BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the...BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.展开更多
AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in v...AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.展开更多
Mounting evidence indicates that amyloid β protein（Aβ） exerts neurotoxicity by disrupting the blood-brain barrier（BBB） in Alzheimer＇s disease. Hyperoside has neuroprotective effects both in vitro and in vivo ag...Mounting evidence indicates that amyloid β protein（Aβ） exerts neurotoxicity by disrupting the blood-brain barrier（BBB） in Alzheimer＇s disease. Hyperoside has neuroprotective effects both in vitro and in vivo against Aβ. Our previous study found that hyperoside suppressed Aβ1-42-induced leakage of the BBB, however, the mechanism remains unclear. In this study, bEnd.3 cells were pretreated with 50, 200, or 500 μM hyperoside for 2 hours, and then exposed to Aβ1-42 for 24 hours. Cell viability was determined using 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay were used to analyze cell apoptosis. Western blot assay was carried out to analyze expression levels of Bax, Bcl-2, cytochrome c, caspase-3, caspse-8, caspase-9, caspase-12, occludin, claudin-5, zonula occludens-1, matrix metalloproteinase-2（MMP-2）, and MMP-9. Exposure to Aβ1-42 alone remarkably induced bEnd.3 cell apoptosis; increased ratios of cleaved caspase-9/caspase-9, Bax/Bcl-2, cleav ed caspase-8/caspase-8, and cleaved caspase-12/caspase-12; increased expression of cytochrome c and activity of caspase-3; diminished levels of zonula occludens-1, claudin-5, and occludin; and increased levels of MMP-2 and MMP-9. However, hyperoside pretreatment reversed these changes in a dose-dependent manner. Our findings confirm that hyperoside alleviates fibrillar Aβ1-42-induced BBB disruption, thus offering a feasible therapeutic application in Alzheimer＇s disease.展开更多
Background: The goat(Caprahircus) is one of the most important livestock animals. Goat milk fat is an important component in the nutritional quality of goat milk. Growing evidence points to the critical roles of micro...Background: The goat(Caprahircus) is one of the most important livestock animals. Goat milk fat is an important component in the nutritional quality of goat milk. Growing evidence points to the critical roles of microRNAs(miRNAs) in lipid metabolism.Results: Using a highly sensitive method of S-poly(T) plus for miRNAs detection, we analyze the expression patterns of 715 miRNAs in goat mammary gland tissues at different stages of lactation. We observed that miR-25 expression had an inverse relationship with milk production. Overexpression of miR-25 significantly repressed triacylglycerol synthesis and lipid droplet accumulation. To explore the regulatory mechanism of miR-25 in milk lipid metabolism,we analyzed its putative target genes with bioinformatics analysis followed by 3′-UTR assays. Peroxisome proliferative activated receptor gamma coactivator 1 beta(PGC-1 beta), a key regulator of lipogenics was identified as a direct target of miR-25 with three specific sites within its 3′-UTR. In addition, miR-25 mimics in goat mammary epithelial cells reduced the expressions of genes involved in lipid metabolism.Conclusions: Taken together, our results show miR-25 is potentially involved in lipid metabolism and we reveal the function of the miR-25/PGC-1 beta regulatory axis during lactation.展开更多
AIM To investigate the relationship between hypoxia-inducible factor-1α(HIF-1α), prolyl 4-hydroxylase beta(P4 HB) expression, and clinicopathologic parameters, as well as the prognostic value of these genes for pati...AIM To investigate the relationship between hypoxia-inducible factor-1α(HIF-1α), prolyl 4-hydroxylase beta(P4 HB) expression, and clinicopathologic parameters, as well as the prognostic value of these genes for patients with gastric cancer(Gc).METHODS Hypoxia is a critical factor that shapes the Gc microenvironment. In previous reports, we have demonstrated that P4 HB is a potential target of HIF-1α. In the present study, gene expression profiling interactive analysis(GEPIA) was used to analyze the relationship between P4 HB and hypoxia-associated genes. To this end, 428 Gc tissue samples were used to analyze the expression of HIF-1α and P4 HB via immunohistochemical staining. Patient samples were classified as having weak-expression or over-expression both in terms of HIF-1α and P4 HB. Correlations between biomarkers and clinicopathological factors were analyzed to predict survival. RESULTS P4 HB demonstrated a positive correlation with hypoxiaassociated genes(P < 0.05). HIF-1α and P4 HB overexpression have a significant correlation with TNM staging(χ2 = 23.32, P = 0.00; χ2 = 65.64, P = 0.00) and peritoneum cavity metastasis(χ2 = 12.67, P = 0.00; χ2 = 39.29, P = 0.00). In univariate analysis, patients with a high HIF-1α expression trend had a shorter disease-free survival(DFS: 44.80 mo vs 22.06 mo) and overall survival(OS: 49.58 mo vs 39.92 mo). P4 HB overexpression reflected similar results: patients with over-expression of P4 HB had a shorter survival time than those with weak-expression(DFS: 48.03 mo vs 29.64 mo, OS: 52.48 mo vs 36.87 mo). Furthermore, HIF-1α is also a clinicopathological predictor of dismal prognosis according to multivariate analysis(DFS, 95%c I: 0.52-0.88, P < 0.00; OS, 95%c I: 0.50-0.85, P < 0.00). However, P4 HB was meaningful in DFS(95%c I: 0.58-1.00, P < 0.05) but not in OS(95%c I: 0.72-1.23, P > 0.05).CONCLUSION Overexpression of HIF-1α and P4 HB is associated with poor prognosis in patients with Gc. Thus, these genes may be potential prognostic biomarker candidates in GC.展开更多
Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ...Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer＇s disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.展开更多
基金supported by grants from the Natural Science Foundation of Jiangsu Province,China (No. BK2006241)the Foundation for Talents in Six Fields of Jiangsu Province (No. 07-B-038)
文摘BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.
基金Shaanxi Province Science and Technology Gongguan Program, China (No.2011-K14-02-03)
文摘AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.
基金financially supported by the National Natural Science Foundation of China,No.81573771the Natural Science Foundation of Jiangsu Province of China,No.BK20151599
文摘Mounting evidence indicates that amyloid β protein（Aβ） exerts neurotoxicity by disrupting the blood-brain barrier（BBB） in Alzheimer＇s disease. Hyperoside has neuroprotective effects both in vitro and in vivo against Aβ. Our previous study found that hyperoside suppressed Aβ1-42-induced leakage of the BBB, however, the mechanism remains unclear. In this study, bEnd.3 cells were pretreated with 50, 200, or 500 μM hyperoside for 2 hours, and then exposed to Aβ1-42 for 24 hours. Cell viability was determined using 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay were used to analyze cell apoptosis. Western blot assay was carried out to analyze expression levels of Bax, Bcl-2, cytochrome c, caspase-3, caspse-8, caspase-9, caspase-12, occludin, claudin-5, zonula occludens-1, matrix metalloproteinase-2（MMP-2）, and MMP-9. Exposure to Aβ1-42 alone remarkably induced bEnd.3 cell apoptosis; increased ratios of cleaved caspase-9/caspase-9, Bax/Bcl-2, cleav ed caspase-8/caspase-8, and cleaved caspase-12/caspase-12; increased expression of cytochrome c and activity of caspase-3; diminished levels of zonula occludens-1, claudin-5, and occludin; and increased levels of MMP-2 and MMP-9. However, hyperoside pretreatment reversed these changes in a dose-dependent manner. Our findings confirm that hyperoside alleviates fibrillar Aβ1-42-induced BBB disruption, thus offering a feasible therapeutic application in Alzheimer＇s disease.
基金supported by the Transgenic Project from the Ministry of Agriculture [2014ZX08009-051B to JL]the National Natural Science Foundation of China [81370151 and 81570046 to DG,31701185 to HQ and81700054 to YZ]+3 种基金the Shenzhen Municipal Basic Research Program[JCYJ20150729104027220 to DG and JCYJ20160520174217859 to HQ]Shenzhen University Interdisciplinary Innovation Team Project [000003 to DG]Natural Science Foundation of Guangdong Province [2017A030310450to HQ]Research Project of Shenzhen Technology University [201731 to HQ]
文摘Background: The goat(Caprahircus) is one of the most important livestock animals. Goat milk fat is an important component in the nutritional quality of goat milk. Growing evidence points to the critical roles of microRNAs(miRNAs) in lipid metabolism.Results: Using a highly sensitive method of S-poly(T) plus for miRNAs detection, we analyze the expression patterns of 715 miRNAs in goat mammary gland tissues at different stages of lactation. We observed that miR-25 expression had an inverse relationship with milk production. Overexpression of miR-25 significantly repressed triacylglycerol synthesis and lipid droplet accumulation. To explore the regulatory mechanism of miR-25 in milk lipid metabolism,we analyzed its putative target genes with bioinformatics analysis followed by 3′-UTR assays. Peroxisome proliferative activated receptor gamma coactivator 1 beta(PGC-1 beta), a key regulator of lipogenics was identified as a direct target of miR-25 with three specific sites within its 3′-UTR. In addition, miR-25 mimics in goat mammary epithelial cells reduced the expressions of genes involved in lipid metabolism.Conclusions: Taken together, our results show miR-25 is potentially involved in lipid metabolism and we reveal the function of the miR-25/PGC-1 beta regulatory axis during lactation.
基金Supported by Liaoning S and T Project,No.2015020269Doctor fund of Liaoning Province Cancer Hospital and Institute,No.Z1410
文摘AIM To investigate the relationship between hypoxia-inducible factor-1α(HIF-1α), prolyl 4-hydroxylase beta(P4 HB) expression, and clinicopathologic parameters, as well as the prognostic value of these genes for patients with gastric cancer(Gc).METHODS Hypoxia is a critical factor that shapes the Gc microenvironment. In previous reports, we have demonstrated that P4 HB is a potential target of HIF-1α. In the present study, gene expression profiling interactive analysis(GEPIA) was used to analyze the relationship between P4 HB and hypoxia-associated genes. To this end, 428 Gc tissue samples were used to analyze the expression of HIF-1α and P4 HB via immunohistochemical staining. Patient samples were classified as having weak-expression or over-expression both in terms of HIF-1α and P4 HB. Correlations between biomarkers and clinicopathological factors were analyzed to predict survival. RESULTS P4 HB demonstrated a positive correlation with hypoxiaassociated genes(P < 0.05). HIF-1α and P4 HB overexpression have a significant correlation with TNM staging(χ2 = 23.32, P = 0.00; χ2 = 65.64, P = 0.00) and peritoneum cavity metastasis(χ2 = 12.67, P = 0.00; χ2 = 39.29, P = 0.00). In univariate analysis, patients with a high HIF-1α expression trend had a shorter disease-free survival(DFS: 44.80 mo vs 22.06 mo) and overall survival(OS: 49.58 mo vs 39.92 mo). P4 HB overexpression reflected similar results: patients with over-expression of P4 HB had a shorter survival time than those with weak-expression(DFS: 48.03 mo vs 29.64 mo, OS: 52.48 mo vs 36.87 mo). Furthermore, HIF-1α is also a clinicopathological predictor of dismal prognosis according to multivariate analysis(DFS, 95%c I: 0.52-0.88, P < 0.00; OS, 95%c I: 0.50-0.85, P < 0.00). However, P4 HB was meaningful in DFS(95%c I: 0.58-1.00, P < 0.05) but not in OS(95%c I: 0.72-1.23, P > 0.05).CONCLUSION Overexpression of HIF-1α and P4 HB is associated with poor prognosis in patients with Gc. Thus, these genes may be potential prognostic biomarker candidates in GC.
基金supported by the National Natural Science Foundation of China,No.31670832,31470807,31270872a grant from the National Key Research and Development Program of China,No.2016YFA0500301a grant from the State Key Laboratory of Protein and Plant Gene Research,College of Life Sciences,Peking University,China
文摘Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer＇s disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.
