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BACKGROUND:Microglia cells play a major role in maintaining the balance as well as the development and function reconstruction of the central nervous system.As a histone deacetylase inhibitor,Scriptaid can inhibit neuroinflammation and enhance neuroprotection. OBJECTIVE:To investigate the effect of silk fibroin methacryloyl hydrogel loaded with Scriptaid on the behaviors of microglia cells. METHODS:Microglia(BV2 cells)were cultured in medium containing different concentrations of Scriptaid(0,0.1,0.5,1,2,5,10 μmol/L).The optimal concentration of Scriptaid was screened by the CCK-8 assay and live/dead cell staining.Silk fibroin methacryloyl hydrogels loaded with or without Scriptaid were prepared using photocuring.The micromorphology,swelling properties,mechanical properties,slow release properties,and hydrophilicity of the hydrogels were characterized.Microglia(BV2 cells)were inoculated in the subventricular region of 24-well Transwell and cultured in five groups.In the control group,the cell culture medium was added to the lower chamber.In the lipopolysaccharide group,Scriptaid group,hydrogel group,and drug-loaded hydrogel group,cell culture media containing lipopolysaccharide were added into the lower chamber.After 24 hours of lipopolysaccharide intervention,in the Scriptaid group,hydrogel group and drug-loaded hydrogel group,Scriptaid,silk fibroin methacryloyl hydrogel,and silk fibroin methacryloyl hydrogel loaded with Scriptaid were added to the upper chamber,respectively.The culture medium was replaced with ordinary culture medium and continued to culture for 24 hours.The cell viability was detected by CCK-8 assay,and the cell phenotype was detected by immunofluorescence staining of induced nitric oxide synthase and arginase 1. RESULTS AND CONCLUSION:(1)Compared with the group without Scriptaid,the viability and number of BV2 cells were decreased after Scriptaid added.When Scriptaid 2 μmol/L or above was added,the cell viability was lower than the standardized cell viability(70%),and the number of BV2 cells was significantly reduced.Therefore,1 μmol/L Scriptaid was selected to be loaded into the hydrogel.(2)Characterization experiments showed that the addition of Scriptaid did not affect the microscopic morphology,swelling rate of water absorption,compression modulus and hydrophilicity of silk fibroin methacryloyl hydrogel,and silk fibroin methacryloyl hydrogel had slow release performance.(3)The result of CCK-8 assay showed that compared with the control group,silk fibroin methacryloyl hydrogel significantly increased the viability of BV2 cells(P<0.001).(4)Immunofluorescence staining showed that compared with the control group,the expression of inducible nitric oxide synthase was increased in the lipopolysaccharide group(P<0.01);the expression of inducible nitric oxide synthase was decreased(P<0.01)and the expression of arginase 1 was increased(P<0.001)in the drug-loaded hydrogel group;the expression of arginase 1 was increased in the Scriptaid group(P<0.01).(5)The results indicate that Scriptaid-loaded silk fibroin methacryloyl hydrogel is able to promote polarization of microglia to the M2 type after lipopolysaccharide induction.
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Liver fibrosis is the healing reaction of chronic liver injury caused by various factors such as viral infection， alcohol， and chemical substances and is a key link in the progression of chronic liver diseases to liver cirrhosis and liver cancer. Liver macrophages are considered important mediators of liver injury and repair， and the polarization trend of macrophages has a bidirectional regulatory effect on liver fibrosis. This article reviews the role of different phenotypes of liver macrophages in the development and progression of liver fibrosis， in order to provide new ideas for the prevention and treatment of fibrosis.
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Objective:To investigate the effects of mild hypothermia on microglia polarization and janus kinase 2/signal transduction and transcriptional activation factor 3 (JAK2/STAT3) signaling pathway during cerebral ischemia-reperfusion (I/R) in rats.Methods:Forty-five clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 3 groups ( n=15 each) by the random number table method: sham operation group (S group), cerebral I/R group (I/R) and mild hypothermia group (H group). In I/R group and H group, cerebral I/R was induced by middle cerebral artery occlusion using a nylon thread in anesthetized animals, the nylon thread was removed to restore the perfusion after 2 h of occlusion, and the rectal temperature was maintained at 36-37 ℃ during the period. Group H was wiped with 75% alcohol for 3 h starting from the time point immediately after reperfusion, and the rectal temperature was maintained at 32-33℃. Modified neurological severity score (mNSS) was evaluated at 24 h of reperfusion. Animals were then sacrificed for determination of the cerebral infarct size (using TTC staining), expression of M1 marker inducible nitric oxide synthase (iNOS), M2 marker arginase 1(Arg-1), phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)(by Western blot), expression of iNOS mRNA and Arg-1 mRNA (by quantitative polymerase chain reaction), and contents of interleukin-6 (IL-6) and IL-10 (by enzyme-linked immunosorbent assay). Results:Compared with group S, mNSS and cerebral infarct size were significantly increased, the expression of iNOS, Arg-1 protein and mRNA in cerebral ischemic penumbral zone was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio, and contents of IL-6 and IL-10 were increased in the other two groups ( P<0.05). Compared with I/R group, mNSS and cerebral infarct size were significantly decreased, the expression of iNOS protein and mRNA in cerebral ischemic penumbral zone was down-regulated, the expression of Arg-1 and mRNA was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio and IL-6 content were decreased, and the IL-10 content was increased in group H ( P<0.05). Conclusions:Mild hypothermia can promote the polarization shift of microglia from M1 to M2 phenotype during cerebral I/R and inhibit the central inflammatory responses, and the mechanism may be related to inhibition of JAK2/STAT3 signaling pathway in rats.
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Objective:To evaluate the role of miR-124-3p in reduction of oxygen-glucose deprivation and restoration (OGD/R) injury by electrostimulation preconditioning in microglia and its relationship with microglial polarization.Methods:The well-growing BV2 cells were divided into 4 groups ( n=30 each) by the random number table method: control group (group C), OGD/R group, electrostimulation preconditioning group (group E) and miR-124-3p inhibitor group (group I). Group C was cultured under normal conditions, and group OGD/R was deprived of oxygen and glucose for 2 h followed by restoration of oxygen and glucose supply for 24 h to develop the OGD/R injury model. In group E, cells were stimulated with 100 mV/mm direct current for 4 h before oxygen-glucose deprivation, and the other treatments were similar to those previously described in group OGD/R. Group I was transfected with micrOFF? mmu-miR-124-3p inhibitor at 48 h before oxygen-glucose deprivation, and the other treatments were similar to those previously described in group E. The cell survival rate was determined by CCK-8 assay, the concentrations of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-10 in the cell supernatant were measured by enzyme-linked immunosorbent assay. The expression of a surface marker of M1 microglia inducible nitric oxide synthase (iNOS) and a surface marker of M2 microglia arginase 1 (Arg-1) was detected by immunofluorescence and Western blot, respectively. The expression of iNOS and Arg-1 mRNA and miR-124-3p was detected by quantitative polymerase chain reaction. Results:Compared with group C, the cell survival rate was significantly decreased, the concentrations of TNF-α, IL-1β and IL-10 in the supernatant were increased, and the expression of iNOS and Arg-1 protein and mRNA and miR-124-3p was up-regulated in the remaining three groups ( P<0.05). Compared with group OGD/R, the cell survival rate was significantly increased, the concentrations of TNF-α and IL-1β in the supernatant were decreased, the IL-10 concentration was increased, the expression of iNOS protein and mRNA was down-regulated, and the expression of Arg-1 protein and mRNA and miR-124-3p was up-regulated in E and I groups ( P<0.05). Compared with group E, the cell survival rate was significantly decreased, the concentrations of TNF-α and IL-1β in the supernatant were increased, the IL-10 concentration was decreased, the expression of iNOS protein and mRNA was up-regulated, and the expression of Arg-1 protein and mRNA and miR-124-3p was down-regulated in group I ( P<0.05). Conclusions:The mechanism by which electrostimulation preconditioning reduces OGD/R injury in microglia is related to up-regulation of the expression of miR-124-3p, promotion of M2 microglia polarization, inhibition of M1 microglia polarization, and thus inhibiting the inflammatory responses.
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This article reviews the research progress in promotion of peripheral nerve regeneration by regulating macrophages, a new idea for the research and treatment of peripheral nerve injury. The Trauma Center of The First People's Hospital Affiliated to Shanghai Jiao Tong University reviewed the relevant literatures in the regulation of macrophages on peripheral nerve regeneration at home and abroad, from 2013 to 2021, were reviewed and analysed. Of the study from 2013 to 2021, autologous nerve transfer was the main option in the treatment of peripheral nerve injury, but it has many setbacks such as insufficient donor, new nerve injury and local neuroma. Modulating macrophage-related function can effectively improve the prognosis of nerve injury. In recent years, the regulation of macrophages in the treatment of nerve injury is mainly through the mechanism of M1 macrophages polarisation to M2 macrophages, increase of phagocytosis, change of the phenotype of macrophages, and so on. By studying the characteristics of macrophages and regulating the function and phenotype of macrophages, it would provide a new idea and important research direction in the treatment of peripheral nerve injury.
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Objective To observe the regulatory effect of olfactory ensheathing cells (OECs) on polarization of M2 macrophages after acute contusion of spinal cords in rats,and explore the mechanism of OECs transplantation in promoting repair of spinal cord injury.Methods The primary OECs were isolated and cultured for transplantation.The T10 spinal cords of SD rats were exposed and impacted by impact stick (10 g) dropping vertically from a height of 25 mm with a NYU-Ⅱ impactor.Twenty-four rats with contusion of spinal cord immediately after injury were randomly divided into DMEM/F12 control group and OECs transplantation group according a random number table.DMEM/F12 culture medium or OECs suspension (3×104/μL,1 μL×3 times) was implanted into the injured spinal cords.At one to 9 weeks after injury,Basso Beattie Bresnahan (BBB) scale was used to evaluate the motor functions in rats.At one week after injury,M2 macrophages and Nogo-A positive cells were labeled by immunofluorescence staining;the expressions of interleukin (IL)-4 and IL-6 were detected by Western blotting.At 9 weeks after injury,the pathological changes of injured spinal cords were observed by HE staining.Results One and 9 weeks after injury,BBB scores in OECs transplantation group were significantly higher than those in control group at each time point after injury (P＜0.05).One week after injury,the number of M2 macrophages in OECs transplantation group was significantly larger than that in control group (3.24％ ±0.56％ vs.0.63％ ±0.21％),and the number of Nogo-A positive cells was significantly smaller and fluorescence intensity was significantly lower in OECs transplantation group than those in control group ([43±24] field vs.([207±88]/field;0.042±0.006 vs.0.062±0.011,P＜0.05);the IL-4 protein level was significantly higher and the IL-6 protein level was significantly lower in OECs transplantation group than those in the control group (0.717±0.152 vs.0.183±0.063;0.550±0.124 vs.1.060±0.209;P＜0.05).Nine weeks after injury,the cytoplasmic cavity area in OECs transplantation group ([1.511±0.581] mm2) was significantly lower than that in control group [2.939±0.823] mm2,P＜0.05).Conclusion OECs transplantation may promote macrophages polarizing to subtype M2,improve the microenvironment of inflammation,reduce Nogo-A secretion,thus promote the structure and spinal cord function recovery.
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Objective To investigate the role and mechanism of B7-H4, a negative costimulatory molecule, in mediating the immunomodulatory effects of mesenchymal stem cells C3H10T1/2 (C3H10) on T cell polarization. Methods The lentiviral vectors that carried the shRNA targeting mouse B7-H4 were transfected into mouse mesenchymal stem cells (C3H10-B7-H4). The cells were co-cultured with PHA-acti-vated mice spleen lymphocytes before and after the transfection. ELISA was performed to detect the concen-trations of cytokines in supernatants of cell culture in order to elucidate the effects of B7-H4 expressed by C3H10 on T cell polarization. A mouse model of experimental allergic encephalitis (EAE) was established. Fifty C57BL/6 mice were divided into five groups including control group, EAE group, C3H10 group (injec-ting EAE mice with C3H10 cells), C3H10-NC group ( injecting EAE mice with C3H10-NC cells) and C3H10-B7-H4 group (injecting EAE mice with C3H10-B7-H4 cells). ELISA was performed to detect the soluble form of IL-2, IL-17, IFN-γ and IL-4 in plasma samples. Results Knocking down the B7-H4 gene with shRNA significantly decreased the expression of B7-H4 on C3H10 cells, which weakened the inhibitory effects of C3H10 cells on the secretion of IL-2, IL-17 and IFN-γ by spleen lymphocytes. The therapeutic effects of C3H10-B7-H4 cells on mice with EAE were weakened after silencing the B7-H4 gene expression, which was manifested as higher nerve function score and earlier onset and bring forwarded peak time of EAE than those of the C3H10 group. Treating EAE mice with C3H10-B7-H4 cells was less efficient in inhibiting the expression of IL-2, IL-17 and IFN-γin plasma. However, knocking down the B7-H4 gene had no signif-icant effect on the expression of IL-4 in terms of treating EAE with C3H10 cells. Conclusion The co-inhib-itor molecule B7-H4 expressed on C3H10 cells mediated the treatment of EAE with C3H10 cells by regula-ting Th1 and Th17 effector T cells.
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Objective To explore the influence of IL-2 pretreatment on splenic lymphocyte following mobilization with G-CSF, which may provide a new approach to attenuate acute graft-versus-host disease (GVHD). Methods Splenic cells and na(i)ve CD4+T cells from C57BL/6N mice receiving G-CSF-mobilized were pretreated with IL-2(50 U/ml) , and cocultured with the allogeneic antigens from BALB/c mice. The proliferation responses and the polarization of T cells were determined. C57BL/6N mice were randomly divided into 4 groups: G-CSF + IL-2 group, G-CSF group, IL-2 group , control group. Results Compared with the control group, IL-4 increased obviously while IFN-γ decreased significantly in the group of G-CSF + IL-2. The proliferation responses were also suppressed in vitro. Conclusion The proliferation responses of splenic cells and naive CD4 + T cells from C57BI/6N mice receiving G-CSF-mobilized to the allogeneic antigens were significantly abrogated by the pretreatment with IL-2, T cells were polarized toward the production of type-2 cytokines. The combination of G-CSF and IL-2 is potentially synergetic in the induction of T lymphocyte immune tolerance.
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Objective:This study was to analyze the changes of CD4~+ T lymphocytes and their subtypes,Th1 and Th2 cells,in the peripheral blood of patients with pulmonary tuberculosis disease and to investigate their clinical significance in the pathologic process of pulmonary tuberculosis.Methods:For polarization measurement of T-helper cells,1∶100 diluted Ionomycin and 1∶10 diluted Monensin were added in sequence into the equivalent volume mixture of heparin anti-coagulated whole blood and RPMI-1640 culture liquid.After being well mixed,the mixture was incubated at 5% CO_2,37℃ for 4 hours or overnight.To 100 μl of the mixture and in sequence,the antibodies of CD3-PerCP,CD8-APC,mIgG1-FITC,Rat IgG1-PE,IL-4-PE or IFN-γ-FITC were added.The stained CD4~+ IL-4~+ (Th2) and CD4~+ IFN-γ~+ (Th1) were analyzed by flow cytometry.Results:Compared with those from healthy controls,the peripheral blood of pulmonary tuberculosis patients contained significantly fewer Th1 (P＜0.01) but significantly more Th2 cells (P＜0.05).Th1 cells in the peripheral blood of the patients with miliary pulmonary tuberculosis were obviously fewer (P＜0.05) than in infiltrative pulmonary tuberculosis patients.The amount of Th2 cells in the peripheral blood of the patients with miliary pulmonary tuberculosis was significantly more (P＜0.05) than in either infiltrative pulmonary tuberculosis or tuberculous pleurisy patients.The ratio of CD4~+/CD3~+ tended to decrease in all these patients,and it was much lower (P＜0.05) in the patients of miliary pulmonary tuberculosis than in infiltrative pulmonary tuberculosis patients.Patients suffering from both pulmonary tuberculosis and diabetes had significantly lower levels of Th1 cells and CD4~+/CD3~+ (P＜0.05) and more Th2 cells,compared with those of pulmonary tuberculosis patients without diabetes.Levels of Th1 and Th2 cells restored significantly (P＜0.05) in 15 severe pulmonary tuberculosis patients after receiving tuberculosis chemotherapy and microcalorie theropy for three months.Patients with positive sputum examination tended to have decreased Th1 and CD4~+/CD3~+ (P＞0.05) and significantly increased Th2 (P＜0.05).Conclusion:Immunosuppression existed,in different extents,in patients of infiltrative pulmonary tuberculosis,tuberculous pleurisy,miliary pulmonary tuberculosis as well as patients with both pulmonary tuberculosis and diabetes.Analysis of Th1,Th2 and CD4~+/CD3~+ may be benefit for the judgments of disease conditions and therapeutic effects.
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Hepatitis C virus cell entry is mediated by multiple factors, including various receptors and cellular factors that trigger virus uptake by the hepatocyte. Occludin is a newly identified essential co-receptor for HCV entry together with CD81, SR-B1 and CLDN1. CLDN1 and occludin highlight the importance of studying the effects of tight junction and cell polarization on HCV entry. Study on cell polarization and tight junction can help to discover new targets for HCV therapy, and therefore interfere the cell entry and cell-cell spread of HCV. This review summarizes the current knowledge of hepatocyte polarization, tight junction and its major integral proteins CLDN1 and occludin, polarized cell culture system and its relation with HCV entry.
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Hepatitis C virus cell entry is mediated by multiple factors,including various receptors and cellular factors that trigger virus uptake by the hepatocyte.Occludin is a newly identified essential co-receptor for HCV entry together with CD81,SR-B1 and CLDN1.CLDN1 and occludin highlight the importance of studying the effects of tight junction and cell polarization on HCV entry.Study on cell polarization and tight junction can help to discover new targets for HCV therapy,and therefore interfere the cell entry and cell-cell spread of HCV.This review summarizes the current knowledge of hepatocyte polarization,tight junction and its major integral proteins CLDN1 and occludin,polarized cell culture system and its relation with HCV entry.