Résumé
Objective:To investigate the effects of rapamycin (RAPA) on the cognitive function of lupus mice by regulating Cysteine rich 61 (Cyr61) and autophagy.Methods:MRL/lpr lupus mice were randomly divided into lupus group and rapamycin + lupus group, wild-type C57BL/6 mice were randomly divided into normal control group and rapamycin group with six mice in each group, RAPA + lupus group and rapamycin group were intraperitoneally injected with RAPA (2.0 mg/kg). The lupus group and the normal control group were injected with equal amounts of dimethyl sulfoxide (DMSO). Morris water maze was used to observe the cognitive function of mice. Western blotting was used to detect the expression of Cyr61, Programmed cell death-1 (Beclin-1), Microtubule associated protein 1 light chain3B (LC3B). Hematoxylin-eosin (HE) staining was used to observe the pathological changes in the hippocampus. The changes of neurons and bodies in hippocampus were observed by Nissl staining. The localization and expression of Cyr61 and LC3B in hippocampus were detected by immunofluorescence staining. One-way analysis of variance (ANOVA) was used between groups, and LSD-T test was used for pairwise comparison.Results:Western blotting results showed thatthe protein expression of Cyr61 (0.99±0.15) was significantly increased ( P=0.011), and the protein expression of Beclin-1 (0.64±0.04) and LC3B(0.54±0.05) was significantly decreased in lupus group ( P=0.025, P= 0.008) when compared with normal control group (0.73±0.08, 0.81±0.12, 0.80±0.03). The expressions of Cyr61 (0.75±0.05, 0.75±0.08), Beclin-1 (0.84±0.08, 0.92±0.04) and LC3B (0.93±0.16, 0.76±0.08) in rapamycin group and rapamycin + lupus group were not significantly changed ( P>0.05). Compared with rapamycin group, the protein expression of Cyr61 (0.99±0.15) was significantly increased ( P=0.016), and Beclin-1 (0.64±0.04), LC3B (0.54±0.05) was significantly decreased in lupus group ( P=0.013, P=0.001). The expressions of Cyr61 (0.75± 0.08), Beclin-1 (0.92±0.04) and LC3B (0.76±0.08) were not significantly changed in rapamycin+lupus group ( P=0.999, P=0.241, P=0.062). Compared with lupus group, the expression of Cyr61 (0.75±0.08) protein in rapamycin+lupus group was significantly decreased ( P=0.016), and the expression of Beclin-1 (0.92±0.04) and LC3B(0.76±0.08) protein were significantly increased ( P=0.002, P=0.017). Immunofluorescence results showed that Cyr61 and LC3B were mainly expressed in the cytoplasm of hippocampal neurons, and the quantitative detection results were consistent with western blot results, the differences were statistically significant ( P=0.025, P=0.032). HE staining showed that the levels and number of cells in the hippocampus of mice with lupus were reduced, and the arrangement was sparse, and the nuclei were hyperchromatic, showing nuclear pyknosis and migration. The results of Nissl staining showed that there were relatively fewer Nissl bodies, loose arrangement of neurons and vacuolar areas in some cells, which were improved after RAPA treatment in lupus mice. Conclusion:RAPA can protect the cognitive function of lupus mice by inhibiting the expression of Cyr61 in hippocampus and promoting autophagy.
Résumé
OBJECTIVE:To study the improvement effect and mechanism of MEBO on lipopolysaccharide (LPS)-induced injury of rat skin fibroblasts. METHODS :Skin fibroblasts of rats were divided into control group ,LPS group (5 μg/mL), Kangfuxin solution group (positive control ,5 μg/mL LPS+1.25% Kangfuxin solution )and MEBO group (5 μg/mL LPS+0.6 mg/mL MEBO),with 6 wells in each group. Inflammatory injury cell model was induced by LPS (except for control group ). After a certain period of cultivation ,the cell survival rate and cell migration rate were detected in each group. The contents of TNF-α and IL-6 in cell supernatant was detected. The localization and fluorescence intensity of IL- 6 protein were detected. The protein expression of PTEN ,p-p65,TNF-α,IL-6,PI3K and Akt in the fibroblasts were also determined. RESULTS :Compared with control group ,survival rate of the fibroblasts was increased significantly in LPS group ,while cell migration was decreased significantly;the contents of TNF-α and IL-6 in cell supernatant as well as relative protein expression of PTEN ,p-p65,TNF-α, IL-6 and PI 3K were increased significantly (P<0.05 or P<0.01);IL-6 protein mainly expressed in the cytoplasm ,and the fluorescence intensity was enhanced. Compared with LPS group ,survival rate of the fibroblasts was decreased significantly in Kangfuxin solution group and MEBO group ,while migration rate was increased significantly ;the contents of TNF-α and IL-6, relative protein expression of PTEN ,p-p65,TNF-α,IL-6(except for Kangfuxin solution group ),PI3K and Akt (except for Kangfuxin solution group ) were decreased significantly (P<0.05 or P<0.01),while fluorescence intensity of IL- 6 protein decreased;relative protein expression of TNF-α,IL-6,PI3K and Akt in MEBO group were significantly lower than Kangfuxin solution group (P<0.05 or P<0.01). CONCLUSIONS :MEBO can inhibit the proliferation of LPS-induced skin fibroblasts , reduce the level of inflammatory factors and the intensity of inflammatory reaction , which may be related to the jiang- down-regulation of PTEN/NF-κB,PI3K/Akt signaling pathway.
Résumé
OBJECTIVE@#To explore the mechanism by which fractalkine (CX3CL1; FKN) inhibits lipopolysaccharide (LPS)-induced immunological response in RAW264.7 cells.@*METHODS@#A RAW264.7 cell model overexpressing FKN was established by transfection with the lentiviral vector CX3CL1. The effects of LPS, ICG-001 (a Wnt/β-catenin signaling pathway inhibitor), either alone or in combination, on M1 polarization of na?ve and FKN-overexpressing RAW264.7 cells were evaluated by detecting of intereukin-6 (IL-6) and tumor necrosis factor-α (TNF-@*RESULTS@#The RAW264.7 cell model of FKN overexpression was successfully established. In na?ve RAW264.7 cells, treatment with both ICG-001 and LPS, as compared with LPS alone, significant promoted TNF-@*CONCLUSIONS@#FKN overexpression suppresses LPS-induced M1 type polarization of RAW264.7 cells by activating Wnt/β-catenin signaling pathway.