Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.
Humans ; Extracellular Traps ; Tetradecanoylphorbol Acetate/pharmacology* ; Neutrophils ; HL-60 Cells ; Tretinoin/pharmacology*

Objective:To evaluate the effect of c-CBL overexpression on activation of astrocytes in the spinal cord of rats with neuropathic pain and the relationship with Kindlin-1.Methods:Eighteen clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 3 groups ( n=6 each) using a random number table method: sham operation group (S group), neuropathic pain group (NP group) and c-CBL overexpression group (c-CBL group). The model of neuropathic pain was developed by chronic compression of the sciatic nerve in anesthetized rats.On 1 day before operation, c-CBL overexpression vector 10 μl was intrathecally injected in group c-CBL, while blank vehicle 10 μl was intrathecally injected in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured on 1 day before operation (T 0) and 1, 4 and 7 days after operation (T 1-3). The rats were sacrificed by decapitation after measurement of the pain threshold at T 3, and the spinal cord of L 4-6 was taken for determination of the expression of Kindlin-1, glial fibrillary acidic protein (GFAP), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β) (by Western blot) and co-expression of Kindlin-1 with c-CBL (by co-immunoprecipitation). Results:Compared with group S, the MWT was significantly decreased and TWL was shortened at T 2, 3 in group NP and group c-CBL, the expression of Kindlin-1, GFAP, TNF-α and IL-1β was significantly up-regulated in group NP, and the expression of c-CBL was significantly up-regulated in group c-CBL ( P<0.05). Compared with group NP, the MWT was significantly increased and TWL was prolonged at T 2, 3, the expression of Kindlin-1, GFAP, TNF-α and IL-1β was down-regulated, and the expression of c-CBL was up-regulated in group c-CBL ( P<0.05). The results of co-immunoprecipitation showed that there was a protein interaction and co-expression relationship between Kindlin-1 and c-CBL in group NP, and the co-expression of Kindlin-1 with c-CBL was enhanced in group c-CBL when compared with group NP. Conclusions:The overexpression of c-CBL can inhibit activation of astrocytes by down-regulating the expression of Kindlin-1 in the spinal cord, thus reducing inflammatory responses and relieving neuropathic pain in rats.

Background::Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated.Methods::Immunoblotting, real-time polymerase chain reaction, in vivo/ in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4 + T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data). Results::The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression ( r= 0.5110) and CD4 + T-cell counts ( r= 0.5083) in HIV-1-infected patients. Conclusions::USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

Objective:To evaluate the relationship between spinal kindlin-1/Wnt3a signaling pathway and inflammatory response in a rat model of neuropathic pain (NP).Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (SH group), NP group, kindlin-1 shRNA group (K group) and Wnt3a inhibition group (W group). NP was induced by chronic constrictive injury in anesthetized animals.At 21 days before operation, kindlin-1 shRNA adenovirus vector 10 μl was intrathecally injected in group K, and empty viral vector 10 μl was intrathecally injected in SH, NP and W groups.Wnt inhibitor IWP-2 10 μl was intrathecally injected in group W, and artificial cerebrospinal fluid 10 μl was intrathecally injected in SH, NP and K groups at 1-3 days after operation.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 4 and 7 days after operation, respectively.At the end of pain threshold measurement at 7 days after operation, the animals were sacrificed and the lumbar segments (L 4-6) of the spinal cord were obtained for determination of the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay) and the expression of kindlin-1 and Wnt3a (by Western blot). Results:Compared with group SH, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, and the contents of TNF-α and IL-1β in spinal cord were increased in NP, K, and W groups, the expression of kindlin-1 and Wnt3a was up-regulated in NP and W groups, and expression of Wnt3a was up-regulated in group K ( P<0.05). Compared with group NP, MWT was significantly increased and TWL was prolonged at 4 and 7 days after operation in K and W groups, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of kindlin-1 and Wnt3a was down-regulated in group K, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of Wnt3a was down-regulated in group W ( P<0.05), and no significant change was found in kindlin-1 expression ( P>0.05). Conclusion:Spinal kindlin-1 regulates the inflammatory response by up-regulating the expression of Wnt3a, and it is involved in the maintenance of NP in rats.

Objective:To investigate the relationship between spinal long chain noncoding RNA (lncRNA) and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain (NP).Methods:The experiment was performed in two parts.Experiment Ⅰ Sprague-Dawley rats of both sexes, aged 7 days, weighing 15-20 g, were selected.Rats were sacrificed, the dorsal horn of spinal cord was removed, and the primary astrocytes were extracted and cultured.Lipopolysaccharide 1 μg/ml was added to induce the activation of astrocytes for 24 h. The lncRNA binding to kindlin-1 was identified using PCR immunoprecipitation method.The localization of lncRNA FOXF1-AS1 in astrocytes was observed by fluorescence in situ hybridization, and the binding between lncRNA FOXF1-AS1 and kindlin-1 was detected by biotin-labeled magnetic bead method.Experiment Ⅱ Thirty clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 5 groups ( n=6 each) using a random number table method: sham operation control group (group C), NP group, lncRNA FOXF1-AS1 overexpression group (group F), lncRNA FOXF1-AS1 overexpression plus kindlin-1 shRNA group (group FK) and lncRNA FOXF1-AS1 overexpression + Wnt inhibitor group (group FW). NP was induced by chronic constrictive injury in anesthetized animals.In group F, lncRNA FOXF1-AS1 overexpression lentivirus 10 μl was intrathecally injected at 28 days before operation, and vector virus 10 μl was intrathecally injected in the other groups.In FK group, kindlin-1 interfering shRNA interference adenovirus 10 μl, and vector virus 10 μl was intrathecally injected in the other groups.In group FW, Wnt inhibitor IWP-2 10 μl was intrathecally injected at 1-3 days after operation, artificial cerebrospinal fluid 10 μl was intrathecally injected at the same time point in the other groups.Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at 1 day before operation, at 4 days and 7 days after operation.The animals were sacrificed at the end of measurement of pain threshold at 7 days after operation, and the spinal cord tissues were taken for determination of the expression of kindlin-1, Wnt3a and glial fibrillary acidic protein (GFAP) (by Western blot) and the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay). Results:ExperimentⅠ lncRNA FOXF1-AS1, which was expressed in the cytoplasm of astrocytes, combined with kindlin-1.Experiment Ⅱ Compared with C group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in group NP ( P<0.05). Compared with NP group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in F group, MWT was increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and the expression of kindlin-1 was up-regulated, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Compared with group F, MWT was significantly increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of spinal kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Conclusion:lncRNA FOXF1-AS1 can up-regulate kindlin-1 expression, activate Wnt3a signaling pathway, promote astrocyte activation, and then regulate inflammatory responses and is involved in the process of neuropathic pain in rats.

Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.
Cytoskeletal Proteins ; Endothelial Cells ; Extracellular Traps ; Integrins ; Intercellular Adhesion Molecule-1 ; Lipopolysaccharides/pharmacology* ; Macrophages ; Neutrophils

Objective To evaluate the role of spinal kindlin-1 in neuropathic pain in rats and the relationship with Wnt3a.Methods Eighteen clean-grade healthy male Sprague-Dawley rats,weighing 250-280 g,aged 10-12 weeks,were divided into 3 groups (n =6 each) using a random number table:sham operation group (group S),neuropathic pain group (group NP) and kindlin-1 inhibitor group (group K).Neuropathic pain was induced by chronic compression of the sciatic nerve.The sciatic nerve was only exposed but not ligated in group S.In group K,shRNA was intrathecally injected at 21 days before operation to inhibit the expression of kindlin-1.Vector virus was intrathecally injected at 21 days before operation in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 1,4,7,10 and 13 days after operation.Rats were sacrificed at 13 days after measurement of pain threshold and the spinal cord was removed for determination of the expression of kindlin-1 and Wnt3a (by Western blot) and expression of Wnt3a mRNA (by real-time polymerase chain reaction).Results Compared with group S,the MWT was significantly decreased and the TWL was shortened at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was up-regulated in group NP (P＜0.05).Compared with group NP,the MWT was significantly increased and the TWL was prolonged at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was down-regulated in group K (P＜0.05).Conclusion Kindlin-1 is involved in the development of neuropathic pain by up-regulating the expression of Wnt3a in rats.

PURPOSE: To investigate the effects of hyperbaric oxygen (HBO) pretreatment on cognitive decline and neuronal damage in an Alzheimer’s disease (AD) rat model. MATERIALS AND METHODS: Rats were divided into three groups: normal saline (NS), AD, and HBO+AD. In the AD group, amyloid β peptide (Aβ)₁₋₄₀ was injected into the hippocampal CA1 region of the brain. NS rats received NS injection. In the HBO+AD group, rats received 5 days of daily HBO therapy following Aβ₁₋₄₀ injection. Learning and memory capabilities were examined using the Morris water maze task. Neuronal damage and astrocyte activation were evaluated by hematoxylin-eosin staining and immunohistochemistry, respectively. Dendritic spine density was determined by Golgi-Cox staining. Tumor necrosis factor-α, interleukin-1β, and interleukin-10 production was assessed by enzyme-linked immunosorbent assay. Neuron apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Protein expression was examined by western blotting. RESULTS: Learning and memory dysfunction was ameliorated in the HBO+AD group, as shown by significantly lower swimming distances and escape latency, compared to the AD group. Lower rates of neuronal damage, astrocyte activation, dendritic spine loss, and hippocampal neuron apoptosis were seen in the HBO+AD than in the AD group. A lower rate of hippocampal p38 mitogen-activated protein kinase (MAPK) phosphorylation was observed in the HBO+AD than in the AD group. CONCLUSION: HBO pretreatment improves cognition and reduces hippocampal damage via p38 MAPK in AD rats.
Alzheimer Disease/*therapy ; Amyloid beta-Peptides/*administration & dosage ; Animals ; Apoptosis ; *Cognition/drug effects ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Hippocampus/*enzymology ; *Hyperbaric Oxygenation ; In Situ Nick-End Labeling ; Interleukin-10/biosynthesis ; Interleukin-1beta/biosynthesis ; Learning/drug effects ; Male ; Memory/drug effects ; Neurons ; Peptide Fragments/*administration & dosage ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride/administration & dosage ; Tumor Necrosis Factor-alpha/biosynthesis ; p38 Mitogen-Activated Protein Kinases/*metabolism

Objective To evaluate the effect of dexmedetomidine on the expression of brain-derived neurotrophic factor (BDNF) during lidocaine-induced spinal neurotoxicity in rats.Methods Fifty-six male Sprague-Dawley rats,weighing 250-280 g,aged 2-3 months,were divided into 7 groups (n =8 each) using a random number table:sham operation group (group S),lidocaine group (group L),normal saline group (group NS),3 different doses of dexmedetomidine groups (D1,D2 and D3 groups),and oα2-adrenoceptor antagonist yohimbine group (group Y).An epidural catheter was placed at L5.6 interspace.Ten percent lidocaine 20 μl was injected intrathecally in all groups except group S.Dexmedetomidine 5,15 and 25 μg/kg were injected intraperitoneally at 30 min before lidocaine injection in D1,D2 and D3 groups,respectively.In group Y,yohimbine 1.0 mg/kg was injected intraperitoneally at 15 min before dexmedetomidine 25 μg/kg was injected.Before intrathecal administration and at 24,48 and 72 h after intrathecal administration (T1-4),the Basso,Beattie,Bresnahan (BBB) Locomotor Rating Scale was used,and the tail flick latency to a thermal nociceptive stimulus (TFL) was measured to assess the locomotor function.The rats were sacrificed after the last behavioral test,and the lumbar segments (L3-5) of the spinal cord were removed for pathological examination and for determination of BDNF expression and cell apoptosis.The apoptosis index was calculated.Results Compared with group S,the BBB score was significantly decreased at T2-4,the TFL was prolonged,the BDNF expression was up-regulated,and apoptosis index was increased in L,NS,D1,D2 and D3 groups (P＜0.05).Compared with group L,the BBB score was significantly increased at T2-4,the TFL was shortened,the BDNF expression was up-regulated,and apoptosis index was decreased in group D3 (P＜0.05),and no significant change was found in the parameters mentioned above in NS,D1,D2 and Y groups (P＞0.05).Compared with group D3,the BBB score was significantly decreased at T2 4,the TFL was prolonged,the BDNF expression was down-regulated,and apoptosis index was increased in group Y (P＜0.05).The pathological changes of the spinal cord were significantly attenuated in group D3 as compared with group L,and there was no significant difference in pathological changes of the spinal cord between group Y and group L.Conclusion The mechanism by which dexmedetomidine reduces lidocaine-induced spinal neurotoxicity may be related to up-regulation of BDNF expression,and the mechanism by which dexmedetomidine up-regulates BDNF expression is completely related to activation of α2-adrenoceptors in rats.

Objective To evaluate the effects of tempol administered via different routes on neuropathic pain (NP) in rats.Methods Thirty-two male Sprague-Dawley rats,weighing 250-280 g,aged 8-10 weeks,were randomly divided into 4 groups (n=8 each) using a random number table:sham operation group (group S),group NP,intrathecal tempol group (group T1),and intraperitoneal tempol group (group T2).Neuropathic pain was induced by chronic constriction injury in chloral hydrate-anesthetized rats.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread.The sciatic nerve was only exposed but not ligated in group S.After successful establishment of the model,a catheter was inserted at L4.5 interspace into the epidural space.In S and NP groups,0.9％ normal saline 20 μl was injected intrathecally,and 0.9％ normal saline 200 μl was injected intraperitoneally once a day for 7 consecutive days.In group T1,tempol 30 μg (in 20 μl of normal saline) was injected intrathecally,and 0.9％ normal saline 200 μl was injected intraperitoneally once a day for 7 consecutive days.In group T2,tempol 30 μg (in 200 μl of normal saline) was injected intraperitoneally,and 0.9％ normal saline 20 μl was injected intrathecally once a day for 7 consecutive days.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 3 days before operation,and at 1,3,5,7,10 and 14 days after operation.The animals were sacrificed after measurement of pain threshold at day 14 after operation.The lumbar segment of the spinal cord was removed to detect malondialdehyde (MDA) content and superoxide dismutase (SOD) activity by enzyme-linked immunosorbent assay.Results Compared with group S,the MWT was significantly decreased,and the TWL was shortened at each time point after operation,the content of MDA in the spinal cord was increased (P＜0.05),and no significant difference was detected in SOD activity in group NP (P＞0.05).Compared with group NP,the MWT was significantly increased at 5,7,10 and 14 days after operation,the TWL was prolonged at 1,3,5,7,10 and 14 days after operation,the content of MDA in the spinal cord was decreased,and the SOD activity was increased in group T1 (P＜0.05),and no significant change was found in the indexes mentioned above in group T2 (P＞0.05).Conclusion Intrathecal tempol can reduce NP in rats,and the mechanism is related to inhibition of lipid peroxidation in the spinal cord.