Establishment of cluster of differentiation 20 immobilized cell membrane chromatography for the screening of active antitumor components in traditional Chinese medicine.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignant tumors occurring in B or T lymphocytes, and no small molecule-positive drugs to treat NHL have been marketed. Cluster of differentiation 20 (CD20) is an important molecule regulating signaling for the life and differentiation of B lymphocytes and possesses the characteristics of a drug target for treating NHL. 2-Methoxyestradiol induces apoptosis in lymphoma Raji cells and CD20 protein is highly expressed by Raji lymphoma cells. Therefore, in this study, a CD20-SNAP-tag/CMC model was developed to validate the interaction of 2-methoxyestradiol with CD20. 2-Methoxyestradiol was used as a small molecule control compound, and the system was validated for good applicability. The cell membrane chromatography model was combined with high-performance liquid chromatography ion trap time-of-flight mass spectroscopy (HPLC-IT-TOF-MS) in a two-dimensional system to successfully identify, analyze, and characterize the potential active compounds of Schisandra chinensis (Turcz.) Baill. extract and Lysionotus pauciflorus Maxim. extract, including Schisandrin A, Schizandrol A, Schizandrol B, Schisantherin B, and Nevadensin, which can act on CD20 receptors. The five potential active compounds were analyzed by non-linear chromatography. The thermodynamic and kinetic parameters of their interaction with CD20 were also analyzed, and the mode of interaction was simulated by molecular docking. Their inhibitory effects on lymphoma cell growth were assessed using a Cell Counting Kit-8 (CCK-8). Nevadensin and Schizandrin A were able to induce apoptosis in Raji cells within a certain concentration range. In conclusion, the present experiments provide some bases for improving NHL treatment and developing small molecule lead compounds targeting CD20 with low toxicity and high specificity.

Secreted phosphoprotein 1 regulates natural compound 3',4',5,7-tetrahydroxyflavone to inhibit mast cell-mediated allergic inflammation.

BACKGROUND: Mast cells (MCs) are important effector cells in anaphylaxis and anaphylactic disease. 3',4',5,7-tetrahydroxyflavone (THF) presents in many medicinal plants and exerts a variety of pharmacological effects. In this study, we evaluated the effect of THF on C48/80-induced anaphylaxis and the mechanisms underlying its effects, including the role of secreted phosphoprotein 1 (SPP1), which has not been reported to IgE-independent MC activation. RESULTS: THF inhibited C48/80-induced Ca2+ flow and degranulation via the PLCγ/PKC/IP3 pathway in vitro. RNA-seq showed that THF inhibited the expression of SPP1 and downstream molecules. SPP1 is involved in pseudo-anaphylaxis reactions. Silencing SPP1 affects the phosphorylation of AKT and P38. THF suppressed C48/80-induced paw edema, hypothermia and serum histamine, and chemokines release in vivo. CONCLUSIONS: Our results validated SPP1 is involved in IgE-independent MC activation anaphylactoid reactions. THF inhibited C48/80-mediated anaphylactoid reactions both in vivo and in vitro, suppressed calcium mobilization and inhibited SPP1-related pathways.

α-Asarone alleviates allergic asthma by stabilizing mast cells through inhibition of ERK/JAK2-STAT3 pathway.

Asthma is a heterogeneous disease related to numerous inflammatory cells, among which mast cells play an important role in the early stages of asthma. Therefore, treatment of asthma targeting mast cells is of great research value. α-Asarone is an important anti-inflammatory component of the traditional Chinese medicine Acorus calamus L, which has a variety of medicinal values. To investigate whether α-asarone can alleviate asthma symptoms and its mechanism. In this study, we investigated the effect of α-asarone on mast cell activation in vivo and in vitro. The release of chemokines or cytokines, AHR (airway hyperresponsiveness), and mast cell activation were examined in a mast cell-dependent asthma model. Western blot was performed to determine the underlying pathway. α-Asarone inhibited the degranulation of LAD2 (laboratory allergic disease 2) cells and decreased IL-8, MCP-1, histamine, and TNF-α in vitro. α-Asarone reduced paw swelling and leakage of Evans blue, as well as serum histamine, CCL2, and TNF-α in vivo. In the asthma model, α-asarone showed an inhibitory effect on AHR, inflammation, mast cells activation, infiltration of inflammatory cells, and the release of IL-5 and IL-13 in lung tissue. α-Asarone decreased the levels of phosphorylated JAK2, phosphorylated ERK, and phosphorylated STAT3 induced by C48/80. Our findings suggest that α-asarone alleviates allergic asthma by inhibiting mast cell activation through the ERK/JAK2-STAT3 pathway.

Licochalcone A inhibits MAS-related GPR family member X2-induced pseudo-allergic reaction by suppressing nuclear migration of nuclear factor-κB.

Licochalcone A (Lico A) is a natural flavonoid belonging to the class of substituted chalcone that has various biological effects. Mast cells (MCs) are innate immune cells that mediate hypersensitivity and pseudo-allergic reactions. MAS-related GPR family member X2 (MRGPRX2) on MCs has been recognized as the main receptor for pseudo-allergic reactions. In this study, we investigated the anti-pseudo-allergy effect of Lico A and its underlying mechanism. Substance P (SP), as an MC activator, was used to establish an in vitro and in vivo model of pseudo-allergy. The in vivo effect of Lico A was investigated using passive cutaneous anaphylaxis (PCA) and active systemic allergy, along with degranulation, Ca2+ influx in vitro. SP-induced laboratory of allergic disease 2 (LAD2) cell mRNA expression was explored using RNA-seq, and Lico A inhibited LAD2 cell activation by reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence staining. Lico A showed an inhibitory effect on SP-induced MC activation and pseudo-allergy both in vitro and in vivo. The nuclear factor (NF)-κB pathway is involved in MRGPRX2 induced MC activation, which is inhibited by Lico A. In conclusion, Lico A inhibited the pseudo-allergic reaction mediated by MRGPRX2 by blocking NF-κB nuclear migration.

Three salvianolic acids inhibit 2019-nCoV spike pseudovirus viropexis by binding to both its RBD and receptor ACE2.

Since December 2019, the new coronavirus (also known as severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, 2019-nCoV])-induced disease, COVID-19, has spread rapidly worldwide. Studies have reported that the traditional Chinese medicine Salvia miltiorrhiza possesses remarkable antiviral properties; however, the anti-coronaviral activity of its main components, salvianolic acid A (SAA), salvianolic acid B (SAB), and salvianolic acid C (SAC) is still debated. In this study, we used Cell Counting Kit-8 staining and flow cytometry to evaluate the toxicity of SAA, SAB, and SAC on ACE2 (angiotensin-converting enzyme 2) high-expressing HEK293T cells (ACE2h cells). We found that SAA, SAB, and SAC had a minor effect on the viability of ACE2h cells at concentrations below 100 µM. We further evaluated the binding capacity of SAA, SAB, and SAC to ACE2 and the spike protein of 2019-nCoV using molecular docking and surface plasmon resonance. They could bind to the receptor-binding domain (RBD) of the 2019-nCoV with a binding constant (KD ) of (3.82 ± 0.43) e-6 M, (5.15 ± 0.64)e-7 M, and (2.19 ± 0.14)e-6 M; and bind to ACE2 with KD (4.08 ± 0.61)e-7 M, (2.95 ± 0.78)e-7 M, and (7.32 ± 0.42)e-7 M, respectively. As a result, SAA, SAB, and SAC were determined to inhibit the entry of 2019-nCoV Spike pseudovirus with an EC50 of 11.31, 6.22, and 10.14 µM on ACE2h cells, respectively. In conclusion, our study revealed that three Salvianolic acids can inhibit the entry of 2019-nCoV spike pseudovirus into ACE2h cells by binding to the RBD of the 2019-nCoV spike protein and ACE2 protein.