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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.
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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.
Subject(s)
Animals , Mice , Chemokine CX3CL1 , Lipopolysaccharides/pharmacology , Macrophages , Tumor Necrosis Factor-alpha , Wnt Signaling PathwayABSTRACT
OBJECTIVE@#To investigate the role of Cyr61 in angiotensin Ⅱ (AngⅡ)-induced functional changes in HEK293 cells and explore the mechanism.@*METHODS@#Cyr61 knockdown in cultured HEK293T cells was achieved by transfection of the cells with CRISPR/Cas9 KO plasmid. The changes in apoptosis and expression levels of Cyr61 and Bcl-2 in the cells with or without Cyr61 knockdown in response to treatment with 10 mol/L AngⅡ for 48 h were analyzed using flow cytometry, qRT-PCR and Western blotting.@*RESULTS@#The cells with Cyr61 knockdown showed significantly decreased expression of Cyr61 protein as compared with the control cells ( < 0.05). AngⅡ treatment for 48 h significantly increased the expression of Cyr61 and lowers the expression of Bcl-2 at both the protein and mRNA levels in HEK293T cells. In HEK293T cells with Cyr61 knockdown, AngⅡ treatment resulted in significantly increased expression of Bcl-2 in HEK293T cells as compared with that of the control group ( < 0.05). AngⅡ treatment caused significantly increased apoptotic rate in HEK293T cells as compared with the cells with Cyr61 knockdown [(26.94 ± 3.73)% (3.87 ± 0.83)%, < 0.05), and the apoptosis rate was significantly lowered to (15.76 ± 1.31)% in HEK293T cells with Cyr61 knockdown following AngⅡ treatment ( < 0.05).@*CONCLUSIONS@#The up-regulation of Cyr61 expression is related with AngⅡ-induced injury in HEK293T cells, and down-regulating Cyr61 expression can effectively protect HEK293T cells against AngⅡ-induced injury.
Subject(s)
Humans , Angiotensin II , Apoptosis , Cysteine-Rich Protein 61 , HEK293 Cells , Up-RegulationABSTRACT
Objective:To explore the possible mechanism of astragaloside involved in the mouse podocytes injury induced by TGF-β1 in vitro.Methods:Mouse podocytes were cultured in vitro and then all cell were divided into 5 groups:normal control group , TGF-β1 treatment group ,TGF-β1 treatment +astragaloside low dose group ,TGF-β1 treatment +astragaloside middle dose group and TGF-β1 treatment +astragaloside high dose group.The proliferation rate of each group was investigated by MTT assay ,the expression of TRPC6 protein and mRNA were measured by Western blot and RT-PCR respectively after 48 hours.Results:TGF-β1 can significantly inhibit the proliferation of podocytes ( P<0.05) ,fusions of foot processes or even effaced of podocytes were observed .TGF-β1 could also increase the expression of TRPC6.Astragaloside could reduce the inhibition of TGF-β1 to the proliferain of podocytes significantly ,make the cell shape tend to be normal,and reduce the expression of TRPC6 mRNA and protein with dose-effect relation.Conclusion:TRPC6 play an impor-tant role in the TGF-β1 induecd podocytes injury .Astragaloside can alleviate podocytes injury by reduce the expression of TRPC 6.
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Objective To investigate the effects of transient receptor potential cation channel 6 (TRPC6) on the expression of nephrin, desmin and caspase 9 and on the apoptosis of podocytes in a mouse model of podocyte injury induced by TGF-β1.Methods Conditionally immortalized mouse podocytes were cultured in vitro and divided into four groups: control, TGF-β1 treatment, TGF-β1+PGPU6/GFP/Neo-TRPC6-mus-581 (TRPC6 knockdown) and TGF-β1+PGPU6/GFP/Neo-NC (negative control).Real-time RT-PCR and Western blot analysis were performed to detect the expression of nephrin, desmin and caspase 9 at mRNA and protein levels, respectively.Flow cytometry was used to analyze the apoptotic rate of podocytes.DAPI fluorescent staining was used to observe the morphological changes of apoptotic podocytes.Results Green fluorescent protein (GFP)-expressing podocytes at 48 hours after transfection were significantly more than those at 24 hours after transfection.The level of TRPC6 in mouse podocytes transfected with PGPU6/GFP/Neo-TRPC6-mus-581 was significantly decreased as compared with that of the control group (P0.05).More apoptotic cells with typical morphological features of apoptosis were observed after exposure to TGF-β1 for 48 hours.Conclusion TGF-β1 could induce the apoptosis of podocytes, inhibit the expression of nephrin and enhance the expression of caspase 9 and desmin, the possible mechanisms of which may be related to TRPC6 signal pathway.These changes in TGF-β1-treated podocytes could be alleviated by inhibiting the expression of TRPC6, which might have a protective effect on podocyte injury.
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<p><b>OBJECTIVE</b>To investigate the expression of fractalkine (FKN) in the blood and renal tissues of patients with lupus nephritis and explore its significance.</p><p><b>METHODS</b>According to the pathological classification, 48 patients with lupus nephritis were divided into mild group (22 cases) and severe group (26 cases), with 26 healthy subjects as the control group. RT-PCR and enzyme-linked immunosorbent assay were employed to detect the expression of FKN mRNA and protein in the blood of the subjects, and FKN expression and localization in the renal tissue of the patients with lupus nephritis were detected using immunohistochemical staining.</p><p><b>RESULTS</b>The patients in both the mild and severe groups showed significantly increased expression of blood FKN mRNA and protein compared with the normal controls, and the increase was more obvious in severe cases (P<0.01). In the renal tissues of the patients, FKN was located mainly in the cytoplasm of the glomerular podocytes and renal tubular epithelial, and the number of positive glomerular cells number was significantly greater in severe cases than in the mild cases (P<0.01); FKN expression in the cortical interstitium did not show a significant difference between the 3 groups.</p><p><b>CONCLUSION</b>FKN expression in the blood and glomeruli of patients with lupus nephritis is related to the severity of renal pathologies.</p>
