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Objective To evaluate the effects of interleukin-22 (IL-22) on the expression of CC chemokine ligand 27 (CCL27) in human epidermal keratinocytes,and to explore its mechanism.Methods Immunohistochemical study was performed to determine the expression of CCL27 in skin lesions of 10 patients with psoriasis and skin tissues of 5 healthy controls.Cultured HaCaT cells were divided into 8 groups:control group treated with PBS,5 IL-22 groups treated with 12.5,25,50,100 and 200 μg/L IL-22 respectively,2 signaling pathway inhibition groups treated with 50 μrmol/L AG490 (JAK2/STAT3 signaling pathway inhibitor) or PD98059 (MAPK-ERK1/2 signaling pathway inhibitor) for 2 hours followed by the treatment with 50 μg/L IL-22 in the 5％ CO2 incubator at 37 ℃.After 24-hour cultivation,total proteins were extracted,and culture supernatants were collected,and both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to determine the expression of CCL27.Results Immunohistochemical study showed that the expression of CCL27 was significantly higher in the skin lesions of the patients with psoriasis than in the skin tissues of the healthy controls.Western blot analysis revealed that the protein expression of CCL27 in the 12.5-,25-,50-,100-and 200-μg/L IL-22 groups was 0.491 ± 0.013,0.620 ± 0.019,0.623 ± 0.014,0.802 ± 0.052 and 1.138 ± 0.013 respectively,which were all higher than that in the control group (0.413 ± 0.013,all P ＜ 0.01).The expression of CCL27 was significantly lower in the IL-22 + AG490 group (0.411 ± 0.019) and IL-22 + PD98059 group (0.280 ± 0.012) than in the 50-μg/L IL-22 group (both P ＜ 0.01).ELISA also showed the same trend of changes in the level of CCL27 in the above groups as Western blot showed.Conclusion IL-22 can promote the expression of CCL27 in HaCaT cells,which may be associated with the MAPK-ERK 1/2 and JAK2/STAT3 signaling pathways.
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Objective To investigate the effect of epidermal growth factor (EGF) on the expression of Matrix metalloproteinase-9 (MMP-9) and the signalling pathways involved in the trophoblast cell line JEG-3. Methods The JEG-3 trophoblast cell line was used in this study. (1) JEG-3 cells were cultured with various concentrations of EGF (0, 1, 10,20 ng/ml) for 24 hours and the expression of MMP-9 was tested by western blotting and reverse transcription PCR (RT-PCR). (2) Western blotting and RT-PCR were also used to investigate the expression of MMP-9 expression after incubation for 0,4,12 and 24 hours with EGF treatment (10 ng/ml) in JEG-3 cells. (3) According to the different added ingredients, JEG-3 cells were divided into some groups: control group (without EGF), EGF group (exposure to l0ng/ml EGF),EGF + inhibitors group (exposure to 10 ng/ml EGF +20 ng/ml SB203580 or exposure to 10 ng/ml EGF + 10ng/ml U0126) inhibitors group (exposure to 20 ng/ml SB203580 or exposure to 10 ng/ml U0126). Western blotting were used to investigate the expression levels of MMP-9, nuclear factor kappa B (NF-kB) ,p38MAPK, phospho-p38MAPK (p-p38MAPK) , extracellular -signal regulated kinase (ERK) and phospho-ERK (p-ERK) protein in JEG-3 cells after incubation for 24 hours. Results (1) The profiles of MMP-9mRNA were increased by various concentrations of EGF (0, 1 , 10, 20 ng/ml) in JEG-3 cells after 24hculture. The expression of MMP-9 mRNA in JEG-3 cells exposure at 1 ng/ml of EGF (0. 567 ±0. 056) , 10ng/ml of EGF (1. 392 ±0. 133) , 20 ng/ml of EGF (1. 971 ±0. 067) were significantly higher respectively (P <0. 05) , compared with 0 ng/ml of EGF treatment (0. 166 ±0. 015). Similarly, MMP-9 mRNAs were also increased with the increasing incubation time. Compared to EGF (10 ng/ml) stimulation for 0 h (0.253 ±0.044), the MMP-9 mRNA profiles were 0. 470 ±0. 026, 1.061 ±0. 115, 1. 453 ±0. 180 for 4,12 and 24 hours, respectively (P < 0. 05). (2) In accordance to the mRNA profiles, the expression of MMP-9 protein was also increased by different concentrations of EGF (0,1, 10, 20 ng/ml) in JEG-3 cells after 24 h-culture. The abundance of MMP-9 protein in the three groups was 0. 043 ±0. 012, 0. 085 ±0. 008, 0. 142 ±0. 015, with a significantly higher expression, compared with 0 ng/ml of EGF treatment (0. 004 ±0.001, P < 0.05) respectively. Similarly, MMP-9 proteins were also increased with the increasing incubation time. Compared to EGF(10 ng/ml) stimulation for 0 h (0. 030 ±0. 009) , the profiles of MMP-9 protein were 0. 137 ± 0. 010, 0. 240 ± 0. 010, 1.240 ±0.061 for 4, 12 and 24 hours, respectively (P < 0.05). (3) Both p38MAPK and ERK signalling pathways were activated by EGF in JEG-3 cells. The expression of p-p38MAPK was significantly higher (without or with 10 ng/ml EGF, 234. 1 ± 4. 1 vs.260. 9 ± 2. 5 , P < 0. 05) , however, the p38 MAPK inhibitor SB203580 markedly suppressed the increase in p-p38MAPK content induced by EGF(227. 9 ±2. 4 vs. 260. 9 ±2. 5, P<0. 05). Similarly, the expression of p-ERK was significantly higher with EGF treatment (812. 2 ±3. 5) vs. without EGF group (453.4±5.8) (P <0. 05) , while the ERK inhibitor U0126 significantly inhibited the increased p-ERK content in response to EGF treatment (71. 0 ± 1. 2 vs. 812. 2 ± 3. 5, P < 0. 05) . (4) The p38MAPK inhibitor SB203580 significantly reduced the expression of EGF-induced MMP-9 (0. 645 ± 0. 270 vs. 1. 476 ± 0. 452, P < 0. 05)and NF-kB (0.530 ± 0.026 vs. 0.959 ± 0. 017, P < 0. 05) . (5) The ERK inhibitor U0126 also significantly reduced the expression of EGF-induced MMP-9 (0. 623 ±0. 030 vs. 2. 112 ±0. 056, P <0. 05)and NF-kB (0. 325 ± 0. 082 vs. 0. 939 ± 0. 153, P < 0. 05). Conclusion EGF induced the expression of MMP-9 in a time and dose-dependant manner in JEG-3 cells. EGF enhanced MMP-9 expression through the activation of p38MAPK and ERK signalling pathways in JEG-3 cells.
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Objective To investigate the role of human leukocyte antigen-G ( HLA-G ) on the invasion and the molecular mechanism involved in this cellular progress in HTR-8/SVneo cell line. Methods There were three groups: groups of transfection, negative control and blank control, which corresponding to treatment by HLA-G specific siRNA, negative siRNA and only lipofectamine 2000 using lipofection technology in HTR-8/SVneo cell line. The efficiency of down-regulated of HLA-G was detected by reverse transcription-polymerase chain reaction and western blot analysis in mRNA and protein level,respectively. Changes of p38 mitogen-activated protein kinases (p-p38MAPK)/p38MAPK protein levels and the cell invasion were respectively detected by western blot analysis and transwell test. Results ( 1 ) The mRNA levels of HLA-G transfection group, negative control group and blank control group were 0. 26 ±0. 08, 0. 71 ±0. 11, 0. 79 ±0. 07, respectively. There was significant difference between transfection group and negative control group ( P ＜ 0. 01 ), while there was no significant difference between negative control group and blank control group ( P ＞ 0. 05 ). The efficiencies of down-regulated of HLA-G were ( 69. 8 ±6. 3)%, ( 14. 9 ± 2. 2 )%, 0 in transfection group, negative control group and blank control group respectively in mRNA level. (2)In protein levels, HLA-G were 0. 20 ±0. 15, 0. 75 ±0. 12, 0. 76 ±0. 21 in transfection group, negative control group and blank control group, respectively. There was significant difference between transfection group and negative control group ( P ＜ 0. 01 ), whereas there was no significant difference between negative control group and blank control group ( P ＞ 0. 05 ). The efficiencies of down-regulated of HLA-G were (81. 1 ± 14.4)%, ( 18.0 ± 7.7)%, 0 in transfection group, negative control group and blank control group respectively. ( 3 ) The invasive number of transfection group, negative control group and blank control group were 57 ± 38,364 ± 79 and 260 ± 84, respectively, with a significant difference between transfection group and negative control group (P ＜ 0. 01 ). There was no significant difference between negative control group and blank control group ( P ＞ 0. 05 ). ( 4 ) The p-p38MAPK/p38MAPK values of the HLA-G transfection group, negative control group and blank control group were 0. 74 ±0.04, 0. 47 ± 0. 09 and 0. 36 ± 0. 21, respectively. HLA-G transfection group was significantly different compared with the other two groups( P ＜0. 01 ). (5)Without or with SB203580, the p-p38MAPK/ p38MAPK values of the HLA-G transfection group were 0. 89 ± 0. 09 and 0. 16 ± 0. 04, the values of negative control group and blank control group were 0.76 ±0.08, 0. 14 ±0.03 and 0.51 ±0.05, 0.03 ±0.01, respectively. There was significant difference between without SB203580 and with SB203580 ( P ＜ 0. 01 ). (6)Without or with SB203580, the invasive number of transfection group were 51 ± 13 and 90 ± 21 ,respectively,which was significantly different ( P ＜ 0. 01 ). The invasive number of negative control group and blank control group were 290 ± 52, 298 ± 33 and 290 ± 73, 264 ± 64, respeczively, which was no significant difference between without SB203580 and with SB203580 (P ＞ 0. 05 ). Conclusions HLA-G gene may regulate invasion of trophoblast-derived cell line HTR-8/SVneo via p38MAPK signaling pathway. The lower expression of HLA-G in trophoblast cells may lead to the occurrence of pathologic pregnancy.
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Objective To observe the influence of hepatic oval cell (HOC) on the expression ERK and P38MAPK signaling pathway protein in liver tissue of murine experimental hepatofibrosis (HF).Method SD rats were fed with 10% ethanol and food with high-fat and low-protein, and were injected subcutaneously with carbontetrachloride once every four days for 8 weeks to establish hepatic fibrosis. HOGs were isolated from male HF rats by collagenase porfusion of the liver. HF rats at 8th week were transplanted with 0. 5 ml HOC suspension medium at a density of 1 × 109 cell /ml via portal vein, and the rats were sacrificed at 8th, 15th, 30th day respectively. Histopathologic changes of liver tissues were observed by HE and Masson. The expression of ERK and P38MAPK signaling pathway protein were determined by Western blotting. Result Hepatofibrosis was reversed and the degree of hyperplasia fibrilcollagen in hepatic fibrosis rats decreased significantly by HOC transplantion. HOC down-regulated the protein expression of Ras, ERK,p-ERK, c-fos, c-jun, STAT3, ALB, FGF-3, PCNA ( F = 91.88,36.28,54.66,93.07,64.76,58.49,52.63,20.45 ,27.03, all P ＜ 0.05 ), up-regulated the protein expression level of HNF-α1, PDGF-Rβ significantly in liver tissues(F = 18.63,25.99,P ＜0.05). Conclusions HOC improves the degree of hepatofibrosis through inhibiting hyperplasia of collagen fibril in liver tissue of hepatofibrosis rats. With the presence of HOC the expression of c-fos,c-jun,STAT3,5 was not activated by p-P38MAPK. The expression of c-kit and HNF-1α increased and that liver tissue injury alleviated, and hepatofibrosis was improved.
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Objective To investigate the signaling mechanisms underlying up-regulation of nontypeable Haemophilus influenzae(NTHi)-induced MUC5AC mucin expression. Methods The expression of MUC 5AC at mRNA level was measured by reverse transcriptase-polymerase chain reaction (RT-PCR)and real-time fluorescent quantitative PCR.Immunoprecipitation and Western blot were performed tO examine the phosphorylation of p38 mitogen-activated protein kinase(p38MAPK)and epidermal growth factor receptor(EGFR)or the effect of dominant negative mutant of EGFR on the phosphorylation of p38MAPK in HM3 cells treated with NTHi.Luciferase assay was also performed to examine the effect of p38MAPK and EGFR inhibitors or dominant negative mutant of EGFR on NTHi-induced MUC5AC expression at transcription level.Results NTHi induced MUC5AC mucin expression at both mRNA and transcription levels.Phosphorylation of p38MAPK and EGFR were observed in HM3 cells treated with NTHi.Either SB203580,a specific inhibitor for p38MAPK or AGl478,a specific inhibitor for EGFR.inhibited NTHi-induced MUC5AC up-regulation at transcription level. Furthermore,Overexpressing dominant negative mutant of EGFR also inhibited NTHi-induced MUC5AC upregulation at transcription 1evel in a dos-dependent manner.EGFR inhibitor reduced NTHi-induced p38MAPK phosphorylation in HM3 cells.Conclusion Bacterium NTHi up-regulates MUC5AC mucin transcription via EGFR-p38MAPK signaling pathway.