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Objective: To explore the effect of Slit2/ROBO1 protein (Slit2/ROBO1) signaling pathway in high glucose-induced epithelial-mesenchymal transdifferentiation (EMT) and its mechanism. Methods: Human renal tubular epithelial cells (HK-2) were cultured in vitro and subjected to high glucose concentration and time gradient experiments. First, for concentration gradient experiment, the sample was randomly divided into normal group, control group 1, control group 2, high glucose group 1, high glucose group 2. While for high glucose time gradient experiment, the sample was randomly divided into normal group, control group, high glucose 24 h group, high glucose 36 h group and high glucose 48 h group. Western blotting was used to detect the expression changes of Slit2, ROBO1, α-smooth muscle actin (α-SMA) and fibronectin in HK-2 cells, and then the optimal high glucose stimulation concentration and time were screened out. Slit2 over-expressed plasmid and negative control plasmid were transfected into HK-2 cells to verify the successful transfection, the cells were then randomly divided into normal group, control group, high glucose group, high glucose empty group and high glucose Slit2 group. The total protein was extracted after stimulation with optimal high glucose concentration and time, and Western blotting was then performed to detect the change in expression of fibronectin and α-SMA. Results: In the high glucose concentration gradient experiment, the expression of Slit2 declined significantly in high glucose group 1(0.647±0.048) and high glucose group 2(0.210±0.023) than in the normal group (1.000±0.050); the expression of ROBO1 declined significantly in high glucose group 1(0.703±0.041) and high glucose group 2(0.303±0.022) than in the normal group (1.000±0.057); while the expression of fibronectin increased significantly in high glucose group 1(1.953±0.042) and high glucose group 2(2.997±0.078) than in the normal group (0.990±0.059), and the expression of α-SMA increased significantly in high glucose group 1(1.767±0.012) and high glucose group 2(2.427±0.059) than in the normal group (1.033±0.067), all the differences were of statistical significance(P<0.05). Compared with the high glucose group 1, the expressions of Slit2 and ROBO1 decreased, and of fibronectin and α-SMA increased significantly in the high glucose group 2(P<0.05). In the high glucose time gradient experiment, compared with the normal group, the expressions of Slit2 in high glucose 36 h group and high glucose 48 h group decreased (0.943±0.032 vs. 0.557±0.020, 0.450±0.055, respectively), and the expression of ROBO1 decreased (1.000±0.058 vs. 0.600±0.023, 0.227±0.028, respectively). Compared with the normal group, the expression of fibronectin increased significantly in high glucose 24 h group, high glucose 36 h group and high glucose 48 h group (0.970±0.040 vs. 1.247±0.052, 1.733±0.084, 2.780±0.090, respectively), and the expression of α-SMA increased significantly in high glucose 24 h group, high glucose 36 h group and high glucose 48 h group (1.033±0.067 vs. 1.277±0.041, 1.767±0.120, 2.537±0.078, respectively), and the difference was statistically significant (P<0.05). Compared with high glucose 24 h group, the expression of Slit2 declined significantly in high glucose 36 h group and high glucose 48 h group(0.893±0.034 vs. 0.557±0.020, 0.450±0.055, respectively), and the expression of ROBO1 declined significantly (0.930±0.025 vs. 0.600±0.023, 0.227±0.028, respectively), the expressions of fibronectin and α-SMA increased significantly with statistical significance (P<0.05). Compared with high glucose 36 h group, the expression of Slit2 and ROBO1 declined significantly, and the expression of fibronectin and α-SMA increased significantly in high glucose 48 h group (P<0.05). In the high glucose environment, and achieving Slit2 overexpression and negative control plasmid transfection, the expression of fibronectin increased significantly in high glucose group, high glucose+empty group and high glucose+Slit2 group (2.760±0.012, 2.667±0.027, 1.460±0.034, respectively) than in normal group (1.000±0.058); the expression of α-SMA increased also in high glucose group, high glucose+empty group and high glucose+Slit2 group (2.487±0.048, 2.557±0.037, 1.270±0.017, respectively) than in normal group (1.000±0.050) with statistical significance (P<0.05). Compared with the high glucose+empty group, the expression of fibronectin and α-SMA declined significantly in the high glucose+Slit2 group(P<0.05). Conclusion: The decreased expression of Slit2 and ROBO1 is involved in the high glucose-induced renal tubular EMT. Overexpression of Slit2 may significantly inhibit the high glucose-induced EMT.
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<p><b>OBJECTIVE</b>To investigate the role of USP33 as an independent prognostic marker in the regulation of SLIT2/ROBO1 signaling pathway to inhibit lung adenocarcinoma invasion and metastasis.</p><p><b>METHODS</b>The expression of USP33 in 20 lung adenocarcinoma specimens was detected by qPCR and immunohistochemistry. A549 and SPC-A-1 cells with small interfering RNA (siRNA)-mediated USP33 silencing were examined for changes in invasion and metastasis abilities using scratch assay and Matrigel assay. Western blotting was used to detect the expression of SLIT2 and ROBO1 in the cells after USP33 silencing and the expression of USP33 after interleukin-6 (IL-6) stimulation.</p><p><b>RESULTS</b>qPCR and immunohistochemistry showed that USP33 was significantly decreased in lung adenocarcinoma tissues as compared with the adjacent tissues. USP33 silencing in A549 and SPC-A-1 cells significantly promoted the cell migration, invasion and metastasis and obviously down-regulated the expressions of SLIT2 and ROBO1. IL-6 stimulation of the cells obviously enhanced the expression of USP33.</p><p><b>CONCLUSIONS</b>USP33 silencing can promote the migration, invasion and metastasis of lung adenocarcinoma cells , and the mechanism may involve IL-6 and SLIT2/ROBO1 signaling pathways.</p>
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Objective The basic biological, echocardiography and gene sequencing parameters of mice overexpressing Slit2 gene (Slit2-Tg mice) were collected and evaluated, and to provide a reference for the application of Slit2-Tg mice in biomedical research. Methods Slit2-Tg and C57BL/6 J mice were inbred. The genotypes of the mice were determined by a PCR assay. The blood samples were collected for blood routine and biochemical tests. The tissues of main organs were collected for protein expression and pathological analysis. Echocardiography and transcriptome sequencing was carried out for analyzing the heart function and gene expression, respectively. Results The litter size was significantly higher in the Slit2-Tg mice than in C57BL/6 J mice. Human Slit2 gene and protein expressions were detected in the main organs of Slit2-Tg mice. Organ coefficient of spleen was significantly increased in Slit2-Tg mice, but the tissue structure appeared normal. There were significant changes in the counts of erythrocytes, platelets, eosinophils, and biochemistry of glucose, globulin, urea nitrogen, triglycerides, HDL, and atherosclerosis index. Echocardiography showed no significant differences in the morphology and function of the Slit2-Tg hearts except in the left ventricular anterior wall thickness at the end-diastolic state. Compared with the C57BL/6 J mice, 535 genes out of 17513 genes in the Slit2-Tg hearts were increased or decreased, mainly involving 15 biological process or signal transduction pathways. Conclusions This study has collected the biological parameters of Slit2-Tg mice and suggests that this model animal is suitable for the studies of cardiovascular diseases.
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Objective Previous studies suggested that overerpression of Slit2 results in abnormal Alzheimer's disease-like behavior and cognition impairment in mice. The aim of this study is to investigate the relationship between overerpression of Slit2 and accumulation and clearance of amyloid-β in aging mice by comparing the differential expression of genes for accumulation and clearance of amyloid-β in aging Tg-Slit2 and Tg-2576 mice. Methods 14-month old male C57BL/6, Tg-Slit2 and Tg-2576 mice were used to detect the expression of Aβ1 - 40 and Aβ1 - 42 in brain by immunohistochemistry. Further, the total RNA in the brain of these mice were extracted, identified and inversely transcripted to cDNA, then the cDNA was detected by PCR array. The expression of genes in the brain of Tg-Slit2 and Tg-2576 mice were analyzed. Results Comparing with the Tg-2576 mice in the same age, accumulation of Aβ was not found in the brain of Tg-Slit2 and C57BL/6 mice. The result from PCR array analysis showed that comparing with the same aged C57BL/6 mice, there were 16 up-regulated genes and 8 down-regulated genes in the brain of Tg-Slit2 mice and 14 up-regulated genes and 17 down-regulated genes in the brain of Tg-Slit2 mice. The expression of amyloid beta precursor protein (APP) in the brain of the three group mice was not changed. The expression of presenilin 2 ( Psen2) related with Aβ production was significantly up-regulated in the Tg-2576 mice. In addition, the expression of low density lipoprotein receptor-related protein ( LRP) 6 and 9 were markedly decreased in the Tg-2576 mice. Notably, these genes were not changed in the brain of the aging Tg-Slit2 mice. Conclusions The accumulation of Aβ in the brain are not found in 14-month Tg-Slit2 mice, In addition, different from Tg-2576 mice, the significant changes of expression of Aβ-related genes is not found in the brain of Tg-Slit2 mice.
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Axon guidance molecules,slit glycoprotein (Slit) and Roundabout receptors (Robo) were firstly identified in the central neural system of Drosophila melanogaster.The Slit-Robo signal plays a crucial role in axon guidance,inflammation,tumor metastasis and angiogenesis,of which the role of Slit2-Robo pathway in angiogenesis has drawn a greater attention and still remains conflicting.Herein,we provide a review on the role of Slit2-Robo pathway in ocular angiogenesis and vascularization of other organs and systems.We hope this review will be the basis of further study on the mechanism of Slit2-Robo signaling on angiogenesis and provide new target for the therapy on ocular vascular disease
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BACKGROUND: Slit2 is a new secreted protein from adipose tissue that improves glucose hemostasis in mice; however, there is no study about the serum levels and precise role of Slit2 in human. The aim of this study is to explore the serum level of Slit2 in human, and to identify the role of Slit2 in diabetes mellitus (DM). METHODS: The participants of this study consist of 38 subjects with newly diagnosed DM, and 75 healthy subjects as a control group. Serum Slit2 levels were measured using an enzyme-linked immunosorbent assay. Relationship between circulating Slit2 and diabetic related factors was investigated in diabetic group compared with non-diabetic group. Additionally, the correlations between the serum level of Slit2 and diverse metabolic parameters were analyzed. RESULTS: Circulating Slit2 level was more decreased in diabetic group than in control group, but there was no significant difference statistically. Interestingly, serum levels of Slit2 were significantly negatively correlated to the serum concentrations of fasting glucose (coefficient r=–0.246, P=0.008), the serum concentrations of postprandial glucose (coefficient r=–0.233, P=0.017), and glycosylated hemoglobin (HbA1c; coefficient r=–0.357, P<0.001). CONCLUSION: From our study, the first report of circulating Slit2 levels in human, circulating Slit2 level significantly negatively correlated with serum glucose and HbA1c. Our results suggest that the circulating Slit2 may play a role in maintainence of glucose homeostasis in human, even though exact contribution and mechanism are not yet known.
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Animals , Humans , Mice , Adipokines , Adipose Tissue , Adipose Tissue, Brown , Blood Glucose , Diabetes Mellitus , Enzyme-Linked Immunosorbent Assay , Fasting , Glucose , Healthy Volunteers , Glycated Hemoglobin , Hemostasis , HomeostasisABSTRACT
Objective To study the effect and mechanism of down-regulating Silt2/Robo 1 signaling pathway on rabbit iliac artery after angioplasty restenosis. Methods The 30 male New Zealand white rabbits were divided randomly into 3 groups , namely the blank group , the control group , and the experimental group , 10 rabbits in each group. Hign-fat feeding , the rabbits were produced endothelial denudation of iliac artery stenosis model. Another 4 weeks of feeding , percutaneous balloon angioplasty was performed. Then R5 antibody was injected into the abdominal cavity. After 4 weeks of feeding ,angiography again. The results of angiography was analysied by image workstation. The concentrations of Slit2 and Robo1 was detected by ELISA. The iliac artery tissue examined by HE staining. Results The rabbit iliac artery after angioplasty restenosis animal model was set up successfully. Compared with the control group and the experimental group , the serum concentration of Slit2 and Robo1 were significantly higher (P < 0.01) than the blank group. But in the experimental group, the Slit2 and Robo1 serum concentrations were significantly lower than those in the control group (P < 0.05) after R5 antibody intervention. The area ratio stenosis and diameter stenosis rate of iliac artery were reduced that confirmed by angiography. Conclusion The expression of Slit2/Robo1 was significantly higher in the rabbit model of vascular restenosis. R5 antibody can effectively inhibit the expression of Slit2/Robo1. Down regulation of Slit2/Robo1 signaling pathway in the treatment of restenosis after angioplasty in rabbits.
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Objective To observe the effect of electro-acupuncture (EA) intervention on expression of Slit2 and SrGAP1 in rats with focal cerebral infarction and to study the underlying mechanism of EA intervention on neuronal plasticity after cerebral ischemia. Methods Sixty male Sprague Dawley rats were randomly divided into Model group(n=30) and EA group (n=30); and according to elctro-apunture treatment duration, both 2 groups were further randomized into 3 subgroups:0 d(n=10),7 d(n=10), and 14 d (n=10).The middle cerebral arteries of the model group and EA group were embolized for 1.5 hours by suture. Nissl staining, immunofluorescence method and Western blotting analysis were sued to observe the histology and morphology change around the cerebral infarction and to assay the expression of Slit2 and SrGAP1 in the cerebral cortex on the ischemic side on day 0, 7, and 14. Results In comparison with the model group, the neurological score of EA group had no obvious difference on day 0 (P>0.05) and was significantly lower on day 7 and day 14 (P0.05); but on day 7 and day 14, the fluorescence intensities and grey levels of EA group were significantly higher than those of the model group(P<0.05). In the model group the above values on day 0 were significantly lower than those on day 7 (P<0.05), and those on day 7 were significantly higher than those on day 14 (P<0.05). In the EA group the fluorescence intensities and grey levels on day 0 were significantly lower than those on day 7 (P<0.05), and those on day 7 were significantly higher than those on day 14 (P<0.05). Conclusion Slit2 and SrGAp1 are lowly expressed following focal cerebral infarction on day 0, and EA intervention for one and two weeks can promote their expression and prolong the duration of high expression, subsequently accelerating axonal regeneration and repair, which may be a mechanism of EA therapy for neurological function recovery following cerebral infarction.
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Objective To observe the effects of Slit2 protein on the proliferation and migration of VSMCs .Methods The VSMCs was cultured in our laboratory .The experiment was divided into two parts ,part one:VSMCs were divided into normal con‐trol group and experimental groups(culture with 50 ,75 ,100 ,125 and 150 ng/mL Slit2 respectively);part two :VSMCs were divided into normal control group ,positive control group(culture with TNF‐α10 ng/mL) and experimental groups(culture with TNF‐α10 ng/mL+Slit2 50 ng/mL ,TNF‐α10 ng/mL+Slit2 75ng/mL ,TNF‐α10 ng/mL+ Slit2 100 ng/mL ,TNF‐α10 ng/mL+ Slit2 125 ng/mL and TNF‐α10 ng/mL+Slit2 150 ng/mL respectively) .To detect proliferation and migration of VSMCs by CCK‐8 and tran‐swell experiment .Results The difference of OD value and numbers of VSMCs has no statistical significance in the presence of Slit 2 (P=0 .516 ,P=0 .52) .The numbers of VSMCs has statistical significance between control and positive control groups (P=0 .00) . The numbers of VSMCs in experimental groups were fewer than positive control group (P<0 .05) ,whereas the difference of OD value still has no statistical significance between experimental and positive groups (P= 0 .173) .Conclusion Recombinant Slit2 could inhibits migration in VSMCs induced by TNF‐α,whereas it has no effect on proliferation of VSMCs .
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Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury (ALI) and explore the function of Slit2 and Robo4 in ALI.Methods Forty-eight Sprague-Dawley rats were randomly (random number) divided into control group (n =24) and ALl group (n =24).ALI model was reproduced by cecum ligation and puncture (CLP).The control group only experienced a simulated operation without CLP.Both groups were further divided into 3 subgroups with 8 rats in each subgroup:12 h,24 h,and 48 h subgroups.artery blood gas analysis,lung tissue wet/dry weight (W/D) ratio,lung histopathologic changes,pulmonary microvascular permeability were observed.The serum tumor nocrosis factor-α (TNF-α) was measured with enzyme linked immunosorbent assay (ELISA).The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR).The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry.Date were analyzed by one-way ANOVA with SPSS version 13.0 software.Statistical significance was established at a P value of less than 0.05.Results Compared with the control group,in ALI rats at different time points,partial pressure of oxygen in arterial blood (PaO2) decreased significantly,lung W/D weight ratio and pulmonary microvascular permeability,the serum TNF-α increased significantly (all P ＜ 0.05),histopathology of lung revealed signs of injury.The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [(0.56±0.13) vs.(0.87±0.05),F=41.39,P＜0.05,(0.42±0.10) vs.(0.85±0.07),F=93.54,P＜0.05,(0.26±0.08) vs.(0.89 ±0.09),F=227.05,P＜0.05].but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [(0.86±0.07) vs.(0.83±0.05),F=0.695,P＞0.05,(0.82±0.05) vs.(0.89±0.08),F=2.061,P ＞ 0.05,(0.86 ± 0.08) vs.(0.86 ± 0.05),F =0.035,P ＞ 0.05].Immunohistochemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells,while lung epithelial cell nuclei and endochylema.Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells.Compared with the control group,expression of Slit2 protein in lung tissue in ALI group decreased markedly [(0.37 ± 0.05) vs.(0.45 ± 0.07),F =6.82,P ＜ 0.05,(0.32±0.06) vs.(0.47±0.09),F=23.54,P＜0.05,(0.28±0.07) vs.(0.46±0.06),F=28.01,P ＜ 0.05].As good as RT-PCR,there were no significant difference in expression of Robo4 protein in lung tissue between two groups [(0.53±0.04) vs.(0.52±0.05),F=0.155,P＞0.05,(0.53± 0.09) vs.(0.50±0.05),F=0.498,P＞0.05,(0.55±0.06) vs.(0.56±0.07),F=0.073,P ＞ 0.05].Conclusions Lung tissues of control group rats express Slit2 and Robo4.The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALI caused by CLP may be associated with the occurrence of ALI.
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Tumor growth and migration are correlated with continuous neovascularization .Slit2-Robo1 signal pathway initially positioned in the nervous system plays an important role in the growth and neuronal axon guidance during migration.However,vascular system resembles the structural features and growth mechanism of neural system .Recent researches have shown that Slit 2-Robo1 signaling pathway plays an important role in vas-cular system,especially in tumor vessel .And Slit2-Robo1 has been shown to play a role in brain metastasis in breast cancer and supporting tumor growth in melanoma .Therefore,the role of Slit2-Robo1 in tumor growth and migration depends on the cancer type .Currently ,researches in this field show controversial results .And the status of Slit2-Robo1 signaling pathway in lung cancer is reviewed in this essay .
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Objective To investigate the expression and significance of Slit2 in different time point after the spinal cord injury of rats.MethodSeventy adult wistar rats were randomly divided into three groups: spinal cord injury by fully transection on T10 level spinal cord (Group A); laminectomy and the spinal cord uninjuried (Group B); natural without operation (Group C).Then the rats were sacrificed and the spinal cord was taken out fresh quickly on different time-point(12h, 1, 3, 5, 7d after operation). The tissues perfusion by formaldehyde were taken out on 3, 5, 7, 14d after operation. The expressions of Slit2 were tested by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical measurement. By means of above,the author investigated the expressions and location of Slit2 after the spinal cord injury.All data were statisticsed by SPSS 11.5 software.ResultThe expression of Slit2 mRNA of appeared in the spinal cord tissue 12 hours after injury, reached peak on the 3rd day, declining gradually later. The positive expression of Slit2 located in the cytoplast on oligodendrocyte and astrocyte.The positive cells were found at 3d after spinal cord injury, reached peak on 7d after injury, declining after 14d. The change of Slit2 was correlative with the rehabilitation and regeneration of the axon on the forepart period.ConclusionAs an important factor in axonal growth-guidance,the author crewed that Slit2 may be participated in the regeneration and rehabilitation of axons after the spinal cord injury.