Abstract：
OBJECTIVE: To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation (EMT) of human peritoneal mesothelial cell (HPMC) via endoplasmic reticulum stress (ERS).
METHODS: HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting.
RESULTS: Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2-ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2-ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2-ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2-ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/β-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/β-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/β-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/β-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2-ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2-ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2-ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2-ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/β-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/β-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/β-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/β-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05].
CONCLUSIONS: High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.
METHODS: HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting.
RESULTS: Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2-ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2-ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2-ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2-ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/β-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/β-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/β-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/β-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2-ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2-ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2-ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2-ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/β-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/β-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/β-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/β-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05].
CONCLUSIONS: High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.
摘要：
目的：研究不同浓度钙离子通过内质网应激(ERS)对人腹膜间皮细胞(HPMC)上皮间质转化(EMT)的影响。
方法：HPMC细胞系HMrSV5体外培养，分组处理。对照组的细胞，高钙组1、高钙组2分别用钙离子浓度分别为1.25、1.75和2.25mmol/L的培养基处理,分别。溶剂对照组用含有1.25mmol/L生理钙离子浓度和0.1%二甲基亚砜(DMSO)的培养基处理,高钙+溶剂组用含2.25mmol/L钙离子浓度和0.1%DMSO的培养基处理,高钙+4-苯基丁酸(4-PBA)组用含2.25mmol/L钙离子浓度和1mmol/LERS抑制剂4-PBA的培养基处理,每组治疗48小时。光镜下观察各组细胞形态变化。免疫荧光染色观察上皮细胞表型标记小带闭塞蛋白1（ZO-1）和间充质细胞表型标记α-平滑肌肌动蛋白（α-SMA）在细胞中的表达。EMT标记基因E-cadherin的表达,荧光定量聚合酶链反应(PCR)检测ZO-1、α-SMA和波形蛋白。ERS标记蛋白磷酸化蛋白激酶R样内质网激酶(p-PERK)的表达,磷酸化真核起始因子2α（p-eIF2α），免疫印迹法检测转录激活因子4（ATF4）和C/EBP同源蛋白（CHOP）。
结果：与对照组相比，HMrSV5细胞的形态变得细长和纤维化，ZO-1的荧光强度增加，高钙1和高钙2组α-SMA荧光强度降低，表明细胞从上皮细胞转化为间充质细胞。E-cadherin和ZO-1的mRNA表达显著降低,而α-SMA和波形蛋白的mRNA表达和p-PERK的蛋白表达,p-eIF2α，ATF4和CHOP显著升高,此外,上述标记基因或蛋白质在高钙2组比高钙1组表达更明显[E-cadherinmRNA（2-ΔCt）：0.53±0.05vs.0.75±0.09，ZO-1mRNA（2-ΔΔCt）：0.42±0.06vs.0.69±0.06，α-SMAmRNA（2-ΔΔCt）：1.81±0.16vs.1.32±0.14，波形蛋白mRNA（2-ΔΔCt）：2.05±0.22vs.1.48±0.16，p-PERK蛋白（p-PERK/β-肌动蛋白）：0.81±0.09vs.0.59±0.06，p-eIF2α蛋白（p-eIF2α/β-肌动蛋白）：0.87±0.10vs.0.50±0.06，ATF4蛋白（ATF4/β-肌动蛋白）：0.93±0.10vs.0.72±0.06，CHOP蛋白（CHOP/β-肌动蛋白）：0.79±0.090.46±0.04,均P<0.05]。与溶剂对照组相比，细胞的形态变化,高钙离子浓度2.25mmol/L后，EMT标记基因和ERS标记蛋白的表达与高钙2组比对照组一致。与高钙+溶剂组相比，高钙+4-PBA组细胞形态恢复多边形和卵石样上皮细胞的特征，ZO-1的荧光强度增加，α-SMA的荧光强度下降，细胞中E-cadherin和ZO-1的mRNA表达明显增加[E-cadherinmRNA（2-ΔCt）：0.86±0.09vs.0.57±0.04，ZO-1mRNA（2-ΔΔCt）：0.81±0.06vs.0.48±0.05，均P<0.05]，α-SMA和波形蛋白的mRNA表达和p-PERK的蛋白表达,p-eIF2α，ATF4和CHOP显着降低[α-SMAmRNA（2-ΔΔCt）：1.21±0.13vs.1.77±0.15，波形蛋白mRNA（2-ΔΔCt）：1.30±0.14vs.1.94±0.20，p-PERK蛋白（p-PERK/β-肌动蛋白）：0.38±0.04vs.0.92±0.11，p-eIF2α蛋白（p-eIF2α/β-肌动蛋白）：0.34±0.05vs.1.05±0.13，ATF4蛋白（ATF4/β-肌动蛋白）：0.57±0.06vs.0.97±0.11，CHOP蛋白（CHOP/β-肌动蛋白）：0.51±0.04vs.0.90±0.12,均P<0.05]。
结论：1.75mmol/L和2.25mmol/L的高钙离子浓度通过激活ERS促进HPMC的EMT。
方法：HPMC细胞系HMrSV5体外培养，分组处理。对照组的细胞，高钙组1、高钙组2分别用钙离子浓度分别为1.25、1.75和2.25mmol/L的培养基处理,分别。溶剂对照组用含有1.25mmol/L生理钙离子浓度和0.1%二甲基亚砜(DMSO)的培养基处理,高钙+溶剂组用含2.25mmol/L钙离子浓度和0.1%DMSO的培养基处理,高钙+4-苯基丁酸(4-PBA)组用含2.25mmol/L钙离子浓度和1mmol/LERS抑制剂4-PBA的培养基处理,每组治疗48小时。光镜下观察各组细胞形态变化。免疫荧光染色观察上皮细胞表型标记小带闭塞蛋白1（ZO-1）和间充质细胞表型标记α-平滑肌肌动蛋白（α-SMA）在细胞中的表达。EMT标记基因E-cadherin的表达,荧光定量聚合酶链反应(PCR)检测ZO-1、α-SMA和波形蛋白。ERS标记蛋白磷酸化蛋白激酶R样内质网激酶(p-PERK)的表达,磷酸化真核起始因子2α（p-eIF2α），免疫印迹法检测转录激活因子4（ATF4）和C/EBP同源蛋白（CHOP）。
结果：与对照组相比，HMrSV5细胞的形态变得细长和纤维化，ZO-1的荧光强度增加，高钙1和高钙2组α-SMA荧光强度降低，表明细胞从上皮细胞转化为间充质细胞。E-cadherin和ZO-1的mRNA表达显著降低,而α-SMA和波形蛋白的mRNA表达和p-PERK的蛋白表达,p-eIF2α，ATF4和CHOP显著升高,此外,上述标记基因或蛋白质在高钙2组比高钙1组表达更明显[E-cadherinmRNA（2-ΔCt）：0.53±0.05vs.0.75±0.09，ZO-1mRNA（2-ΔΔCt）：0.42±0.06vs.0.69±0.06，α-SMAmRNA（2-ΔΔCt）：1.81±0.16vs.1.32±0.14，波形蛋白mRNA（2-ΔΔCt）：2.05±0.22vs.1.48±0.16，p-PERK蛋白（p-PERK/β-肌动蛋白）：0.81±0.09vs.0.59±0.06，p-eIF2α蛋白（p-eIF2α/β-肌动蛋白）：0.87±0.10vs.0.50±0.06，ATF4蛋白（ATF4/β-肌动蛋白）：0.93±0.10vs.0.72±0.06，CHOP蛋白（CHOP/β-肌动蛋白）：0.79±0.090.46±0.04,均P<0.05]。与溶剂对照组相比，细胞的形态变化,高钙离子浓度2.25mmol/L后，EMT标记基因和ERS标记蛋白的表达与高钙2组比对照组一致。与高钙+溶剂组相比，高钙+4-PBA组细胞形态恢复多边形和卵石样上皮细胞的特征，ZO-1的荧光强度增加，α-SMA的荧光强度下降，细胞中E-cadherin和ZO-1的mRNA表达明显增加[E-cadherinmRNA（2-ΔCt）：0.86±0.09vs.0.57±0.04，ZO-1mRNA（2-ΔΔCt）：0.81±0.06vs.0.48±0.05，均P<0.05]，α-SMA和波形蛋白的mRNA表达和p-PERK的蛋白表达,p-eIF2α，ATF4和CHOP显着降低[α-SMAmRNA（2-ΔΔCt）：1.21±0.13vs.1.77±0.15，波形蛋白mRNA（2-ΔΔCt）：1.30±0.14vs.1.94±0.20，p-PERK蛋白（p-PERK/β-肌动蛋白）：0.38±0.04vs.0.92±0.11，p-eIF2α蛋白（p-eIF2α/β-肌动蛋白）：0.34±0.05vs.1.05±0.13，ATF4蛋白（ATF4/β-肌动蛋白）：0.57±0.06vs.0.97±0.11，CHOP蛋白（CHOP/β-肌动蛋白）：0.51±0.04vs.0.90±0.12,均P<0.05]。
结论：1.75mmol/L和2.25mmol/L的高钙离子浓度通过激活ERS促进HPMC的EMT。
