The bioinformatics analysis of quercetin in octagonal lotus for the screening of breast cancer MYC, CXCL10, CXCL11, and E2F1.

BACKGROUND: Comprehensive bioinformatics analysis of the effective molecular screening of Podophyllum octagonal in breast cancer treatment by using network pharmacology. METHODS: We collected the active ingredients and target genes of Chinese medicine octagonal lotus through the Traditional Chinese Medicine System Pharmacology Analysis Platform (TCMSP); downloaded human protein annotation information on the protein database Uniport; and collected data from five databases: GeneCards, OMIM, PharmGkb, TDD, and DrugBank. Construct the practical ingredient-target gene data intersection to obtain the target gene-disease gene and draw the Venn diagram. We use Cytoscape 3.8.0 software to construct the effective component-target gene-disease gene network. The STRING database protein interaction (PPI) networks were erected, and we used Cytoscape 3.8.0 software to screen out its core sub-networks and hub gene networks. Through survival analysis, core genes and hub genes were screened to identify several key genes. We performed key target gene ontology (GO) analysis and gene interaction (KEGG) analysis, which were followed by molecular docking of the key active ingredients in the star anise corresponding to several key genes. RESULTS: 19 active ingredients, 444 drug targets, and 10,941 disease-related genes were obtained. The key active ingredient was quercetin. GO analysis revealed 2471 affected biological processes, and 167 pathways were obtained in KEGG enrichment analysis. CONCLUSION: This study initially screened the key active ingredients of star aniseed lotus and analyzed key genes and several essential pathways. Traditional Chinese medicine is expected to provide new evidence and research ideas to prevent and treat breast cancer.

Inhibition of macrophage migration in zebrafish larvae demonstrates in vivo efficacy of human CCR2 inhibitors.

The chemokine signaling axes CCR2-CCL2 and CXCR3-CXCL11 participate in the inflammatory response by recruiting leukocytes to damaged tissue or sites of infection and are, therefore, potential pharmacological targets to treat inflammatory disorders. Although multiple CCR2 orthosteric and allosteric inhibitors have been developed, none of these compounds has been approved for clinical use, highlighting the need for a fast, simple and robust preclinical test system to determine the in vivo efficacy of CCR2 inhibitors. Herein we show that human CCL2 and CXCL11 drive macrophage recruitment in zebrafish larvae and that CCR2 inhibitors designed for humans also limit macrophage recruitment in this model organism due to the high conservation of the chemokine system. We demonstrated anti-inflammatory activities of three orthosteric and two allosteric CCR2 inhibitors using macrophage recruitment to injury as a functional read-out of their efficiency, while simultaneously evaluating toxicity. These results provide proof-of-principle for screening CCR2 inhibitors in the zebrafish model.

Bu-Shen-Fang-Chuan formula attenuates T-lymphocytes recruitment in the lung of rats with COPD through suppressing CXCL9/CXCL10/CXCL11-CXCR3 axis.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by irreversible airflow limitation. The current medications show limited effects on the decline of pulmonary function in COPD. Our multicenter clinical trial found that Bu-Shen-Fang-Chuan fomula (BSFCF), a Chinese herbal formula, markedly reduced the frequencies of acute exacerbation of COPD and delayed lung function decline. However, the underlying mechanisms are still unclear. In this study, we established a COPD rat model through a 6-month exposure to cigarette smoke (CS) and found that BSFCF (7.2 g/kg) effectively improved CS-induced reduction in pulmonary function and remarkably decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid (BALF). Importantly, BSFCF treatment notably prevented the accumulation of T-lymphocytes (especially CD8+ T-cells) in the lung of COPD rats. RNA sequencing analysis of lung tissue demonstrated that CXCL9/CXCL10/CXCL11-CXCR3 chemokine axis in the lung of CS-exposed rats was significantly suppressed by BSFCF. Moreover, our Real-time PCR data verified that BSFCF evidently inhibited the mRNA expressions of CXCL9, CXCL10, CXCL11 and CXCR3. Conclusively, BSFCF markedly improved pulmonary function and attenuated CD8+ T-cells recruitment in the lung of CS-exposed rats, which were partially through inhibition of CXCL9/CXCL10/CXCL11-CXCR3 axis.

Selenium supplementation in patients with subclinical hypothyroidism affected by autoimmune thyroiditis: Results of the SETI study.

OBJECTIVE: The purpose of this prospective study was to assess the effects of selenium supplementation on TSH and interferon-γ inducible chemokines (CXCL9, CXCL10 and CXCL11) levels in patients with subclinical hypothyroidism due to Hashimoto's thyroiditis. PATIENTS AND METHODS: Patients with subclinical hypothyroidism due to Hashimoto thyroiditis were prospectively enrolled in the SETI study. They received 83mcg of selenomethionine/day orally in a soft gel capsule for 4 months with water after a meal. No further treatment was given. All patients were measured thyroid hormone, TPOAb, CXCL9, CXCL10, CXCL11, iodine, and selenium levels at baseline and at study end. RESULTS: 50 patients (43/7 female/male, median age 43.9±11.8 years) were enrolled, of which five withdrew from the study. At the end of the study, euthyroidism was restored in 22/45 (48.9%) participants (responders), while 23 patients remained hypothyroid (non-responders). There were no significant changes in TPOAb, CXCL9, CXCL10, CXCL11, and iodine levels from baseline to the end of the study in both responders and non-responders. TSH levels were re-tested six months after selenomethionine withdrawal: 83.3% of responding patients remained euthyroid, while only 14.2% of non-responders became euthyroid. CONCLUSIONS: The SETI study shows that short-course supplementation with selenomethionine is associated to a normalization of serum TSH levels which is maintained 6 months after selenium withdrawal in 50% of patients with subclinical hypothyroidism due to chronic autoimmune thyroiditis. This TSH-lowering effect of selenium supplementation is unlikely to be related to changes in humoral markers of autoimmunity and/or circulating CXCL9.

Xia-Yu-Xue Decoction Inhibits Intestinal Epithelial Cell Apoptosis in CCl4-Induced Liver Fibrosis.

BACKGROUND/AIMS: Intestine-derived endotoxin is thought to play a role in the development of liver fibrosis. However, the pathological change in the intestine during liver fibrosis is still poorly understood. Here, we investigated the effects of Xia-yu-xue decoction (XYXD) on intestinal inflammation, apoptosis, and tight junction integrity in the carbon tetrachloride (CCl4)-induced liver fibrosis. METHODS: Murine liver fibrosis was developed by CCI4 treatment three times per week over a 6-week period. The CCl4-treated mice were divided into two groups: the CCl4-water group (n=8, CCl4) and the CCl4-XYXD group (n=8, CCl4+XYXD). The CCl4+XYXD mice were treated with XYXD from the beginning of the first week. The expression of inflammatory cytokines and apoptotic molecules were examined using immunohistochemistry, real-time PCR, and western blot. The intestinal epithelial cell apoptosis was examined by TUNEL staining. The tight junction-related molecules, such as ZO-1, claudin, and occludin in the gut were measured by real-time PCR. RESULTS: In CCl4-treated mice damage of the intestinal epithelia and infiltration of inflammatory cells into the lamina propria and muscular layer were observed. Proinflammatory markers MCP-1, TNF-α, CXCL11, IL-6, and CD68 were significantly increased in the intestinal epithelia in CCI4-treated mice. The expression of pro-apoptotic molecules including Fas and Bax was increased in the intestinal epithelia in CCI4-treated mice compared with that in control. The number of TUNEL-positive intestinal epithelial cells was also markedly increased in CCl4-treated mice. The expression of the tight junction proteins including ZO-1, claudin, and occludin was significantly decreased in CCI4-treated mice compared with that in control mice. Notably, XYXD treatment ameliorated increased inflammatory markers and apoptosis-related molecules and decreased tight-junction proteins in CCl4-treated mice. CONCLUSION: CCl4-treatment increased expression of proinflammatory cytokines and pro-apoptotic molecules and disrupted tight junction integrity in the intestine. XYXD treatment ameliorated intestinal inflammation, cell death, and tight junction disintegrity induced by CCl4 treatment, suggesting that XYXD inhibits CCl4-mediated liver fibrosis at least in part by ameliorating the intestinal epithelial damage.

The CXCL10/CXCR3 Axis and Cardiac Inflammation: Implications for Immunotherapy to Treat Infectious and Noninfectious Diseases of the Heart.

Accumulating evidence reveals involvement of T lymphocytes and adaptive immunity in the chronic inflammation associated with infectious and noninfectious diseases of the heart, including coronary artery disease, Kawasaki disease, myocarditis, dilated cardiomyopathies, Chagas, hypertensive left ventricular (LV) hypertrophy, and nonischemic heart failure. Chemokine CXCL10 is elevated in cardiovascular diseases, along with increased cardiac infiltration of proinflammatory Th1 and cytotoxic T cells. CXCL10 is a chemoattractant for these T cells and polarizing factor for the proinflammatory phenotype. Thus, targeting the CXCL10 receptor CXCR3 is a promising therapeutic approach to treating cardiac inflammation. Due to biased signaling CXCR3 also couples to anti-inflammatory signaling and immunosuppressive regulatory T cell formation when activated by CXCL11. Numbers and functionality of regulatory T cells are reduced in patients with cardiac inflammation, supporting the utility of biased agonists or biologicals to simultaneously block the pro-inflammatory and activate the anti-inflammatory actions of CXCR3. Other immunotherapy strategies to boost regulatory T cell actions include intravenous immunoglobulin (IVIG) therapy, adoptive transfer, immunoadsorption, and low-dose interleukin-2/interleukin-2 antibody complexes. Pharmacological approaches include sphingosine 1-phosphate receptor 1 agonists and vitamin D supplementation. A combined strategy of switching CXCR3 signaling from pro- to anti-inflammatory and improving Treg functionality is predicted to synergistically lessen adverse cardiac remodeling.

Andrographolide inhibits prostate cancer by targeting cell cycle regulators, CXCR3 and CXCR7 chemokine receptors.

Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB(-/-)) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.

Anti-inflammatory effect of 635 nm irradiations on in vitro direct/indirect irradiation model.

Low-level laser therapy (LLLT) has been promoted for its beneficial effects on tissue healing and pain relief. As during laser treatment it is possible to irradiate only a small area of the surface body or wound and, correspondingly, of a very small volume of the circulating blood, it is necessary to explain how its photomodification can lead to a wide spectrum of therapeutic effects. To establish the experimental model for indirect irradiation, irradiation with 635 nm was performed on immortalized human gingival fibroblasts (IGFs) in the presence of Porphyromonas gingivalis lipopolysaccharides (LPS). The irradiated medium was transferred to non-irradiated IGFs which were compared with direct irradiated IGFs. The protein expressions were assessed by Western blot, and prostaglandin E2 (PGE2 ) was measured using an enzyme-linked immunoassay. Reactive oxygen species (ROS) were measured by DCF-DA; cytokine profiles were assessed using a human inflammation antibody array. Cyclooxygenase-2 (COX-2) protein expression and PGE2 production were significantly increased in the LPS-treated group and decreased in both direct and indirect irradiated IGFs. Unlike direct irradiated IGFs, ROS level in indirect irradiated IGFs was decreased by time-dependent manners. There were significant differences of released granulocyte colony-stimulating factor (G-CSF), regulated on activated normal T-cell expressed and secreted (RANTES), and I-TAC level observed compared with direct and indirect irradiated IGFs. In addition, in the indirect irradiation group, phosphorylations of C-Raf and Erk1/2 increased significantly compared with the direct irradiation group. Thus, we suggest that not only direct exposure with 635 nm light, but also indirect exposure with 635 nm light can inhibit activation of pro-inflammatory mediators and may be clinically useful as an anti-inflammatory tool.

Anti-cytomegalovirus effects of tricin are dependent on CXCL11.

It has been reported that treatment with tricin (4',5,7-trihydroxy-3',5'-dimethoxyflavone), a derivative of Sasa albo-marginata, after human cytomegalovirus (HCMV) infection significantly suppressed both infectious virus production and HCMV replication in the human embryonic fibroblast cell line MRC-5. In this paper, we examined the mechanisms for the anti-HCMV effects of tricin in MRC-5 cells. Exposure of fibroblasts to tricin inhibited infectious HCMV production, with concomitant decreases in levels of transcripts of the CXC chemokine IFN-inducible T cell alpha chemoattractant (I-TAC or CXCL11) gene. We also found that the transcripts of the HCMV immediate early (IE) gene and replication of HCMV were lower in CXCL11 gene-knockdown cells. These results suggest that tricin is a novel compound with potential anti-HCMV activity and that CXCL11 is one of the chemokines involved in HCMV replication. In addition, it is possible that CXCL11 is the one of the targets of tricin.

The yin and yang of chemokine receptor activation.

Chemokines represent a class of cytokines that control the migration of leucocytes. The human chemokine system comprises 44 ligands and 21 receptors that have evolved to control leucocyte migration. Although chemokines are an attractive therapeutic target for anti-inflammatory intervention, clinical trials of small molecule receptor antagonists have failed to demonstrate efficacy. One often cited explanation for this is the apparent redundancy within the chemokine system, wherein several ligands bind and activate each receptor. The work of Scholten et al. and Nedjai et al. reported in this issue of the British Journal of Pharmacology demonstrates that this redundancy does not exist at the molecular level and provides a powerful insight into the complex nature of chemokine receptor activation.

Inhibition of the CXCR3-mediated pathway suppresses ultraviolet B-induced pigmentation and erythema in skin.

BACKGROUND: Skin pigmentation by ultraviolet (UV) B radiation is caused in part by inflammation mediated by chemokines and cytokines secreted by keratinocytes in the irradiated area. However, such inflammatory processes have not been well documented. OBJECTIVES: To elucidate the inflammation processes caused by UVB irradiation using skin-lightening agents that suppress melanin synthesis after UVB irradiation. METHODS: Utilizing a three-dimensional (3D) skin model, agents that suppressed formation of sunburn cells (SBC) after UVB irradiation were screened. Molecules whose expression was upregulated by UVB irradiation and attenuated by pretreatment with the agent were then screened by gene microarray to explore the mechanism of UVB irradiation. Messenger RNA expression of the molecules identified to be responsible for melanin biosynthesis was knocked down with a Tet-off shRNA lentivirus construct to confirm the involvement of the molecule in the pigmentation pathway following UVB irradiation. RESULTS: Paeonia suffruticosa Andrews (PSA) pretreatment suppressed SBC formation in the 3D skin model, and erythema formation and pigmentation in volunteers exposed to UVB irradiation. Comprehensive gene analysis after UVB irradiation showed upregulation of CXCR3 and its ligands, CXCL9/monokine induced by interferon (IFN)-γ (MIG), CXCL10/10-kDa IFN-γ-induced protein (IP-10) and CXCL11/inducible T-cell α-chemoattractant (I-TAC). Upregulation of these genes was partially suppressed by PSA pretreatment. Melanin biosynthesis increased upon stimulation of CXCR3 ligands (MIG, IP-10 or I-TAC) and decreased following CXCR3 downregulation by shRNA knockdown. CONCLUSIONS: UVB irradiation activates CXCR3-mediated signalling that leads to melanin synthesis. PSA pretreatment shows a lightening effect partly by attenuating CXCR3-mediated signalling at the transcriptional level.

Identification and initial evaluation of 4-N-aryl-[1,4]diazepane ureas as potent CXCR3 antagonists.

The identification and evaluation of aryl-[1,4]diazepane ureas as functional antagonists of the chemokine receptor CXCR3 are described. Specific examples exhibit IC(50) values of approximately 60 nM in a calcium mobilization functional assay, and dose-dependently inhibit CXCR3 functional response to CXCL11 (interferon-inducible T-cell alpha chemoattractant/I-TAC) as measured by T-cell chemotaxis, with a potency of approximately 100 nM.

Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function.

The chemokine receptor CXCR3 is a G protein-coupled receptor found predominantly on T cells that is activated by three ligands as follows: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Previously, we have found that of the three ligands, CXCL11 is the most potent inducer of CXCR3 internalization and is the physiologic inducer of CXCR3 internalization after T cell contact with activated endothelial cells. We have therefore hypothesized that these three ligands transduce different signals to CXCR3. In light of this hypothesis, we sought to determine whether regions of CXCR3 are differentially required for CXCL9, CXCL10, and CXCL11 function. Here we identified two distinct domains that contributed to CXCR3 internalization. The carboxyl-terminal domain and beta-arrestin1 were predominantly required by CXCL9 and CXCL10, and the third intracellular loop was predominantly required by CXCL11. Chemotaxis and calcium mobilization induced by all three CXCR3 ligands were dependent on the CXCR3 carboxyl terminus and the DRY sequence in the third trans-membrane domain. Our findings demonstrate that distinct domains of CXCR3 mediate its functions and suggest that the differential requirement of these domains contributes to the complexity of the chemokine system.

IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines.

IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, IL-10, and IL-17 to modulate IP-10, Mig, and I-TAC in cultured human keratinocytes and CXCR3 expression in T cells from allergic contact dermatitis (ACD). IL-4, but not IL-10 or IL-17, significantly up-regulated IFN-gamma- or TNF-alpha-induced IP-10, Mig, and I-TAC mRNA accumulation in keratinocytes and increased the levels of IP-10 and Mig in keratinocyte supernatants. Immunohistochemistry of skin affected by ACD revealed that >70% of infiltrating cells were reactive for CXCR3 and that CXCR3 staining colocalized in CD4+ and CD8+ T cells. Nickel-specific CD4+ and CD8+ T cell lines established from ACD skin produced IFN-gamma and IL-4 and expressed moderate to high levels of CXCR3. Finally, CXCR3 agonistic chemokines released by stimulated keratinocytes triggered calcium mobilization in skin-derived nickel-specific CD4+ T cells and promoted their migration, with supernatant from keratinocyte cultures stimulated with IFN-gamma and IL-4 attracting more efficaciously than supernatant from keratinocytes activated with IFN-gamma alone. In conclusion, IL-4 exerts a proinflammatory function on keratinocytes by potentiating IFN-gamma and TNF-alpha induction of IP-10, Mig, and I-TAC, which in turn may determine a prominent recruitment of CXCR3+ T lymphocytes at inflammatory reaction sites.

Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.

Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2-activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-gamma-inducible 10-kD protein (IP-10) and IFN-gamma- induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-gamma and IL-1 together results in an approximately 400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1-stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.

The CXC-chemokine, H174: expression in the central nervous system.

H174 is a new member of the CXC-chemokine family. A cDNA probe containing the entire H174 coding region recognized a predominant inducible transcript of approximately 1.5 kb expressed in interferon (IFN) activated astrocytoma and monocytic cell lines. H174 message can be induced following IFN-alpha, IFN-beta, or IFN-gamma stimulation. H174 message was also detected in IFN treated cultures of primary human astrocytes, but was absent in unstimulated astrocytes. H174, like IP10 and Mig, lacks the ELR sequence associated with the neutrophil specificity characteristic of most CXC-chemokines. Preliminary experiments suggest H174, IP10 and Mig are independently regulated. Recombinant H174 is a weak chemoattractant for monocyte-like cells. H174 can also stimulate calcium flux responses. The data support the classification of H174 as a member of a subfamily of interferon-gamma inducible non-ELR CXC-chemokines. Brain tissues were obtained at autopsy from one patient with AIDS dementia, one patient with multiple sclerosis, and two normal control patients. H174 and Mig were detected by RT-PCR in brain tissue cDNA derived from the patients with pathological conditions associated with activated astrocytes but not in cDNA from control specimens.