Objective To investigate the effect of gentiopicroside on osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs), and to determine whether its mechanism involves the stromal cell-derived factor 1(SDF-1)/C-X-C chemokine receptor 4 (CXCR4) pathway. Methods BMSCs were divided into six groups: normal culture control group, osteogenic induction model group, low-dose gentiopicroside (L-gentiopicroside, 10 μmol/L) group, medium-dose gentiopicroside (M-gentiopicroside, 20 μmol/L) group, high-dose gentiopicroside (H-gentiopicroside, 40 μmol/L) group, and H-gentiopicroside+SDF-1/CXCR4 pathway inhibitor (AMD3100) group (H-gentiopicroside+AMD3100, 40 μmol/L gentiopicroside+10 μg/mL AMD3100). Cell viability, apoptosis, ALP activity, mineralized nodule formation, and protein levels of the SDF-1/CXCR4 pathway were assessed using the CCK-8 assay, flow cytometry, ALP staining, Alizarin Red S staining, and Western blotting, respectively. Results No mineralized nodules were observed in either the control and model group, although the color of the model group deepened. Compared with the control group, the model group showed significantly increased A value, ALP activity, expression levels of Runt related transcription factor 2 (RUNX2), osteopontin (OPN), SDF-1, CXCR4 proteins, along with a lower apoptosis rate. Compared with the model group, the L-gentiopicroside, M-gentiopicroside and H-gentiopicroside groups showed dose-dependently (L Humans ; Receptors, CXCR4/genetics* ; Mesenchymal Stem Cells/metabolism* ; Chemokine CXCL12/genetics* ; Iridoid Glucosides/pharmacology* ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Signal Transduction/drug effects* ; Cells, Cultured ; Apoptosis/drug effects* ; Bone Marrow Cells/metabolism*

OBJECTIVES: To observe the role of miR-139-5p and Notch1 signaling pathway in regulation of homing of bone mesenchymal stem cells (BMSCs) of asthmatic rats. METHODS: Normal rat BMSCs were co-cultured with bronchial epithelial cells from normal or asthmatic rats, followed by transfection with miR-139-5p mimics or a negative control sequence. The changes in cell viability and cell cycle were analyzed, and the cellular expressions of CXCR4 and SDF-1 were detected using immunofluorescence staining. The changes of BMSC homing after the transfection were observed, and the expressions of Notch1, RBP-J, and Hes1 mRNAs and proteins and Th1/Th2 cytokines were detected with RT-qPCR, Western blotting or ELISA. RESULTS: The co-cultures of BMSCs and asthmatic bronchial epithelial cells showed significantly decreased expressions of miR-139-5p, IL-2 and IL-12 and increased expressions of CXCR4, SDF-1, IL-5, IL-9, Notch1, RBP-J, and Hes1. Transfection with miR-139-5p mimics significantly increased the expressions of miR-139-5p, IL-2, CXCR4 and SDF-1 and lowered the expression levels of IL-5, IL-9, Notch1, activated Notch1, and Hes1 in the co-cultured cells. Correlation analysis showed that BMSC homing was positively correlated with miR-139-5p and IL-12 and negatively correlated with IL-5 expression. The expression of CXCR4 was negatively correlated with activated Notch1, and SDF-1 was positively correlated with miR-139-5p but negatively correlated with Notch1 expression. CONCLUSIONS High expression of miR-139-5p promotes homing of BMSCs in asthma by targeting the Notch1 signaling pathway to regulate the expressions of Th1/Th2 cytokines, thereby alleviating airway inflammation.
Asthma/genetics* ; Animals ; Mesenchymal Stem Cells/cytology* ; MicroRNAs/metabolism* ; Rats ; Transcription Factor HES-1/genetics* ; Signal Transduction ; Receptor, Notch1/genetics* ; Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics* ; Receptors, CXCR4/genetics* ; Coculture Techniques ; Rats, Sprague-Dawley ; Chemokine CXCL12/genetics* ; Epithelial Cells/metabolism*

Based on the CX3C chemokine ligand 1(CX3CL1)-CX3C chemokine receptor 1(CX3CR1) axis, this study explored the potential mechanism by which Zuogui Jiangtang Jieyu Formula(ZGJTJY) improved neuroinflammation and enhanced neuroprotective effect in a rat model of diabetes mellitus complicated with depression(DD). The DD rat model was established by feeding a high-fat diet combined with streptozotocin(STZ) intraperitoneal injection for four weeks and chronic unpredictable mild stress(CUMS) combined with isolated cage rearing for five weeks. The rats were divided into a control group, a model group, a positive control group, an inhibitor group, and a ZGJTJY group. The open field test and forced swimming test were used to assess the depression-like behaviors of the rats. Enzyme-linked immunosorbent assay(ELISA) was performed to measure the expression levels of the pro-inflammatory cytokines interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) in plasma. Immunofluorescence staining was used to detect the expression of ionized calcium-binding adapter molecule 1(Iba1), postsynaptic density protein-95(PSD95), and synapsin-1(SYN1) in the hippocampus. Hematoxylin-eosin(HE) staining, Nissl staining, and TdT-mediated dUTP nick end labeling(TUNEL) fluorescence staining were performed to assess hippocampal neuronal damage. Western blot was used to measure the expression levels of CX3CL1, CX3CR1, A2A adenosine receptor(A2AR), glutamate receptor 2A(NR2A), glutamate receptor 2B(NR2B), and brain-derived neurotrophic factor(BDNF) in the hippocampus. Compared with the model group, the ZGJTJY group showed improved depression-like behaviors in DD rats, enhanced neuroprotective effect, increased expression of PSD95, SYN1, and BDNF(P<0.01), and decreased expression of Iba1, IL-1β, and TNF-α(P<0.01), as well as the expression of CX3CL1, CX3CR1, A2AR, NR2A, and NR2B(P<0.01). These results suggest that ZGJTJY may exert its neuroprotective effect by inhibiting the CX3CL1-CX3CR1 axis and activation of hippocampal microglia, thereby improving neuroinflammation and abnormal activation of N-methyl-D-aspartate receptor(NMDAR) subunits, and ultimately enhancing the expression of synaptic-related proteins PSD95, SYN1, and BDNF in the hippocampus.
Rats ; Animals ; Depression/drug therapy* ; Brain-Derived Neurotrophic Factor ; Neuroprotective Agents ; Tumor Necrosis Factor-alpha/metabolism* ; Neuroinflammatory Diseases ; Diabetes Mellitus ; Receptors, Glutamate ; CX3C Chemokine Receptor 1/genetics*

BACKGROUND: There is growing evidence that 5-fluorouracil (5-FU) combined with therapeutic trauma can effectively induce skin repigmentation in vitiligo patients who are unresponsive to conventional treatments. Previous studies have mainly focused on identifying the antimitotic activity of 5-FU for the treatment of skin cancer, but few studies have investigated its extra-genotoxic actions favoring melanocyte recruitment. METHODS: We utilized the full thickness excisional skin wound model in Dct-LacZ transgenic mice to dynamically assess the migration of melanocytes in the margins of wounds treated with or without 5-FU. The in-situ expression of CXCL12 was examined in the wound beds using immunofluorescence staining. Quantitative real-time polymerase chain reaction and Western blotting analyses were performed to detect the expression levels of CXCL12 mRNA and protein in primary mouse dermal fibroblasts treated with or without 5-FU. Transwell assays and fluorescein isothiocyanate (FITC)-phalloidin staining were used to observe cell migration and filamentous actin (F-actin) changes of melan-a murine melanocytes. RESULTS: Whole mount and cryosection X-gal staining showed that the cell numbers of LacZ-positive melanocytes were much higher in the margins of dorsal and tail skin wounds treated with 5-FU compared with the controls. Meanwhile, CXCL12 immunostaining was significantly increased in the dermal compartment of wounds treated with 5-FU (control vs. 5-FU, 22.47 ± 8.85 vs. 44.69 ± 5.97, P < 0.05). Moreover, 5-FU significantly upregulated the expression levels of CXCL12 mRNA (control vs. 5-FU, 1.00 ± 0.08 vs. 1.54 ± 0.06, P < 0.05) and protein (control vs. 5-FU, 1.00 ± 0.06 vs. 2.93 ± 0.10, P < 0.05) in cultured fibroblasts. Inhibition of the CXCL12/CXCR4 axis suppressed melanocyte migration in vitro using a CXCL12 small interfering RNA (siRNA) or a CXCR4 antagonist (AMD3100). CONCLUSION 5-FU possesses a pro-pigmentary activity through activation of the CXCL12/CXCR4 axis to drive the chemotactic migration of melanocytes.
Animals ; Cell Movement ; Cell Proliferation ; Chemokine CXCL12/genetics* ; Fibroblasts ; Fluorouracil/therapeutic use* ; Humans ; Mice ; RNA, Messenger ; Receptors, CXCR4

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.
Animals ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Chemokine CCL19 ; metabolism ; Glypicans ; metabolism ; HEK293 Cells ; Humans ; Interleukin-7 ; metabolism ; Lentivirus ; genetics ; Liver Neoplasms ; Mice ; Receptors, Chimeric Antigen ; metabolism ; T-Lymphocytes ; metabolism ; Xenograft Model Antitumor Assays

Metastasis is the leading cause of mortality for cancer patients, and lymphatic metastasis is one of the main ways of tumor metastasis. The role of CCL21 and its receptor CCR7 in lymphatic metastasis has been increasingly concerned in recent years. CCR7 is mainly expressed by both dendritic cells and T cells for immune responses. CCL21, the chemokine ligand for CCR7, secreted from lymphatic endothelial cells binds CCR7 and recruits immune cells toward lymphatic vessels and lymphatic nodes. CCR7 expressed tumor cells can also metastasize to lymphatic system by the similar way as immune cells. Targeting CCL21/CCR7 axis to inhibit lymphatic metastasis but remain potent anti-tumor immune response has increasingly become a spot light of tumor immunotherapy. In this review, we summarize the role of CCL21/CCR7 axis in lymphatic metastasis, as well as preclinical trials and clinical trials in targeting CCL21/CCR7 axis for tumor metastasis therapy, hoping to accelerate the progress on tumor metastasis therapy by targeting CCL21/CCR7 axis.
Cell Line, Tumor ; Chemokine CCL21 ; Endothelial Cells ; Humans ; Lymphatic Metastasis ; Neoplasms/therapy* ; Receptors, CCR7/genetics*

BACKGROUND: The epidermal growth factor receptor (EFGR) mutation was closely related to the invasion and metastasis of lung adenocarcinoma and the biological axis of CXCR4/CXCL12 (chemokine receptor 4/chemokine ligand 12) played an important role in the organ-specific metastasis of the tumor. It was a question surrounding whether there is interaction between them in the process of lung adenocarcinoma metastasis. To investigate the potential molecular mechanisms of EGFR over-expression and EFGR-mutations effects on cell proliferation, migration and invasion, we constructed EGFR over-expression and three EFGR-mutant human lung adenocarcinoma H1299 cell sublines. METHODS: EGFR over-expression and three EFGR-mutant (EGFR-E746-A750del, EGFR-T790M and EGFR-L858R) plasmid were designed and transfected H1299 cells with Lipofectamine 2000. H1299 cells transfected with empty vector were negative control (NC), and H1299 cells without transfection were set as blank control (BC). The effects of EGFR over-expression and mutations on the proliferation, migration and invasion of H1299 cells were detected by cell cloning assay, wound healing assay and Transwell assay. The mRNA and protein expression levels of MMP-2, MMP-9, CXCR4 and CXCL12 were detected by RT-PCR and Western blot. RESULTS: Compared with negative control group and blank control group, EGFR over-expression and EGFR-E746-A750 deletion have significantly higher colony formation (28±2, 28.33±4.16; respectively) (P<0.05) and the cell migration and invasion ability were significantly increased (P<0.05). RT-PCR and Western blot assay showed that the mRNA and protein expression of MMP-2, MMP-9, CXCR4 and CXCL12 in EGFR over-expression and EGFR-E746-A750 deletion group were remarkably higher than that in negative control and blank control group (P<0.05). CONCLUSIONS EGFR over-expression and 19 exon deletion can promote the expression of MMP-2 and MMP-9 by up-regulating CXCR4/CXCL12 signaling pathway, leading to the change of tumor biological characteristics such as higher proliferation, migration and invasion ability.
Adenocarcinoma ; pathology ; Adenocarcinoma of Lung ; Cell Line, Tumor ; Cell Movement ; genetics ; Chemokine CXCL12 ; metabolism ; ErbB Receptors ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; pathology ; Mutation ; Neoplasm Invasiveness ; Receptors, CXCR4 ; metabolism ; Signal Transduction ; genetics

Chemokine 12 (CXCL12), also known as stromal cell derived factor-1 (SDF-1) and a member of the CXC chemokine subfamily, is ubiquitously expressed in many tissues and cell types. It interacts specifically with the ligand for the transmembrane G protein-coupled receptors CXCR4 and CXCR7. The CXCL12/CXCR4 axis takes part in a series of physiological, biochemical, and pathological process, such as inflammation and leukocyte trafficking, cancer-induced bone pain, and postsurgical pain, and also is a key factor in the cross-talking between tumor cells and their microenvironment. Aberrant overexpression of CXCR4 is critical for tumor survival, proliferation, angiogenesis, homing and metastasis. In this review, we summarized the role of CXCL12/CXCR4 in cancer, CXCR4 inhibitors under clinical study, and natural product CXCR4 antagonists. In conclusion, the CXCL12/CXCR4 signaling is important for tumor development and targeting the pathway might represent an effective approach to developing novel therapy in cancer treatment.
Animals ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Biological Products ; chemistry ; pharmacology ; Chemokine CXCL12 ; genetics ; metabolism ; Humans ; Molecular Targeted Therapy ; Neoplasms ; drug therapy ; genetics ; metabolism ; Receptors, CXCR4 ; antagonists & inhibitors ; genetics ; metabolism

OBJECTIVE: To investigate the association of CXCL12 and CXCR4 polymorphisms with the genetic risk and severity of coronary stenosis in patients with coronary artery disease (CAD). METHODS: Competitive allele specific PCR(KASP) was performed to identify the genotypes of rs2297630 and rs2322864 polymorphisms in 302 CAD patients and 302 age-and gender-matched healthy controls. The severity of CAD patients was assessed by the Gensini scoring system according to the results of coronary arteriography. The association of rs2297630 and rs2322864 polymorphisms with genetic risk of CAD and Gensini scores were analyzed by unconditional logistic regression and multivariate linear regression respectively. RESULTS: There were significant differences in the genotype and allele frequencies of both rs2297630 and rs2322864 between the CAD group and healthy control (all <0.01). Regression analysis showed that rs2297630 polymorphism was associated with genetic risk of CAD and Gensini scores (all <0.01). People who carried the AA genotype suffered higher risk of CAD susceptibility and more serious coronary stenosis (all <0.01), compared with GG genotype carriers. There was also significant association between rs2322864 polymorphism and genetic risk of CAD (<0.01); those who carried the CT genotype had higher risk of CAD (<0.01), compared with TT genotype carriers. However, rs2322864 polymorphism was not associated with the severity of coronary stenosis (>0.05). CONCLUSIONS Gene polymorphism of CXCL12 rs2297630 is associated with the genetic risk of CAD and the severity of coronary stenosis. Moreover, the gene polymorphism of CXCR4 rs2322864 is associated with genetic risk of CAD, but not with the severity of coronary stenosis.
Chemokine CXCL12 ; genetics ; Coronary Angiography ; Coronary Artery Disease ; complications ; Coronary Stenosis ; complications ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Polymorphism, Genetic ; Receptors, CXCR4 ; genetics ; Risk Factors

Recent studies have shown that the chemokine receptor CXCR3 and its ligand CXCL10 in the dorsal root ganglion mediate itch in experimental allergic contact dermatitis (ACD). CXCR3 in the spinal cord also contributes to the maintenance of neuropathic pain. However, whether spinal CXCR3 is involved in acute or chronic itch remains unclear. Here, we report that Cxcr3 mice showed normal scratching in acute itch models but reduced scratching in chronic itch models of dry skin and ACD. In contrast, both formalin-induced acute pain and complete Freund's adjuvant-induced chronic inflammatory pain were reduced in Cxcr3 mice. In addition, the expression of CXCR3 and CXCL10 was increased in the spinal cord in the dry skin model induced by acetone and diethyl ether followed by water (AEW). Intrathecal injection of a CXCR3 antagonist alleviated AEW-induced itch. Furthermore, touch-elicited itch (alloknesis) after compound 48/80 or AEW treatment was suppressed in Cxcr3 mice. Finally, AEW-induced astrocyte activation was inhibited in Cxcr3 mice. Taken together, these data suggest that spinal CXCR3 mediates chronic itch and alloknesis, and targeting CXCR3 may provide effective treatment for chronic pruritus.
Acetamides ; therapeutic use ; Animals ; Chemokine CXCL10 ; metabolism ; Chloroquine ; toxicity ; Chronic Disease ; Cyclopropanes ; adverse effects ; Dehydration ; complications ; Dinitrofluorobenzene ; adverse effects ; Disease Models, Animal ; Formaldehyde ; toxicity ; Freund's Adjuvant ; toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Motor Activity ; drug effects ; Pain ; chemically induced ; Pruritus ; chemically induced ; pathology ; Pyrimidines ; therapeutic use ; Receptors, CXCR3 ; antagonists & inhibitors ; genetics ; metabolism ; Skin ; pathology ; Spinal Cord ; drug effects ; metabolism ; pathology ; Time Factors ; p-Methoxy-N-methylphenethylamine ; toxicity