Netrin-1 Concentrations Research Articles

Evaluation of Serum Cystatin-C and Netrin-1 as Predictive Biomarkers for Renal Injury in Children with Beta-thalassemia Major

BACKGROUND: Despite optimal transfusion and chelation therapy, beta-thalassemia patients still commonly experience various complications, including renal complications, which may affect their survival. This case–control study involved pediatric patients with beta-thalassemia major (β-TM). MATERIALS AND METHODS: This case–control study involved pediatric patients with β-TM, a total of 60 patient samples were randomly collected from the Genetic Hematology Center at the Dhi-Qar Health Directorate, with an age range of 1–14 years. In addition, 60 samples were collected from healthy children in the same age range as the control group. In this study, the glomerular injury was predicted by measuring the concentration of serum cystatin-C (Cys-C), and the tubular injury was predicted by measuring the concentration of serum netrin-1 (NTN-1). Routine markers, such as hemoglobin (Hb) level, ferritin, serum albumin, and creatinine, were also examined for the patients and the control group. RESULTS: The results showed a statistically significant increase in serum Cys-C in the patient group compared to the control group (P < 0.001) and a significant increase of serum NTN-1 in β-TM patients compared to the control group (P < 0.001). In addition, the study showed a statistically significant decrease in Hb, serum albumin, and creatinine and a significant increase in serum ferritin in the patients group compared to the control group. In conclusion, from the results of this study, we can conclude that the serum Cys-C and NTN-1 measurements can be used as predictive biomarkers for glomerular and tubular injury and increase with age. CONCLUSION: Predicting glomerular and tubular injury using the measurement of serum Cys-C and NTN-1 has improved significantly and advanced with age.

Dephosphorylation of neural wiring protein shootin1 by PP1 phosphatase regulates netrin-1-induced axon guidance

Axon pathfinding is an essential step in neuronal network formation. Shootin1a is a clutch-linker molecule that is mechanically involved in axon outgrowth and guidance. It was previously shown that concentration gradients of axon guidance molecule netrin-1 in the extracellular environment elicit asymmetrically localized Pak1 kinase-mediated phosphorylation of shootin1a within axonal growth cones, which is higher on the netrin-1 source side. This asymmetric phosphorylation promotes shootin1a-mediated local actin-adhesion coupling within growth cones, thereby generating directional forces for turning the growth cone toward the netrin-1 source. However, how the spatial differences in netrin-1 concentration are transduced into the asymmetrically localized signaling within growth cones remains unclear. Moreover, the protein phosphatases that dephosphorylate shootin1a remain unidentified. Here, we report that protein phosphatase-1 (PP1) dephosphorylates shootin1a in growth cones. We found that PP1 overexpression abolished the netrin-1-induced asymmetric localization of phosphorylated shootin1a as well as axon turning. In addition, we show PP1 inhibition reversed the asymmetrically localized shootin1a phosphorylation within growth cones under netrin-1 gradient, thereby changing the netrin-1-induced growth cone turning from attraction to repulsion. These data indicate that PP1-mediated shootin1a dephosphorylation plays a key role in organizing asymmetrically localized phosphorylated shootin1a within growth cones, which regulates netrin-1-induced axon guidance.

Effect of Netrin-1 on VEGFA Expression in T-ALL Cells and Its Related Mechanism

ELISA assays were applied to detect the levels of Netrin-1 and VEGFA in the bone marrow (BM) samples from children in the T-ALL and control group. The level of Netrin-1 and VEGFA were compared between control children and patients, and the liner correlation between Netrin-1 and VEGFA was analyzed. The T-ALL cells Jurkat and Molt-4 were culture in vitro, and the cells were treated with different concentration of Netrin-1 (0, 25, 50, 100 ng/ml) for 24 h, quantitative RT-PCR (qRT-PCR) and Western blot were used to detect the VEGFA expression in Jurkat, Molt-4 cells. The expression of Netrin-1 receptors in T-ALL cells was detected by qRT-PCR and the interaction between Netrin-1 and receptor in each cells was detected by co-IP. Furthermore, Western blot was used to detect the phosphorylation level of key prateins of AKT signal transduction pathway including Akt and mTOR in T-ALL cells treated with Netrin-1 (100 ng/ml). The expression of VEGFA and phosphorylation of AKT pathway transducers were detected by Western blot, after T-ALL cells treated with Netrin-1 (100 ng/ml) combined with inhibitors specific to Akt or mTOR. The expression level of Netrin-1 and VEGFA in T-ALL patients BM samples were both signi-ficantly higher than that of control group. And the expression level of Netrin-1 was positively correlated with that of VEGFA（r2=0974). With the increase of Netrin-1 concentration, the expression level of VEGFA also increased(P<0.05）. Netrin-1 interacted with its receptor, integrin-β4 at the Netrin-1 concentration of 100 ng/ml. Further, the treatment of Netrin-1 could increase the phosphorylation of Akt and mTOR, which were the key transducers of AKT pathway. After treatment of T-ALL cells with Netrin-1 (100 ng/mL) and Akt inhibitor, the expression of VEGFA and phosphorylation of Akt or mTOR decreased. When the cells were treated with Netrin-1(100 ng/ml) and mTOR inbititor, the phosphorylation level of mTOR and the expression of VEGFA decreased, the phosphorylation level of Akt increased. The expression of Netrin-1 and VEGFA in bone marrow of childred with T-ALL were abnormal, and there was a linear relationship between them. Netrin-1 can interact with its receptor, integrin-β4 and activate AKT transduction pathway to elevate the expression of VEGFA in T-ALL cells.

The Prognostic Value of Deleted in Colorectal Cancer (DCC) Receptor and Serum Netrin-1 in Severe Traumatic Brain Injury.

Traumatic brain injury (TBI) is a common neurological disease. Netrin-1 and deleted in colorectal cancer (DCC) receptor are potential biomarkers associated with nerve regeneration and immune regulation. We aimed to investigate the ability of the DCC receptor and Netrin-1 to predict a high ICP level after operation in severe traumatic brain injury and their prognostic significance. This study is a prospective observational study. We selected 23 patients with traumatic brain injury who had undergone surgical operations as subjects. Immunohistochemical staining was performed on the contusion tissue that was removed by the operation to determine the expression of DCC receptor. At the same time, enzyme-linked immunosorbent assay (ELISA) kits were used to detect the serum Netrin-1 content. Determination of intracranial pressure (ICP) value was measured by intraventricular catheter. The Glasgow Outcome Scale (GOS) score at six months after trauma was defined as the main study endpoint. The results showed that serum Netrin-1 concentrations of patients in the critical TBI group (GCS 3–5 points) was significantly lower than that in the severe TBI group (GCS 6–8 points). The ICP peak and average mannitol consumption in the high Netrin-1 group were significantly lower than those in the low Netrin-1 group. DCC receptor-positive patients had a significantly lower ICP peak. There was no significant difference in six month-GOS scores between patients in the high and low Netrin-1 groups, while DCC receptor concentrations below 3.82 ng/mL predicted poor prognosis (GOS 1–3 points). In conclusion, the expression level of the DCC receptor can better evaluate the postoperative high ICP level and prognosis than the level of serum Netrin-1 in severe traumatic brain injury.

Conductive Hydrogel Conduits with Growth Factor Gradients for Peripheral Nerve Repair in Diabetics with Non-Suture Tape.

Diabetic patients suffer from peripheral nerve injury with slow and incomplete regeneration owing to hyperglycemia and microvascular complications. This study develops a graphene-based nerve guidance conduit by incorporating natural double network hydrogel and a neurotrophic concentration gradient with non-invasive treatment for diabetics. GelMA/silk fibroin double network hydrogel plays quadruple roles for rapid setting/curing, suitable mechanical supporting, good biocompatibility, and sustainable growth factor delivery. Meanwhile, graphene mesh can improve the toughness of conduit and enhance conductivity of conduit for regeneration. Here, novel silk tapes show quick and tough adhesion of wet tissue by dual mechanism to replace suture step. The in vivo results demonstrate that gradient concentration of netrin-1 in conduit have better performance than uniform concentration caused by chemotaxis phenomenon for axon extension, remyelination, and angiogenesis. Altogether, GelMA/silk graphene conduit with gradient netrin-1 and dry double-sided adhesive tape can significantly promote repairing of peripheral nerve injury and inhibit the atrophy of muscles for diabetics.

Decreased Netrin-1 in Mild Cognitive Impairment and Alzheimer's Disease Patients.

Background and ObjectiveInflammatory mediators are closely associated with the pathogenesis of Alzheimer’s disease (AD) and mild cognitive impairment (MCI). Netrin-1 is an axon guidance protein and despite its capacity to function as a neuroimmune guidance signal, its role in AD or MCI is poorly understood. In addition, the association among netrin-1, cognitive impairment and serum inflammatory cytokines such as interleukin-17 (IL-17) and tumor necrosis (TNF-α) remains unclear. The aim of this study was to determine serum levels of IL-17, TNF-α and netrin-1in a cohort of AD and MCI patients, and to study the relationship between these cytokines and cognitive status, as well as to assess the possible relationships between netrin-1 levels and inflammatory molecules.MethodsSerum concentrations of netrin-1, TNF-α and IL-17 were determined in 20 AD patients, 22 MCI patients and 22 healthy controls using an enzyme-linked immunosorbent assay (ELISA). In addition, neuropsychological evaluations and psychometric assessments were performed in all subjects.ResultsSerum netrin-1 levels were decreased in AD and MCI patients and were positively correlated with Mini Mental State Examination (MMSE) scores. In contrast, serum TNF-α and IL-17 levels were elevated in AD and MCI cohorts and negatively correlated with MMSE scores. Serum netrin-1 levels were inversely related with TNF-α and IL-17 levels in AD, but not MCI, patients.ConclusionBased on the findings reported here, netrin-1 may serve as a marker for the early recognition of dementia and predict cognitive impairment.

Relationship between Netrin-1 Levels and Diabetes Mellitus Type 2

Objects: The study's purpose was to see if there was a link between netrin.1 and type II diabetes patients. Methodology: The present study carried out on (45) patients affected with diabetes mellitus from (120) persons were examined in the education of Diwaniyah hospital from 6th September to the 4th December 2020. The study includes measurement of the body mass index, fasting blood glucose level, HbA1c, lipid profile and level of netrin-1. Results: Our findings were revealed the netrin-1 level was significantly lower in the diabetes (1205.36±753.09) compared to control group (1477.79±700.26; P &lt; 0.01). Our observations were appeared Significantly higher levels of blood cholesterol, TG, LDL, and VLDL-C and In diabetes individuals, there was a significant drop There was a 0.05 difference in HDL-C levels when compared to healthy controls. Conclusion: These findings indicate that low netrin1 concentration in serum are strongly linked to the occurrence of type II diabetes.

The expression of Netrin-1 in the MIA-induced osteoarthritic temporomandibular joint in mice

Subchondral bone degeneration is the main pathological change during temporomandibular joint (TMJ) osteoarthritis (OA) development. Netrin-1, an axon-guiding factor, might play roles in OA development and pain. The purpose of this study was to investigate the expression of Netrin-1 in TMJ OA and its possible role in the progression of TMJ OA and pain. The synovial fluids of temporomandibular joint disorders (TMDs) patients were collected for Netrin-1 by enzyme linked immunosorbent assay (ELISA). TMJ OA model was built by MIA joint injection, and then the von Frey test, hematoxylin & eosin (H&E) staining, toluidine blue (TB) staining, immunohistochemical (IHC) staining and micro-CT were performed. After induction of osteoclast differentiation of raw264.7 cells, immunofluorescence (IF) was used to detect the Netrin-1 and its receptors on osteoclast membrane. The concentration of Netrin-1 increased in the synovial fluid of TMJ OA patients. After MIA injection to TMJ, the head withdrawal threshold (HWT) was significantly decreased. Microscopically, the structural disorder of subchondral bone was the most obvious at the 2nd week after MIA injection. In addition, Netrin-1 expression increased in the subchondral bone at the 2nd week after MIA injection. In vitro, the expressions of Netrin-1 and its receptor Unc5B were upregulated on the osteoclast membrane. Netrin-1 might be an important regulator during bone degeneration and pain in the process of TMJ OA.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems.Key points• Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1• Biolayer interferometry allows target protein quantitation in expression media• High production yields in the bioreactor do not impair Netrin-1 proteoglycan qualityGraphical abstract

Increased serum concentration of netrin-1 after intravitreal bevacizumab injection: is it a compensatory mechanism to counteract drug side effects?

BackgroundTo evaluate alterations in the serum concentrations of vascular endothelial growth factor (VEGF) and netrin-1 after intravitreal bevacizumab (BCZ) injection for the treatment of diabetic macular edema (DME).MethodsThis prospective case-control study included a total of 50 participants assigned to one of three groups, including 10 individuals with DME and non-proliferative diabetic retinopathy (NPDR), 13 with DME, and proliferative diabetic retinopathy (PDR), and 27 healthy individuals as a control group. Serum VEGF and netrin-1 concentrations were measured by enzyme-linked immunosorbent assays (ELISAs) immediately before, as well as 1 week and 1 month after, intravitreal BCZ injection.ResultsThe mean VEGF serum concentrations in the PDR and NPDR groups were 388.4 and 196.9 pg/mL at baseline, respectively. After 1 week, these concentrations changed to 193.41 and 150.23 pg/mL, respectively (P = 0.001 and P = 0.005, respectively); after 1 month, the concentrations were 97.89 and 76.46 pg/mL, respectively (P = 0.001 and P = 0.009, respectively). The mean netrin-1 serum concentrations in the PDR patients and NPDR groups were 318.2 and 252.7 pg/mL at baseline, respectively. After 1 week, these concentrations increased to 476.6 and 416.3 pg/mL, respectively (P = 0.033 and P = 0.005, respectively), and after 1 month, they were 676.6 and 747.5 pg/mL, respectively (P = 0.001 and P = 0.005, respectively). The correlation analysis revealed a significant inverse relationship between changes in serum VEGF and netrin-1 concentrations in both the PDR and NPDR groups (r = − 0.685, P = 0.029).ConclusionsIntravitreal BCZ injections work systemically to significantly decrease serum VEGF levels, leading to a significant upregulation in the concentration of another angiogenic mediator, netrin-1.

Serum netrin-1 as a potential biomarker for functional outcome of traumatic brain injury

BackgroundDecreased serum netrin-1 concentrations have been found after acute brain injury. We investigated the role of serum netrin in prognosis of traumatic brain injury (TBI). MethodsIn this prospective and observational study, enzyme-linked immunosorbent assay was used to detect serum netrin-1 concentrations in 50 mild TBI patients (Glasgow coma scale (GCS) score, 13–15), 83 moderate TBI patients (GCS score, 9–12), 69 severe TBI patients (GCS score, 3–8) and 50 healthy controls. Glasgow outcome scale score of 1–3 at 6 months after trauma was defined as poor outcome. ResultsSerum netrin-1 concentrations were significantly lower in moderate or severe TBI patients than in controls and in severe TBI patients than in moderate TBI patients, while not in mild TBI patients than in controls. GCS score and Rotterdam computed tomography classification were closely correlated with serum netrin-1 concentrations among TBI patients. Forty-two (20.8%) patients had poor outcome. Receiver operating characteristic curve analysis revealed that serum netrin-1 concentrations could distinguish patients with poor outcome from the other remainders significantly. In addition, serum netrin-1 concentrations were independently associated with poor outcome. ConclusionsSerum netrin-1 might serve as a potential biomarker for prognosis of TBI.

Is Urinary Netrin-1 a Good Marker of Tubular Damage in Preterm Newborns?

There is a lack of a good marker for early kidney injury in premature newborns. In recent publications, netrin-1 seems to be a promising biomarker of kidney damage in different pathological states. The study aimed to measure the urinary level of netrin-1 depending on gestational age. A prospective study involved 88 newborns (I-60 premature newborns, II-28 healthy term newborns). Additionally, premature babies were divided for 2 groups: IA-28 babies born between 30–34 weeks of gestation and IB-32 born at 35–36 weeks. The median urinary concentration of netrin-1 was: IA-(median, Q1–Q3) 63.65 (56.57–79.92) pg/dL, IB-61.90 (58.84–67.17) pg/dL, and II-60.37 (53.77–68.75) pg/dL, respectively. However urinary netrin-1 normalized by urinary concentration of creatinine were IA-547.9 (360.2–687.5) ng/mg cr., IB-163.64 (119.15–295.96) ng/mg cr., and II-81.37 (56.84–138.58) ng/mg cr., respectively and differ significantly between the examined groups (p = 0.00). The netrin-1/creatinine ratio is increased in premature babies. Further studies examining the potential factors influencing kidney function are necessary to confirm its potential value in the diagnosis of subclinical kidney damage in premature newborns.

Urinary netrin-1 concentration in healthy full-term newborns.

IntroductionMonitoring of renal function in acute kidney injury in the pediatric population is complicated by the lack of age-related reference values of new biomarkers. Urinary netrin-1 is a new marker to demonstrate early kidney damage. Netrin-1 has a molecular mass of 72 kDa. It is therefore unlikely that it is filtered by the glomerulus under normal conditions. However, netrin-1 is highly induced after acute and chronic kidney injury and excreted in urine in humans. The aim of the study was to determine the normal concentrations of urinary netrin-1 in healthy full-term newborns.Material and methodsThe study included 88 healthy full-term neonates (51 boys and 37 girls) born from normal, uncomplicated pregnancies. The concentration of netrin-1 was determined in urine obtained on the first or second day of life with a commercially available ELISA kit.ResultsThe urinary concentration of netrin-1 in newborns was independent of gender and time of urine collection. We found a negative correlation between both the urinary netrin-1 concentration and urinary netrin-1 concentration after normalization for urinary creatinine and the birth weight.ConclusionsThis is the first study showing the urinary netrin-1 concentration in healthy full-term newborns. Future investigation is needed to confirm its potential role as a marker of kidney function in this age group.

Macrophage-derived netrin-1 contributes to endometriosis-associated pain.

BackgroundEndometriosis-associated pain can be considered a type of neuropathic pain. Netrin-1 is an axon guidance cue that regulates axonal attraction or rejection in neural injury and regeneration. However, whether netrin-1 plays a role in endometriosis-associated pain remains unclear. This study aimed to determine the role of netrin-1 in endometriosis-related pain.MethodsPeripheral blood, peritoneal fluid, and endometrial tissues were sampled from women with (n=37) and without endometriosis (n=23). Lipopolysaccharide (LPS) and interferon gamma (IFN-γ) were used to stimulate human monocytic cell lines (THP-1) and rat alveolar macrophage-derived cell lines (NR8383) to induce M1 phenotype macrophages. Serum netrin-1 concentrations, endometrial expression levels of netrin-1, and its receptors including deleted in colorectal cancer (DCC), A2B adenosine receptor (A2BAR), uncoordinated B receptor (UNC5B), uncoordinated C receptor (UNC5C) and Down’s syndrome cell adhesion molecule (DSCAM) were assessed. The polarization phenotypes of the peritoneal macrophages were identified by detecting the marker expression of M1/M2 macrophages via flow cytometry. The expression levels of M1 markers and netrin-1 in THP-1/NR8383 cells were determined.ResultsThe expression levels of netrin-1 in serum and endometriotic lesions were significantly higher in women with endometriosis, and were positively correlated with the severity of endometriosis-associated pain. Netrin-1 was co-expressed with CD68 (a macrophage marker) in endometriotic lesions and was synthesized and secreted by THP-1 and NR8383 cells in the process of M1 polarization. In women with endometriosis, peritoneal macrophages were polarized towards the M1 phenotype. In addition, increased expression of DCC and A2BAR, and decreased expression of UNC5B, UNC5C and DSCAM were found in endometriotic lesions.ConclusionsThese results suggest that netrin-1 production by macrophages in endometriotic lesions may play an important role in endometriosis-associated pain.

Decreased serum netrin-1 is associated with ischemic stroke: A case–control study

Background and aimsNetrin-1 was a laminin-related protein involved in neurovascular protection, and we previously discovered that decreased serum netrin-1 was associated with poor prognosis of ischemic stroke. However, the relationship between serum netrin-1 level and the risk of ischemic stroke remains unclear. The aim of this study was to investigate the association between netrin-1 level and risk of ischemic stroke. Methods and resultsA case–control study including 591 ischemic stroke patients and 591 age- and sex-matched healthy individuals was conducted, and serum netrin-1 concentrations were quantitatively determined via enzyme-linked immunosorbent assay for all participants. The serum netrin-1 levels were significantly lower in the ischemic stroke patients than those in matched controls (median, 496.4 vs 652.1 pg/mL; P < 0.001). After adjustment for potential confounders, the odds ratio of ischemic stroke associated with the highest quartile of netrin-1 was 0.07 (95% CI: 0.01–0.65; Ptrend = 0.018) compared with the lowest quartile of netrin-1. Each 1-standard deviation increase of log-transformed netrin-1 was associated with a lower odds of ischemic stroke (odds ratio: 0.45, 95% CI: 0.22–0.94; P = 0.032), and a dose–response relationship between serum netrin-1 and ischemic stroke was observed (Plinearity = 0.003). Incorporating netrin-1 to conventional risk factors improved the discriminatory power for ischemic stroke (net reclassification index = 98.0%, P < 0.001; integrated discrimination improvement = 0.28%, P = 0.027). ConclusionsSerum netrin-1 was decreased in patients with ischemic stroke compared with healthy controls, suggesting that there was a potential role of netrin-1 in the pathogenesis of ischemic stroke.

Serum netrin-1 concentrations are associated with clinical outcome in acute intracerebral hemorrhage

BackgroundNetrin-1 is an axon guidance protein, which can inhibit inflammatory reaction and stabilize the blood-brain barrier to protect against experimental brain injury. We evaluated the concentration of netrin-1 in acute intracerebral hemorrhage (ICH) patients and explored whether netrin-1 is a potential prognostic biomarker for ICH. MethodsThis study recruited a total of 126 ICH patients and 126 healthy controls. Netrin-1 concentration was determined using a commercially available human enzyme-linked immune sorbent assay kit. National Institutes of Health Stroke Scale (NIHSS) score and hematoma volume were used to assess hemorrhagic severity. An unfavorable outcome was defined as modified Rankin Scale >2 at 90 days. ResultsICH patients showed significantly lower serum netrin-1 concentrations than controls. Serum netrin-1 concentrations were strongly and inversely correlated with serum C-reactive protein concentrations, NIHSS score and hematoma volume. Multivariate analyses revealed that low netrin-1 concentration was associated with 90-day death, unfavorable outcome and overall survival after adjustment for other confounding variables. Under the receiver operating characteristic curve, serum netrin-1 remarkably discriminated patients at risk of 90-day death and unfavorable outcome. ConclusionsSerum netrin-1 concentrations are decreased in patients with ICH, and the concentrations of netrin-1 were intimately associated with inflammation, hemorrhagic severity and clinical outcome of ICH.

Expression of Netrin-1 in Patients with Acute Lymphoblastic Leukemia and Its Clinical Significance

To investigate the correlation of the Netrin-1 expression level with the clinical characteristics in children with acute lymphoblastic leukemia (ALL) and to explore its possible regulatory mechanism. ELISA was used to detect the expression level of Netrin-1 in peripheral blood serum from 48 child ALL patients (newly diagnosed, recurrent), and its relevance with clinical indicators was statistically analyzed. The blood serum samples from 27 children with non malignant hematological diseases were choosen as controls. Leukemia cell lines of Jurkat,Molt-4,SUP-B15 and Raji were cultivated in vitro, after treated with different concentrations of recombinant human Netrin-1 protein， the invasive ability of the cells was detected by Transwell method； the effect of Netrin-1 to the proli feration of cells was detected by CCK-8 method； The expression and phosphorylation level of key molecules, such as FAK,Erk1/2,PI3K and Akt signaling pathway were detected by Western blot. The expression of Netrin-1 in child patients was significantly higher than that of the control group (P<0.05). With the increasing of Netrin-1 level, the level of Plt (r=0.483, P<0.05) increased, while the level of WBC (r=-0.290, P<0.05) decreased, and there were no significant correlation with age, Hb level and the proportion of immature cells in bone marrow. When the concentration of Netrin-1 was 25-50 ng/ml, the level of Netrin-1 positively correlated with WBC (r=0.886, P<0.05) ; the level of Netrin-1 significantly decreased when the patient's WBC was ＞50×109/L and Plt ＞20×109/L(P=0.042，P=0.001); The expression level of Netrin-1 was significantly different in the risk group(P=0.017), and level of Netrin-1 in high-risk group was significantly higher than that in low risk group and middle risk group, but there was no significant difference of Netrin-1 expression in sex, hepatosplenomegaly, MRD, recurrence and chromosome abnormality. Netrin-1 could promote the invasiveness of the four kinds of cells (P<0.05). With the increase of Netrin-1 concentration, the number of cells increased at first and then decreased, and the number of cells in the invading chamber was the highest when the concentration of Netrin-1 was 100 ng/ml; the survival rate of the four kinds of cells significantly increased when the concentration of Netrin-1 was 25 ng/ml(P<0.05), and SUP-B15 cells showed the highest cell survival rate at a concentration of 100 ng/ml; The survival rate of the four kinds of cells showed a tendency : survival of cells increased at low concentration of Netrin-1 and survival of cells decreased at high concentration of Netrin-1. The results of Western blot showed that Netrin-1 activated the phosphorylation level of key molecules such as FAK,Erk1/2,PI3K,Akt signaling pathway (P<0.05). There is abnormal expression of Netrin-1 in serum of children with ALL. Netrin-1 may affect the occurrence and development of ALL by increasing the proliferation and invasiveness of leukemia cells, and may become a risk factor of ALL or a potential target in biotherapy.

Netrin-1 and the Grade of Atherosclerosis Are Inversely Correlated in Humans.

Netrin-1 has been shown to play a role in the initiation of atherosclerosis in mice models. However, little is known about the role of Netrin-1 in humans. We set out to study whether Netrin-1 is associated with different stages of atherosclerosis. Approach and Results: Plasma Netrin-1 levels were measured in different patient cohorts: (1) 22 patients with high cardiovascular risk who underwent arterial wall inflammation assessment using positron-emission tomography / computed tomography, (2) 168 patients with a positive family history of premature atherosclerosis in whom coronary artery calcium scores were obtained, and (3) 104 patients with chest pain who underwent coronary computed tomography angiography imaging to evaluate plaque vulnerability and burden. Netrin-1 plasma levels were negatively correlated with arterial wall inflammation (β, -0.01 [95% CI, 0.02 to -0.01] R2, 0.61; P<0.0001), and concentrations of Netrin-1 were significantly lower when atherosclerosis was present compared with individuals without atherosclerosis (28.01 versus 10.51 ng/mL, P<0.001). There was no difference in Netrin-1 plasma concentrations between patients with stable versus unstable plaques (11.17 versus 11.74 ng/mL, P=0.511). However, Netrin-1 plasma levels were negatively correlated to total plaque volume (β, -0.09 [95% CI, -0.11 to -0.08] R2, 0.57, P<0.0001), calcified plaque volumes (β, -0.10 [95% CI, -0.12 to -0.08] R2, 0.53; P<0.0001), and noncalcified plaque volumes (β, -0.08 [95% CI, -0.10 to -0.06] R2, 0.41; P<0.0001). Treatment of inflammatory stimulated endothelial cells with plasma with high Netrin-1 level resulted in reduced endothelial inflammation and consequently, less monocyte adhesion. Netrin-1 plasma levels are lower in patients with subclinical atherosclerosis and in patients with arterial wall inflammation. Netrin-1 is not associated with plaque vulnerability; however, it is negatively correlated to plaque burden, suggesting that Netrin-1 is involved in some, but not all, stages of atherosclerosis.

Netrin-1 and its receptor Unc5b as markers of periodontal disease.

The aim of this present study was to evaluate the levels of Netrin-1 and Unc5b in periodontal health and disease. Netrin-1, acting via its receptor UNC5b, regulates the inflammatory response and takes apart in bone destructive diseases. Samples of gingival crevicular fluid (GCF), whole saliva, and serum were taken from systemically healthy, nonsmoking 20 periodontitis , 20 gingivitis patients, and 20 periodontally healthy subjects at baseline and 4weeks after nonsurgical periodontal treatment (NSPT). Whole-mouth and site-specific clinical periodontal parameters were recorded. Netrin-1 and Unc5b levels were measured by enzyme-linked immunosorbent assay. Data were analyzed by nonparametric tests. Total amount of Netrin-1 in GCF was significantly higher in periodontitis than the others, and the levels were significantly reduced after NSPT. Salivary and serum concentrations of Netrin-1 were significantly different among the study groups (P=.000), and NSPT significantly increased the concentration levels of both salivary and serum Netrin-1 (P<.05). Healthy subjects had significantly lower GCF (P=.001) and conversely, higher salivary and serum levels of Unc5b than the other groups (P=.002). The GCF levels of Unc5b were significantly reduced (P<.01), and conversely, serum concentrations were significantly increased after NSPT (P<.05). GCF Netrin-1 and Unc5b total amounts were positively correlated with clinical parameters (P<.01 and P<.05) whereas salivary Netrin-1 and Unc5b concentrations were negatively correlated with clinical parameters (P<.01 and P<.05). The results of this study indicate that Netrin-1 and its receptor Unc5b may have essential roles in periodontal inflammation and those can be assumed as useful therapeutic agent to control inflammation and periodontal breakdown.

Decreased Netrin-1 and Correlated Th17/Tregs Balance Disorder in Aβ1-42 Induced Alzheimer's Disease Model Rats.

There is increasing evidence indicating that inflammation represents a key pathological component of Alzheimer’s disease (AD). A possible factor that may contribute to this process is netrin-1, a neuronal guidance molecule. This molecule has been shown to exert an unexpected immunomodulatory function. However, the potential changes and correlations of netrin-1 with T helper 17/regulatory T cells (Th17/Tregs) as related to inflammation in AD has yet to be examined. In this study, netrin-1 and Th17/Tregs balance were investigated, and the relationship among netrin-1, Th17/Tregs and cognitive function were analyzed in a rat model of AD. In this model, a bilateral intracerebroventricular administration of Amyloid β1-42 (Aβ1–42) was used to produce spatial learning and memory deficits, as well as increased neuronal apoptosis, which were detected 7 days after injection for AD7d group and 14 days for AD14d group. Netrin-1 concentrations were significantly down regulated in both serum and cerebrospinal fluid (CSF) of these AD rats, effects which were strongly correlated with cognitive deficits. Increased levels of interleukin (IL)-17 and deceased IL-10 were observed in both the circulation and CSF and were also correlated with the percent of time spent in the target quadrant of AD in these rats. These changes resulted in netrin-1 concentrations being negatively correlated with IL-17 but positively correlated with IL-10 concentrations in the serum and CSF. We also found that the Th17/Tregs balance was disrupted in these AD rats. Collectively, these findings reveal that the reduction in netrin-1 and the correlated disruption of Th17/Tregs balance in AD rats may diminish the immunosuppressive effect of netrin-1 on Th17/Tregs in AD pathogenesis.