This study aims to investigate the effect of Chaijin Jieyu Anshen Formula on the neuroinflammation of rats with insomnia complicated with depression through the regulation of triggering receptor expressed on myeloid cells 2(TREM2)/complement protein C1q signaling pathway. Rats were randomly divided into a normal group, a model group, a positive drug group, as well as a high, medium, and low-dose groups of Chaijin Jieyu Anshen Formula, with 10 rats in each group. Except for the normal group, the other groups were injected with p-chlorophenylalanine and exposed to chronic unpredictable mild stress to establish the rat model of insomnia complicated with depression. The sucrose preference experiment, open field experiment, and water maze test were performed to evaluate the depression in rats. Enzyme-linked immunosorbent assay was employed to detect serum 5-hydroxytryptamine(5-HT), dopamine(DA), and norepinephrine(NE) levels. Hematoxylin and eosin staining and Nissl staining were used to observe the damage in nucleus accumbens neurons. Western blot and immunofluorescence were performed to detect TREM2, C1q, postsynaptic density 95(PSD-95), and synaptophysin 1(SYN1) expressions in rat nucleus accumbens, respectively. Golgi-Cox staining was utilized to observe the synaptic spine density of nucleus accumbens neurons. The results show that, compared with the model group, Chaijin Jieyu Anshen Formula can significantly increase the sucrose preference as well as the distance and number of voluntary activities, shorten the immobility time in forced swimming test and the successful incubation period of positioning navigation, and prolong the stay time of space exploration in the target quadrant test. The serum 5-HT, DA, and NE contents in the model group are significantly lower than those in the normal group, with the above contents significantly increased after the intervention of Chaijin Jieyu Anshen Formula. In addition, Chaijin Jieyu Anshen Formula can alleviate pathological damages such as swelling and loose arrangement of tissue cells in the nucleus accumbens, while increasing the Nissl body numbers. Chaijin Jieyu Anshen Formula can improve synaptic damage in the nucleus accumbens and increase the synaptic spine density. Compared to the normal group, the expression of C1q protein was significantly higher in the model group, while the expression of TREM2 protein was significantly lower. Compared to the model group, the intervention with Chaijin Jieyu Anshen Formula significantly downregulated the expression of C1q protein and significantly upregulated the expression of TREM2. Compared with the model group, the PSD-95 and SYN1 fluorescence intensity is significantly increased in the groups receiving different doses of Chaijin Jieyu Anshen Formula. In summary, Chaijin Jieyu Anshen Formula can reduce the C1q protein expression, relieve the TREM2 inhibition, and promote the synapse-related proteins PSD-95 and SNY1 expression. Chaijin Jieyu Anshen Formula improves synaptic injury of the nucleus accumbens neurons, thereby treating insomnia complicated with depression.
Animals ; Male ; Rats ; Nucleus Accumbens/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Depression/complications* ; Membrane Glycoproteins/genetics* ; Rats, Sprague-Dawley ; Sleep Initiation and Maintenance Disorders/complications* ; Neurons/metabolism* ; Receptors, Immunologic/genetics* ; Signal Transduction/drug effects* ; Synapses/metabolism*

OBJECTIVE: To investigate the expression of co-inhibitory molecules TIGIT/CD155 and PD-1 on CD4⁺T cells and Treg cells in peripheral blood of patients with chronic lymphocytic leukemia (CLL) and analyze their clinical significance. METHODS: The expression of PD-1 and TIGIT on CD4⁺T cells and Treg cells was detected by flow cytometry in 40 CLL patients and 20 healthy controls. Additionally, the expression of CD155 on peripheral blood B cells and DC cells of the enrolled subjects was detected. RESULTS: The proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells and CD155⁺DC cells in peripheral blood of CLL patients were significantly higher than those of healthy controls ( P < 0.05). The proportions of PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells in CLL patients were significantly higher than those of PD-1⁺TIGIT^-CD4⁺T cells and PD-1⁺TIGIT^-Treg cells, respectively ( P < 0.05). Both PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells were positively correlated with the level of CD155⁺DC cells (r =0.742, r =0.766). With the progression of Binet stage, the proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells, and CD155⁺DC cells gradually increased ( P < 0.05), and the aforementioned three types cells were all increased in patients with CD38≥30%, IGVH unmutated, or poor prognosis due to chromosomal abnormalities ( P < 0.05). CONCLUSION Co-inhibitory molecules PD-1 and TIGIT may be involved in immunodepletion in patients with advanced CLL, which has clinical prognostic value. Dual inhibitor molecular targeted therapy provides a new direction for the individualized treatment of CLL.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; Receptors, Immunologic/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; T-Lymphocytes, Regulatory/metabolism* ; Receptors, Virus/metabolism* ; CD4-Positive T-Lymphocytes/metabolism* ; Male ; Female ; Middle Aged ; Flow Cytometry ; Clinical Relevance

Triggering receptor expressed on myeloid cells 2 (TREM2)-mediated microglial phagocytosis is an energy-intensive process that plays a crucial role in amyloid beta (Aβ) clearance in Alzheimer's disease (AD). Energy metabolic reprogramming (EMR) in microglia induced by TREM2 presents therapeutic targets for cognitive impairment in AD. Jiawei Xionggui Decoction (JWXG) has demonstrated effectiveness in enhancing energy supply, protecting microglia, and mitigating cognitive impairment in APP/PS1 mice. However, the mechanism by which JWXG enhances Aβ phagocytosis through TREM2-mediated EMR in microglia remains unclear. This study investigates how JWXG facilitates microglial phagocytosis and alleviates cognitive deficits in AD through TREM2-mediated EMR. Microglial phagocytosis was evaluated through immunofluorescence staining in vitro and in vivo. The EMR level of microglia was assessed using high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) kits. The TREM2/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/hypoxia-inducible factor-1α (HIF-1α) signaling pathway was analyzed using Western blotting in BV₂ cells. TREM2^-/- BV₂ cells were utilized for reverse validation experiments. The Aβ burden, neuropathological features, and cognitive ability in APP/PS1 mice were evaluated using ELISA kits, immunohistochemistry (IHC), and the Morris water maze (MWM) test. JWXG enhanced both the phagocytosis of EMR disorder-BV₂ cells (EMRD-BV₂) and increased EMR levels. Notably, these effects were significantly reversed in TREM2^-/- BV₂ cells. JWXG elevated TREM2 expression, adenosine triphosphate (ATP) levels, and microglial phagocytosis in APP/PS1 mice. Additionally, JWXG reduced Aβ-burden, neuropathological lesions, and cognitive deficits in APP/PS1 mice. In conclusion, JWXG promoted TREM2-induced EMR and enhanced microglial phagocytosis, thereby reducing Aβ deposition, improving neuropathological lesions, and alleviating cognitive deficits.
Drugs, Chinese Herbal/pharmacology* ; Microglia/drug effects* ; Phagocytosis ; Cognitive Dysfunction/drug therapy* ; Metabolic Reprogramming ; Animals ; Mice ; Cell Line ; Receptors, Immunologic/metabolism* ; Membrane Glycoproteins/metabolism* ; Signal Transduction ; Amyloid beta-Peptides/metabolism* ; Energy Metabolism

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.
Humans ; HEK293 Cells ; CRISPR-Cas Systems ; DEAD Box Protein 58/metabolism* ; Signal Transduction ; Receptors, Immunologic/metabolism* ; Gene Knockout Techniques ; Transfection ; DEAD-box RNA Helicases/metabolism* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Interferon-Induced Helicase, IFIH1/metabolism* ; Transcription Factor RelA/metabolism* ; Interferon-beta/metabolism*

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

Humans ; Nerve Tissue Proteins ; Epithelial-Mesenchymal Transition/genetics* ; Constriction, Pathologic ; Receptors, Immunologic ; Epithelial Cells/metabolism* ; Smad3 Protein/metabolism* ; Transforming Growth Factor beta1/metabolism*

RNA helicases, the largest family of proteins that participate in RNA metabolism, stabilize the intracellular environment through various processes, such as translation and pre-RNA splicing. These proteins are also involved in some diseases, such as cancers and viral diseases. Autophagy, a self-digestive and cytoprotective trafficking process in which superfluous organelles and cellular garbage are degraded to stabilize the internal environment or maintain basic cellular survival, is associated with human diseases. Interestingly, similar to autophagy, RNA helicases play important roles in maintaining cellular homeostasis and are related to many types of diseases. According to recent studies, RNA helicases are closely related to autophagy, participate in regulating autophagy, or serve as a bridge between autophagy and other cellular activities that widely regulate some pathophysiological processes or the development and progression of diseases. Here, we summarize the most recent studies to understand how RNA helicases function as regulatory proteins and determine their association with autophagy in various diseases.
Animals ; Antiviral Agents/pharmacology* ; Autophagy ; Beclin-1/metabolism* ; Carcinogenesis ; Cell Survival ; DEAD Box Protein 58/metabolism* ; Disease Progression ; Gene Expression Regulation ; Homeostasis ; Humans ; Immune System/physiology* ; Neoplasms/metabolism* ; RNA Helicases/metabolism* ; RNA Splicing ; Receptors, Immunologic/metabolism*

Objective To investigate the expression and regulation of programmed cell death protein 1 (PD1), B lymphocyte and T lymphocyte attenuator (BTLA) in peripheral blood of patients with non-small cell lung cancer (NSCLC); to examine the correlation of the mRNA levels between PD and BTLA in NSCLC. Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8⁺ T cells and γδ⁺ T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals. We compared the expression of PD1 and BTLA on the surfaces of γδ⁺ T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid. The correlations of PD1 and BTLA, as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform. Results The frequency of PD1 on the surfaces of CD8⁺ T cells was significantly higher than that of the γδT cells in both healthy controls (t=2.324, P=0.024) and NSCLC patients（t=2.498, P=0.015). The frequency of PD1 on CD8⁺ T cells, rather than on γδ⁺ T cells, was significantly upregulated in advanced NSCLC patients compared with that in healthy controls (t=4.829, P<0.001). The PD1⁺ BTLA⁺γδT cells of the healthy controls were significantly lower than that of the NSCLC patients (t=2.422, P=0.0185). No differences in percentage of PD1⁺γδ⁺ and BTLA⁺γδ⁺ T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment. PD1 was positively correlated with BTLA in both lung adenocarcinoma (r=0.54; P<0.05) and lung squamous cell carcinoma (r=0.78; P<0.05). Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8⁺ T cells and γδT cells in advanced NSCLC, suggesting that these molecules were involved in regulating the inactivation of CD8⁺ T cells and γδ⁺ T cells, immune escape and tumor invasion.
Bone Neoplasms/secondary* ; CD8-Positive T-Lymphocytes ; Carcinoma, Non-Small-Cell Lung/immunology* ; Case-Control Studies ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Ligands ; Lung Neoplasms/immunology* ; Lymphocyte Subsets/immunology* ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor/metabolism* ; RNA, Messenger/metabolism* ; Receptors, Antigen, T-Cell, gamma-delta ; Receptors, Immunologic/metabolism*

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.
Antigens ; metabolism ; CTLA-4 Antigen ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Lymphocyte Subsets ; drug effects ; metabolism ; Phenotype ; Proteomics ; Receptors, Chimeric Antigen ; metabolism ; Single-Cell Analysis ; methods ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; cytology ; drug effects ; Th2 Cells ; cytology ; drug effects ; Transcription, Genetic ; drug effects ; Up-Regulation ; drug effects

Epilepsy is a chronic and severe neurological disorder that has negative effects on the autonomous activities of patients. Functionally, Trem2 (triggering receptor expressed on myeloid cells-2) is an immunoglobulin receptor that affects neurological and psychiatric genetic diseases. Based on this rationale, we aimed to assess the potential role of Trem2 integration with the PI3K/Akt pathway in epilepsy. We used microarray-based gene expression profiling to identify epilepsy-related differentially-expressed genes. In a mouse hippocampal neuron model of epilepsy, neurons were treated with low-Mg extracellular fluid, and the protein and mRNA expression of Trem2 were determined. Using a gain-of-function approach with Trem2, neuronal apoptosis and its related factors were assessed by flow cytometry, RT-qPCR, and Western blot analysis. In a pilocarpine-induced epileptic mouse model, the malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) content and superoxide dismutase (SOD) and glutathione-peroxidase (GSH-Px) activity in the hippocampus were determined, and the protein expression of Trem2 was measured. In addition, the regulatory effect of Trem2 on the PI3K/Akt pathway was analyzed by inhibiting this pathway in both the cell and mouse models of epilepsy. Trem2 was found to occupy a core position and was correlated with epilepsy. Trem2 was decreased in the hippocampus of epileptic mice and epileptic hippocampal neurons. Of crucial importance, overexpression of Trem2 activated the PI3K/Akt pathway to inhibit neuronal apoptosis. Moreover, activation of the PI3K/Akt pathway through over-expression of Trem2 alleviated oxidative stress, as shown by the increased expression of SOD and GSH-Px and the decreased expression of MDA and 8-OHdG. The current study defines the potential role of Trem2 in inhibiting the development of epilepsy, indicating that Trem2 up-regulation alleviates hippocampal neuronal injury and oxidative stress, and inhibits neuronal apoptosis in epilepsy by activating the PI3K/Akt pathway.
Animals ; Apoptosis ; Cells, Cultured ; Epilepsy ; metabolism ; Gene Expression Profiling ; Hippocampus ; metabolism ; Male ; Membrane Glycoproteins ; metabolism ; Mice, Inbred ICR ; Neurons ; metabolism ; Oxidative Stress ; Phosphatidylinositol 3-Kinase ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptors, Immunologic ; metabolism ; Signal Transduction ; Up-Regulation