti-cancer effects, in addition to protecting and supporting the immune system. The present study investigated the effects of AM on renal fibrosis. A mouse model of unilateral ureteral obstruction (UUO) was established and treated with various concentrations of AM (100, 200 or 400 mg/kg/day). Interstitial fibrosis markedly increased in the UUO mice. AM significantly reduced the obstruction-induced upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin in the kidneys of the UUO mice (P<0.05). Furthermore, AM treatment significantly inhibited the induction of EMT and the deposition of extracellular matrix. In addition, a transforming growth factor (TGF)-ß1-stimulated murine renal proximal tubule cell line (NRK-52E) was treated with various concentrations of AM (10, 20, and 40 µg/ml). E-cadherin expression levels significantly decreased and those of α-SMA significantly increased in NRK-52E cells stimulated with TGF-ß1 in vitro (P<0.05). Co-treatment with AM reversed these effects (P<0.05), and AM treatment reduced TGF-ß1-induced expression and Smad2/3 phosphorylation (P<0.05). These results suggested that AM antagonizes tubular EMT by inhibiting the Smad signaling pathway.

Baicalein attenuates renal fibrosis by inhibiting inflammation via down-regulating NF-κB and MAPK signal pathways.

Baicalein is a natural flavonoid that possesses notable anti-inflammatory effects. In this study, we detected whether baicalein protects against inflammatory response in unilateral ureteral obstruction mice model to ameliorate tubulointerstitial fibrosis. Baicalein treatment significantly attenuated tubulointerstitial fibrosis by markedly reducing fibronectin and collagen-I. The downregulation of alpha-smooth muscle actin and upregulation of E-cadherin indicated that the epithelial-mesenchymal transition process was suppressed. Furthermore, baicalein administration blocked the infiltration of macrophages and lymphocytes, as evidenced by the significantly reduced CD68 and CD3 positive cells. Meanwhile, the mRNA expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, and monocyte chemotactic protein in baicalein-treated groups was markedly reduced compared with the vehicle-treated group. More importantly, unilateral ureteral obstruction induced the activation of NF-κB and mitogen-activated protein kinase signal pathways to switch on inflammatory response to aggravate kidney fibrosis, but these effects were mitigated by baicalein. These data demonstrate that baicalein could inhibit inflammatory process via inactivation of NF-κB and MAPK signal pathways to execute its anti-fibrotic actions in obstructive kidney disease.

Regulatory control of high levels of carotenoid accumulation in potato tubers.

Potato (Solanum tuberosum L.) tubers contain a wide range of carotenoid contents. To decipher the key factors controlling carotenoid levels in tubers, four potato lines (Atlantic, Désirée, 91E22 and POR03) were examined by a combination of biochemical, molecular and genomics approaches. These lines contained incremental levels of carotenoids, which were found to be associated with enhanced capacity of carotenoid biosynthesis as evident from norflurazon treatment. Microarray analysis of high and low carotenoid lines (POR03 versus Atlantic) revealed 381 genes that showed significantly differential expression. The carotenoid metabolic pathway genes ß-carotene hydroxylase 2 (BCH2) and ß-carotene hydroxylase 1 (BCH1), along with zeaxanthin epoxidase (ZEP), and carotenoid cleavage dioxygenase 1A (CCD1A) were among the most highly differentially expressed genes. The transcript levels of BCH2 and BCH1 were lowest in Atlantic and highest in POR03, whereas those of ZEP and CCD1A were high in low carotenoid lines and low in high carotenoid lines. The high expression of BCH2 in POR03 line was associated with enhanced response to sugars. Our results indicate that high levels of carotenoid accumulation in potato tubers were due to an increased metabolic flux into carotenoid biosynthetic pathway, as well as the differential expression of carotenoid metabolic genes.

Modulation of carotenoid accumulation in transgenic potato by inducing chromoplast formation with enhanced sink strength.

An increasing interest in carotenoids as nutritional sources of provitamin A and health-promoting compounds has prompted a significant effort in metabolic engineering of carotenoid content and composition in food crops. The strategy commonly used in plants is to increase the biosynthetic capacity by altering the carotenogenic enzyme activities. The recent isolation of the Or gene from a cauliflower orange mutant has brought a new endeavor for carotenoid enhancement by increasing the sink strength to sequester and store the synthesized carotenoids. Potato as one of the major staple crops usually accumulates low levels of carotenoids. In this chapter, we describe a detailed protocol for metabolic engineering of carotenoids in potato plants with the Or gene and the analysis of the Or transformants.

Inhibition of D-amino-Acid oxidase activity induces pain relief in mice.

(1). We investigated the effects of inhibiting D: -amino-acid oxidase (DAO) activity on nociceptive responses through the use of mutant ddY/DAO(-) mice, which lack DAO activity, and through the application of a selective inhibitor of DAO, sodium benzoate, in the tail flick test, hot-plate test, formalin test, and acetic acid-induced writhing test. (2). Compared with normal ddY/DAO+ mice, ddY/DAO(- )mice showed significantly prolonged tail withdrawal latency in the tail flick test and licking/jumping latency in the hot-plate test, as well as significantly reduced duration of licking/biting in the late phase of the formalin test and the number of abdominal writhing in the acetic acid-induced writhing test. (3). In addition, we investigated the effects of sodium benzoate in Kunming mice having normal DAO activity. (4). Intravenous administration of sodium benzoate (400 mg/kg) significantly inhibited pain responses of the late phase of the formalin test and abdominal writhing responses in the acetic acid-induced writhing test, with no effects on the early phase flinch responses in the formalin test, nociceptive responses in the tail flick test, or hot-plate test. (5). These results suggest that DAO acts as a pro-nociceptive factor in pain, particularly chronic pain, transmission and modulation, and may be a target for pain treatment.

The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation.

Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of beta-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.