Chemokine CX3CL1 (Fractalkine) Signaling and Diabetic Encephalopathy.

Diabetes mellitus (DM) is the most common metabolic disease in humans, and its prevalence is increasing worldwide in parallel with the obesity pandemic. A lack of insulin or insulin resistance, and consequently hyperglycemia, leads to many systemic disorders, among which diabetic encephalopathy (DE) is a long-term complication of the central nervous system (CNS), characterized by cognitive impairment and motor dysfunctions. The role of oxidative stress and neuroinflammation in the pathomechanism of DE has been proven. Fractalkine (CX3CL1) has unique properties as an adhesion molecule and chemoattractant, and by acting on its only receptor, CX3CR1, it regulates the activity of microglia in physiological states and neuroinflammation. Depending on the clinical context, CX3CL1-CX3CR1 signaling may have neuroprotective effects by inhibiting the inflammatory process in microglia or, conversely, maintaining/intensifying inflammation and neurotoxicity. This review discusses the evidence supporting that the CX3CL1-CX3CR1 pair is neuroprotective and other evidence that it is neurotoxic. Therefore, interrupting the vicious cycle within neuron-microglia interactions by promoting neuroprotective effects or inhibiting the neurotoxic effects of the CX3CL1-CX3CR1 signaling axis may be a therapeutic goal in DE by limiting the inflammatory response. However, the optimal approach to prevent DE is simply tight glycemic control, because the elimination of dysglycemic states in the CNS abolishes the fundamental mechanisms that induce this vicious cycle.

CX3CL1 (Fractalkine)-CX3CR1 Axis in Inflammation-Induced Angiogenesis and Tumorigenesis.

The chemotactic cytokine fractalkine (FKN, chemokine CX3CL1) has unique properties resulting from the combination of chemoattractants and adhesion molecules. The soluble form (sFKN) has chemotactic properties and strongly attracts T cells and monocytes. The membrane-bound form (mFKN) facilitates diapedesis and is responsible for cell-to-cell adhesion, especially by promoting the strong adhesion of leukocytes (monocytes) to activated endothelial cells with the subsequent formation of an extracellular matrix and angiogenesis. FKN signaling occurs via CX3CR1, which is the only known member of the CX3C chemokine receptor subfamily. Signaling within the FKN-CX3CR1 axis plays an important role in many processes related to inflammation and the immune response, which often occur simultaneously and overlap. FKN is strongly upregulated by hypoxia and/or inflammation-induced inflammatory cytokine release, and it may act locally as a key angiogenic factor in the highly hypoxic tumor microenvironment. The importance of the FKN/CX3CR1 signaling pathway in tumorigenesis and cancer metastasis results from its influence on cell adhesion, apoptosis, and cell migration. This review presents the role of the FKN signaling pathway in the context of angiogenesis in inflammation and cancer. The mechanisms determining the pro- or anti-tumor effects are presented, which are the cause of the seemingly contradictory results that create confusion regarding the therapeutic goals.

Emerging evidence for endogenous neurosteroid modulation of pro-inflammatory and anti-inflammatory pathways that impact neuropsychiatric disease.

This mini-review presents emerging evidence that endogenous neurosteroids modulate both pro- and anti-inflammatory signaling by immune cells and brain cells that contribute to depression, alcohol use disorders, and other inflammatory conditions. We first review the literature on pregnenolone and allopregnanolone inhibition of proinflammatory neuroimmune pathways in the periphery and the brain - effects that are independent of GABAergic mechanisms. We follow with evidence for neurosteroid enhancement of anti-inflammatory and protective pathways in brain and immune cells. These studies draw clinical relevance from a large body of evidence that pro-inflammatory immune signaling is dysregulated in many brain disorders and the fact that neurosteroids inhibit the same inflammatory pathways that are activated in depression, alcohol use disorders and other inflammatory conditions. Thus, we describe evidence that neurosteroid levels are decreased and neurosteroid supplementation has therapeutic efficacy in these neuropsychiatric conditions. We conclude with a perspective that endogenous regulation of immune balance between pro- and anti-inflammatory pathways by neurosteroid signaling is essential to prevent the onset of disease. Deficits in neurosteroids may unleash excessive pro-inflammatory activation which progresses in a feed-forward manner to disrupt brain networks that regulate stress, emotion and motivation. Neurosteroids can block various inflammatory pathways in mouse and human macrophages, rat brain and human blood and therefore provide new hope for treatment of intractable conditions that involve excessive inflammatory signaling.

Blockage of the fractalkine pathway reduces hyperalgesia and prevents morphological glial alterations-Comparison between inflammatory and neuropathic orofacial pain in male rats.

This study aimed to evaluate the effects of inhibitors of the fractalkine pathway in hyperalgesia in inflammatory and neuropathic orofacial pain in male rats and the morphological changes in microglia and satellite glial cells (SGCs). Rats were submitted to zymosan-induced arthritis of the temporomandibular joint or infraorbital nerve constriction, and treated intrathecally with a P2 X7 antagonist, a cathepsin S inhibitor or a p-38 mitogen-activated protein kinase (MAPK) inhibitor. Mechanical hyperalgesia was evaluated 4 and 6 h following arthritis induction or 7 and 14 days following nerve ligation. The expression of the receptor CX3 CR1 , phospho-p-38 MAPK, ionized calcium-binding adapter molecule-1 (Iba-1), and glutamine synthetase and the morphological changes in microglia and SGCs were evaluated by confocal microscopy. In both inflammatory and neuropathic models, untreated animals presented a higher expression of CX3 CR1 and developed hyperalgesia and up-regulation of phospho-p-38 MAPK, which was prevented by all drugs (p < .05). The number of microglial processes endpoints and the total branch length were lower in the untreated animals, but the overall immunolabeling of Iba-1 was altered only in neuropathic rats (p < .05). The mean area of SGCs per neuron was significantly altered only in the inflammatory model (p < .05). All morphological alterations were reverted by modulating the fractalkine pathway (p < .05). In conclusion, the blockage of the fractalkine pathway seemed to be a possible therapeutic strategy for inflammatory and neuropathic orofacial pain, reducing mechanical hyperalgesia by impairing the phosphorylation of p-38 MAPK and reverting morphological alterations in microglia and SGCs.

Extract of Curcuma zedoaria R. prevents atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND/OBJECTIVES: Curcuma zedoaria R. (Zingiberaceae) has been used to treat headache, fever, and hypertension-related symptoms in Asian countries, including Korea, China, and Japan. We investigated whether dietary intake of a C. zedoaria extract (CzE) affected atherosclerosis in vivo. MATERIALS/METHODS: Apolipoprotein E-deficient (ApoE-/-) mice (n = 32) were fed a normal diet (ND), a high-cholesterol diet (HCD), an HCD containing CzE (100 mg/kg/day), or an HCD containing simvastatin (10 mg/kg/day) for 12 weeks. The anti-atherosclerotic effects were evaluated by observing changes in fatty streak lesions, immunohistochemical analysis, ex vivo fluorescence imaging, lipid profiles, and western blot analysis. RESULTS: The CzE-fed group showed a 41.6% reduction of atherosclerosis. Furthermore, CzE significantly reduced the levels of serum triglyceride, high-density lipoprotein, the chemokine (C-X3-C-motif) ligand 1, the adhesion molecules vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin; down-regulation of tumor necrosis factor-α, interleukin-6, high mobility group box-1, and cathepsin levels in the aortic sinuses and aortas of ApoE-/- mice were also observed. CONCLUSIONS: The results suggest that the inclusion of a water extract of C. zedoaria in a HCD is closely correlated with reducing the risk of vascular inflammatory diseases in an ApoE mouse model.

Effect of CX3CL1/CX3CR1 in cerebral ischemia and its mechanisms / 国际脑血管病杂志

CX3CL1, also known as Fractalkine, is the only member of chemokines CX3C subclass. It plays an important role in a variety of central nervous system diseases and ischemic cerebrovascular diseases by binding to its specific receptor CX3CR1. In recent years, a large number of studies have investigated the specific role and related molecular mechanism of CX3CL1/CX3CR1. This article reviews the effect and molecular mechanism of CX3CL1/CX3CR1 in ischemic cerebrovascular disease, aiming to expand the understanding of the mechanism of CX3CL1/CX3CR1, and provide new ideas and intervention targets for the prevention, diagnosis and treatment of ischemic cerebrovascular disease.

Comparison of bead array and glass nanoreactor multi-analyte platforms for the evaluation of CNS and peripheral inflammatory markers during HIV infection.

While human immunodeficiency virus (HIV) infection has become a treatable disease with the development of combination antiretroviral therapy (cART), chronic inflammation that affects the central nervous system and other organs is still common. Reliable methods are needed to study HIV-associated inflammatory biomarkers. In this study involving both plasma and cerebrospinal fluid (CSF), we compared multiplex bead array (MBA) to a relatively new technology based on microfluidics and glass nanoreactor (GNR) technology for the measurement of three commonly studied markers from HIV-infected individuals. We found that results correlated between the two platforms for MCP-1 in both fluids as well as for plasma TNFα (all p < .005). However, results between the two platforms did not correlate for CSF TNFα or fractalkine from plasma or CSF. A statistically significant decrease in CSF TNFα over time (p < .0001) was only detectable with the MBA platform, and TNFα on the MBA was the only CSF biomarker to correlate with CSF HIV RNA (rho = 0.71, p < .0001). Meanwhile, the GNR platform was superior in terms of intra-assay fractalkine (FKN) variability and the detection of a significant FKN decrease over time. Additionally, the only significant correlation between blood biomarkers and plasma HIV RNA was with FKN on the GNR platform (rho = 0.38, p = .015). Given the variability in results between platforms, more research is needed on methods to quantitate HIV-associated inflammation.

Roles and mechanisms of chemokine CX3CL1 in cerebral ischemia / 国际脑血管病杂志

Chemokine CX3CL1 mainly participates in the physiological and pathological processes of nervous system by activating its receptor CX3CR1.Under physiological conditions,CX3CL1 can inhibit the activation of microglia;when cerebral ischemia and hypoxia,CX3CL1 can affect the expression of multiple downstream target genes involved in activation of adenosine receptor,inhibition of Ca 2+ influx,and promotion of blood vessel growth.It is of great significance to improve the energy metabolism disorder and to establish microcirculation around infarcts after cerebral ischemia.

Roles and mechanisms of chemokine CX3CL1 in cerebral ischemia / 国际脑血管病杂志

Chemokine CX3CL1 mainly participates in the physiological and pathological processes of nervous system by activating its receptor CX3CR1. Under physiological conditions, CX3CL1 can inhibit the activation of microglia; when cerebral ischemia and hypoxia, CX3CL1 can affect the expression of multiple downstream target genes involved in activation of adenosine receptor, inhibition of Ca2+ influx, and promotion of blood vessel growth. It is of great significance to improve the energy metabolism disorder and to establish microcirculation around infarcts after cerebral ischemia.

Anti-inflammatory Action of Metformin with Respect to CX3CL1/CX3CR1 Signaling in Human Placental Circulation in Normal-Glucose Versus High-Glucose Environments.

Upregulation of chemokine CX3CL1 and its receptor CX3CR1 occurs in the diabetic human placenta. Metformin, an insulin-sensitizing biguanide, is used in the therapy of diabetic pregnancy. By preventing the activation of NF-κB, metformin exhibits anti-inflammatory properties. We examined the influence of hyperglycemia (25 mmol/L glucose; HG group; N = 36) on metformin-mediated effects on CX3CL1 and TNF-α production by placental lobules perfused extracorporeally. Additionally, CX3CR1 expression and contents of CX3CR1, TNF-α receptor 1 (TNFR1), and NF-κB proteins in the placental tissue were evaluated. Placentae perfused under normoglycemia (5 mmol/L glucose; NG group; N = 36) served as the control. Metformin (2.5 and 5.0 mg/L; subgroups B and C) lowered the production of CX3CL1 and TNF-α in a dose-dependent and time-dependent manner. Hyperglycemia did not weaken the strength of these metformin effects. Moreover, CX3CL1 levels after perfusion with 5.0 mg/L metformin were reduced by 33.28 and 33.83% (at 120 and 150 min, respectively) in the HG-C subgroup versus 24.98 and 23.66% in the NG-C subgroup, which indicated an augmentation of the metformin action over time in hyperglycemia. CX3CR1 expression was significantly higher in the HG-B and HG-C subgroups compared to that in the NG-B and NG-C subgroups. Increased CX3CR1 protein content in the placental lysates was observed in subgroups B and C. The two higher metformin concentrations significantly decreased the levels of NF-κBp65 protein content in both groups. However, the decrease was significantly stronger in hyperglycemia. TNFR1 upregulation in the HG group was not affected by metformin. Further studies on metformin therapy during pregnancy are needed, including safety issues.

NF-kB mediated CX3CL1 activation in the dorsal root ganglion contributes to the maintenance of neuropathic pain induced in adult male Sprague Dawley rats

Abstract Purpose: To evaluate the role of CX3CL1 and NF-κB in the lumbar disc herniation induced neuropathic pain. Methods: After LDH induced by implantation of autologous nucleus pulposus (NP) on the left L5 nerve root was established, mechanical thresholds and thermal hyperalgesia were tested at relevant time points during an observation period of 28 days. Expression of CX3CL1 and NF-κBin the dorsal root ganglion (DRG) were performed by using Western blotting and RT-PCR. Results: Implantation of autologous nucleus pulposus (NP) induced neuropathic pain, associated with increased mRNA and protein expression of CX3CL1 in the DRG. Moreover, intrathecal injection of neutralizing antibody against CX3CL1 could attenuates LDH-induced persistent pain hypersensitivity. Interestingly, NF-κB activation in the DRGs were found in LDH-induced neuropathic pain. Furthermore, NF-κB downregulation by p65 inhibitor PDTC markedly alleviated LDH-induced mechanical allodynia and thermal hyperalgesia in rat. Importantly, CX3CL1 neutralizing antibody (10 μg/10 μl, i.t.) reduces p-p65 protein level in DRG Conclusions: CX3XL1 could regulate LDH-induced neuropathic pain through NF-κB pathway. Targeting CX3CL1 and NF-κB may represent a potential treatment for neuropathic pain caused by LDH.

Research progress of CX3CL1-CX3CR1 and the pathogenesis of sepsis / 天津医药

Sepsis has poor prognosis, and its pathogenesis is not clear. Chemokine CX3CL1 (Fractalkine, FNK) has many functions such as chemotaxis, adhesion and mediate immune injury. CX3CR1 is the only receptor of CX3CL1 and participates in the development of sepsis. Here we review the structure, biological function and possible mechanism of CX3CL1 and CX3CR1 in the pathogenesis of sepsis.

The influence of IL-18 on pancreatic stellate cells and CX3CL1 expression / 中华胰腺病杂志

Objective To evaluate the effect of IL-18 on the expression of E-cadherin,α-SMA and CX3 CL1 in pancreatic stellate cells (PSCs).Methods The human PSC line HPaSteC was routinely cultured and passaged.Five,25,50 and 100 μg/L IL-18 was used to treat PSCs for 72 h and the untreated PSCs were used as control.Treated and untreated cells were both collected,and RT-PCR and Western blot were used to detect mRNA and protein expression of E-cadherin,α-SMA and CX3CL1 respectively.Results The mRNA expressions of E-cadherin in control group and 5,25,50 and 100 μg/L IL-18 treated group were 1.03 ± 0.17,0.77 ±0.15,0.89 ± 0.12,0.54 ± 0.11 and 0.46 ± 0.06.The mRNA expression of α-SMA were 1.03 ± 0.19,0.85 ± 0.14,1.33 ± 0.22,1.60 ± 0.14 and 1.94 ± 0.09;The mRNA expression of CX3CL1 were 1.01 ±0.08,0.88 ±0.25,0.86 ±0.17,1.58 ±0.26 and 1.83 ±0.13.The mRNA expression of E-cadherin in IL-18 treated group were down-regulated,while the mRNA expression of α-SMA and CX3CL1 were up-regulated,and the differences between control and IL-18 100 μg/L treated group were statistically significant (P ＜ 0.05 or ＜ 0.01).The protein expression of E-cadherin in control group and 5,25,50 and 100 μg/L IL-18 treated group were 1.00 ±0.14,1.14 ±0.04,1.14 ±0.07,0.85 ±0.08 and 0.80 ±0.06.The protein expression of α-SMA were 1.00 ± 0.02,0.77 ± 0.07,1.29 ± 0.02,1.59 ± 0.07 and 1.70 ± 0.02;The protein expression of CX3CL1 were 1.00 ± 0.05,1.03 ± 0.05,1.37 ± 0.06,1.46 ± 0.18 and 1.45 ± 0.12.The protein expression of E-cadherin was down-regulated but no significant differences were observed among different groups.The protein expression of α-SMA was up-regulated and the differences between control group and 25,50 and 100 μg/L IL-18 treated groups were statistically significant (all P ＜0.01).The protein expression of CX3CL1 was up-regulated and the differences between control group and 100 ng/ml IL-18 treated group were statistically significant (P ＜0.05).Conclusions IL-18 can activate PSCs and up-regulate the expression of chemokine CX3CL1.

In vitro effects of bone marrow-derived mesenchymal stem cells on the biological behavior of lipopolysaccharide-activated retinal microglia / 中华实验眼科杂志

Background Retinal microglia (RMG) plays an important role in the pathogenesis of retinal degenerative diseases,while chemokine CX3CL1 participates in the regulation of steady-state of microglia.It has been determined that bone marrow-derived mesenchymal stem cells (BMSCs) have a remarkable role to modulate the immune response and protect the central nervous system through the release of soluble factors in a paracrine fashion and further affect the functional behavior of cells.However,whether BMSCs are able to interact with RMG and activate related signaling pathway for the maintaining of homeostasis in the retina is still unclear.Objective The aim of this study was to investigate the interaction between BMSCs and lipopolysaccharide (LPS)-activated RMG in vitro,and dissect the effects of CX3CL1/CX3CR1 signaling pathway on the biological behavior of BMSCs and RMG.Methods RMG was isolated from SD rats,cultured with mixed culture of retinal glial cells and purified by shaking.The cells were identified by detecting the expression of CD111b,Iba1 and glutamamine synthetase (GS) with indirect immunofluorescence assay.LPS (1 mg/ml,2 μl) was added in the medium for 24 hours to stimulate RMG,and then the cells were divided into LPS control group,BMSCs group (cocultured with BMSCs for 24 hours) and CB-BMSCs group (cocultured with CX3CL1-blocking-BMSCs for 24 hours).The cells without LPS stimulation served as the blank control group.The functions of RMG,including the release content of tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β),the proliferation,phagocytosis,and migration of RMG were examined.Results RMG was successfully isolated and harvested from SD rats by using mixed culture of retinal glial cells and purified by shaking.CD11b and Iba1 showed the positive expression with the green fluorescence in the cells and GS was absent.The contents of TNF-αt in the cell supernatant were (2.55 ±0.97) ng/ml,(24.91 ±3.07) ng/ml,(20.38 ±2.97) ng/ml and (24.90 ± 1.88) ng/ml in the blank control group,LPS control group,BMSCs group and CB-BMSCs group,respectively,showing a significant difference among the groups (F=119.90,P＜0.05).The contents of IL-1 β in the cell supernatant were (1.12±0.36) ng/ml,(10.40±2.76) ng/ml,(7.00± 1.75) ng/ml and (9.55 ± 1.11) ng/ml in the blank control group,LPS control group,BMSCs group and CB-BMSCs group,respectively,showing a significant difference among the groups(F =34.96,P＜0.05).The secretory volume of TNF-α and IL-1 β were evidently lower in the BMSCs group than those in the LPS control group (both at P＜0.05),and no significant differences were found in the secretory volume of TNF-α and IL-1β between CB-BMSCs group and LPS control group (both at P＞0.05).The proliferative rate of RMG was lower in the BMSCs group than that in the LPS control group (P＜0.05),while there was no statistical difference between BMSCs group and CB-BMSCs group (P＞0.05).The mean fluorescence intensity (MFI) and the number of migrated RMG were considerably different among the four groups (F=70.55,15.49,both at P＜0.05),and those in the BMSCs group were significantly increased in comparison with the LPS control group (both at P＜0.05),while there was no significant difference between CB-BMSCs group and LPS control group (both at P＞0.05).Conclusions BMSCs could suppress the proliferation of LPS-activated RMG.Moreover,BMSCs might inhibit proinflammatory cytokines releasing,enhance phagocytosis and migration capabilities of RMG via CX3CL1/CX3CR1 signaling pathway.

Serum cytokine profiles of patients with interstitial lung disease associated with anti-CADM-140/MDA5 antibody positive amyopathic dermatomyositis.

BACKGROUND: Patients with amyopathic dermatomyositis (ADM) sometimes develop rapidly progressive interstitial lung disease (ILD) predominantly in Asia. Although anti-CADM-140/MDA5 antibody titer could correlate with disease activity and predict the course of ILD associated with ADM, it is not clear how this antibody is involved in the pathogenesis of ILD in ADM. METHODS: We retrospectively collected clinical records and preserved serum before treatment of consecutive patients with ADM-ILD treated in the Niigata University Medical and Dental Hospital since 2000. We measured anti-CADM-140/MDA5 antibody titer and compared it between survivors and non-survivors. Serum cytokine/growth factor protein concentration was measured using a multiplex immunoassay system. The associations between anti-CADM-140/MDA5 antibody titer and each cytokine/growth factor protein concentration were evaluated. RESULTS: Thirteen patients were enrolled into the study. Among them, four patients did not respond to intensive immunosuppressive therapy and died. The mean anti-CADM-140/MDA5 antibody titer was significantly higher in patients who did not responded to therapy than in those who survived (p < 0.05). Relationship analyses between the antibody titer and each cytokine/GF protein concentration revealed that Spearman's rank correlation coefficients were more than 0.4 in thirteen cytokine/GF proteins. In particular, the strongest correlation was found between anti-CADM-140/MDA5 antibody titer and CX3CL1 (r = 0.8897). CONCLUSIONS: These results confirmed that anti-CADM-140/MDA5 antibody levels could predict outcomes of ADM-ILD. Relationship analyses suggested that CX3CL1 might be involved in the pathogenesis of anti-CADM-140/MDA5 antibody positive ADM-ILD.

Effects of expression silencing of chemokine receptor CX3CR1 on human hepatocellular carcinoma Huh7 cells and its machanism / 肿瘤

Objective: To investigate the effect of chemokine CX3C receptor 1 (CX3CR1) on human hepatocellular carcinoma Huh7 cells and its probable machanism. Methods: Hepatocellular carcinoma Huh7 cells were transfected with the recombinant adenovirus Ad-siCX3CR1 targeting and silencing CX 3CR 1 gene. Then the expression of CX3CR1 was detected by reverse transcription-PCR (RT-PCR) and Western blotting. The proliferation, apoptosis, migration and invasion of Huh7 cells were tested by MTT, flow cytometry, wound-healing and Transwell assay, respectively. The expression and phosphorylation of Akt which was related to phosphoinositide 3-kinase (PI3K)-Akt signal pathway were detected by Western blotting. Meanwhile, Huh7 cells transfected with AdsiCX3CR1 were treated with different concentrations of PI3K inhibitor LY294002, then the expression of Akt and cell invasion ability induced by CX3CR1 were detected by Western blotting and Transwell assay, respectively. Results: After Ad-siCX3CR1 was stably transfected into Huh7 cells, the expression of CX3CR1 was significantly inhibited (P < 0.001), and the cell proliferation and migration and invasion abilities were significantly increased (P < 0.001), but the apoptosis rate was significantly decreased (P < 0.01). When the expression of CX3CR1 was silenced, the level of phosphorylated Akt (p-Akt) in Huh7 cells was increased (P < 0.001), and LY294002 could reverse CX3CR1-induced phosphorylation of Akt and invasion of Huh7 cells effectively (P < 0.001). Conclusion: CX 3CR 1 gene silencing can promote proliferation, migration and invasion of hepatocellular carcinoma Huh7 cells, and also can suppress their apoptosis. The activation of PI3K-Akt signaling pathway may be involved in this process.

Effect of fractalkine, IP-10 and different signal pathway inhibitors on NK cells in the tumor microenvironment / 解放军医学杂志

Objective To investigate the inducing effects of chemokines [fractalkine (FKN), IP-10] and different signal pathway inhibitors on NK cells in the tumor microenvironment (TME). Methods Immunohistochemistry was performed using antibodies for CD56 and DAP10 respectively on human breast carcinoma. Murine macrophages (RAW 264.7) and breast cancer cells (4T1) were co-cultivated at a 1:4 ratio to imitate the TME with NK cells (KY-1) set as the object. RT-PCR was used to determine the mRNA expressions of CD16, NKG2D and NK1.1, and the content of CD107a in the supernatants was determined by ELISA. 10ng/ ml FKN and 10ng/ml IP-10 were added into the TME, NK1.1+CD16+KY-1 cells were counted with flow cytometry, migration and adhesion assays were used to assess the related function of KY-1 cells. 4T1 cells were incubated in 10nmol/L of rapamycin, 30μmol/ L of LY294002, 500ng/μl of andrographolide and 2mmol/L of wortmannin, the 4T1 tumor supernatants (TSNs) were harvested separately and used to incubate RAW 264.7 for 48h, then the expressions of Rae1α and H60a mRNA in 4T1, RAW 264.7 and their mixture were determined by RT-PCR. Results The related indicators of KY-1 cells such as NK1.1+ number, chemotaxis rate, and adhesion function decreased obviously in TME, and the above indices increased after the addition of FKN and IP-10, and some signal pathway inhibitors indirectly promoted NK cells' function in TME, and among them rapamycin was the most efficient one (P<0.05). Conclusion FKN and IP-10 may up-regulate the number and function of NK cells in TME, and rapamycin can promote NK cells' killing function by inducing high expression of NKG2DLs (Rae1, H60a) on tumor cells.

Fractalkine promotes human monocyte survival via a reduction in oxidative stress.

OBJECTIVE: The CX3C chemokine fractalkine (CX3CL1) has a critical role in the development of atherogenesis because apolipoprotein-E-deficient mice lacking CX3CL1 or its receptor CX3CR1 develop smaller plaques and polymorphisms in CX3CR1 are associated with altered risk of cardiovascular disease. CX3CR1 is found on numerous cell types involved in atherogenesis but seems to have a key role in monocyte function. We aimed to elucidate the role of CX3CL1 in human monocyte survival and determine the mechanism by which CX3CL1 spares monocytes from apoptosis. APPROACH AND RESULTS: Primary human monocytes were prepared from healthy donors and subjected to serum-starvation to induce spontaneous apoptosis. The addition of CX3CL1, but not other chemokines tested, promoted monocyte survival in a dose-dependent manner with full-length CX3CL1 (including the mucin stalk) having a more potent antiapoptotic effect than chemokine-domain CX3CL1. The prosurvival effect of CX3CL1 was evident in both monocyte subsets although nonclassical monocytes were more prone to spontaneous apoptosis. In addition, we found that the effect of CX3CL1 was independent of CX3CR1 genotype. Serum-starvation increased the level of intracellular reactive oxygen species, and this was reduced by the addition of CX3CL1. Inhibition of oxidative stress with an antioxidant prevented monocyte apoptosis, indicating that this is the dominant mechanism of cell death targeted by CX3CL1. CONCLUSIONS: CX3CL1 has a substantial and highly reproducible antiapoptotic effect on human monocytes, via a mechanism involving a reduction in oxidative stress. This suggests that CX3CL1 is likely to play a key role in human atherogenesis and may provide a novel therapeutic target in cardiovascular disease.

Effects of interfering chemotactic factor Fractalkine RNA expression on biological functions of human pancreatic cancer cell lines / 中华消化杂志

Objective To explore the effects of Fractalkine (FKN) on the biological functions of human pancreatic cancer cell lines SW-1990 and PNAC-1.Methods Adenovirus mediated FKN-small interfering RNA (siRNA) was transfected into human pancreatic cancer cell lines SW 1990 and PNAC-1.The differences in proliferation and invasion ability between before and after FKN-siRNA transfection were determined by clone formation assay,MTT assay and cells invasion assay.After FKN-siRNA transfection,the expression of FKN,tumor necrosis factor (TNF)-α and interleukin (IL)-6 at protein and mRNA level in human pancreatic cancer cell were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR).The data were analyzed by one way analysis of variance.Results After human pancreatic cancer cell lines SW-1990 and PNAC-1 transfected with FKN-siRNA,the clone numbers (5.27 ％ ± 0.35 ％ and 4.60 ％ ± 0.30％ ) increased compared with those of control group ( 1.97％ ±0.25％ and 1.77％ ± 0.25％ ) and negative FKN-siRNA group (2.10％±0.30％ and 1.97％±0.25％),and the difference was statistically significant (F=113.51,103.86; both P＜0.05).The clone size was also enlarged.After human pancreatic cancer cell lines SW-1990 and PNAC-1 transfected with FKN-siRNA for 48 hours and 72 hours,the MTT test results showed the absorbance value (48 h:1.28±0.07 and 1.19±0.14; 72 h:1.49±0.11 and 1.52±0.16) was higher than that of control group (48 h:0.80±0.03 and 0.74±0.11;72 h:0.89±0.03 and 0.93±0.04) and negative FKN-siRNA group (48 h:0.85±0.02 and 0.76±0.05; 72 h:0.89±0.02 and 1.07±0.09),and the difference was statistically significant (F=83.80,71.99,17.19,23.51; all P＜0.05).The invasion ability assay showed that the invasion ability of FKN-siRNA transfected cells was stronger than that of control group and negative FKN-siRNA group,and the difference was statistically significant (F=37.37,9.08; both P＜0.05).After FKN-siRNA transfection,the expression of FKN at protein and mRNA level in SW-1990 and PNAC-1 cell line decreased (protein:F=118.93 and 88.62,mRNA:F=47.91 and 72.59),at the same time the expression of TNF-α and IL-6 at protein and mRNA level increased (protein:FTNF-α =112.90 and 77.88,FIL-6 =165.27 and 286.49,mRNA:FTNF-α ==47.93 and 45.19,FIL-6 =36.41 and 23.67),and the differences were statistically significant (all P values＜0.05).Conclusion With siRNA technology to silent FKN function,the proliferation and invasion ability of pancreatic cancer cell lines increased,which indicated FKN might inhibit certain biological functions of pancreatic cancer cells.

Effects of diazoxide pretreatment on the expression of NF-κB mRNA and fractalkine mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation / 中华麻醉学杂志

Objective To investigate the effects of diazoxide pretreatment on the expression of NF-κB mRNA and fractalkine (FKN) mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R). Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates (100μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into4 groups (n = 24 each): Ⅰ normal control group (group C), Ⅱ H/R group, Ⅲ diazoxide pretreatment group (group DZ), Ⅳ diazoxide pretreatment + mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) group (group DZ + 5-HD). The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100μmol/L were added to the cultured medium 2 h before hypoxia in group DZ and DZ + 5-HD respectively. The cell vitality, apoptotic rate and expression of NF-κB mRNA and FKN mRNA were detected at end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of NF-κB mRNA and FKN mRNA up-regulated in H/R group. Compared with group H/R, the cell vitality was significantly increased,apoptotic rate decreased and the expression of NF-κB mRNA and FKN mRNA down-regulated in group DZ. 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above. Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through down-regulating the expression of NFκB and FKN, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.