Research progress of drugs for cancer immunotherapy based on CCL2/CCR2 signaling axis1 / 中国药科大学学报

@#C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 are closely related to tumorigenesis and tumor progression. The CCL2/CCR2 signaling axis promotes tumor progression through multiple mechanisms: CCL2 binds to CCR2 on the surface of tumor cells, and thus promotes tumor growth/survival and metastasis; more importantly, CCL2 recruits a variety of immunosuppressive cells to aggregate in the tumor microenvironment, and inhibits the function and activity of immune cells, promoting tumor progression. The article reviews the CCL2/CCR2 signaling axis and its role in tumors and tumor microenvironment, with particular focus on the advances in clinical research on drugs targeting CCL2/CCR2 signaling axis, in order to gain an in-depth and overall understanding of the mechanism of action of CCL2/CCR2 axis in tumor progression and develop more effective anti-tumor immunotherapeutic agents.

Mechanism of crocin alleviating monocrotaline-induced right ventricular injury in rats with pulmonary arterial hypertension / 中国病理生理杂志

AIM:To investigate whether crocin alleviates right ventricular injury induced by monocrotaline(MCT)in rats with pulmonary arterial hypertension(PAH),and to explore the underlying mechanisms.METHODS:Forty male SD rats were randomly divided into 4 groups:normal group,PAH group,crocin group and sildenafil group,with 10 rats in each group.The rats in PAH,crocin and sildenafil groups received subcutaneous injection of MCT(50 mg/kg)to establish the PAH model.Starting from the day of MCT injection,the rats in crocin group received crocin(200 mg/kg),the rats in sildenafil group received sildenafil(30 mg/kg),and those in PAH and normal groups were orally gavaged with an equal volume of saline once daily.After 4 weeks,measurements of right ventricular systolic pressure(RVSP),mean pulmonary artery pressure(mPAP),right ventricular hypertrophy index(RVHI)and right ventricular mass index(RVMI)were taken for the rats in each group.Tissue staining was conducted to observe pathological changes in the right ventricle,and the expression levels of inflammatory factors(IL-1β,IL-6 and TNF-α),the p38 MAPK/NF-κB inflammato-ry pathway,CCL2,CCR2,and the macrophage marker CD68 were assessed.RESULTS:Compared with PAH group,the rats in crocin and sildenafil groups exhibited significant reductions in RVSP,mPAP,RVHI and RVMI(P＜0.05).Right ventricular tissue displayed no evident infiltration of inflammatory cells or proliferation of collagen fibers.The down-regulation of the p38 MAPK/NF-κB pathway and inflammatory factors(IL-1β,IL-6 and TNF-α)was significant(P＜0.05).Additionally,the CCL2/CCR2 pathway and the infiltration of CD68+ macrophages were markedly decreased(P＜0.05).CONCLUSION:Crocin effectively mitigates right ventricular damage in MCT-induced PAH rats,with its effica-cy comparable to that of sildenafil at the dosage utilized in this experiment.Some protective mechanisms of crocin may be attributed to its regulatory effects on inflammation.

Research progress of drugs for cancer immunotherapy based on CCL2/CCR2 signaling axis / 中国药科大学学报

C-C motif chemokine ligand 2(CCL2)and its receptor CCR2 are closely related to tumorigenesis and tumor progression.The CCL2/CCR2 signaling axis promotes tumor progression through multiple mechanisms:CCL2 binds to CCR2 on the surface of tumor cells,and thus promotes tumor growth/survival and metastasis;more importantly,CCL2 recruits a variety of immunosuppressive cells to aggregate in the tumor microenvironment,and inhibits the function and activity of immune cells,promoting tumor progression.The article reviews the CCL2/CCR2 signaling axis and its role in tumors and tumor microenvironment,with particular focus on the advances in clinical research on drugs targeting CCL2/CCR2 signaling axis,in order to gain an in-depth and overall understanding of the mechanism of action of CCL2/CCR2 axis in tumor progression and develop more effective anti-tumor immunotherapeutic agents.

Mangiferin alleviates renal inflammatory injury in spontaneously hypertensive rats by inhibiting MCP-1/CCR2 signaling pathway / 中草药·英文版

OBJECTIVE@#Hypertension is a low-grade inflammation state of the disease and was easily complicated by kidneys' inflammatory response. Mangiferin (MGF), a pharmacologically active compound in various plants including Mangifera indica, has a strong anti-inflammatory activity. However, the effects of MGF on renal inflammatory injury in spontaneously hypertensive rats (SHRs) remain unclear. The purpose of this study was to investigate the protective effects and mechanisms of MGF on renal inflammatory injury in SHRs.@*METHODS@#MGF was used in SHRs at the doses of 10, 20, 40 mg/kg/d for 8 weeks consecutively. The blood and urine were collected for assessment of renal function. Renal tissues were collected for histological, immunohistochemistry, ELISA, Western blot and real time reverse transcription PCR (RT-PCR) analysis.@*RESULTS@#The results showed that the levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and recombinant chemokine C-C-Motif receptor 2 (CCR2) were increased in SHRs, meanwhile, the level of IL-10 was decreased in SHR. Treatment of MGF inhibited the expression of IL-6, TNF-α, MCP-1 and CCR2, and promoted the expression of IL-10. Furthermore, the content of blood urea nitrogen (BUN) and serum uric acid (SUA) was significantly increased in the model group, and treatment of MGF had no obvious effects on these parameters at all dose levels.@*CONCLUSION@#Our study proved that the kidneys of SHRs had significant inflammatory injury, and MGF had the protective effects on renal inflammatory injury in SHRs; The protective mechanism may be mediated partly by the MCP-1/CCR2 signaling pathway. Thus, it is a potential new drug for the treatment of hypertension.

CCL2/CCR2 Contributes to the Altered Excitatory-inhibitory Synaptic Balance in the Nucleus Accumbens Shell Following Peripheral Nerve Injury-induced Neuropathic Pain / 神经科学通报·英文版

The medium spiny neurons (MSNs) in the nucleus accumbens (NAc) integrate excitatory and inhibitory synaptic inputs and gate motivational and emotional behavior output. Here we report that the relative intensity of excitatory and inhibitory synaptic inputs to MSNs of the NAc shell was decreased in mice with neuropathic pain induced by spinal nerve ligation (SNL). SNL increased the frequency, but not the amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs), and decreased both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in the MSNs. SNL also decreased the paired-pulse ratio (PPR) of evoked IPSCs but increased the PPR of evoked EPSCs. Moreover, acute bath application of C–C motif chemokine ligand 2 (CCL2) increased the frequency and amplitude of sIPSCs and sEPSCs in the MSNs, and especially strengthened the amplitude of N-methyl-D-aspartate receptor (NMDAR)-mediated miniature EPSCs. Further Ccl2 overexpression in the NAc in vivo decreased the peak amplitude of the sEPSC/sIPSC ratio. Finally, Ccr2 knock-down improved the impaired induction of NMDAR-dependent long-term depression (LTD) in the NAc after SNL. These results suggest that CCL2/CCR2 signaling plays a role in the integration of excitatory/inhibitory synaptic transmission and leads to an increase of the LTD induction threshold at the synapses of MSNs during neuropathic pain.

Cionbufagin attenuates bone cancer pain by blocking the activation of microglia in the spinal cord / 药学学报

Cionbufagin has anti-inflammatory and analgesic effects. It is of great value in the treatment of bone cancer pain, but its mechanism is still unclear. To generate a bone metastasis model of breast cancer, 4×105 Walker-256 cells were inoculated into the left hind limb of SD rats. The experimental protocol was approved by the Medical Laboratory Animal Ethics Committee of Medical College of China Three Gorges University. Rats were randomly divided into sham, model, cionbufagin, morphine, saline, minocycline, microglia inhibitor (RS102895) and co-treatment with cionbufagin and minocycline group. The cionbufagin (5 mL·kg-1, i.p.), morphine (8 mg·kg-1, i.p.) and co-treatment groups (included cionbufagin 5 mL·kg-1, i.p.) received continuous administration from day 9 to day 21. The saline, minocycline (2.5 μg·μL-1, 20 μL), RS102895 (1.5 μg·μL-1, 20 μL) and co-treatment groups (included minocycline 2.5 μg·μL-1, 20 μL) received continuous administration by intrathecal cannulation from day 12 to day 21. Bone destruction of the left hind limb of rats was detected by hematoxylin-eosin staining (H&E). The pain threshold before treatment and at day 2, 5, 7, 9, 12, 14, 17 and 20 was measured by behavioral indexes. Activation and expression of a microglia marker (Iba-1) was determined by immunofluorescence and Western blot. The level of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) in rat spinal cord was measured by enzyme-linked immunosorbent assay (ELISA). H&E results showed that cionbufagin effectively inhibited the destruction of bone marrow in rats with bone cancer pain; cionbufagin treatment significantly increased the mechanical and thermal pain threshold. Immunofluorescence showed that cionbufagin effectively inhibited the activation of microglia in the spinal dorsal horn. Western blot analysis confirmed that the activation of microglia in the spinal dorsal horn was inhibited by cionbufagin treatment. It was also found that the CCL2/CCR2 pathway may be involved in the analgesic effect of cionbufagin. These results suggest that cionbufagin can effectively alleviate bone cancer pain, possibly by inhibiting the release of inflammatory factors and the activation of spinal microglia cells through the CCL2/CCR2 pathway.

Mechanism of spinal chemokine C-C motif receptor 2-mediated maintenance of bone cancer pain in rats / 上海交通大学学报（医学版）

Objective • To investigate the mechanism of spinal chemokine C-C motif receptor 2 (CCR2)-mediated maintenance of bone cancer pain (BCP) in rats. Methods • Fifty-four SD rats were divided into BCP group, sham operation group, BCP+INCB3344 (CCR2 specific antagonist) group, and BCP+vehicle control group. Walker256 breast cancer cells were injected into the tibia medullary cavity of rats in the BCP group to establish the BCP model, while the rats in the sham operation group were injected with the same amount of saline. The rats in the BCP+INCB3344 group received intrathecal injection of INCB3344 on the 14th day after the establishment of BCP model, while the BCP+vehicle control group rats were injected with the same amount of vehicle. The mechanical pain thresholds of BCP group rats and sham operation group rats were measured to judge the success of BCP model. The expressions of CCR2 in the dorsal horn of spinal cord in the sham operation group rats and the BCP group rats were detected by Western blotting. The effects of intrathecal administration of INCB3344 on the mechanical pain threshold of BCP rats were observed by mechanical pain behavior test. Whole-cell patch-clamp recordings were used to investigate the differences of spontaneous excitatory postsynaptic currents (sEPSCs), α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA) and N-methyl-D-aspartic acid (NMDA)-induced currents of spinal substantia gelatinosa (SG) neurons of rats in the BCP group, the BCP+INCB3344 group and the BCP+vehicle control group. Results • Compared with the sham operation group, the mechanical pain threshold of BCP group rats reduced significantly on the 14th day after operation (P=0.000), and the expression of CCR2 in ipsilateral spinal cord of BCP group rats increased significantly (P=0.009). After intrathecal injection of INCB3344 for 4 h, the mechanical pain threshold of BCP+INCB3344 group rats was significantly increased (P=0.002). The frequency and amplitude of sEPSCs and the amplitude of AMPA and NMDA-induced currents in SG neurons of BCP group rats were significantly higher than those of the sham operation group rats (all P=0.000), while intrathecal administration of INCB3344 could significantly inhibit the above-mentioned indices in the BCP+INCB3344 group (all P<0.05). In addition, extracellular perfusion of INCB3344 could also significantly inhibit the frequency (P=0.001) and amplitude (P=0.020) of sEPSCs in SG neurons in BCP rats. Conclusion • CCR2 expressing in the spinal cord mediates the enhancement of excitatory synaptic transmission efficacy in the spinal dorsal horn of BCP rats by enhancing the functions of AMPA and NMDA receptors, which may be an important mechanism for the maintenance of BCP.

Effect of Mangiferin on Morphology of Relevant Vessels and MCP-1/CCR-2 Pathway in Spontaneously Hypertensive Rats / 中国实验方剂学杂志

Objective:To observe the morphological changes of carotid artery, thoracic aorta and superior mesenteric artery in spontaneously hypertensive rats(SHR), in order to further study the effect of Mangiferin on the expressions of inflammatory factors and monocyte chemoattract protein-1 (MCP-1)/c-chemokine receptor type 2 (CCR-2) pathway in SHR. Method:Forty spontaneously hypertensive rats were randomly divided into model group, benazepril group (10 mg·kg-1·d-1) and low, medium and high-dose mangiferin groups (25, 50, 100 mg·kg-1·d-1). Eight male WKY rats of the same age were selected as normal control group. Systolic blood pressure was observed every two weeks after eight weeks of administration. Morphology of carotid artery, thoracic aorta and superior mesenteric artery was observed by hematoxylin-eosin (HE) staining. Immunohistochemical assay (IHC) and Western blot were used to detect MCP-1 and CCR-2 protein expressions in thoracic aorta. MCP-1 and CCR-2 mRNA expression levels in thoracic aorta were detected by Real-time quantitative fluorescence PCR (Real-time PCR). Result:Compared with the normal group, the inflammatory cells in the model group increased significantly, the systolic blood pressure was significantly higher than that in the WKY group (PPPPConclusion:There are inflammation damages in carotid artery, thoracic aorta and superior mesenteric artery of spontaneously hypertensive rats. Mangiferin has an anti-inflammatory effect by possibly inhibiting the expressions of MCP-1/CCR-2 pathway in SHR vessels.

Role of monocyte chemoattractant protein 1 and its receptor in the progression of liver fibrosis / 临床肝胆病杂志

Liver fibrosis is a common reversible pathological change in chronic liver injury and may progress to liver cirrhosis, liver failure, and portal hypertension. In recent years, several studies have shown a significant change in chemokine profiles in patients with liver fibrosis, which is closely associated with the progression of liver fibrosis. Monocyte chemoattractant protein 1 (MCP-1) belongs to the family of CC chemokines and can induce the activation, recruitment, and migration of inflammatory cells during liver fibrosis. MCP-1 may be involved in the activation of hepatic stellate cells, the development of insulin resistance, and the progression to hepatocellular carcinoma. This article mainly reviews the potential role of MCP-1 and CC chemokine receptor in the progression of liver fibrosis and related therapies.

Role of monocyte chemoattractant factor∕chemokine C-C receptor 2 pathway in amygdala in neuro-pathic pain in rats / 中华麻醉学杂志

Objective To evaluate the role of monocyte chemotactic factor-1 (MCP-1)∕chemokine C-C receptor 2 ( CCR2) in amygdala in neuropathic pain ( NP) in rats. Methods Clean-grade healthy male Sprague-Dawley rats, weighing 200-260 g, aged 2 months, in which NP model was established by ligating the left L5,6spinal nerve, were used in this study. The experiment was performed in two parts. Ex-periment Ⅰ Thirty-two rats were divided into 2 groups using a random number table method: control group (C group, n＝8) and NP group (n＝24). Rats were sacrificed at 7, 14 and 21 days after establis-hing NP model in group NP or at the corresponding time points before establishing NP model in group C, and the amygdala was removed for determination of the expression of MCP-1 and CCR2 mRNA and positive cell count using quantitative real-time polymerase chain reaction and immunohistochemistry. Experiment ⅡThirty-two rats were divided into 4 groups ( n＝8 each) using a random number table method: control group (C group), NP group, MCP-1 group and specific CCR2 antagonist RS102895 group (RS group). MCP-1 (50 pmol for each side) or RS102895 (100 pmol for each side) was injected into the bilateral a-mygdala at days 3, 6, 13 and 20 after establishing NP model in MCP-1 and RS groups, respectively. The thermal paw withdrawal latency (TWL) and mechanical paw withdrawal threshold (MWT) were measured at days 4, 7, 14 and 21 after establishing NP model (T1-4). Rats were sacrificed at T4and the L5segment of the spinal cord was removed for determination of interleukin-1beta (IL-1β), IL-6 and tumor necrosis fac-tor-alpha ( TNF-α) contents by enzyme-linked immunosorbent assay. Results Experiment Ⅰ Compared with group C, the expression of MCP-1 and CCR2 mRNA in amygdala was significantly up-regulated, and the number of MCP-1 and CCR2 positive cells was increased in group NP ( P＜0. 05). Experiment ⅡCompared with group C, the MWT was significantly decreased and TWL was shortened at T1-4, and the contents of IL-1β, IL-6 and TNF-α were increased in the other three groups ( P＜0. 05). Compared with group NP, the MWT was significantly decreased and TWL was shortened at T1, and the contents of IL-1β, IL-6 and TNF-α were increased in group MCP-1, and the MWT was significantly increased and TWL was prolonged at T1-4, and the contents of IL-1β, IL-6 and TNF-α were decreased in group RS ( P＜0. 05 or 0. 01). Conclusion Enhanced function of MCP-1∕CCR2 in amygdala may be involved in the pathophysio-logical process of NP in rats.

Expression and function of MCP-1 and its receptor in wAMD model mouse / 临床与实验病理学杂志

Purpose To detect the expression and the function of MCP-1 and its receptor CCR2 in wet agerelated macular degenerative (wAMD) model mouse retina. Methods C57BL/6J mouse were enrolled into the study. Model mouse of wAMD was induced with laser. Frozen sections were prepared for histopathological tests. Immunofluorescence study for MCP-1 and CCR2 was carried out. Co-expression study for CCR2/ CDllb or CCR2/CD68 was carried out. Total protein and total mRNA from the eyes of both wAMD and wild type mouse were extracted. The expression of mRNA and protein of MCP-1 and CCR2 in the eyes were determined by reverse transcription-poly-merase chain reaction (RT-PCR) and Western blots test, respectively. Results In wild type mouse, both MCP-1 and its receptor CCR2 were not detected in the retina. However in wAMD mouse, an obvious up-regulated MCP-1 and CCR2 expression was seen in the retinal pigment epithelium (RPE) cells accompanied with the increased expression of their mRNA and protein. The co-expression study showed that CCR2 co-ex-pressed with CDllb, but not with CD68. Conclusion MCP-1 and its receptor CCR2 may play a role in the wAMD through stimulation of microglia.

Inhibition of Human Cytomegalovirus Replication by Compound Derivative from Sasa albomarginata is Associated with CCL2/CCR2 Interaction / 日本補完代替医療学会誌

We previously reported that treatment with tricin (4’,5,7-trihydroxy-3’,5’-dimethoxyflavone) after human cytomegalovirus (HCMV) infection significantly suppressed both infectious virus production and HCMV replication in human embryonic lung fibroblast (HEL) cells. Moreover, we recently revealed that HCMV infection can increase the expression of CC-motif ligand 2 (CCL2/MCP-1) and CCR2, a specific receptor for CCL2, which can enhance HCMV infection and replication, in turn. In this study, we examined whether CCL2 and/or CCR2 are involved in the anti-HCMV effects of tricin in HEL cells. Exposure of fibroblasts to tricin inhibited infectious HCMV production, with concomitant decreases in CCL2 and CCR2 transcript levels and CCL2 protein levels in a dose-dependent manner. Propagermanium, an inhibitor of CCR2 function, has also been shown to inhibit infectious HCMV production with concomitant decreases in CCL2 protein levels. We further observed that tricin and propagermanium reduced mRNA expression of HCMV immediate early gene and DNA polymerase in a dose-dependent manner. These results suggest that tricin is a novel anti-inflammatory compound with potential anti-HCMV activity, and CCL2/CCR2 interactions are associated with HCMV replication.

Protective effects of mangiferin on inflammatory injury of cerebral tissue in spontaneously hypertensive rats / 中成药

AIM To observe the protective effects of mangiferin on the inflammatory injury and expression of the inflammatory factor in the cerebral tissue of spontaneously hypertensive rats and on MCP-1/CCR2 signal pathway.METHODS Forty spontaneously hypertensive rats were randomly divided into model,benazepril [10 mg/(kg · d)] and mangiferin high,middle and low dose [40,20,10 mg/(kg · d)] groups and other eight rats of same week age served as control group.After consecutive intragastric administration for eight weeks,morphology of the rats' cerebral tissue was observed;their levels of ICAM-1,IL-6 and TNF-α in cerebral tissue were determined by ELISA;their expressions of MCP-1 and CCR2 protein in brain tissue of rats were detected by immunohistochemistry and Western blot and the detection of mRNA expressions of MCP-1 and CCR2 in cerebral tissue of rats were carried out by RT-PCR.RESULTS Compared with the model group,the blood pressure of mangiferin in each dosage group decreased slightly,but there was no significant statistical difference.In the control group and the model group,there was no obvious morphological change in the cerebral tissue.The morphology of rats in the benazepril group,each dose of mangiferin group were all normal.The contents of IL-6,TNF-α,ICAM-1 and MCP1,CCR2 protein and mRNA expression were significantly decreased in the cerebral tissues of spontaneously hypertensive rats.CONCLUSION Mangiferin has obvious anti-inflammatory effects on inflammatory reaction in spontaneously hypertensive rats,its mechanism may be related to inhibiting the expression of MCP/CCR2 signaling pathway.

Antagonistic effects of extracts from Artemisia rupetris L. and Leontopodium leontopodioides to CC chemokine receptor 2b (CCR2b) / 中国天然药物

The present study was designed to establish a suitable assay to explore CCR2b receptor antagonists from the natural products of Artemisia rupetris and Leontopodium leontopodioides. An aequorin assay was developed as a cell-based assay suitable for 384-well microplate and used for screening CCR2b receptor antagonists from natural products. Through establishing suitable conditions, the assay was shown to be suitable for screening of CCR2b receptor antagonists. Seven compounds were identified in preliminary screening. Five of them showed evident dose-response relationship in secondary screening. The structure-activity relationship study suggested that 7-position hydroxyl group of flavonoids was necessary, a polar group should be introduced on the 3-position, and the substituents on 2-position benzene ring of flavonoids have little influence on the potentency of the inhibition activity on CCR2b receptor. The ortho-position dihydroxyl structure in quinic acid compounds may be important. In conclusion, Compounds HR-1, 5, 7, and AR-20, 35 showed activity as antagonist of CCR2b receptor, which shed lights on the development of novel drugs as CCR2b receptor antagonists for preventing inflammation related diseases.

Relationship between hippocampal monocyte chemotactic protein-1 and its receptor and postoperative cognitive dysfunction in aged rats / 中华麻醉学杂志

Objective To evaluate the relationship between hippocampal monocyte chemotactic protein-1 (MCP-1) and its receptor C-C chemokine receptor 2 (CCR2) and postoperative cognitive dysfunction (POCD) in aged rats.Methods Forty-eight male Sprague-Dawley rats,aged 20-22 months,weighing 480-550 g,were randomly divided into 2 groups (n =24 each) using a random number table:control group (group C) and POCD group.POCD group inhaled 2.0％ isoflurane and underwent splenectomy.Before surgery and at 1,3 and 7 days after surgery,Morris water maze test was performed to evaluate the spatial learning and memory ability.The escape latency and swimming distance were recorded.Eight rats were sacrificed after the end of Morris water maze test performed at 1,3 and 7 days after surgery.Then the brains were removed,and the hippocampi were isolated for detection of the expression of MCP-1 and CCR2 by Western blot.Results Compared with group C,the escape latency and swimming distance were significantly prolonged,and the expression of MCP-1 and CCR2 in hippocampi was up-regulated at 1,3 and 7 days after surgery in POCD group.Conclusion Up-regulation of hippocampal MCP-1 and CCR2 expression may be involved in the mechanism of POCD in aged rats.

Effect of contrast-enhanced ultrosound imaging with CCR2 targeted microbubbles on murine breast tumor model / 中华超声影像学杂志

Objective To develop and validate a ultrasonographic (US)imaging agent with targeted microbubbles that attaches to chemokine receptor 2 (CCR2)and to compare the US single obtained from targeted microbubble with that from control microbubble in murine breast tumor model.Methods The microbubble which carried CCR2 antibody (MBCCR2 )and isotype-macthed immunoglobulin G-labled control microbubble (MBcontrol ) were prepared. The microbubble size and distribution were assessed by AccuSizer780.Binding specificities of targeted microbubble compared with control microbubble were tested with murine microvascular endothelial cells (bEnd.3 ).Orthotopic breast tumor model was estabished in BALB/c mice with mouse breast cancer 4T1 cell.In vivo imaging signals of contrast material-enhanced ultrasound by use these two different types of microbubble which were injected respectively into each mouse at random order and 30 min interval.Tumor tissue was stained for CCR2 and CD3 1 .Results Automatic Particle Sizer showed size uniform of two kinds of microbubbles,and narrow distribution of particle size (mean diameter of about 1 -2 μm),which were not significantly different (P >0.05).Adhension to bEnd. 3 endothelial cells was significantly higher (P < 0.001 )for MBCCR2 (mean,9.50 ± 1 .5 1 )than that for MBcontrol (mean,0.01 ±0.01).Imaging signal in the murine tumor model was significantly higher for MBCCR2 [mean,(6.76±0.26)dB]than that for MBcontrol [mean,(1 .06 ±0.62)dB,P <0.001 ].Immunofluorescence confirmed expression of CCR2 on tumor vasculature.Conclusions The targeted microbubbles with CCR2 monoclonal antibody had been successfully prepared,which precisely targeted to CCR2 of tumor angiogenesis in the murine breast cancer xenograft tumor models in vivo.These results suggest that the targeted microbubbles as a kind of ultrasound molecular imaging agent with a better specificity can be used for both evaluating tumor neovascularization and monitoring therapeutic effect of anti-angiogenesis.

Effects of intrathecal TRESK gene recombinant adenovirus on inflammatory responses mediated by chemokine in spinal cord of rats with neuropathic pain / 中华麻醉学杂志

Objective To evaluate the effects of intrathecal TRESK gene recombinant adenovirus on inflammatory responses mediated by chemokine in the spinal cord of rats with neuropathic pain ( NP ) . Methods Thirty?six male Sprague?Dawley rats, weighing 200-250 g, were randomly divided into 6 groups (n=6 each) using a random number table: control group (group C); sham operation group (group S);NP group; TRESK?overexpressed adenovirus group ( group TRESK ); negative adenovirus group ( group Virus); normal saline group ( group NS) . Spinal nerve injury was produced by exposing the sciatic nerve and its branches and ligation and transection of tibial nerve and common fibular nerve in anesthetized rats. In TRESK, Virus and NS groups, pAd∕CMV∕V5?DEST?TRESK 25 μl (109IU∕ml), negative adenovirus 25 μl and normal saline 25 μl were intrathecally injected, respectively. At 1 day before operation ( base?line, T0 ) and 1, 3, 7 and 14 days after operation ( T1-4 ) , the mechanical paw withdrawal threshold ( MWT) and thermal paw withdrawal latency were measured. Six rats in each group were sacrificed after measurement of pain threshold at T3 . The L4,5 segments of the spinal cords were removed for determination of monocyte chemotactic protein?1 ( MCP?1) , MIP?2, tumor necrosis factor?alpha ( TNF?α) , interleukin?1 beta ( IL?1β) and IL?6 mRNA expression by real?time PCR. Results There was no significant difference in thermal paw withdrawal latency at each time point between groups. Compared with C and S groups, MWT at T1-4 in NP and TRESK groups and at T1-3 in Virus and NS groups were significantly decreased, and the expression of MCP?1, MIP?2, TNF?α, IL?1βand IL?6 mRNA was up?regulated in NP, TRESK, Virus and NS groups. Compared with group NP, MWT was significantly increased at T1-4, and the expres?sion of MCP?1, MIP?2, TNF?α, IL?1β and IL?6 mRNA was down?regulated in group TRESK. Conclusion The mechanism by which intrathecal TRESK gene recombinant adenovirus reduces NP is re?lated to inhibition of inflammatory responses mediated by chemokine in the spinal cord of rats.

Relationship between the expression of chemokines and their receptors in the maternal-fetal ;interface and pathogenesis of unexplained recurrent spontaneous abortion / 中华妇产科杂志

Objective To explore the relationship between the expression of chemokines and their receptors in the maternal-fetal interface and the pathogenesis of unexplained recurrent spontaneous abortion (URSA). Methods 8-10 weeks CBA/J female mice were mated with DBA/2 and BALB/c male mice at the ratio of 2∶1 to establish the model of normal pregnant mice (CBA/J × BALB/c) and URSA mice (CBA/J × DBA/2). Sixty mice were divided into 6 groups, with ten in each group. The mice in the normal unpregnancy group were executed for endometrial tissues; the mice in the embryonic implantation normal pregnancy group were executed for endometrial tissues at the sixth day of gestation; the mice in the embryonic development normal pregnancy group were executed for decidua and chorionic tissues at the fourteenth day of gestation. While, the mice in the embryonic implantation URSA group were executed for endometrial tissues at the sixth day of gestation;the mice in the pre-abortion URSA group were executed for decidua and chorionic tissues at the ninth day of gestation;the mice in the post-abortion URSA group were executed for decidua and chorionic tissues at the fourteenth day of gestation. The chemokines and their receptors in different tissues of the mice were determined by western blot, including the protein expression of stromal cell derived factor (CXCL12), monocyte chemotactic protein 1 (CCL2), regulated upon activation normal T cell expressed and secreted(RANTES) and their receptor CXCR4, CCR2, CCR5 in maternal-fetal interface. Results (1) The protein expression of CXCL12 and CXCR4, CCL2 and CCR2, RANTES and CCR5 in endometrial tissues of the normal unpregnant group were 0.13±0.04 and 0.18±0.09, 0.057±0.023 and 0.39± 0.08, 0.034 ± 0.012 and 0.22 ± 0.05, respectively. They were 0.35 ± 0.09 and 0.93 ± 0.15, 0.349 ± 0.056 and 0.91 ± 0.15, 0.336 ± 0.089 and 0.44 ± 0.05 in endometrial tissues in the embryonic implantation normal pregnancy group;and were 0.62±0.15 and 1.23±0.28, 0.283±0.051 and 0.55±0.09, 0.225±0.065 and 0.35± 0.07 in decidua tissues in the embryonic development normal pregnancy group. The protein expression of chemokines and their receptors in endometrial tissues in the embryonic implantation normal pregnancy group and in decidua tissues in the embryonic development normal pregnancy group were higher than those in the normal unpregnancy group, with statistically significant difference(P<0.05). Compared with the embryonic implantation normal pregnancy group, CXCL12 and CXCR4 in decidual tissues in the embryonic development normal pregnancy group were significantly higher(P<0.05), while CCL2 and CCR2, RANTES and CCR5 were significantly lower (P<0.05). (2) Compared with the embryonic implantation normal pregnancy group, CXCL12 and CXCR4 (0.20±0.06 and 0.44±0.11) in endometrial tissues in the embryonic implantation URSA group were significantly lower (P<0.01), while CCL2 and CCR2(0.451±0.133 and 1.32± 0.20), RANTES and CCR5(0.488 ± 0.137 and 0.61 ± 0.18)were higher (P<0.05). (3) Compared with the embryonic development normal pregnancy group, CXCL12 and CXCR4 in decidual tissues of pre-abortion URSA group(0.27 ± 0.09 and 0.26 ± 0.10) , post-abortion URSA group (0.25 ± 0.08 and 0.23 ± 0.08) were significantly lower (P<0.01), while CCL2 and CCR2 (0.576±0.123 and 0.92±0.15 in the pre-abortion URSA group;0.748±0.112 and 1.56±0.34 in the post-abortion URSA group), RANTES and CCR5(0.294±0.054 and 0.59 ± 0.18 in the pre-abortion URSA group;0.363 ± 0.058 and 0.78 ± 0.14 in the post-abortion URSA group) were significantly higher(P<0.05). CCL2 and CCR2, RANTES and CCR5 in decidual tissues in the post-abortion URSA group was obviously higher than those of the pre-abortion URSA group, with statistically significant difference (P<0.05). Couclusions The accurate expression of CXCL12, CCL2, RANTES and their receptors CXCR4, CCR2, CCR5 play important roles in the embryonic implantation and development. The lower expression of CXCL12 and CXCR4 protein and higher expression of CCL2 and CCR2, RANTES and CCR5 in decidua and chorionic tissues are closely related to the pathogenesis of URSA.

The role of MCP-1-CCR2 ligand-receptor axis in chondrocyte degradation and disease progress in knee osteoarthritis

BACKGROUND: Osteoarthritis (OA) is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented. RESULTS: In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1) and receptor chemokine (C-C motif) receptor 2 (CCR2) in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001) and the expression of MMP-13 also increased (P < 0.05). MCP-1 stimulation also induced (or enhanced) the apoptosis of OA chondrocytes (P < 0.05). Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13) in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA) model. CONCLUSIONS: The results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis.

Role of spinal microglial C-C chemokine receptor type 2 in maintenance of bone cancer pain in rats / 中华麻醉学杂志

Objective To evaluate the role of spinal microglial C-C chemokine receptor type 2 (CCR2) in the maintenance of bone cancer pain (BCP) in rats.Methods Fifty unmated female Sprague-Dawley rats,aged 2 months,weighing 160-180 g,were randomly divided into 5 groups (n =10 each):sham operation group (group Ⅰ),sham operation + RS102895 (CCR2 antagonist) group (group Ⅱ),BCP group (group Ⅲ),BCP + dimethylsulfoxide (DMSO) group (group Ⅳ),and BCP + RS102895 group (group Ⅴ).The rats were anesthetized with intraperitoneal chloral hydrate.BCP was induced by intra-tibial inoculation of 1 × 105 Walker 256 mammary gland carcinoma cells into the medullary cavity of the left tibial metaphysis.On 10-12 days after operation,3 μg/μl RS102895 10 μd was injected intrathecally once a day in Ⅱ and Ⅴ groups,10％ DMSO 10 μl was injected intrathecally once a day in Ⅳ group,and normal saline 10 μl was injected intrathecally once a day in Ⅰ and Ⅲ groups.Mechanical paw withdrawal threshold was measured at 1 day before operation and 3,6,9,10,11 and 12 days after operation.After measurement of pain threshold at day 12 after operation,the lumbar segments (L4-6) of the spinal cord were removed for determination of the level of ox-42 (spinal microglial activation marker) (by immuno-histochemistry) and contents of IL1-β,IL-6 and TNF-α (by ELISA).Results Compared with group Ⅰ,mechanical paw withdrawal threshold was significantly decreased at 6-12 days after operation,the number of ox-42 positive cells and contents of IL1-β,IL-6 and TNF-α were increased in Ⅲ,Ⅳ and Ⅴ groups,and no significant change was found in the parameters mentioned above in group Ⅱ.Compared with group Ⅲ,mechanical paw withdrawal threshold was significantly increased at 10-12 days after operation,the number of ox-42 positive cells and contents of IL1-β,IL-6 and TNF-α were decreased in group Ⅴ,and no significant change was found in the parameters mentioned above in group Ⅳ.Conclusion Spinal microglial CCR2 is involved in the maintenance of BCP via activating microglia and promoting the release of inflammatory cytokines of rats.