CD20 is a surface marker protein of B-cell lymphoma, and its extracellular region is the target of specific antibodies and drugs. To obtain a cheap and easily modified specific preparation targeting CD20, we optimized the gene of CD20 extracellular region according to codon degeneracy to facilitate its expression in Escherichia coli. The optimized gene was cloned into pGEX-4T-1 vector, and the recombinant vector was transformed into E. coli BL21(DE3) for expression. The purified protein was identified by SDS-PAGE and Western blotting. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to screen the ssDNA aptamer that specifically binds to the fusion protein, and the affinity of the aptamer to CD20 was detected by flow cytometry. Then, the cytotoxicity test was carried out to examine the inhibitory effect of the aptamer on B lymphoma cells. In this study, we established the prokaryotic expression method of CD20 and obtained the aptamer specifically binding to the extracellular region of CD20, which laid a foundation for the development of therapeutic drugs targeting CD20.
Humans ; Escherichia coli/metabolism* ; SELEX Aptamer Technique/methods* ; Aptamers, Nucleotide/genetics* ; Antigens, CD20/metabolism* ; Ligands

Neuronomodulation refers to the modulation of neural conduction and synaptic transmission (i.e., the conduction process involved in synaptic transmission) of excitable neurons via changes in the membrane potential in response to chemical substances, from spillover neurotransmitters to paracrine or endocrine hormones circulating in the blood. Neuronomodulation can be direct or indirect, depending on the transduction pathways from the ligand binding site to the ion pore, either on the same molecule, i.e. the ion channel, or through an intermediate step on different molecules. The major players in direct neuronomodulation are ligand-gated or voltage-gated ion channels. The key process of direct neuronomodulation is the binding and chemoactivation of ligand-gated or voltage-gated ion channels, either orthosterically or allosterically, by various ligands. Indirect neuronomodulation involves metabotropic receptor-mediated slow potentials, where steroid hormones, cytokines, and chemokines can implement these actions. Elucidating neuronomodulation is of great significance for understanding the physiological mechanisms of brain function, and the occurrence and treatment of diseases.
Ligands ; Neurons/metabolism* ; Synaptic Transmission/physiology* ; Ion Channels/metabolism* ; Hormones/metabolism*

Proto-Oncogene Proteins c-akt/metabolism* ; Butyrates ; Ligands ; Glucose ; Receptors, G-Protein-Coupled/metabolism* ; Glycogen Synthase Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta ; Phosphorylation

The development of chronic liver disease can be promoted by excessive fat accumulation, dysbiosis, viral infections and persistent inflammatory responses, which can lead to liver inflammation, fibrosis and carcinogenesis. An in-depth understanding of the etiology leading to chronic liver disease and the underlying mechanisms influencing its development can help identify potential therapeutic targets for targeted treatment. Orphan nuclear receptors (ONRs) are receptors that have no corresponding endogenous ligands to bind to them. The study of these ONRs and their biological properties has facilitated the development of synthetic ligands, which are important for investigating the effective targets for the treatment of a wide range of diseases. In recent years, it has been found that ONRs are essential for maintaining normal liver function and their dysfunction can affect a variety of liver diseases. ONRs can influence pathophysiological activities such as liver lipid metabolism, inflammatory response and cancer cell proliferation by regulating hormones/transcription factors and affecting the biological clock, oxidative stress, etc. This review focuses on the regulation of ONRs, mainly including retinoid related orphan nuclear receptors (RORs), pregnane X receptor (PXR), leukocyte cell derived chemotaxin 2 (LECT2), Nur77, and hepatocyte nuclear factor 4α (HNF4α), on the development of different types of chronic liver diseases in different ways, in order to provide useful references for the therapeutic strategies of chronic liver diseases based on the regulation of ONRs.
Humans ; Orphan Nuclear Receptors/metabolism* ; Receptors, Steroid/physiology* ; Ligands ; Liver ; Liver Diseases ; Intercellular Signaling Peptides and Proteins

Proteolysis targeting chimera (PROTAC) refers to heterobifunctional small molecules that can simultaneously bind an E3 ubiquitin ligase and a target protein, enabling specific degradation of the target protein with the aid of the ubiquitin proteasome system. At present, most PROTAC drugs are in the clinical trial stage, and the ligands are mainly non-covalent compounds. PROTAC drugs have the advantage of overcoming drug resistance and degrading "undruggable" target proteins, but non-covalent ligands could lead to the hook effect that undermines drug efficacy. With its own advantages, covalent ligands can avoid the occurrence of this phenomenon, which is of great help to the development of PROTAC. This review summarizes the progress in preclinical and clinical research and application of PROTAC molecules targeting three different classes of protein targets, including intranuclear, transmembrane, and cytosolic proteins. We also offer perspective discussions to provide research ideas and references for the future development of PROTAC.
Proteolysis ; Proteolysis Targeting Chimera ; Proteasome Endopeptidase Complex/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; Proteins/metabolism* ; Ligands

CD226 is an activated receptor on the surface of natural killer (NK) cells. It competes with TIGIT and CD96 to bind to ligands such as CD155 on the surface of tumor cells and mediates the killing function of NK cells. Although TIGIT and CD96 have other binding ligands in the tumor microenvironment, they compete to bind CD115 ligands with higher affinity and inhibit the activity of NK cells, which allows tumor cells to evade killing. Therefore, studying the expression patterns of these three NK cell surface receptors in different tumors and monitoring their binding ability with ligands will help us to explore new tumor treatment strategies. This article reviews the role and mechanism of CD226, TIGIT, CD96 and other NK cell receptor molecules in regulating NK cell function in anti-tumor immune response.
Ligands ; Receptors, Immunologic ; Receptors, Natural Killer Cell ; Killer Cells, Natural ; Antigens, CD

Lectins are proteins responsible for recognizing the signals of sugar molecules in the body. Sialic acid-binding immunoglobulin-like lectins (Siglecs) regulate the innate and adaptive immune responses in the tumor microenvironment by recognizing the glycan structure containing sialic acid and mediating downstream signals through immune receptor tyrosine inhibitory motifs. In recent years, a variety of tumor treatment strategies targeting the sialic acid-Siglecs axis have been introduced, including sialoglycoprotein-mediated drug delivery and antibody mediated inhibition of Siglecs from recognizing tumor surface ligands. In the future, by combining with glycoprotein nanotherapy, antibody therapy and gene therapy, Siglecs can be used to accurately locate tumor targets and release the anti-tumor immunity, so as to achieve the purpose of effective cure of tumors.
Sialic Acid Binding Immunoglobulin-like Lectins/metabolism* ; N-Acetylneuraminic Acid ; Immunoglobulins/metabolism* ; Receptors, Immunologic ; Ligands

OBJECTIVE: To investigate the significance of Tim-3 and Galectin-9 in Th1/Th2 imbalance in patients with multiple myeloma (MM). METHODS: 55 newly diagnosed MM patients and 20 healthy controls were included. Flow cytometry was used to detect the expression of Tim-3 on CD4⁺T cells, the proportion of Th1, Th2, Tim-3⁺Th1 and Tim-3⁺Th2 cells in peripheral blood. ELISA was used to detect the levels of cytokines IFN-γ and IL-4 in serum, and PCR was used to detect the level of Galectin-9 mRNA. Then the correlations between Galectin-9 mRNA expression and Th-cell subsets and related cytokine levels, as well as the relationship between Tim-3⁺Th1/Tim-3⁺Th2 ratio and corresponding clinical features were analyzed. RESULTS: Compared with the control group, the expression of Tim-3 on CD4⁺T cells in peripheral blood of MM patients was significantly increased (P<0.05), the proportions of Tim-3⁺Th1 cells, Tim-3⁺Th2 cells and Tim-3⁺Th1/Tim-3⁺Th2 ratio in MM patients were also increased (P<0.05), while the proportion of Th1 cells and Th1/Th2 ratio in MM patients were significantly decreased (P<0.05). The level of cytokine IFN-γ and IFN-γ/IL-4 ratio in MM patients were significantly decreased (P<0.05), while the level of cytokine IL-4 was increased (P<0.05). The mRNA levels of Galectin-9 in MM patients were significantly increased (P<0.05). The levels of Galectin-9 mRNA were positively correlated with Tim-3⁺CD4⁺T cells (r=0.663), Tim-3⁺Th2 cells (r=0.492) and IL-4 (r=0.470), while negatively correlated with IFN-γ (r=-0.593). The ratios of Tim-3⁺Th1/Tim-3⁺Th2 in MM patients were positively correlated with ISS stage (r=0.511), osteolytic damage (r=0.556) and chromosome abnormality (r=0.632). CONCLUSION These results suggest that Tim-3 and Galectin-9 are involved in Th1/Th2 imbalance in MM patients, and the high ratio of Tim-3⁺Th1/Tim-3⁺Th2 is associated with poor clinical prognosis.
Humans ; Cytokines/metabolism* ; Galectins/metabolism* ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Interleukin-4/metabolism* ; Ligands ; Multiple Myeloma/metabolism* ; RNA, Messenger/metabolism* ; Th1 Cells/metabolism* ; Th2 Cells/metabolism*

OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.
Mice ; Animals ; Receptors, Tumor Necrosis Factor/physiology* ; Receptors, OX40 ; Membrane Glycoproteins ; Ligands ; Antibodies, Monoclonal/pharmacology* ; Antineoplastic Agents/pharmacology*

OBJECTIVE: Aconite is a traditional Chinese herbal medicine that has been found to inhibit the development of liver cancer; however, its exact molecular mechanisms in this process remain unclear. This study explores how aconite aqueous extract (AAE) inhibits hepatocellular carcinoma (HCC). METHODS: An in vivo mouse model of subcutaneous liver cancer was established. After AAE treatment, immunohistochemistry (IHC) was used to determine the effect of AAE on natural killer (NK) cells. Subsequently, C57BL/6 mice were used to establish the subcutaneous tumor model, and a group of these mice were treated with anti-PK163 antibody to remove NK cells, which was verified by flow cytometry and IHC. The effect of AAE on the proliferation of HCC cells in vitro was determined using cell counting kit-8. The effect of AAE on chemokine production in HCC cells was measured using real-time quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay. The effect of AAE on the migration of NK cells was determined using a transwell assay. Finally, the molecular mechanism was investigated using the Western blotting method. RESULTS: We demonstrated that the ability of AAE to induce overexpression of the cytokine C-C motif chemokine ligand 2 (CCL2) in HCC cells is fundamental to the infiltration of NK cells into the tumor bed. Mechanistically, we found that the upregulation of CCL2 was achieved by the activation of c-Jun N-terminal kinase but not extracellular regulated protein kinase or p38. CONCLUSION Our findings suggest that AAE can be used as an effective immune adjuvant to enhance antitumor immunity by increasing NK cell infiltration into tumors, which could help to improve the efficacy of HCC treatments. Please cite this article as: Yang KD, Zhang X, Shao MC, Wang LN. Aconite aqueous extract inhibits the growth of hepatocellular carcinoma through CCL2-dependent enhancement of natural killer cell infiltration. J Integr Med. 2023; 21(6): 575-583.
Animals ; Mice ; Carcinoma, Hepatocellular/drug therapy* ; Liver Neoplasms/drug therapy* ; Aconitum ; Ligands ; Mice, Inbred C57BL ; Killer Cells, Natural/metabolism* ; Chemokines/pharmacology* ; Cell Line, Tumor