AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the ...AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL63 mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer. Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-LS-LOX-for Ad-vectorvia Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR(2) could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.展开更多
从Nrf2信号通路探讨淫羊藿苷(icariin,ICA)对D-半乳糖(D-galactose,D-Gal)诱导的小鼠睾丸支持细胞株15P-1细胞损伤的保护作用。将15P-1细胞分为对照组(control)、150 mM D-Gal处理组、D-Gal+ICA(0.5μM)组和D-Gal+ICA(1.0μM)组。RT-PC...从Nrf2信号通路探讨淫羊藿苷(icariin,ICA)对D-半乳糖(D-galactose,D-Gal)诱导的小鼠睾丸支持细胞株15P-1细胞损伤的保护作用。将15P-1细胞分为对照组(control)、150 mM D-Gal处理组、D-Gal+ICA(0.5μM)组和D-Gal+ICA(1.0μM)组。RT-PCR法和Western blot法检测15P-1细胞分泌的相关因子GDNF、BMP4和SCF mRNA和蛋白的表达水平。Western blot法检测15P-1细胞紧密连接相关蛋白Occludin和Claudin-1、Nrf2信号通路相关蛋白Nrf2、HO-1和NQO-1的蛋白表达水平。免疫荧光法检测Nrf2表达及定位。与对照组相比,D-Gal处理组15P-1细胞分泌的相关因子GDNF、BMP4和SCF的mRNA和蛋白表达水平均显著下降,而给予ICA后均显著升高。此外,与对照组相比,D-Gal处理组15P-1细胞Occludin、Claudin-1、Nrf2、HO-1和NQO-1的蛋白表达水平均显著下降,而ICA可显著上调上述相关蛋白的表达。免疫荧光结果进一步显示,ICA可上调15P-1细胞核内Nrf2蛋白表达。ICA可减轻D-Gal诱导的15P-1细胞损伤,并改善其功能,其机制可能与上调Nrf2信号通路相关蛋白有关。展开更多
AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-...AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.展开更多
目的探讨下调15-脂氧酶-1(15-LOX-1)对脂多糖(LPS)作用下肺动脉内皮细胞(PAECs)和炎性因子分泌的影响。方法体外培养PAECs,并随机分为4组:对照组、LPS组、15-LOX-1 si RNA对照组(scramble组)和15-LOX-1 si RNA组。采用Real-time PCR检测...目的探讨下调15-脂氧酶-1(15-LOX-1)对脂多糖(LPS)作用下肺动脉内皮细胞(PAECs)和炎性因子分泌的影响。方法体外培养PAECs,并随机分为4组:对照组、LPS组、15-LOX-1 si RNA对照组(scramble组)和15-LOX-1 si RNA组。采用Real-time PCR检测15-LOX-1表达,MTT法检测PAECs增殖变化,ELISA法检测产物13-HODE以及炎性因子白细胞介素1α(IL-1α)和白细胞介素6(IL-6)的变化。结果与LPS组比较,15-LOX-1si RNA组15-LOX-1 m RNA表达降低,PAECs增殖增多,13-HODE、IL-1α和IL-6分泌减少,差异有统计学意义(P<0.05)。结论下调15-LOX-1可抑制炎性因子分泌,促进PAEC增殖,改善LPS对内皮的损伤。展开更多
基金National Natural Science Foundation of China (No. 81000393)
文摘AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL63 mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer. Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-LS-LOX-for Ad-vectorvia Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR(2) could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.
基金Supported by the National Natural Science Foundation of Shanghai,No.201540068
文摘AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.
文摘目的探讨下调15-脂氧酶-1(15-LOX-1)对脂多糖(LPS)作用下肺动脉内皮细胞(PAECs)和炎性因子分泌的影响。方法体外培养PAECs,并随机分为4组:对照组、LPS组、15-LOX-1 si RNA对照组(scramble组)和15-LOX-1 si RNA组。采用Real-time PCR检测15-LOX-1表达,MTT法检测PAECs增殖变化,ELISA法检测产物13-HODE以及炎性因子白细胞介素1α(IL-1α)和白细胞介素6(IL-6)的变化。结果与LPS组比较,15-LOX-1si RNA组15-LOX-1 m RNA表达降低,PAECs增殖增多,13-HODE、IL-1α和IL-6分泌减少,差异有统计学意义(P<0.05)。结论下调15-LOX-1可抑制炎性因子分泌,促进PAEC增殖,改善LPS对内皮的损伤。
