Safranal alleviates pentetrazole-induced epileptic seizures in mice by inhibiting the NF-κB signaling pathway and mitochondrial-dependent apoptosis through GSK-3ß inactivation.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, a traditional Chinese medicine, is derived from Crocus sativus L. stigmas and has been reported to possess neuroprotective properties and potentially contribute to the inhibition of apoptosis and inflammation. Safranal, a potent monothyral aldehyde, is a main component of saffron that has been reported to have antiepileptic activity. However, the specific mechanism by which safranal suppresses epileptic seizures via its antiapoptotic and anti-inflammatory properties is unclear. AIM: To evaluate the effect of safranal on seizure severity, inflammation, and postictal neuronal apoptosis in a mouse model of pentetrazole (PTZ)-induced seizures and explore the underlying mechanism involved. MATERIALS AND METHODS: The seizure stage and latency of stage 2 and 4 were quantified to assess the efficacy of safranal in mitigating PTZ-induced epileptic seizures in mice. Electroencephalography (EEG) was employed to monitor epileptiform afterdischarges in each experimental group. The cognitive abilities and motor functions of the mice were evaluated using the novel object recognition test and the open field test, respectively. Neurons were quantified using hematoxylin and eosin staining. Additionally, bioinformatics tools were utilized to predict the interactions between safranal and specific target proteins. Glycogen synthase kinase-3ß (GSK-3ß), mitochondrial apoptosis-related proteins, and inflammatory factor levels were analyzed through western blotting. Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) concentrations in brain tissue were assessed by ELISA. RESULTS: Safranal decreased the average seizure stage and increased the lantency of stage 2 and 4 seizures in PTZ-induced epileptic mice. Additionally, safranal exhibited neuroprotective effects on hippocampal CA1 and CA3 neurons and reduced hyperactivity caused by postictal hyperexcitability. Bioinformatics analysis revealed that safranal can bind to five specific proteins, including GSK-3ß. By promoting Ser9 phosphorylation and inhibiting GSK-3ß activity, safranal effectively suppressed the NF-κB signaling pathway. Moreover, the findings indicate that safranal treatment can decrease TNF-α and IL-1ß levels in the cerebral tissues of epileptic mice and downregulate mitochondrial apoptosis-related proteins, including Bcl-2, Bax, Bak, Caspase 9, and Caspase 3. CONCLUSION: Safranal can suppress the NF-κB signaling pathway and mitochondrial-dependent apoptosis through GSK-3ß inactivation, suggesting that it is a promising therapeutic agent for epilepsy treatment.

Assessment of the anti-rheumatoid arthritis activity of Gastrodia elata (tian-ma) and Radix aconitic lateralis preparata (fu-zi) via network pharmacology and untargeted metabolomics analyses.

AIM: Gastrodia elata and Radix aconiti lateralis preparrata are respectively named as Tian-Ma and Fu-Zi (TF) in Chinese. We explored the active components against rheumatoid arthritis (RA) from an extensively used couplet of Chinese herbs, Gastrodia elata and Radix aconiti lateralis preparata (TF) via untargeted metabolomics and network pharmacological approaches. METHODS: Water extracts of TF were mixed at ratios 1:1, 3:2 and 2:3 (w/w). Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was then utilized as metabolomics screening. Human Metabolome (http://www.hmdb.ca/) and Lipidmaps (http://www.lipidmaps.org/) databases were used to annotate detected compounds. Further identification of vital genes and important pathways associated with the anti-RA properties of the TF preparations was done via network pharmacology, and verified by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Four key compounds involved in unsaturated fatty acid biosynthesis and isoflavonoid biosynthesis were identified through metabolomics analyses. Three key components of TF associated with anti-RA activity were linoleic acid, daidzein, and daidzin. Results of RT-qPCR revealed that all 3 tested TF couplets (1:1, 3:2, and 2:3) markedly suppressed the transcription of PTGS2. These results were consistent with our network pharmacological predictions. CONCLUSIONS: The anti-RA properties of Tian-Ma and Fu-Zi are associated with the inhibition of arachidonic acid metabolism pathway.

Comprehensive Analysis of Transcriptomics and Metabolomics between the Heads and Tails of Angelica Sinensis: Genes Related to Phenylpropanoid Biosynthesis Pathway.

BACKGROUND: In Traditional Chinese Medicine (TCM), the heads and tails of Angelica sinensis (Oliv.) Diels (AS) is used in treating different diseases due to their different pharmaceutical efficacies. The underline mechanisms, however, have not been fully explored. OBJECTIVE: Novel mechanisms responsible for the discrepant activities between AS heads and tails were explored by a combined strategy of transcriptomes and metabolomics. METHODS: Six pairs of the heads and tails of AS roots were collected in Min County, China. Total RNA and metabolites, which were used for RNA-seq and untargeted metabolomics analysis, were respectively isolated from each AS sample (0.1 g) by Trizol and methanol reagent. Subsequently, differentially expressed genes (DEGs) and discrepant pharmaceutical metabolites were identified for comparing AS heads and tails. Key DEGs and metabolites were quantified by RT-qPCR and targeted metabolomics experiment. RESULTS: Comprehensive analysis of transcriptomes and metabolomics results suggested that five KEGG pathways with significant differences included 57 DEGs. Especially, fourteen DEGs and six key metabolites were related to the metabolic regulation of Phenylpropanoid biosynthesis (PB) pathway. Results of RT-qPCR and targeted metabolomics indicated that higher levels of expression of crucial genes in PB pathway, such as PAL, CAD, COMT and peroxidase in the tail of AS, were positively correlated with levels of ferulic acid-related metabolites. The average content of ferulic acid in tails (569.58±162.39 nmol/g) was higher than those in the heads (168.73 ± 67.30 nmol/g) (P.

A multi-lock inhibitory mechanism for fine-tuning enzyme activities of the HECT family E3 ligases.

HECT E3 ligases control the degradation and functioning of numerous oncogenic/tumor-suppressive factors and signaling proteins, and their activities must be tightly regulated to prevent cancers and other diseases. Here we show that the Nedd4 family HECT E3 WWP1 adopts an autoinhibited state, in which its multiple WW domains sequester HECT using a multi-lock mechanism. Removing WW2 or WW34 led to a partial activation of WWP1. The structure of fully inhibited WWP1 reveals that many WWP1 mutations identified in cancer patients result in a partially active state with increased E3 ligase activity, and the WWP1 mutants likely promote cell migration by enhancement of ∆Np63α degradation. We further demonstrate that WWP2 and Itch utilize a highly similar multi-lock autoinhibition mechanism as that utilized by WWP1, whereas Nedd4/4 L and Smurf2 utilize a slightly variant version. Overall, these results reveal versatile autoinhibitory mechanisms that fine-tune the ligase activities of the HECT family enzymes.

Early-life high-fat diet-induced obesity programs hippocampal development and cognitive functions via regulation of gut commensal Akkermansia muciniphila.

Obesity is one of the most serious public health challenges in the world. Obesity during early life has been associated with an increased risk of neurodevelopmental disorders, including deficits in learning and memory, yet the underlying mechanisms remain unclear. Here, we show that early life high-fat diet (HFD) feeding impairs hippocampus-dependent contextual/spatial learning and memory, and alters the gut microbiota, particularly by depleting Akkermansia muciniphila (A. muciniphila), in mice. Transplantation of the HFD microbiota confers hippocampus-dependent learning and memory deficits to mice fed a chow diet. Oral treatment of HFD-fed mice with the gut commensal A. muciniphila corrects gut permeability, reduces hippocampal microgliosis and proinflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6) expression, and restores neuronal development and synapse plasticity, thus ameliorates defects in learning and memory. Interestingly, treatment of mice with lipopolysaccharide (LPS) mimics HFD-induced hippocampus-dependent cognitive impairment in chow-fed mice. In line with these findings, pharmacologic blockade of Toll-like receptor 4 (TLR4) signalling or antibiotics treatment both effectively prevent hippocampus-dependent learning and memory deficits in HFD-fed mice. Collectively, our findings demonstrate an unexpected pivotal role of gut microbiota in HFD-induced cognitive deficits and identify a potential probiotic therapy for obesity associated with cognitive dysfunction during early life.

WW domain-mediated regulation and activation of E3 ubiquitin ligase Suppressor of Deltex.

The Nedd4 family E3 ligases Itch and WWP1/2 play crucial roles in the regulation of cell cycle progression and apoptosis and are closely correlated with cancer development and metastasis. It has been recently shown that the ligase activities of Itch and WWP1/2 are tightly regulated, with the HECT domain sequestered intramolecularly by a linker region connecting WW2 and WW3. Here, we show that a similar autoinhibitory mechanism is utilized by the Drosophila ortholog of Itch and WWP1/2, Suppressor of Deltex (Su(dx)). We show that Su(dx) adopts an inactive steady state with the WW domain region interacting with the HECT domain. We demonstrate that both the linker and preceding WW2 are required for the efficient binding and regulation of Su(dx) HECT. Recruiting the multiple-PY motif-containing adaptor dNdfip via WW domains relieves the inhibitory state of Su(dx) and leads to substrate (e.g. Notch) ubiquitination. Our study demonstrates an evolutionarily conservative mechanism governing the regulation and activation of some Nedd4 family E3 ligases. Our results also suggest a dual regulatory mechanism for specific Notch down-regulation via dNdfip-Su(dx)-mediated Notch ubiquitination.

Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·α-adaptin complex.

Traffic of cargo across membranes helps establish, maintain, and reorganize distinct cellular compartments and is fundamental to many metabolic processes. The cargo-selective endocytic adaptor Numb participates in clathrin-dependent endocytosis by attaching cargoes to the clathrin adaptor α-adaptin. The phosphorylation of Numb at Ser265 and Ser284 recruits the regulatory protein 14-3-3, accompanied by the dissociation of Numb from α-adaptin and Numb's translocation from the cortical membrane to the cytosol. However, the molecular mechanisms underlying the Numb-α-adaptin interaction and its regulation by Numb phosphorylation and 14-3-3 recruitment remain poorly understood. Here, biochemical and structural analyses of the Numb·14-3-3 complex revealed that Numb phosphorylation at both Ser265 and Ser284 is required for Numb's efficient interaction with 14-3-3. We also discovered that an RQFRF motif surrounding Ser265 in Numb functions together with the canonical C-terminal DPF motif, required for Numb's interaction with α-adaptin, to form a stable complex with α-adaptin. Of note, we provide evidence that the phosphorylation-induced binding of 14-3-3 to Numb directly competes with the binding of α-adaptin to Numb. Our findings suggest a potential mechanism governing the dynamic assembly of Numb with α-adaptin or 14-3-3. This dual-site recognition of Numb by α-adaptin may have implications for other α-adaptin targets. We propose that the newly identified α-adaptin-binding site surrounding Ser265 in Numb functions as a triggering mechanism for the dynamic dissociation of the Numb·α-adaptin complex.

Allosteric auto-inhibition and activation of the Nedd4 family E3 ligase Itch.

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto-inhibition. However, the molecular mechanism underlying Nedd4 E3 auto-inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2-E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1-mediated phosphorylation relieves the auto-inhibition of Itch in a WW2-dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch.

Common susceptibility variants are shared between schizophrenia and psoriasis in the Han Chinese population.

BACKGROUND: Previous studies have shown that individuals with schizophrenia have a greater risk for psoriasis than a typical person. This suggests that there might be a shared genetic etiology between the 2 conditions. We aimed to characterize the potential shared genetic susceptibility between schizophrenia and psoriasis using genome-wide marker genotype data. METHODS: We obtained genetic data on individuals with psoriasis, schizophrenia and control individuals. We applied a marker-based coheritability estimation procedure, polygenic score analysis, a gene set enrichment test and a least absolute shrinkage and selection operator regression model to estimate the potential shared genetic etiology between the 2 diseases. We validated the results in independent schizophrenia and psoriasis cohorts from Singapore. RESULTS: We included 1139 individuals with psoriasis, 744 with schizophrenia and 1678 controls in our analysis, and we validated the results in independent cohorts, including 441 individuals with psoriasis (and 2420 controls) and 1630 with schizophrenia (and 1860 controls). We estimated that a large fraction of schizophrenia and psoriasis risk could be attributed to common variants (h2SNP = 29% ± 5.0%, p = 2.00 × 10-8), with a coheritability estimate between the traits of 21%. We identified 5 variants within the human leukocyte antigen (HLA) gene region, which were most likely to be associated with both diseases and collectively conferred a significant risk effect (odds ratio of highest risk quartile = 6.03, p < 2.00 × 10-16). We discovered that variants contributing most to the shared heritable component between psoriasis and schizophrenia were enriched in antigen processing and cell endoplasmic reticulum. LIMITATIONS: Our sample size was relatively small. The findings of 5 HLA gene variants were complicated by the complex structure in the HLA region. CONCLUSION: We found evidence for a shared genetic etiology between schizophrenia and psoriasis. The mechanism for this shared genetic basis likely involves immune and calcium signalling pathways.

Association analysis revealed one susceptibility locus for vitiligo with immune-related diseases in the Chinese Han population.

Generalized vitiligo is an autoimmune disease characterized by melanocyte loss, which results in patchy depigmentation of skin and hair, and is associated with an elevated risk of other immune-related diseases. However, there is no reported study on the associations between immune susceptibility polymorphisms and the risk of vitiligo with immune-related diseases. The aim of this study was to evaluate the potential influence of 10 single-nucleotide polymorphisms (SNPs) at 18q21.31 (rs10503019), 4p16.1 (rs11940117), 3q28 (rs1464510), 14q12 (rs2273844), 12q13.2 (rs2456973), 16q12.2 (rs3213758), 10q25.3 (rs4353229), 3q13.33 (rs59374417), and 10p15.1 (rs706779 and rs7090530) on vitiligo with immune-related diseases in the Chinese Han population. All SNPs were genotyped in 552 patients with vitiligo-associated immune-related diseases and 1656 controls using the Sequenom MassArray system. Data were analyzed with PLINK 1.07 software. The C allele of rs2456973 at 12q13.2 was observed to be significantly associated with vitiligo-associated immune-related diseases (autoimmune diseases and allergic diseases) (P = 0.0028, odds ratio (OR) = 1.27). In subphenotype analysis, the rs2456973 C allele was also significantly associated with early-onset vitiligo by comparing with controls (P = 0.0001) and in the case-only analysis (P = 0.0114). We confirmed that 12q13.2 was an important candidate locus for vitiligo with immune-related diseases (autoimmune diseases and allergic diseases) and affected disease phenotypes with early onset.

Filaggrin gene mutation c.3321delA is associated with various clinical features of atopic dermatitis in the Chinese Han population.

BACKGROUND: We confirmed that the filaggrin gene mutation c.3321delA is associated with atopic dermatitis in our previous genome wide association study of the Chinese Han population. c.3321delA is the most common filaggrin gene mutation in Chinese atopic dermatitis patients but is not present in European populations. OBJECTIVE: To investigate the genetic model for the c.3321delA mutation and to determine the correlation between c.3321delA and atopic dermatitis clinical phenotypes in the Chinese Han population. METHOD: The filaggrin gene mutation c.3321delA was sequenced in 1,080 atopic dermatitis patients and 908 controls from the Chinese population. The χ2 test, ANOVA,nonparametric tests and logistic regression were used to investigate the relationship between the c.3321delA genotype and atopic dermatitis clinical phenotypes in the Chinese Han population. RESULTS: Analyses of the genetic model revealed that the additive model best described the c.3321delA mutation (P = 3.09E-11, OR = 3.43, 95%CI = 2.38-4.96). Stratified analyses showed that the c.3321delA allele frequency distribution is significantly associated with concomitant skin xerosis (P = 1.68E-03, OR = 2.13,95%CI = 1.32-3.46), palmar hyperlinearity (P = 3.64E-17, OR = 4.0,95%CI = 2.86-5.70), white dermatographism (P = 4.25E-03, OR = 1.82,95%CI = 1.22-2.71), food intolerance (P = 1.51E-03, OR = 1.76,95%CI = 1.23-2.50) and disease severity ( P = 9.67E-05). CONCLUSION: Our study indicates that the filaggrin gene mutation c.3321delA is associated with clinical phenotypes of atopic dermatitis in the Chinese Han population, which might help us gain a better understanding on the pathogenesis of atopic dermatitis.