The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

Effects of vitamin D and/or calcium intervention on sleep quality in individuals with prediabetes: a post hoc analysis of a randomized controlled trial.

PURPOSE: The aim of this study was to evaluate the effects of vitamin D and/or calcium supplementation on sleep quality in individuals with prediabetes. METHODS: A 24-week randomized controlled trial (RCT) was conducted in a 212 Chinese population with prediabetes. Participants were randomly assigned to four groups: vitamin D + calcium group (1600 IU/day + 500 mg/day, n = 53), vitamin D group (1600 IU/day, n = 54), calcium group (500 mg/day, n = 51), and control group (placebo, n = 54). The Pittsburgh Sleep Quality Index (PSQI) was used as the primary outcome to assess sleep quality. Questionnaires and fasting blood samples were collected at baseline and post-intervention for demographic assessment and correlation index analysis. RESULTS: After a 24-week intervention, a significant difference was observed in serum 25(OH)D concentration among the four groups (P < 0.05), and the total PSQI score in vitamin D + calcium group was lower compared to the preintervention levels. Subgroup analyses revealed improved sleep quality with calcium supplementation (P < 0.05) for specific groups, including women, individuals with a low baseline 25(OH)D level (< 30 ng/mL), and individuals in menopause. Moreover, correlation analysis revealed a negative correlation between the extent of change in sleep efficiency scores before and after the calcium intervention and the degree of change in insulin efficiency scores (r = - 0.264, P = 0.007), as well as the magnitude of change in islet beta cell function (r = - 0.304, P = 0.002). CONCLUSIONS: The combined intervention of vitamin D and calcium, as well as calcium interventions alone, exhibits substantial potential for improving sleep quality in individuals with prediabetes. CLINICAL TRIAL REGISTRATION: The trial was registered in August 2019 as ChiCTR190002487.

Leonurine alleviates rheumatoid arthritis by regulating the Hippo signaling pathway.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause joint inflammation and damage. Leonurine (LE) is an alkaloid found in Leonurus heterophyllus. It has anti-inflammatory effects. HYPOTHESIS/PURPOSE: The molecular mechanisms by which LE acts in RA are unclear and further investigation is required. METHODS: Mice with collagen-induced arthritis (CIA), and RA-fibroblast-like synoviocytes (FLSs) isolated from them were used as in vivo and in vitro models of RA, respectively. The therapeutic effects of LE on CIA-induced joint injury were investigated by micro-computed tomography, and staining with hematoxylin and eosin and Safranin-O/Fast Green. Cell Counting Kit-8, a Transwell® chamber, enzyme-linked immunosorbent assays, RT-qPCR, and western blotting were used to investigate the effects of LE on RA-FLS viability, migratory capacity, inflammation, microRNA-21 (miR-21) levels, the Hippo signaling pathway, and the effects and intrinsic mechanisms of related proteins. Dual luciferase was used to investigate the binding of miR-21 to YOD1 deubiquitinase (YOD1) and yes-associated protein (YAP). Immunofluorescence was used to investigate the localization of YAP within the nucleus and cytoplasm. RESULTS: Treatment with LE significantly inhibited joint swelling, bone damage, synovial inflammation, and proteoglycan loss in the CIA mice. It also reduced the proliferation, cell colonization, migration/invasion, and inflammation levels of RA-FLSs, and promoted miR-21 expression in vitro. The effects of LE on RA-FLSs were enhanced by an miR-21 mimic and reversed by an miR-21 inhibitor. The dual luciferase investigation confirmed that both YOD1 and YAP are direct targets of miR-21. Treatment with LE activated the Hippo signaling pathway, and promoted the downregulation and dephosphorylation of MST1 and LATS1 in RA, while inhibiting the activation of YOD1 and YAP. Regulation of the therapeutic effects of LE by miR-21 was counteracted by YOD1 overexpression, which caused the phosphorylation of YAP and prevented its nuclear ectopic position, thereby reducing LE effect on pro-proliferation-inhibiting apoptosis target genes. CONCLUSION: LE regulates the Hippo signaling pathway through the miR-21/YOD1/YAP axis to reduce joint inflammation and bone destruction in CIA mice, thereby inhibiting the growth and inflammation of RA-FLSs. LE has potential for the treatment of RA.

ß-asarone improves cognitive impairment and alleviates autophagy in mice with vascular dementia via the cAMP/PKA/CREB pathway.

BACKGROUND: Vascular dementia (VD) is the second most common type of dementia after Alzheimer's disease. ß-asarone, a major component of Acorus tatarinowii Schott, is important in neurodegenerative and neurovascular diseases. Studies have confirmed that ß-asarone can mitigate autophagy and reduce damage in hypoxic cells. We also reported that ß-asarone improves learning and memory. This study further clarifies whether ß-asarone attenuates cerebral ischaemic injury by acting through the cAMP/PKA/CREB pathway in VD model mice. METHODS: Here, genes and potential pathways that may be targeted by ß-asarone for the treatment of transient cerebral ischaemia (TCI) and cognitive impairment (CI) were obtained using network pharmacology. The two-vessel occlusion method was used to establish the VD model. The Morris water maze test was used to evaluate the effects on memory. Then, the protein levels of mitofusin-2 (Mfn2), brain-derived neurotrophic factor (BDNF), optic atrophy 1 (OPA1), cyclic adenosine monophosphate (cAMP), myelin basic protein (MBP), matrix metalloproteinase-9 (MMP9) and neuron specific enolase (NSE) were determined by ELISA. The levels of superoxide dismutase (SOD) and malonaldehyde (MDA) were measured using commercial kits. Then, qRT-PCR was employed to investigate the expression of the candidate genes screened from the protein-protein interaction (PPI) network. Furthermore, the expression of the autophagy-related proteins Beclin-1, (microtubule-associated protein light chain 3) LC3, p62, postsynaptic density protein 95 (PSD95), protein kinase A (PKA), pPKA, cyclic-AMP response binding protein (CREB), and pCREB was determined by western blotting. The expression of autophagy-related proteins, PSD95 and translocase of outer mitochondrial membrane 20 (TOM20) was determined by immunofluorescence analyses. RESULTS: The network pharmacological analysis showed 234 targets related to ß-asarone, 1,118 genes related to TCI and 2,039 genes associated with CI. Our results confirm that ß-asarone treatment not only alleviated brain damage in the VD model by improving mitochondrial and synaptic function, reducing neuronal injury and upregulating the expression of antioxidants but also effectively improved the cognitive behaviour of VD model mice. Moreover, ß-asarone downregulated VD-induced RELA and CCND1 mRNA expression. In addition, we validated that ß-asarone increased the phosphorylation of PKA and CREB and upregulated cAMP protein expression. The results showed that the cAMP/PKA/CREB signalling pathway was upregulated. Moreover, ß-asarone administration decreased the protein expression levels of Beclin-1 and LC3 and increased the expression levels of p62 in VD model mice. CONCLUSIONS: ß-asarone inhibits Beclin-1-dependent autophagy and upregulates the cAMP/PKA/CREB signalling pathway to attenuate mitochondrial and synaptic damage from cerebral ischaemia and improve learning and cognitive abilities in VD model mice.

[Mechanism of Zhongfeng Xingnao Decoction in improving microcirculatory disorders in cerebral hemorrhage based on network pharmacology and molecular docking techniques].

This study aimed to explore the mechanism of Zhongfeng Xingnao Decoction(ZFXN) in intervening microcirculatory di-sorders in cerebral hemorrhage by network pharmacology and molecular docking techniques. The information on the components of ZFXN was obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and the predicted targets of chemical components were obtained from PubChem and SwissTargetPrediction. The relevant targets of cerebral hemorrhage and microcirculatory disorders were collected from the GeneCards database, and the common targets of the components and diseases were analyzed by the Database for Annotation, Visualization, and Integrated Discovery(DAVID) for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. Visualization of the correlation network was carried out using Cytoscape software to further screen important chemical components for molecular docking prediction with disease targets. The animal experiment validation was performed using modified neurological severity score(mNSS), enzyme-linked immunosorbent assay(ELISA), quantitative real-time polymerase chain reaction(qRT-PCR), immunofluorescence, and Western blot to detect the effects of ZFXN intervention in mice with cerebral hemorrhage. The results showed that there were 31 chemical components and 856 targets in the four drugs contained in ZFXN, 173 targets for microcirculatory disorders in cerebral hemorrhage, and 57 common targets for diseases and components. The enrichment analysis showed that common targets were mainly involved in biological processes, such as cell proliferation and apoptosis, and signaling pathways, such as tumor pathway, viral infection, phosphoinositide-3-kinase/protein kinase B(PI3K/AKT) signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway. Molecular docking results revealed that the common components ß-sitosterol of Rhei Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Ginseng Radix et Rhizoma Rubra showed good docking with proto-oncogene tyrosine-protein kinase(SRC), signal transducer and activator of transcription 3(STAT3), phosphoinositide-3-kinase catalytic alpha polypeptide gene(PIK3CA), recombinant protein tyrosine phosphatase non receptor type 11(PTPN11), AKT1, epidermal growth factor receptor(EGFR), calcium adhesion-associated protein beta 1(CTNNB1), vascular endothelial growth factor A(VEGFA), and tumor protein p53(TP53). Moreover, sennoside E of Rhei Radix et Rhizoma showed good docking with MAPK1. The results revealed that the ZFXN relieved the neural injury in mice with cerebral hemorrhage, decreased the expression of S100 calcium-binding protein B(S100ß), neuron specific enolase(NSE), matrix metalloproteinase 9(MMP9), tumor necrosis factor α(TNF-α), interleukin 1ß(IL-1ß), SRC, EGFR, CTNNB1, VEGFA, TP53, glial fibrillary acidic protein(GFAP), and leukocyte differentiation antigen 86(CD86), and increased the expression of p-PI3K, p-AKT, and zona occludens 1(ZO-1). The results indicate that ZFXN may inhibit neuronal apoptosis and inflammatory response through PI3K/AKT/p53 pathway to protect the blood-brain barrier, thereby slowing down microcirculatory impairment in cerebral hemorrhage.

Effect of Vitamin D and/or Calcium Supplementation on Pancreatic ß-Cell Function in Subjects with Prediabetes: A Randomized, Controlled Trial.

So far, the potential role of vitamin D in ß-cell function remains a matter of debate. Therefore, a randomized, placebo-controlled trial (RCT) was conducted to evaluate the effect of a vitamin D supplement with or without calcium on ß-cell function in a Chinese population with prediabetes. Two hundred and forty-three subjects were randomly assigned in a 2-by-2 factorial-design RCT to receive either 1600 IU/day vitamin D3 with/or 500 mg/day calcium for 24 weeks. The results showed that oral administration of vitamin D and calcium could increase the secretion of insulin. Vitamin D-insufficient individuals displayed an increment in the disposition index (adjusted change = 0.31, 95%CI: 0.07, 0.56) after treatment by vitamin D + calcium. It illustrated that supplementation with vitamin D and calcium might improve the function of pancreatic ß-cell in prediabetes with low serum 25(OH)D levels. However, further studies are needed to confirm the findings. Given the low vitamin D content in natural foods, it is necessary to fortify processed foods with vitamin D.

Study on the effect and mechanism of quercetin in treating gout arthritis.

Quercetin is widely found in natural plants, especially Chinese herbal plants. It has been used to treat arthritis in China for thousands of years. However, the effects and mechanisms of quercetin in the treatment of gout arthritis (GA) remain unclear. We aimed to verify the treatment of GA with quercetin and investigate the underlying mechanism. A combination of network pharmacology and experiments was used to reveal the mechanism of quercetin in the treatment of GA. Potential targets of quercetin and gout were identified. Then, the protein-protein interaction network for the common targets between quercetin and gout was constructed and the core targets were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses for the common targets were performed to elucidate the pharmacological functions and mechanisms associated with quercetin treatment in GA. Finally, a monosodium urate-induced GA rat model was used to validate the predicted mechanisms in network pharmacology. Seventy-two common targets were identified. KEGG analysis revealed that treatment of GA with quercetin predominantly involved the interleukin (IL)-17, tumor necrosis factor (TNF), mitogen-activated protein kinase, and phosphoinositide 3-kinase-Akt signaling pathways. In an experimental validation, quercetin attenuated ankle joint inflammation-induced bone destruction and histological lesions. It also diminished the expression of IL-6, IL-17A, and IL-17F in the IL-17 pathway, and regulated the release of RAR-related orphan receptor gamma t,IL-17E, IL-1ß, IL-6, TNF-α, Foxp3, and transforming growth factor-beta 1. The collective findings implicate quercetin as a valuable alternative drug for the treatment of GA.

[Galangin alleviates learning and memory impairments in APP/PS1 double- transgenic mice by regulating Akt/MEF2D/Beclin-1 signaling pathway].

The study aimed to investigate the effects of galangin on learning and memory impairments and Akt/MEF2 D/Beclin-1 signaling pathway in APP/PS1 double-transgenic mice. The mice in this experiment were divided into the normal group, model group, low-(25 mg·kg~(-1)), medium-(50 mg·kg~(-1)), and high-dose(100 mg·kg~(-1)) galangin groups, donepezil(3 mg·kg~(-1)) group, Akt inhibitor(25 mg·kg~(-1)) group, and autophagy inhibitor(30 mg·kg~(-1)) group, with ten in each group, and administered with the corresponding drugs for 30 successive days. On the 24 th day of medication, the water maze and dark avoidance tests were performed. The levels of p-tau, ß-amyloid peptide 1-42(Aß_(42)), acetylcholinesterase(AChE), ß-site amyloid precursor protein cleaving enzyme 1(BACE1), and amyloid precursor protein(APP) in hippocampus were detected by ELISA, the Beclin-1 mRNA expression by RT-PCR, the expression of Aß_(42) and glial fibrillary acidic protein(GFAP) by immunohistochemistry, and the expression of myocyte enhancer factor 2 D(MEF2 D) by immunofluorescence assay. The pathological changes in hippocampus were observed after HE staining, and the expression of Akt, MEF2 D, and Beclin-1 in hippocampus were assayed by Western blot. These results showed that compared with the normal group, the model group exhibited prolonged swimming time, increased number of errors and electric shocks, up-regulated p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, shortened incubation period, decreased p-Akt and MEF2 D, and obvious hippocampal injury. Compared with the model group, donepezil and galangin shortened the swimming time, reduced the number of errors and electric shocks, down-regulated the expression of p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, prolonged the incubation period, up-regulated p-Akt and MEF2 D, and improved the pathological changes in hippocampus. Compared with the autophagy inhibitor group, galangin prolonged the swimming time, elevated the number of errors and electric shocks, enhanced the expression of p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, shortened the incubation period, and diminished the expression of p-Akt and MEF2 D. In conclusion, galangin improves the learning and memory impairments and hippocampal neuron injury of APP/PS1 mice, which may be related to its regulation of Akt/MEF2 D/Beclin-1 signaling pathway.

PSORI-CM02 formula alleviates imiquimod-induced psoriasis via affecting macrophage infiltration and polarization.

AIMS: Psoriasis is a refractory skin disease characterized by macrophage cell infiltrated in the dermal layer. Macrophages can simultaneously polarize into two distinct functional subtypes, M1 and M2, and this process is affected by the microenvironment, cytokines and JAK/STAT pathways. Formula PSORI-CM02 is a novel Chinese medicine used to alleviate psoriasis symptoms and regulate T cell differentiation and epithelial cell proliferation. However, the effects of PSORI-CM02 in imiquimod (IMQ)-induced psoriasis and macrophage infiltration and polarization in the dermis remain unknown. MAIN METHODS: Imiquimod induced psoriasis mice model and M1/M2 polarization model on mice peritoneal macrophages cell line RAW264.7 in vitro were used to observe the therapeutic effect of PSORI-CM02 on skin and its molecular mechanisms. KEY FINDINGS: PSORI-CM02 can significantly improve skin lesions and reduce macrophage infiltration in mice induced by imiquimod. After treatment with PSORI-CM02 formula, M1 macrophage mediators were significantly reduced, while M2 mediators were significantly increased in mice. Similarly in vitro, M1 macrophage proliferation was suppressed and M2 macrophage proliferation was elevated by PSORI-CM02 in the presence of LPS and IL-4, respectively. The elevated expression of TNF-α, iNOS, and IL-1ß induced by LPS was reduced, while the expression of Arg-1, Fizz-1, Ym-1, and IL-10 induced by IL-4 was elevated in PSORI-CM02-treated cells. Finally, we found that the effects of PSORI-CM02 in macrophage polarization were associated with regulation of STAT1 and STAT6 expression, which were activated by LPS and IL-4, respectively. SIGNIFICANCE: Our novel findings reveal that PSORI-CM02 may possess therapeutic action in psoriasis treatment by regulating the infiltration and polarization of macrophages in the dermal layer.

Genistein suppresses psoriasis-related inflammation through a STAT3-NF-κB-dependent mechanism in keratinocytes.

Psoriasis is a chronic recurrent skin inflammatory disease, and inhibition of inflammation may be an effective means of treating psoriasis. The flavonoid genistein has a clear anti-inflammatory effect. However, the anti-psoriatic effects of genistein and their underlying mechanisms remain unclear. In this study, we investigated the effects of genistein on imiquimod (IMQ)-induced psoriasis-like skin lesions in vivo and explored the mechanisms underlying those effects in vitro. It was found that genistein can significantly improve IMQ-induced pathological scores of cutaneous skin lesions in mice, reduce epidermal thickness, and inhibit the expression of inflammatory factors，including interleukin (IL)-1ß, IL-6, tumour necrosis factor-alpha (TNF-α), chemokine ligand 2 (CCL2), IL-17 and IL-23. In vitro studies, genistein inhibited the proliferation of human keratinocyte HaCaT cells and inhibited the expression of inflammatory factors in a dose-dependent manner which induced by TNFα. Further researches showed that genistein could also significantly inhibit phosphorylated STAT3 (pSAT3) expression in IMQ mice dorsal skin and in TNF-α-induced HaCaT cells. The inhibitory effect of genistein on the expression of IL-6, IL-23 and TNF-α was weakened after Stat3 siRNA in HaCaT cells. Genistein could also significantly inhibit TNF-α induced the nuclear translocation of NF-κB, and inhibit the phosphorylation of I-kBα (pI-kBα). After combining with NF-κB blocker BAY 11-7082, the effect of genistein down-regulate the expression of TNF-α and VEGFA was attenuated in HaCaT cells. The results suggest that genistein may be developed for the treatment of psoriasis lesions.

Total polysaccharides of the Sijunzi decoction attenuate tumor necrosis factor-α-induced damage to the barrier function of a Caco-2 cell monolayer via the nuclear factor-κB-myosin light chain kinase-myosin light chain pathway.

AIM: To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction (TPSJ) on the epithelial barriers in vitro. METHODS: Caco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance (TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respectively used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. Western blotting was also used to evaluate the cellular expression of myosin light chain (MLC), phosphorylated MLC (pMLC), MLC kinase (MLCK), and nuclear factor (NF)-κB p65. RESULTS: TPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cytokines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor (TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65. CONCLUSION: Our results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.

Electrostatic Spray Ionization from 384-Well Microtiter Plates for Mass Spectrometry Analysis-Based Enzyme Assay and Drug Metabolism Screening.

We have realized the direct ionization of samples from wells of microtiter plates under atmospheric conditions for mass spectrometry analysis without any liquid delivery system or any additional interface. The microtiter plate is a commercially available 384-well plate without any modification, working as a container and an emitter for electrostatic spray ionization of analytes. The approach provides high throughput for the large batches of reactions and both the qualitative and quantitative analysis of a single compound or mixture. The limits of detection in small drug molecules, peptides, and proteins are similar in comparison with standard direct infusion electrospray ionization. The analysis time per well is only seconds. These analytical merits benefit many microtiter plate-based studies, such as combinatorial chemistry and high throughput screening in enzyme assay or drug metabolism. Herein, we illustrate the application in enzyme assay using tyrosine oxidation catalyzed by tyrosinase in the presence or absence of inhibitors. The potential application in drug development is also demonstrated with cytochrome P450-catalyzed metabolic reactions of two drugs in microtiter plates followed with direct ESTASI-MS/MS-based characterization of the metabolism products.

Effect of sodium ferulate on the hyperalgesia mediated by P2X3 receptor in the neuropathic pain rats.

Neuropathic pain is usually persistent and there is no effective treatment. Activation of P2X(3) receptor subtype in primary sensory neurons is involved in neuropathic pain. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the hyperalgesia mediated by P2X(3) receptor of rats after chronic constriction injury (CCI). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and the expression of P2X(3) receptor immunoreactivity and protein in dorsal root ganglion (DRG) neurons was analyzed by immunohistochemistry and western blotting. In CCI rats treated with SF, the MWT and TWL were increased compared with CCI rats treated with normal saline. The expression of P2X(3) receptor in DRG neurons was increased after CCI. In CCI rats treated with SF, the up-regulated expression of P2X(3) receptor in DRG neurons was reduced. SF may reduce the thermal and mechanical hyperalgesia in CCI rat model by decreasing the pain transmitted by primary afferant neurons mediated by P2X(3) receptor during the chronic neuropathic pain injury.