ABSTRACT
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.
ABSTRACT
OBJECTIVE@#Psoriasis is a common chronic inflammatory skin disease that is prone to recurrence, and the proinflammatory factor, cysteine-rich protein 61 (Cyr61), is important in its pathophysiology. Long-term clinical practice has shown that Sancao Formula (SC), a Chinese herbal compound, is effective in the treatment of psoriasis, but the precise mechanism remains unknown. In this study, we investigate the mechanism by which SC extract alleviates imiquimod (IMQ)-induced psoriasis.@*METHODS@#The expression of Cyr61 in psoriatic lesions and normal healthy skin was detected using immunohistochemical analysis to investigate the biological role of Cyr61 in models of psoriatic inflammation. A psoriatic mouse model was established by topical application of IMQ, and the effect of topical application of SC extract was evaluated using the psoriasis area and severity index (PASI) score, hematoxylin-eosin staining, and histopathological features of the skin. Next, a HaCaT cell inflammation model was established using interferon-γ (IFN-γ), and the effect of SC extract on the mRNA and protein levels of Cyr61 and intercellular cell adhesion molecule-1 (ICAM-1) was confirmed using Western blot and quantitative real-time polymerase chain reaction analyses.@*RESULTS@#Immunohistochemical staining showed that the expression of Cyr61 in psoriatic lesions was higher than that in normal skin samples (78.26% vs 41.18%, P < 0.05), and the number of Cyr61-positive cells in psoriatic lesions was also significantly higher than in normal skin (18.66 ± 2.51 vs 4.33 ± 1.52, P < 0.05). Treatment in mice with IMQ-induced psoriasis showed that SC extract could significantly improve the inflammatory phenotype, PASI score (10.875 ± 0.744 vs 3.875 ± 0.582, P < 0.05), and pathological features compared with those in IMQ model group; SC treatment was also associated with decreased levels of Cyr61 and ICAM-1. In the IFN-γ-induced inflammatory cell model, the mRNA and protein levels of Cyr61 and ICAM-1 were upregulated, while the SC extract downregulated the levels of Cyr61 and ICAM-1.@*CONCLUSION@#The results provide a theoretical basis for the involvement of Cyr61 in the pathogenesis of psoriasis, and suggest that SC should be used to target Cyr61 for the prevention of psoriasis recurrence.
Subject(s)
Animals , Mice , China , Cysteine-Rich Protein 61/metabolism , Disease Models, Animal , Drugs, Chinese Herbal/therapeutic use , Imiquimod/adverse effects , Inflammation/drug therapy , Intercellular Adhesion Molecule-1/genetics , Interferon-gamma , Mice, Inbred BALB C , Psoriasis/pathology , RNA, Messenger/therapeutic useABSTRACT
Objective To observe the expression of cysteine-rich protein 61 (Cyr61) in transforming growth factor-β1 (TGF-β1)-activated renal fibroblasts (NRK-49F),and to explore its effect and mechanism.Methods (1) NRK-49F cells were activated by TGF-β1 with different concentrations (0.0,0.5,1.0,2.0,5.0 μg/L).Western blotting was used to detect the expression of Cyr61 protein,and CCK-8 assay was used to test the proliferative activity of NRK-49F cells.(2) NRK-49F cells with low expression and over expression of Cyr61 were established by plasmid transfection.The cells were divided into control group (null vector transfection),over-expression group and lowexpression group.The proliferation was discovered by CCK-8 assay after 24,48 and 72 h.Further,5.0 μg/L TGF-β1 activated these three groups.The proliferation was also discovered by CCK-8 assay and the cell cycle was analyzed by flow cytometry.The mRNA expressions of fibrosis markers (Collα1,Col3αl,MMP9,MMP13) and factors of cell senescence signal pathway (p53,p21,Rb,p16) were ascertained by real time PCR,and the protein expressions of Col3 and MMP9 were detected by Western blotting.Results (1) Compared with 0.0 μg/L TGF-β1 group,the proliferation of NRK-49F cells was enhanced in 0.5,1.0,2.0 and 5.0 μg/L TGF-β1 groups (all P ＜ 0.05),while the expression of Cyr61 protein was decreased in 1.0 μg/L group and increased in 5.0 μg/L group (all P ＜0.05).(2) The proliferation of over-expression group was lower than that of control group after 24,48and 72 h (all P＜ 0.05),which was in a time-dependent manner.(3) Compared with control group activated by TGF-β1,the over-expression group expressed less fibrosis factors (Col1α1 and Col3α1)and more anti-fibrosis factors (MMP9 and MMP13) with decreased proliferation (all P ＜ 0.05).Simultaneously,the proportion of cells bogged down in G1 phases,as well as the expressions of p53,p21 and Rb mRNA increased (all P ＜ 0.05).The above effects of low-expression group were just opposite to over-expression group.Moreover,there was no significant difference in the expression of p16 gene among the three groups (P ＞ 0.05).Conclusions Cyr61 can curb the proliferation and fibrotic phenotypes of fibroblasts,thereafter slowing down the process of renal fibrosis.The p53/p21/Rb interrelated cell senescence signal pathway may be involved in the anti-fibrosis process.
ABSTRACT
Objective To observe the expression of cysteine-rich protein 61 (Cyr61) in transforming growth factor-β1 (TGF-β1)-activated renal fibroblasts (NRK-49F),and to explore its effect and mechanism.Methods (1) NRK-49F cells were activated by TGF-β1 with different concentrations (0.0,0.5,1.0,2.0,5.0 μg/L).Western blotting was used to detect the expression of Cyr61 protein,and CCK-8 assay was used to test the proliferative activity of NRK-49F cells.(2) NRK-49F cells with low expression and over expression of Cyr61 were established by plasmid transfection.The cells were divided into control group (null vector transfection),over-expression group and lowexpression group.The proliferation was discovered by CCK-8 assay after 24,48 and 72 h.Further,5.0 μg/L TGF-β1 activated these three groups.The proliferation was also discovered by CCK-8 assay and the cell cycle was analyzed by flow cytometry.The mRNA expressions of fibrosis markers (Collα1,Col3αl,MMP9,MMP13) and factors of cell senescence signal pathway (p53,p21,Rb,p16) were ascertained by real time PCR,and the protein expressions of Col3 and MMP9 were detected by Western blotting.Results (1) Compared with 0.0 μg/L TGF-β1 group,the proliferation of NRK-49F cells was enhanced in 0.5,1.0,2.0 and 5.0 μg/L TGF-β1 groups (all P ＜ 0.05),while the expression of Cyr61 protein was decreased in 1.0 μg/L group and increased in 5.0 μg/L group (all P ＜0.05).(2) The proliferation of over-expression group was lower than that of control group after 24,48and 72 h (all P＜ 0.05),which was in a time-dependent manner.(3) Compared with control group activated by TGF-β1,the over-expression group expressed less fibrosis factors (Col1α1 and Col3α1)and more anti-fibrosis factors (MMP9 and MMP13) with decreased proliferation (all P ＜ 0.05).Simultaneously,the proportion of cells bogged down in G1 phases,as well as the expressions of p53,p21 and Rb mRNA increased (all P ＜ 0.05).The above effects of low-expression group were just opposite to over-expression group.Moreover,there was no significant difference in the expression of p16 gene among the three groups (P ＞ 0.05).Conclusions Cyr61 can curb the proliferation and fibrotic phenotypes of fibroblasts,thereafter slowing down the process of renal fibrosis.The p53/p21/Rb interrelated cell senescence signal pathway may be involved in the anti-fibrosis process.
ABSTRACT
Cysteine-rich protein 61 (CYR61) is a secreted protein.It consists of four different structural domains and plays an important role in all kinds of cell life activities,such as proliferation,migration,adhesion,angiogenesis,inflammation regulation,embryogenesis and cartilage formation,by the means of controlling variety of cytokines and signaling pathways.Meanwhile,it also has been widely concerned because of its critical function in neovascularization.In this paper,we present the research progress in CYR61 and its application in ocular fundus neovascular disease,such as diabetic retinopathy,retinopathy of prematurity and wet age-related macular degeneration,in the structure of review,so as to provide a new perspective on the pathogenesis and therapy of ocular fundus neovascular disease.
ABSTRACT
Cyr61/CCN1 is a secreted extracellular matrix ( ECM) protein, which has been shown to regulate a multitude of cellular responses.Many researches indicate that Cyr61 plays important roles in oncogenesis and development of tumor and is considered to be a novel potential oncogene.This review would provide a comprehensive summary about the roles of Cyr61 in the diagnosis and treatment of tumor.
ABSTRACT
PURPOSE: One of the features in cancer development is the migration of cancer cells to form metastatic lesions. CYR61 protein promotes migration and the epithelial-mesenchymal transition in several cancer cell types. Evidence suggests that CYR61 and dexamethasone are relevant to colorectal cancer. However, relationships between them and colorectal cancer are still unclear. Understanding the molecular mechanism of colorectal cancer progression related with CYR61 and dexamethasone, which is widely used for combination chemotherapy, is necessary for improved therapy. MATERIALS AND METHODS: We used colorectal cancer cells, HCT116, co-treated with transforming growth factor β1 (TGF-β1) and dexamethasone to examine the inhibitory migration effect of dexamethasone by migratory assay. Alternatively, both migratory pathways, expression of AKT and ERK, and the target factor CYR61 was also tested by co-treatment with TGF-β1 and dexamethasone. RESULTS: We report that dexamethasone significantly inhibited TGF-β1-induced cell migration, without affecting cell proliferation. Importantly, we observed that TGF-β1 promoted the epithelial-mesenchymal transition process and that dexamethasone co-treatment abolished this effect. ERK and AKT signaling pathways were found to mediate TGF-β1-induced migration, which was inhibited by dexamethasone. In addition, TGF-β1 treatment induced CYR61 expression whereas dexamethasone reduced it. These observations were compatible with the modulation of migration observed following treatment of HCT116 cells with human recombinant CYR61 and anti-CYR61 antibody. Our results also indicated that TGF-β1 enhanced collagen I and reduced matrix metalloproteinase 1 expression, which was reversed by dexamethasone treatment. CONCLUSION: These findings suggested that dexamethasone inhibits AKT and ERK phosphorylation, leading to decreased CYR61 expression, which in turn blocks TGF-β1-induced migration.
Subject(s)
Humans , Cell Movement , Cell Proliferation , Collagen , Colon , Colonic Neoplasms , Colorectal Neoplasms , Cysteine-Rich Protein 61 , Dexamethasone , Drug Therapy, Combination , Epithelial-Mesenchymal Transition , HCT116 Cells , Matrix Metalloproteinase 1 , Phosphorylation , Transforming Growth FactorsABSTRACT
Objective To explore the inhibition effect of Cysteine-rich 61 (CCN1; Cyr61) specific siRNA expression vector on RNV in a mouse model of oxygen-induced retinopathy (OIR).Methods One hundred and twenty healthy C57BL/6J mice were chosen and randomly divided into the experimental group and control group,with 60 mice in each group.The experimental group was intravitreously injected with CCN1siRNA recombinant plasmids.The control group was injected with vector plasmids.Adenosine diphosphate-ase stained retina flat-mounts was performed to assess the retinal vascular profiles,retinal section with HE staining was applied to count the number of new vascular cell nuclei and the protein and mRNA expression of CCN1 and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry,Western blot and Real-time RT-PCR.Results Compared with control group,regular distributions,good branches and reduced density of retinal neovascularization were observed in the experimental group.The number of nucleus of vascular endothelial cells breaking through the inner limiting membrane was obviously less in the experimental group than that in the control group (t=8.756,P＜ 0.05).The expression of CCN1 and VEGF were obviously decreased in the experimental group compared with the control group (all P＜0.05).Conclusion The development of RNV of ROP can be markedly inhibited by RNA interference targeting CCN1,and CCNlsiRNA may provide an effective method for preventing vascular proliferative retinopathy.
ABSTRACT
Objective To investigate the effect of cysteine-rich protein 61 (Cyr61) on proliferation and cell cycle in human renal tubular epithelial cells (HK-2).Methods Cyr61 cDNA was cloned into pEGFP-N2,then HK-2 cells were transfected with the recombinant plasmid pEGFP-N2-Cyr61 by Lipofectamine.The cell proliferation was measured by MTT.The expression level of Cyr61,p-FAK and cyclin dependent cyclin-dependent kinase 2 (CDK2) protein were detected by Western blotting.The cell cycle and cell apoptosis were analyzed by flow cytometry.Results The recombinant plasmid pEGFP-N,-Cyr61 could be transfected into HK-2 efficiently.After transfection,the proliferative activity was significantly increased,the proportion of HK-2 cells in G1 phase decreased and in S-phase increased significantly,the level of cell apoptosis decreased markedly (all P ＜ 0.01).The expressions of Cyr61,p-FAK and CDK2 in Cyr61-transfected group were all amplified significantly (all P ＜ 0.01).Conclusions Cyr61 protein over-expressed in HK-2 cells can increase CDK2 expression throngh FAK pathway,resulting in the promotion of HK-2 cells entering into S phase,cell proliferation and the reduction of cell apoptosis.
ABSTRACT
Objective To investigate the relationship between cysteine-rich protein 61 ( Cyr61 ) and phosphatidylinositol 3-kinase ( PI3K ) signal pathway on cell proliferation and apoptotic in human ovarian carcinoma cells.Methods Recombinant human Cyr61 (rhCyr61) was pretreated with ovarian carcinoma cells.The expression of Cyr61 protein was detected by confocal spectral microscopy.Then treated the ovarian carcinoma cells with PI3K transduction inhibitors (LY294002) for 24 hours.Cell apoptosis was detected by flow cytometry (FCM).Cell viability was determined by methyl thiazolyl tetrazolium (MTT) method.The mRNA expressions of Cyr61,the protein levels of protein kinase B ( PKB),phospho-PKB and Cyr61 were assaved by real time-PCR and western blot analysis,respectively.Results The Cyr61 and phospho-PKB protein expression in two ovarian carcinoma cells (OV2008 and OVCAR-3 ) were increased in rhCyr61pretreated group.The decreasing of cell apoptosis [ ( 1.4 ±0.9)％,(2.1 ± 1.0)％ ] and increasing of cell proliferation [ ( 124.0 ± 1.8)％,( 133.0 ±2.2)％ ] was detected in the same time,compared with negative control group,there were significant difference ( P ＜ 0.05 ).After exposed to LY294002 for 24 hours,the apoptosis rate of OV2008 and OVCAR-3 in pretreated with rhCyr61 group exposed to LY294002 were (21.1 ± 1.6)％ and (26.4 ± 1.5 )％,respectively.Cells viability [ (59.0 ± 2.3 )％,(51.0 ± 2.0)％ ]was also significantly decreased in OV2008 and OVCAR-3 pretreated with rhCyr61 cells.Meanwhile,the mRNA expressions of Cyr61 (3.2 ± 0.8,6.2 ± 1.1 ) and the protein levels of phospho-PKB and Cyr61 were greatly decreased.Compared with negative control group,there were significant difference in OV2008 and OVCAR-3 cells (all P ＜ 0.0l ).Conclusions The activation of PI3K intracellular signaling pathways may lead to up-regulation of Cyr61 expression.Block PI3K signal pathway could significantly inhibit the expression of Cyr61,and may promote the apoptotic effects and inhibit the cell growth of ovarian carcinoma cells.
ABSTRACT
Objective To investigate the different expression of microRNA-155 (miR-155) and cysteine-rich 61 (CYR61) in human placentas between severe preeclamptic and normal pregnancies.Methods Placentas were obtained from severe preeclamptic and healthy control pregnant women (n=18 for each group) at 36～40 gestational weeks. The expressions of miR-155 and CYR61 mRNA were assessed by real-time quantitative reverse transcription-polymerase chain reaction, and the levels of CYR61 protein were tested by Western blot. Results Compared with the control group, the miR-155 expression was increased in placentas from severe preeclampsia groups ( 165. 7 ± 16. 4 vs 527.9±49.1,t=7.00, P＜0.01), and the CYR61 mRNA expression (31.7±2.7 vs 16.4±1.2,t=5.10,P＜0. 01), as well as the CYR61 protein expression (36.4±1.5 vs 19.7±1.2,t=36.26, P＜0.01 ) were decreased. There was a significantly negative correlation between the expression of miR-155 and CYR61 mRNA within both groups (preeclamptic group: r=-0.52, P＜0.05;control:r=-0.57, P＜0.05). Conclusions Up-regulation of placental miR-155 in severe preeclampsia may be related to the decreased expression of CYR61. Both miR-155 and CYR61 may contribute to the disorders of placental angiogenesis in severe preeclampsia in human.