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Objective:To explore the effect of estrogen-related receptor α (ERRα) on lipopolysaccharide (LPS)-induced vascular endothelial cell apoptosis and tight junction protein degradation.Methods:RPMVECs transfected with shERRα were cultured in vitro and divided into four groups: Normal control group (Ctr group); shERRα knockdown group (shERRα group); normal cells + LPS treated group (LPS group): The cells in the six-well plates were cultured in serum-free medium for 12 h, and then treated with 20 μg/mL LPS for 12 h; and shERRα+LPS group: ERRα knockdown cells were treated as the LPS group. ROS fluorescence kit was used to detect the intracellular ROS levels . Apoptosis ratio was detected by TUNEL staining, AnnexinV-FITC and PI. Cell membrane ZO-1 expression was detected by cellular immunofluorescence, and the levels of apoptosis-related proteins Bcl-2, Bax, Smac, Cytochrome c, and tight junction protein ZO-1, as well as the expression of Occludin, JAM-A and E-Ca at molecular level were detected by Western blot.Results:Compared with the Ctr group and the shERRα group, the ROS level, apoptosis rate (TUNEL test: 16.44 ± 2.55; and flow cytometry test: 23.56 ± 2.22), the expression of pro-apoptotic proteins Bax, Smac and Cytochrome c were increased in the LPS group, while the expression of anti-apoptotic proteins Bcl-2 and tight junction protein were decreased. In the LPS group. Cellular immunofluorescence results showed that the ZO-1 was degraded in the cell membrane and the network structure was broken. Compared with the LPS group, inhibition of ERRα in the shERRα+LPS group increased cell damage.Conclusions:ERRα can negatively regulate the apoptosis and affect the function of pulmonary microvascular endothelial cells, thereby regulating sepsis-induced acute lung injury.
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Objective To investigate influences of estrogen-related receptor α(ERRα) on pulmonary vascular endothelium of rats undergoing sepsis. Methods Male Sprague-Dawley (SD) rats were divided into four groups according to the random number table method (12 in each group): normal control group (NC group), sham operation group (Sham group), sepsis model caused by cecal ligation and puncture (CLP) group (CLP group), XCT790 intervention group (XCT790 group, given the XCT790 2.5 mg/kg via intraperitoneal injection 30 minutes before CLP). After 24 hours, rats were sacrificed and the organs were harvested. The pathological changes of lung tissue were observed using hematoxylin and eosin (HE) staining, and the ultrastructural changes of lung tissue were observed by double staining of uranium citrate with lead acetate, the degree of apoptosis of pulmonary capillary endothelial cells were observed by TdT-mediated dUTP nike end labeling stain (TUNEL), the permeability of lung vascular endothelial was detected by Evans blue (EB) staining, the levels of serum cytokines were detected by enzyme linked immunosorbent assay (ELISA), and white blood cell count in bronchial alveolar lavage fluid (BALF) was detected. Results Compared with NC group and Sham group, the CLP group and XCT790 group had severe pathological damage and increased lung tissue permeability, the levels of serum cytokines and white blood cell count in BALF were increased. Compared with CLP group, the pathological changes of lung tissue, the degree of ultrastructural damage of lung tissue, the degree of apoptosis of lung capillary endothelial cells in XCT790 group further intensified, the permeability of lung endothelial barrier further increased [the content of EB (μg/g): 116.00±15.46 vs. 60.19±19.79, P < 0.05], and the level of serum cytokines further increased [interleukin-1β(IL-1β, ng/L): 71.38±4.01 vs. 56.58±2.45, interleukin-6 (IL-6, ng/L): 741.62±88.94 vs. 534.22±72.70, tumor necrosis factor-α(TNF-α, ng/L):188.55±7.41 vs. 143.33±11.27, all P < 0.05], the white blood cell count in the BALF increased further (×104/L:193.79±27.46 vs. 99.34±36.41, P < 0.05). Conclusion ERRα can aggravate inflammation in sepsis rats, destroy lung tissue and increase pulmonary permeability.
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Objective To observe the changes of proliferation and apoptosis of colon cancer cell line treated with oxaliplatin after the downregulation of ERRα and to investigate the mechanism.Methods Colon cancer cell lines Colo-205,HCT-116,SW620 and HT-29 were cultured by adherent cells and in accordance with the given intervention,they were divided into group XCT790-OHP-HCT-116(after oxaliplatin treatment,ERR αinhibitor XCT790 was administered),group siERRα-OHP-HCT-116(after oxaliplatin treatment,siERR α was transfected into HCT-116 cells and downregulated ERR αexpression),oxaliplatin intervention group(group OHP-HCT-116)and the control group(NC group)which was given no intervention.The experiment was divided into siERR αgroup with siERR α transfected with HCT-116 cells,downregulated ERR αexpression and the negative control group(siNC group)transfected with siNegative Control.Using Western blot method and real-time quantitative(qRT)-PCR for the detection of colorectal cancer cell ERRαprotein and mRNA expression,the expression of ERR αwas downregulated by ERR αinhibitors XCT790 and siERR,and apoptosis and proliferation of colon cancer cells were detected by flow cytometry and MTT.Western blot and qRT-PCR were used to detect apoptosis and proliferation-related gene proteins and mRNA expression.Results ERR αand mRNA protein in HCT-116 were higher than those of Colo-205,SW620 and HT-29 cell lines(P<0.05); in the XCT790-OHP-HCT-116 group,the early apoptosis rate was higher than those of the NC group and OHP-HCT-116 group(P<0.05),the survival rate of cell culture in 72 and 96 h in the XCT790-OHP-HCT-116 group was lower than those in the NC group and OHP-HCT-116 group(P<0.05).The siERR α HCT-116 cells transfected with down-regulation of ERR expression,siERR α -OHP-HCT-116 group early apoptosis rate was lower than those of NC group and OHP-HCT-116 group(P<0.05),siERR -OHP-HCT-116 group cells cultured for 72 and 96 h after the survival rate was lower than the NC group and OHP-HCT-116 group(P<0.05);After the downregulation of ERRαby siERR alpha transfected with HCT-116 cells,the early apoptotic rate in the group siERRα-OHP-HCT-116 was lower than that in the group NC and group OHP-HCT-116(P<0.05),the survival rate of the group siERRα-OHP-HCT-116 after 72 and 96 h were lower than those in the group NC and group OHP-HCT-116(P<0.05),siERR α was transfected into HCT-116 cells,compared with the siNC group,YAP1,p73,p63,MDM2, Capase 8,Capase 9 protein in the siERR group decreased(P<0.01),there was no significant difference in the level of mRNA(P>0.05).Conclusion The downregulation the expression of ERRαcan promote colon cancer cell apoptosis,inhibit proliferation,and enhance the killing effect of oxaliplatin on colon cancer cells.
ABSTRACT
Objective To observe the changes of proliferation and apoptosis of colon cancer cell line treated with oxaliplatin after the downregulation of ERRα and to investigate the mechanism.Methods Colon cancer cell lines Colo-205,HCT-116,SW620 and HT-29 were cultured by adherent cells and in accordance with the given intervention,they were divided into group XCT790-OHP-HCT-116(after oxaliplatin treatment,ERR αinhibitor XCT790 was administered),group siERRα-OHP-HCT-116(after oxaliplatin treatment,siERR α was transfected into HCT-116 cells and downregulated ERR αexpression),oxaliplatin intervention group(group OHP-HCT-116)and the control group(NC group)which was given no intervention.The experiment was divided into siERR αgroup with siERR α transfected with HCT-116 cells,downregulated ERR αexpression and the negative control group(siNC group)transfected with siNegative Control.Using Western blot method and real-time quantitative(qRT)-PCR for the detection of colorectal cancer cell ERRαprotein and mRNA expression,the expression of ERR αwas downregulated by ERR αinhibitors XCT790 and siERR,and apoptosis and proliferation of colon cancer cells were detected by flow cytometry and MTT.Western blot and qRT-PCR were used to detect apoptosis and proliferation-related gene proteins and mRNA expression.Results ERR αand mRNA protein in HCT-116 were higher than those of Colo-205,SW620 and HT-29 cell lines(P<0.05); in the XCT790-OHP-HCT-116 group,the early apoptosis rate was higher than those of the NC group and OHP-HCT-116 group(P<0.05),the survival rate of cell culture in 72 and 96 h in the XCT790-OHP-HCT-116 group was lower than those in the NC group and OHP-HCT-116 group(P<0.05).The siERR α HCT-116 cells transfected with down-regulation of ERR expression,siERR α -OHP-HCT-116 group early apoptosis rate was lower than those of NC group and OHP-HCT-116 group(P<0.05),siERR -OHP-HCT-116 group cells cultured for 72 and 96 h after the survival rate was lower than the NC group and OHP-HCT-116 group(P<0.05);After the downregulation of ERRαby siERR alpha transfected with HCT-116 cells,the early apoptotic rate in the group siERRα-OHP-HCT-116 was lower than that in the group NC and group OHP-HCT-116(P<0.05),the survival rate of the group siERRα-OHP-HCT-116 after 72 and 96 h were lower than those in the group NC and group OHP-HCT-116(P<0.05),siERR α was transfected into HCT-116 cells,compared with the siNC group,YAP1,p73,p63,MDM2, Capase 8,Capase 9 protein in the siERR group decreased(P<0.01),there was no significant difference in the level of mRNA(P>0.05).Conclusion The downregulation the expression of ERRαcan promote colon cancer cell apoptosis,inhibit proliferation,and enhance the killing effect of oxaliplatin on colon cancer cells.