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Objective To study the effects of estrogen on bicarbonate secretion of duodenal mucosal , and to observe estrogen receptor (ER) subtypes of estrogen.Methods Sixteen 4-week-old male C57 mice were divided into control group and estrogen group , with eight mice in each group .The mice serum level of estrogen was detected by chemiluminescence .The expression of cystic fibrosis transmembrane conductance regulator (CFTR), solute carrier family 26 (SLC26) A3 and SLC26A6 in the duodenum tissues were determined by real-time polymerase chain reaction ( RT-PCR).After SCBN cells treated with estrogen , ERαand ERβblocking agent, and transfected with silenced ER αand ERβfor 24 and 48 hours, the expression levels of CFTR, SLC26A3 and SLC26A6 mRNA in cells were detected by RT-PCR.The effects of estrogen before and after silenced ER αand ERβon bicarbonate secretion of SCBN cells were observed by high-speed ion imaging system.T test and rank sum test were used for statistical analysis .Results Compared with that of control group , the serum estrogen level of estrogen group was significantly high ((4 874 ±942) pmol/L vs.(657 ±187) pmol/L,t＝-11.579, P?0.01). The expression levels of CFTR, SLC26A3 and SLC26A6 mRNAs in duodenum tissues of estrogen group were higher than those of control group (0.856 ±0.302 vs.0.452 ±0.246, 2.910 ±1.680 vs.1.120 ±0.540, 1.272 ± 0.667 vs.0.319 ±0.114), and the differences were statistically significant ( t ＝-2.317,-2.483 and-3.721, all P?0.05).Compared with those treated with estrogen for 24 and 48 hours, the levels of CFTR mRNA and SLC26A6 mRNA were lower after the ERβblocking agent were added into estrogen for 24 and 48 hours (CFTR mRNA: 0.171 ±0.059 vs.0.522 ±0.260 and 0.111 ±0.014 vs.0.578 ±0.297; SLC26A6 mRNA:0.486 ±0.289 vs.1.118 ±0.571 and 0.492 ±0.231 vs.1.551 ±0.605), and the differences were statistically significant (tCFTR mRNA＝2.974 and 2.655, tSLC26A6 mRNA＝2.393 and 3.272; all P?0.05).Compared with those of silenced ERαgroup, the levels of CFTR mRNA, SLC26A3 mRNA and SLC26A6 mRNA were higher after ERα silenced and then added estrogen for 24 and 48 hours (24 h: 5.073 ±2.270 vs.1.185 ±0.494, 1.796 ±1.168 vs.0.468 ±0.108 and 3.085 ±1.357 vs.0.706 ±0.347; 48 h: 5.025 ±1.998 vs.1.185 ±0.494, 1.557 ± 0.653 vs.0.468 ±0.108 and 3.290 ±1.750 vs.0.706 ±0.347), and the differences were statistically significant (t24 h＝-4.122,-2.773 and -4.162, t48 h ＝-4.604,-4.034 and -3.250; all P?0.05).Compared with that of silenced ERαgroup, the bicarbonate secretion increased after ER αsilenced and then added estrogen for 24 and 48 hours (0.72 ±0.17 and 1.15 ±0.25 vs.0.35 ±0.17), and pH also elevated, and the differences were statistically significant (t＝-6.516 and -12.387, both P?0.01).Conclusion Estrogen mainly up-regulates the expression of bicarbonate transporter protein in duodenal mucosal epithelial cells through ER β, and promotes the bicarbonate secretion of duodenal mucosa .
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Objective@#To study the effects of estrogen on bicarbonate secretion of duodenal mucosal, and to observe estrogen receptor (ER) subtypes of estrogen.@*Methods@#Sixteen 4-week-old male C57 mice were divided into control group and estrogen group, with eight mice in each group. The mice serum level of estrogen was detected by chemiluminescence. The expression of cystic fibrosis transmembrane conductance regulator (CFTR), solute carrier family 26 (SLC26) A3 and SLC26A6 in the duodenum tissues were determined by real-time polymerase chain reaction (RT-PCR). After SCBN cells treated with estrogen, ERα and ERβ blocking agent, and transfected with silenced ERα and ERβ for 24 and 48 hours, the expression levels of CFTR, SLC26A3 and SLC26A6 mRNA in cells were detected by RT-PCR. The effects of estrogen before and after silenced ERα and ERβ on bicarbonate secretion of SCBN cells were observed by high-speed ion imaging system. T test and rank sum test were used for statistical analysis.@*Results@#Compared with that of control group, the serum estrogen level of estrogen group was significantly high ((4 874±942) pmol/L vs. (657±187) pmol/L, t=-11.579, P<0.01). The expression levels of CFTR, SLC26A3 and SLC26A6 mRNAs in duodenum tissues of estrogen group were higher than those of control group (0.856±0.302 vs. 0.452±0.246, 2.910±1.680 vs. 1.120±0.540, 1.272±0.667 vs. 0.319±0.114), and the differences were statistically significant (t=-2.317, -2.483 and -3.721, all P<0.05). Compared with those treated with estrogen for 24 and 48 hours, the levels of CFTR mRNA and SLC26A6 mRNA were lower after the ERβ blocking agent were added into estrogen for 24 and 48 hours (CFTR mRNA: 0.171±0.059 vs. 0.522±0.260 and 0.111±0.014 vs. 0.578±0.297; SLC26A6 mRNA: 0.486±0.289 vs. 1.118±0.571 and 0.492±0.231 vs. 1.551±0.605), and the differences were statistically significant (tCFTR mRNA=2.974 and 2.655, tSLC26A6 mRNA=2.393 and 3.272; all P<0.05). Compared with those of silenced ERα group, the levels of CFTR mRNA, SLC26A3 mRNA and SLC26A6 mRNA were higher after ERα silenced and then added estrogen for 24 and 48 hours (24 h: 5.073±2.270 vs. 1.185±0.494, 1.796±1.168 vs. 0.468±0.108 and 3.085±1.357 vs. 0.706±0.347; 48 h: 5.025±1.998 vs. 1.185±0.494, 1.557±0.653 vs. 0.468±0.108 and 3.290±1.750 vs. 0.706±0.347), and the differences were statistically significant (t24 h=-4.122, -2.773 and -4.162, t48 h=-4.604, -4.034 and -3.250; all P<0.05). Compared with that of silenced ERα group, the bicarbonate secretion increased after ERα silenced and then added estrogen for 24 and 48 hours (0.72±0.17 and 1.15±0.25 vs. 0.35±0.17), and pH also elevated, and the differences were statistically significant (t=-6.516 and -12.387, both P<0.01).@*Conclusion@#Estrogen mainly up-regulates the expression of bicarbonate transporter protein in duodenal mucosal epithelial cells through ERβ, and promotes the bicarbonate secretion of duodenal mucosa.
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Objective: To observe the influence of TRPV6 gene silence on SW480 colon cancer cell biological behavior, change in the in-tracellular concentration of calcium, as well as influence of 1,25 (OH)2D3CaCl2and CuCl2on SD rat colonic neoplasm models. Method: SW480 colon cancer cells were infected using lentivirus particles. TRPV6 protein and mRNA expression was detected using immunohistochemical tests, Western blot, and PCR. Moreover, the proliferation, metastasis, and apoptosis of SW480 colon cancer cells were detected through MTT assay and metastasis and apoptosis experiments, and the concentration of Ca2+in SW480 colon cancer cells was measured using high-speed ionic imaging. The SD rat colon cancer model was established based on DMH, and were assigned into experimental group (DMH group, 15) and intervention group (DMH+1,25 (OH)2D3group, DMH+CuCl2group) and control group, 10 in each group. The SD rat colon cancer model is established based on DMH, given 1,25(OH)2D3(37.5 nmol/kg) and CuCl2(375 μmol/kg) separately as intervention. The occurrence of colonic neoplasms and glandular cancers in each group of rats was observed, and Western blot was employed for detection of the TRPV6 protein expression. Results: After the transfection of SW480 colon cancer cells by TRPV6-RNAi, the expression of TRPV6 mRNA and protein decreased, intracellular concentration of Ca2+decreased, proliferation and metastasis rate of SW480 colon cancer cells decreased, and apoptosis rate of these cells increased. The differences between the groups with intervention and the blank control group and negative control group showed statistical significance (P<0.05). The colon cancer occurrence rate in the control group was 0, while that of the DMH+1,25 (OH)2D3 group, DMH group, and DMH+CuCl2were 100%, 84.62%, and 33.33%, respectively. The TRPV6 protein expression was detected in all groups, while DMH+1,25(OH)2D3group was observed to exhibit the highest level of expression, followed by the DMH group, DMH+CuCl2group, and control group. The differences were of statistical significance (P<0.05). Conclusions: The proliferation and metastasis of SW480 colon cancer cells can be prohibited by lowering the concentration of Ca2+in the cells. Thus, the apoptosis of the cells can be induced. 1,25 (OH)2D3can help improve the expression of TRPV6 protein in experimental rat colon tissues and promote the formation of colon neoplasms. CuCl2can help lower the expression of TRPV6 protein in experimental rat colon tissues and prevent the formation of colon neoplasms.
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Objective:To investigate the role of TRPV5 and TRPV6 in intracellular calcium regulation and biological behaviors of SW480 colon cancer cells. Methods:qRT-PCR, Western blot, and immunocytochemistry were applied to determine the mRNA and protein ex-pression levels of TRPV5 and TRPV6 in SW480 colon cancer cell line upon treatment with TRPV5 and TRPV6 agonist, 1-25(OH)2D3, and inhibitor, CuCl2. The change of intracellular Ca2+level was examined with a confocal laser scanning microscope. Scratch test, MTT, and TUNEL assays were used to analyze the cell migration, proliferation, and apoptosis, respectively. Results:As an agonist of TRPV5 and TRPV6, 1-25(OH)2D3 significantly up-regulated the mRNA and protein expression levels of TRPV5 and TRPV6 in SW480 cell lines. On the other hand, CuCl2, being an inhibitor of TRPV5 and TRPV6, effectively down-regulated the TRPV5 and TRPV6 mRNA and protein expres-sion levels (P<0.05). The intracellular calcium concentration in SW480 cell line significantly increased upon treatment with 1-25 (OH)2D3, and significantly decreased with CuCl2 treatment (P<0.05). 1-25(OH)2D3 promoted cell proliferation and migration, and inhibit-ed apoptosis of SW480 cell in a time-and dose-dependent manner (P<0.05). However, CuCl2 significantly repressed cell proliferation and migration and induced apoptosis (P<0.05). Conclusion: TRPV5 and TRPV6 can affect the biological behaviors of colon cancer SW480 cells by regulating intracellular Ca2+level.
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Background and purpose:Previous studies have suggested Na+-Ca2+ exchanger isoform 1 (NCX1) as a key component of calcium homeostasis was involved in the tumorigenesis. However, the role of NCX1 and calcium signal in tumorigenesis of hepatocellular carcinoma (HCC) has not been explored. This study aimed to investigate the effect of NCX1 on cell proliferation and migration of HCC HepG2 cells in vitro and the possible mechanism.Methods:Both the real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were applied to assess the expression of NCX1 mRNA and protein in normal hepatic cells (LO2), HCC cell line (HepG2), human normal hepatic tissues and hepatocellular carcinoma tissues. The change of intracellular calcium signal in LO2 and HepG2 cells via acti-vated NCX1 channel in the presence or absence of Na+ was examined by a confocal laser scanning microscope. The effects of NCX1 special inhibitor KB-R7943 on cell proliferation and migration of HepG2 cells were measured by MTT and cellscratch test.Results:Both mRNA and protein expression of NCX1 were higher in HCC tissues and cell line HepG2 than in the normal tissues and cell line LO2 (P<0.05). The activation of NCX1 channel induced a slight rise in cytoplasmic Ca2+concentration ([Ca2+]cyt) in normal cells, but caused a marked increase in cancer cells. And the NCX1 activation induced intracellular calcium increase was significantly reversed by NCX1 inhibitor KB-R7943 (P<0.05). Both NCX1-mediated proliferation and migration of HepG2 were also significantly attenuated by the KB-R7943 (P<0.05).Conclusion:NCX1 is up-regulated in HCC cells and tissues. The activation of NCX1 mediates intracellular calcium homeostasis. The inhibition of NCX1 activity can suppress the proliferation and migration of HepG2 cells. It is suggested that NCX1 may be involved in the development and progression of HCC.
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Objective To investigate the expression of Aiolos transcription factor in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE),and to explore their clinical significance.Methods Western blot was performed to detect the protein expression of Aiolos transcription factor in PBMCs from 19 patients with active SLE,13 patients with stable SLE and 30 healthy volunteers.The relationship between the expression of Aiolos transcription factor and SLE disease activity index (SLEDAI) was analyzed.Results A significant difference was noted in the relative expression of Aiolos transcription factor between SLE patients and normal controls(0.56±0.17 vs 0.81±0.09,P<0.01) and between patients in active stage and those in stable stage (0.52±0.14 vs 0.65±0.19,P<0.05).In addition,the expression level of Aiolos protein was negatively correlated with SLEDAI in patients (r=-0.65,P<0.01).Conclusion A decrease is observed in the expression of Aiolos transcription factor in SLE patients.which is correlated with the disease activity in SLE.