Construction of Cell Membrane Chromatography Screening Materials Based on Avi-Tag Fused G Protein-Coupled Receptors.

Mas-related G protein-coupled receptor X2 (MrgprX2) plays a crucial role in anaphylactoid reactions and allergic diseases. Some antagonists with reasonable potency and selectivity have been reported. Cell membrane chromatography (CMC) is effective for discovering ligands. Protein-tag-based CMC models (e.g., SNAP tags and HALO tags) have enhanced performance but also increased nonspecific adsorption of small molecules. The Avi tag, a short peptide sequence, binds biotin specifically via BirA catalysis. Our study showed that 2-iminobiotin (IB) can be a BirA substrate, enabling the development of a new cell membrane stationary phase (CMSP) based on the chemical properties (modifying carboxyl silica gel and specifically labeling the Avi tag) of IB. First, we constructed the MrgprX2-Avi-tag HEK293T cell line. Next, we synthesized IB-modified silica gel (SiO2-IB) stepwise. Finally, we immobilized Avi-tagged MrgprX2 cell membranes on SiO2-IB under BirA catalysis. We characterized the developed CMSP and used it to establish a MrgprX2-Avi-tag/CMC-HPLC/MS two-dimensional screening platform, successfully screening vitexicarpin fromViticis Fructus extract via a 2D/CMC platform. In vitro and in vivo experiments confirmed that vitexicarpin targets the MrgprX2 receptor, demonstrating antiallergic effects. Our IB-Avi tag-based CMC approach effectively decreased nonspecific adsorption of the screening materials. The Avi-tag-based 2D/CMC platform is suitable for screening potential drug candidates.

Developing neural network diagnostic models and potential drugs based on novel identified immune-related biomarkers for celiac disease.

BACKGROUND: As one of the most common intestinal inflammatory diseases, celiac disease (CD) is typically characterized by an autoimmune disorder resulting from ingesting gluten proteins. Although the incidence and prevalence of CD have increased over time, the diagnostic methods and treatment options are still limited. Therefore, it is urgent to investigate the potential biomarkers and targeted drugs for CD. METHODS: Gene expression data was downloaded from GEO datasets. Differential gene expression analysis was performed to identify the dysregulated immune-related genes. Multiple machine algorithms, including randomForest, SVM-RFE, and LASSO, were used to select the hub immune-related genes (HIGs). The immune-related genes score (IG score) and artificial neural network (ANN) were constructed based on HIGs. Potential drugs targeting HIGs were identified by using the Enrichr platform and molecular docking method. RESULTS: We identified the dysregulated immune-related genes at a genome-wide level and demonstrated their roles in CD-related immune pathways. The hub genes (MR1, CCL25, and TNFSF13B) were further screened by integrating several machine algorithms. Meanwhile, the CD patients were divided into distinct subtypes with either high- or low-immunoactivity using single-sample gene set enrichment analysis (ssGSEA) and consensus clustering. By constructing IG score based on HIGs, we found that patients with high IG score were mainly attributed to high-immunoactivity subgroups, which suggested a strong link between HIGs and immunoactivity of CD patients. In addition, the novel constructed ANN model showed the sound diagnostic ability of HIGs. Mechanistically, we validated that the HIGs play pivotal roles in regulating CD's immune and inflammatory state. Through targeting the HIGs, we also found potential drugs for anti-CD treatment by using the Enrichr platform and molecular docking method. CONCLUSIONS: This study unveils the HIGs and elucidates the networks regulated by these genes in the context of CD. It underscores the pivotal significance of HIGs in accurately predicting the presence or absence of CD in patients. Consequently, this research offers promising prospects for the development of diagnostic biomarkers and therapeutic targets for CD.

KIT-SNAP-tag/cell membrane chromatography model coupled with liquid chromatography-mass spectrometry for anti-GIST compound screening from Evodia rutaecarpa.

Gastrointestinal mesenchymal tumors, as the most common mesenchymal tumors in the gastrointestinal tract, are adjuvantly treated with multi-targeted tyrosine kinase inhibitors, such as imatinib and sunitinib, but there are problems of drug resistance and complex methods of monitoring therapeutic agents. The pathogenesis of this disease is related to mutations in tyrosine kinase (KIT) or platelet-derived growth factor receptor α, an important target for drug therapy. In recent years, the screening of relevant tyrosine kinase inhibitors from traditional Chinese medicine has become a hotspot in antitumor drug research. In the current study, the KIT-SNAP-tag cell membrane chromatography (KIT-SNAP-tag/CMC) column was prepared with satisfying specificity, selectivity, and reproducibility by chemically bonding high KIT expression cell membranes to the silica gel surface using the SNAP-tag technology. The KIT-SNAP-tag/CMC-HPLC-MS two-dimensional coupling system was investigated using the positive drug imatinib, and the results showed that the system was a reliable model for screening potential antitumor compounds from complex systems. This system screened and identified three potential active compounds of evodiamine (EVO), rutaecarpin (RUT), and dehydroevodiamine (DEVO), which possibly target the KIT receptor, from the alcoholic extract of the traditional Chinese medicine Evodia rutaecarpa. Then, the KD values of the interaction of EVO, RUT, and DEVO with KIT receptors measured using nonlinear chromatography were 7.75 (±4.93) × 10-6, 1.42 (±0.71) × 10-6, and 2.34 (±1.86) × 10-6 mol/L, respectively. In addition, the methyl thiazolyl tetrazolium assay validated the active effects of EVO and RUT in inhibiting the proliferation of high KIT-expressing cells in the ranges of 0.1-10 µmol/L and 0.1-50 µmol/L, respectively. In conclusion, the KIT-SNAP-tag/CMC could be a reliable model for screening antitumor components from complex systems.

Single-cell RNA sequencing unveils Lrg1's role in cerebral ischemia‒reperfusion injury by modulating various cells.

BACKGROUND AND PURPOSE: Cerebral ischemia‒reperfusion injury causes significant harm to human health and is a major contributor to stroke-related deaths worldwide. Current treatments are limited, and new, more effective prevention and treatment strategies that target multiple cell components are urgently needed. Leucine-rich alpha-2 glycoprotein 1 (Lrg1) appears to be associated with the progression of cerebral ischemia‒reperfusion injury, but the exact mechanism of it is unknown. METHODS: Wild-type (WT) and Lrg1 knockout (Lrg1-/-) mice were used to investigate the role of Lrg1 after cerebral ischemia‒reperfusion injury. The effects of Lrg1 knockout on brain infarct volume, blood‒brain barrier permeability, and neurological score (based on 2,3,5-triphenyl tetrazolium chloride, evans blue dye, hematoxylin, and eosin staining) were assessed. Single-cell RNA sequencing (scRNA-seq), immunofluorescence, and microvascular albumin leakage tests were utilized to investigate alterations in various cell components in brain tissue after Lrg1 knockout. RESULTS: Lrg1 expression was increased in various cell types of brain tissue after cerebral ischemia‒reperfusion injury. Lrg1 knockout reduced cerebral edema and infarct size and improved neurological function after cerebral ischemia‒reperfusion injury. Single-cell RNA sequencing analysis of WT and Lrg1-/- mouse brain tissues after cerebral ischemia‒reperfusion injury revealed that Lrg1 knockout enhances blood‒brain barrier (BBB) by upregulating claudin 11, integrin ß5, protocadherin 9, and annexin A2. Lrg1 knockout also promoted an anti-inflammatory and tissue-repairing phenotype in microglia and macrophages while reducing neuron and oligodendrocyte cell death. CONCLUSIONS: Our results has shown that Lrg1 mediates numerous pathological processes involved in cerebral ischemia‒reperfusion injury by altering the functional states of various cell types, thereby rendering it a promising therapeutic target for cerebral ischemia‒reperfusion injury.

Construction and application of covalently bonded CD147 cell membrane chromatography model based on polystyrene microspheres.

In this study, a novel cell membrane chromatography (CMC) model was developed to investigate cluster of differentiation 147 (CD147) targeted anti-tumor drug leads for specific screening and ligand-receptor interaction analysis by SNAP-tagged CD147 fusion protein conjugation and polystyrene microspheres (PS) modification. Traditional Chinese medicines (TCMs) are widely used in the treatment of cancer. CD147 plays important roles in tumor progression and acts as an attractive target for therapeutic intervention; therapeutic drugs for CD147-related cancers are limited to date. Thus, a screening method for active components in TCMs is crucial for the further research and development of CD147 antagonists. However, improvement is still needed to perform specific and accurate drug lead screening using the CMC-based method. Recently, our group developed a covalently immobilized receptor-SNAP-tag/CMC model using silica gel as carrier. Besides the carboxyl group on multi-step modified silica particles, the amino group of benzyl-guanine (BG, substrate of SNAP-tag) also possesses reactivity towards the carboxyl group on available carboxyl-modified PS. Herein, we used PS as carrier and an extended SNAP-tag with CD147 receptor to construct the PS-BG-CD147/CMC model for active compound investigation coupled with HPLC/MS and applied this coupled PS-BG-CD147/CMC-HPLC/MS two-dimensional system to drug lead screening from Nelumbinis Plumula extract (NPE) sample. In addition, to comprehensively verify the pharmacological effects of screened ingredients, a cell proliferation inhibition assay was performed, and the interaction between the ingredients and CD147 was studied by the frontal analysis method. This study developed a high-throughput PS-based CMC screening platform, which could be widely applied and utilized in chromatographic separation and drug lead discovery.

Quantitative Analysis and Structural Elucidation of Fatty Acids by Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags and m-CPBA Epoxidation.

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.

NMMHC IIA triggered lipid metabolize reprogramming resulting in vascular endothelial cellular tight junction injury.

BACKGROUND AND PURPOSE: Nonmuscle myosin heavy chain IIA, played an essential role in the promotion of tight junction injury in vascular endothelial cells under oxygen glucose deprivation condition. Rat microvascular endothelial cells had been confirmed to have the susceptibility to ox-LDL stimulation under OGD condition. We proposed the hypothesis that lipid metabolic reprogramming might be the root cause for damage to RBMCs tight junction. METHODS: Untargeted shotgun and targeted lipid metabolomics mass spectrometry approaches combined with principal component analysis was applied to better define the lipids contributing to the variance observed between control and different OGD time. The protein expression of tight junction of RBMCs: occludin, claudin-5, and ZO-1 were detected with immunofluorescence staining and western blot. The proof of the interaction between NMMHC IIA and SREBP1 was investigated via GST-pull down, while their specific binding fragments were also confirmed. The regulation mechanism of NMMHC IIA on SREBP1 was investigated to explore downstream regulatory signaling pathways. RESULTS: Untargeted and targeted shotgun lipidomics data revealed that OGD might be the conditional factor in reshaping lipid components. Mechanistic studies showed that with the increase of OGD time, PCA analysis of lipidomics obtained from RBMCs indicated their specificity in reshaping lipid components, while ≥80% major lipid components phospholipids and sphingolipids transferred from phospholipids, sphingolipids, and neutral lipids, of which neutral lipids taken the largest proportion with OGD time course. Perturbing reprogramming of lipid composition was less susceptible to OGD condition via knockdown of NMMHC IIA of vascular endothelial cells. Knockdown of NMMHC IIA could promote tight junction defense to OGD condition. NMMHC IIA could directly bind with SREBP1, then could affect sterol regulatory element binding protein-1 to adjust lipid metabolize reprogramming of RBMCs. CONCLUSIONS: Mechanistic studies showed that perturbing reprogramming of lipid composition could enhance tight junction damage, which was mediated by the opposing effects of NMMHC IIA.

MrgX2-SNAP-tag/cell membrane chromatography model coupled with liquid chromatography-mass spectrometry for anti-pseudo-allergic compound screening in Arnebiae Radix.

Pseudo-allergic reactions (PARs) are IgE-independent hypersensitivity reactions. Mas-related G protein-coupled receptor-X2 (MrgX2) was proved the key receptor of PAR. The anti-pseudo-allergic compound discovery based on MrgX2 was of great value. Cell membrane chromatography (CMC) based on MrgX2 provides a convenient and effective tool in anti-pseudo-allergic compound screening and discovery, and further improvements of this method are still needed. In this work, SNAP-tag was introduced at C-terminal of Mas-related G protein-coupled receptor (MrgX2-SNAP-tag), and an MrgX2-SNAP-tag/CMC model was then conducted using CMC technique. Comparative experiments showed that the new model not only satisfied the good selectivity and specificity of screening but also exhibited more stable and longer life span than traditional MrgX2/CMC model. By coupling with HPLC-MS, two compounds were screened out from Arnebiae Radix and identified as shikonin and acetylshikonin. Nonlinear chromatography was performed to study the interactions between two screened compounds and MrgX2, and binding constant (KA) of shikonin and acetylshikonin with MrgX2 were 2075.67 ± 0.34 M-1 and 32201.36 ± 0.35 M-1, respectively. Furthermore, ß-hexosaminidase and histamine release assay in vitro demonstrated that shikonin (1-5 µM) and acetylshikonin (2.5-10 µM) could both antagonize C48/80-induced allergic reaction. In conclusion, the MrgX2-SNAP-tag/CMC could be a reliable model for screening pseudo-allergy-related components from complex systems.

Tamoxifen Regulates Epithelial-Mesenchymal Transition in Endometrial Cancer via the CANP10/NRP1 Signaling Pathway.

Tamoxifen, which is used to treat advanced gynecological tumors, has been associated with tumor cell metastasis. Herein, we investigated the effect of tamoxifen on epithelial-mesenchymal transition in endometrial cancer and the associated signaling mechanism. Wound healing and invasion chamber assays, respectively, were performed to determine the migrative capacity and invasiveness of tamoxifen-stimulated endometrial carcinoma (RL95-2) cells. Western blotting and immunofluorescence were used to evaluate the expression of vimentin, E-cadherin, calpain 10 (CANP10), and neuropilin-1 (NRP1). Transfection of a CAPN10-harboring plasmid was used to overexpress CANP10 in RL95-2 cells, and small interfering RNAs were used to silence CANP10 and NRP1 expression. Tamoxifen induced migration, invasion, and morphological changes in RL95-2 cells. It also downregulated E-cadherin expression and upregulated vimentin, CANP10, and NRP1 expression. CANP10 silencing inhibited tamoxifen-induced NRP1 upregulation, and CANP10 or NRP1 silencing inhibited the migration and invasion of RL95-2 cells. CANP10 overexpression upregulated vimentin expression and downregulated that of E-cadherin and also increased cell migration and invasion. Silencing NRP1 protein expression inhibited the induction effect of CANP10 overexpression. In conclusion, tamoxifen promotes the epithelial-mesenchymal transition of RL95-2 cells via the CANP10/NRP1 signaling pathway. Thus, targeting CANP10 or NRP1 may be a novel strategy for preventing tamoxifen-induced endometrial cancer metastasis.

Enhanced stability designs of cell membrane chromatography for screening drug leads.

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.

Investigating interactions between chloroquine/hydroxychloroquine and their single enantiomers and angiotensin-converting enzyme 2 by a cell membrane chromatography method.

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate).

Targeting and Covalently Immobilizing the EGFR through SNAP-Tag Technology for Screening Drug Leads.

Membrane protein immobilization is particularly significant in in vitro drug screening and determining drug-receptor interactions. However, there are still some problems in the immobilization of membrane proteins with controllable direction and high conformational stability, activity, and specificity. Cell membrane chromatography (CMC) retains the complete biological structure of membrane proteins. However, conventional CMC has the limitation of poor stability, which results in its limited life span and low reproducibility. To overcome this limitation, we propose a method for the specific covalent immobilization of membrane proteins in cell membranes. We used the SNAP-tag as an immobilization tag fused to the epidermal growth factor receptor (EGFR), and Cys145 located at the active site of the SNAP-tag reacted with the benzyl group of O6-benzylguanine (BG). The SNAP-tagged EGFR was expressed in HEK293 cells. We captured the SNAP-tagged EGFR from the cell membrane suspension onto a BG-derivative-modified silica gel. Our immobilization strategy improved the life span and specificity of CMC and minimized loss of activity and nonspecific attachment of proteins. Next, a SNAP-tagged EGFR/CMC online HPLC-IT-TOF-MS system was established to screen EGFR antagonists from Epimedii folium. Icariin, magnoflorine, epimedin B, and epimedin C were retained in this model, and pharmacological assays revealed that magnoflorine could inhibit cancer cell growth by targeting the EGFR. This EGFR immobilization method may open up possibilities for the immobilization of other membrane proteins and has the potential to serve as a useful platform for screening receptor-binding leads from natural medicinal herbs.

A Mast Cell-Specific Receptor Is Critical for Granuloma Induced by Intrathecal Morphine Infusion.

Intrathecal morphine infusion is often applied to treat chronic pain related to cancer and other conditions. However, persistent pain can be caused by nerve compression because of granuloma formation. In this study, a mouse model of morphine-induced granuloma formation by intrathecal catheterization morphine infusion into the atlanto-occipital membrane of the foramen magnum was established in wild-type mice, MrgprB2 mutant (MrgprB2-/-) mice, and in mast cell-deficient W-sash c-kit mutant (KitW-sh/W-sh) mice. Heat-related pain after surgery was performed to investigate the antipain effect of morphine. H&E staining and immunofluorescence staining of the spinal cord were applied to analyze the mechanism of granuloma formation. Morphine-induced mast cell degranulation was assessed by measuring the Ca2+ influx and mediator release. Anaphylactoid reactions were measured after s.c. morphine infusion to the paws. Chemokine release by mast cells was determined by Human XL Cytokine Array. Experiments with wild-type, MrgprB2 mutant, and mast cell-deficient W-sash c-kit mutant mice demonstrated that morphine activated mast cells and inflammatory cell aggregation through MrgprB2 in intrathecal infusion sites. The chemokine production of human mast cells demonstrated that granuloma formation is correlated with chemokines release. In addition, morphine activated mouse primary mast cells and de novo chemokine synthesis via the MRGPRX2 in human LAD2 cells. We concluded that granuloma formation during intrathecal morphine infusion was associated with MrgprB2/X2. Reducing MRGPRX2 potentially blocks morphine-induced side effects, including granuloma formation.

Construction of graphene quantum dots-decorated EGFR cell membrane chromatography for screening active components from Peucedanum praeruptorum Dunn.

A novel stability-enhanced graphene quantum dot (GQD)-decorated epidermal growth factor receptor (EGFR) cell membrane chromatography was constructed to study the potential application of GQDs in bioaffinity chromatography, and to screen active components acting on EGFR from traditional Chinese medicine (TCM). The carboxyl groups on the surface of GQDs reacted with the amino groups of the amino-silica gel (SiO2-NH2) to form a covalent bond, thereby preparing the GQD-decorated silica gel (SiO2-GQDs). The EGFR cell membrane was further immobilized on the SiO2-GQDs through the same covalent binding method to obtain the GQD-decorated cell membrane stationary phase (SiO2-GQDs-CMSP). In this way, the cell membrane was firmly immobilized on the decorated silica carrier. The life span and stability of the GQD-decorated cell membrane chromatographic (SiO2-GQDs-CMC) column were both enhanced, and the optimal immobilization conditions of the EGFR cell membrane were also determined. This model was then verified by establishing a SiO2-GQDs-CMC online liquid chromatography-ion trap-time-of-flight (LC-IT-TOF) system to screen possible active components in Peucedanum praeruptorum Dunn. As a result, praeruptorin B (Pra-B) was screened out, and its inhibitory effect against EGFR cell growth was evaluated by the cell counting kit-8 (CCK-8) assay. Molecular docking assay was also conducted to further estimate the interaction between Pra-B and EGFR. Overall, this research indicated that GQDs may be a promising nanomaterial to be used in prolonging the life span of the CMC column, and Pra-B could be a potential EGFR inhibitor so as to treat cancer.

Screening and evaluation of anti-SARS-CoV-2 components from Ephedra sinica by ACE2/CMC-HPLC-IT-TOF-MS approach.

Traditional Chinese medicines played an important role in the treatment of COVID-19 in 2020. Ephedra sinica, one of the major constituent herbs of multi-component herbal formula, has been widely used to treat COVID-19 in China. However, its active components are still unclear. The objectives of this study are to screen and evaluate active components from the traditional Chinese medicine Ephedra sinica for the treatment of COVID-19. In our study, we established an ACE2/CMC bioaffinity chromatography model, and then developed an ACE2/CMC-HPLC-IT-TOF-MS system for the active compounds screening and identification from Ephedra sinica extract. We performed molecular docking and surface plasmon resonance (SPR) assays to assess the binding characteristics (binding mode and KD value). We used CCK-8 staining to assess the toxicity of screened compounds, and also used SARS-CoV-2 pseudovirus to observe the viropexis effect of screened compounds in ACE2h cells. In this current work, one fraction was fished out, separated and identified as ephedrine (EP), pseudoephedrine (PEP), and methylephedrine (MEP). Binding assays showed that the three compounds could bind with ACE2 in a special way to some amino acid residues, similar to the way SARS-CoV-2 bound with ACE2. Additionally, the three compounds, especially EP, can inhibit the entrance of SARS-CoV-2 spike pseudovirus into ACE2h cells because they can reduce the entrance ratio of pseudovirus in the pseudovirus model. Overall, the ACE2/CMC-HPLC-IT-TOF-MS system was established and verified to be suitable for ACE2-targeted bioactive compound screening. EP, PEP, and MEP with ACE2-binding features were screened out from Ephedra sinica, and acted as blockers inhibiting SARS-CoV-2 spike pseudovirus entering ACE2h cells.

Pseudo-allergic compounds screened from Shengmai injection by using high-expression Mas-related G protein-coupled receptor X2 cell membrane chromatography online coupled with liquid chromatography and mass spectrometry.

Adverse drug reactions of traditional Chinese medicine injection mainly manifested as pseudo-allergic reactions. In the present study, ginsenoside Rd, Ro, and Rg3 were identified as pseudo-allergic components in Shengmai injection by a high-expression Mas-related G protein-coupled receptor X2 cell membrane chromatography coupled online with high-performance liquid chromatography and mass spectrometry. Their pseudo-allergic activities were evaluated by in vitro and in vivo assay. The three compounds were further found to induce pseudo-allergic reaction through Mas-related G protein-coupled receptor X2. Therefore, we concluded that ginsenoside Rd, Ro and Rg3 may be potential allergens that cause pseudo-allergic reactions. This study might be helpful for the safe use of Shengmai injection.

Azithromycin inhibits the production of MUC5AC in the airway mucosa of patients with bronchiectasis induced by Pseudomonas aeruginosa.

To explore the clinical benefits of azithromycin in the treatment of Pseudomonas aeruginosa induced bronchiectasis and to evaluate its effect on MUC5AC. From April 2018 to June 2020, 160 patients with bronchiectasis due to Pseudomonas aeruginosa infection were selected. The patients were divided into a control groupand an azithromycin group. Statistics of patients' general clinical data, lung function indexes, sputum volume, oxidative stress level, Bhalla score before and after treatment; Western blot analysis of MUC5AC expression; RT-PCR analysis of TNF-α, IL-8, IL- 1ß mRNA expression. The mRNA expression of TNF-α, IL-8 and IL-1ß in the azithromycin group was lower than that in the control group (P<0.05). After treatment, the protein expression of MUC5AC in the azithromycin group was lower than that in the control group (P<0.05). The improvement rate in the azithromycin group was significantly higher than that in the control group (P<0.05). The azithromycin group had a lower lung infection rate than the control group (P<0.05). The azithromycin group had a lower dyspnea rate than the control group (P<0.05). Azithromycin treatment has certain clinical benefits for patients with bronchiectasis induced by Pseudomonas aeruginosa and inhibits the MUC5AC expression.

Cell membrane chromatography coupled online with LC-MS to screen anti-anaphylactoid components from Magnolia biondii Pamp. targeting on Mas-related G protein-coupled receptor X2.

Mas-related G protein-coupled receptor X2 was a mast cell-specific receptor mediating anaphylactoid reactions by activating mast cells degranulation, and it was also identified as a target for modulating mast cell-mediated anaphylactoid and inflammatory diseases. The anti-anaphylactoid drugs used clinically disturb the partial effect of partial mediators released by mast cells. The small molecule of Mas-related G protein-coupled receptor X2 specific antagonists may provide therapeutic action for the anaphylactoid and inflammatory diseases in the early stage. In this study, the Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography was coupled online with liquid chromatography and mass spectrometry and successfully used to screen anti-anaphylactoid components from Magnolia biondii Pamp. Fargesin and pinoresinol dimethyl ether were identified as potential anti-anaphylactoid components. Bioactivity of these two components were investigated by ß hexosaminidase and histamine release assays on mast cells, and it was found that these two components could inhibit ß hexosaminidase and histamine release in a concentration-dependent manner. This Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography coupled online with liquid chromatography and mass spectrometry system could be applied for screening potential anti-anaphylactoid components from natural medicinal herbs. This study also provided a powerful system for drug discovery in natural medicinal herbs.

The Protein Kinase R Inhibitor C16 Alleviates Sepsis-Induced Acute Kidney Injury Through Modulation of the NF-κB and NLR Family Pyrin Domain-Containing 3 (NLPR3) Pyroptosis Signal Pathways.

BACKGROUND Protein kinase R (PKR) is implicated in the inflammatory response to bacterial infection while the role of PKR in sepsis-induced acute kidney injury (AKI) is largely unknown. This study aimed to investigate the effects of the specific PKR inhibitor C16 (C13H8N4OS) on lipopolysaccharide (LPS)-induced AKI, and its mechanisms of action. MATERIAL AND METHODS C57BL/6J mice were injected intraperitoneally with C16 or vehicle 1 h before the LPS challenge and then injected intraperitoneally with LPS or 0.9% saline. After the LPS challenge, histopathological damage, renal function, and levels of proinflammatory cytokines were assessed. All the related signaling pathways were analyzed. RESULTS C16 effectively inhibited LPS-induced renal elevation of proinflammatory cytokines and chemokines. C16 prevented NF-kappaB activation and suppressed the PKR/eIF2alpha signaling pathway in AKI after the LPS challenge. Furthermore, C16 significantly inhibited pyroptosis during AKI, as evidenced by decreased renal levels of apoptosis-associated speck-like protein; NACHT, LRR, NLR Family Pyrin Domain-Containing 3; caspase-1; interleukin (IL)-1ß; and IL-18. CONCLUSIONS Our findings suggest that inhibition by C16 ameliorated LPS-induced renal inflammation and injury, at least partly through modulation of the pyroptosis signal pathway in the kidney.

Myosin IIA Regulated Tight Junction in Oxygen Glucose-Deprived Brain Endothelial Cells Via Activation of TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 Signal Transduction Pathway.

Non-muscle myosin heavy chain IIA (NMMHC IIA), a member of Myosin II family, plays a critical role in various cellular physiological processes. Our previous research had suggested that NMMHC IIA could participate in regulating tight junction morphological changes induced by ischemia stroke. Thus, in the current study, we attempted to uncover the regulation pattern of NMMHC IIA on tight junction dysfunction in oxygen glucose-deprived (OGD) mouse brain bEND.3 endothelial cells. The regulation of NMMHC IIA on tight junction in OGD-stimulated bEND.3 cells was evaluated by western blotting assay. Morphologic change of occludin, claudin-5, and ZO-1 tight junction proteins was compared with pretreatment with NMMHC II inhibitor blebbistatin via immunohistochemical staining. Detection of activation of NMMHC IIA on OGD-mediated tight junction transduction pathway was investigated via Koch's postulate using corresponding protein inhibitor. Our results showed that NMMHC IIA was activated in OGD-stimulated bEND.3 endothelial cells. The inhibition of NMMHC IIA could attenuate the morphologic change of occludin, claudin-5, and ZO-1 tight junction proteins. NMMHC IIA participated in regulating downstream transduction pathway TLR4, phosphatidylinositol 3-kinase (PI3K), Akt, JNK1/2, 14-3-3ε, nuclear factor kappa B (NF-кB) and matrix metalloprotein 9 (MMP9). Blocking of these pathways using indicated inhibitors demonstrated that NMMHC IIA destroyed the connection of tight junction via the activation of TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 pathway. Our study described the key role of NMMHC IIA in OGD-stimulated mouse brain bEND.3 endothelial cells, while also exhibited the molecule effect on tight junction dysfunction via TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 signal transduction pathway.