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Inhibition of neurite outgrowth using comme(3)
MAG was primarily distributed along cell membranes in a circular manner. As time progressed, the membranous circular appearance of MAG positivity broadened and increased in intensity, and was particularly distinctive after 50 to 60 minutes of MAG treatment. A few localized, strong immunofl uorescent MAG-positive dots of various sizes were observed both on the surface of cell membranes and in the cytoplasm(Figure 1). The cytoplasmic visualization of MAG in neuro-2a cells by addition of MAG-Fc to the medium indicated that MAG-Fc was possibly endocytosed through specific MAG receptors on the plasma membrane of neuro-2a cells.
Expression NgR and PirB and their colocalization with MAG
The immuno fluorescence of both NgR and PirB was evaluated to verify whether neuro-2a cells expressed NgR and PirB after incubation with MAG-Fc for one hour. The results revealed that neuro-2a cells were positive for both NgR and PirB. Neuro-2a cells cultured for 4 days exhibited relatively weak NgR immuno fluorescence and moderate PirB immunoreactivity. In addition, NgR staining was mainly distributed along the plasma membrane and also appeared as strong immuno fluorescent foci along the membrane. However, the appearance of PirB staining was different from that of NgR;it was primarily cytoplasmic and diffuse (Figure 2).
Incubation of cells with MAG-Fc (20 nM) for 1 hour enhanced the expression of NgR. Co-localization of NgR and MAG was clearly observed both on plasma membranes and in the cytoplasm. Furthermore, the addition of MAGFc did not alter the expression of PirB, although there appeared to be more double labeling of PirB and MAG based on immuno fluorescence (Figure 2).
Figure 1 Immuno fluorescence of MAG in neuro-2a cells treated with 20 nM MAG-Fc for 10 minutes to 6 hours.(A) Intracellular level and distribution of MAG detected by immuno fluorescence staining (scale bar: 50 μm). Arrowheads: Positive MAG dots in the cytoplasm; arrows: positive MAG dots near the plasma membrane (green: Alexa Fluorescence-488). (B) MAG levels detected by western blotting. (C) MAG immunoreactivity. Data are expressed as the mean ± SE (one-way analysis of variance followed by t-test). **P < 0.01, ***P < 0.001, vs. 0 min (control group).MAG: Myelin-associated glycoprotein; min: minute(s); h: hour(s).
Figure 2 Expression of NgR and PirB in neuro-2a cells with or without MAG treatment.(A) Cultured neuro-2a cells showed NgR-positive immunofluorescence staining, which mainly appeared along the cell membrane. (B) There was increased NgR expression when MAG-Fc was added to the medium at cell plating. NgR staining was clearly observed in the cytoplasm. (C) Double labeling of NgR and MAG was observed in most of the NgR-positive cells. (D)Cultured neuro-2a cells also exhibited positive PirB immuno fluorescence staining. In contrast to NgR, PirB immunoreactivity was primarily distributed in the cytoplasm. (E) The level of PirB staining did not increase further with MAD incubation, and was similar to the pattern observed without MAG-Fc treatment. (F) A few cells were double-labeled for PirB and bars: 50 μm. Green: Anti-NgR or anti-PirB labeled with Alexa Fluorescence-488; red: anti-MAG labeled with Alexa Fluorescence-594; blue:DAPI counterstaining for nuclei; arrowhead: location of double-labeling for NgR/MAG or PirB/MAG. w/o: Without; MAG: myelin-associated glycoprotein; PirB: paired immunoglobulin-like receptor B; NgR: Nogo receptor.
These findings indicated that MAG-Fc was endocytosed into neuro-2a cells via binding to its receptor, either NgR or PirB. Endocytosis of MAG-Fc upregulated the expression of NgR but not PirB in neuro-2a cells, or increased the rafting/clustering of NgR on the plasma membrane. Therefore, exogenous MAG may affect neuritogenesis by interacting with NgR and its relevant intracellular signaling pathways.
Effect of MAG-Fc on RhoA activity and phosphorylation of ROCK
Neuro-2a cells displayed a basal level of RhoA activation during culture. After 20 nM MAG-Fc was added to cells for 5, 10, 20, or 40 minutes, the activity of RhoA was 20 minutes of MAG-Fc treatment, the activity of RhoA was increased to nearly 1.5 times that of the 0 nM MAG-Fc group (P < 0.01). However, RhoA activity dropped to the level observed after 5 minutes of treatment when the MAGFc incubation time reached 40 minutes. This result indicated that activation of RhoA by MAG-NgR/PirB binding was a short-acting intracellular response; however, other intracellular events should proceed after RhoA activation.
Figure 3 Effects of exogenous MAG treatment on RhoA activity in neuro-2a cells (ELISA assay).Data are expressed as the mean ± SE (n = 6 per group; one-way analysis of variance followed by t-test). **P < 0.01, ***P < 0.001. ELISA: Enzyme-linked immunosorbent assay; MAG: myelin-associated glycoprotein;ns: not significant; min: minutes.
Figure 4 Effects of exogenous MAG-Fc on the phosphorylation of ROCK II in neuro-2a cells.(A, B) Western blot assay showed that after MAG-Fc was administered,increased p-ROCK II levels were observed compared to the control(*P < 0.05, vs. control group), but the increased level dropped after MAG treatment for 40 minutes, although the level remained higher than are expressed as the mean ± SE(one-way analysis of variance followed by t-test). (C) Immunofluorescence of cytoplasmic p-ROCK II in the MAG treatment group was generally stronger than that in controls. Some p-ROCK II foci were clearly identified in the cytoplasm, especially in the area adjacent to the membrane (arrows).(a–e) DAPI counterstaining; (f–g)anti-p-ROCK; (k–o) merged images of DAPI (blue) and anti-p-ROCK(green). Scale bar: 50 μm. Green color: Alexa Fluorescence-488; blue color: DAPI counterstaining of : myelin-associated glycoprotein;p-ROCK II: phosphorylated Rho-associated protein kinase II; DAPI:4′,6-diamidino-2-phenylindole; min:minutes.
文章来源:《新商务周刊》 网址: http://www.xswzkzzs.cn/qikandaodu/2020/1021/526.html