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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.
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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.
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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.
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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.
Subject(s)
Animals , Male , NF-kappa B/metabolism , Chemokine CX3CL1/metabolism , Ganglia, Spinal/metabolism , Intervertebral Disc Displacement/metabolism , Neuralgia/etiology , Neuralgia/metabolism , Time Factors , Behavior, Animal , Down-Regulation , Blotting, Western , NF-kappa B/analysis , Rats, Sprague-Dawley , Disease Models, Animal , Chemokine CX3CL1/analysis , Real-Time Polymerase Chain Reaction , Hyperalgesia/metabolism , Intervertebral Disc Displacement/complicationsABSTRACT
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.
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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.
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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.
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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.
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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.
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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.
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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.