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Objective:To study the role and possible molecular mechanism of neural precursor cell-expressed developmentally down-regulated gene 4-like ( NEDD4 L) in the replication of hepatitis B virus (HBV). Methods:Small interfering RNA (siRNA) targeting NEDD4 L, plasmid expressing NEDD4 L with hemagglutinin(HA) C-terminal tag (pcDNA3.1- NEDD4 L-HA), plasmid expressing 1.3×HBV genome (pGEM-HBV1.3) and poly (dAT: dAT) were respectively transfected into HepG2 cells using Lipofectamine2000. HepG2.2.15 cells, a cell line that can stably express HBV, were used as control. The mRNA levels of NEDD4 L, interferon (IFN)-α, IFN-β, interferon-stimulated gene 56 ( ISG56), myxovirus resistance protein A ( MxA), oligoadenylate synthetase ( OAS), and the levels of HBV DNA or 3.5 kb HBV RNA were detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the silence and over-expression of NEDD4 L, and the protein levels of the related signaling molecules. The amount of IFN-β in the cellular supernatant was measured by enzyme linked immunosorbent assay (ELISA). Student t test was used for comparison of continuous data between groups. Results:The levels of NEDD4L mRNA and protein in HepG2.2.15 cells were 10.53±0.47 and 4.17±0.43, respectively, which were both statistically higher than those in HepG2 cells (1.00±0.05, t=3.27, P=0.008 and 1.26±0.25, t=1.68, P=0.030, respectively). In HepG2 cells with knockdown of NEDD4 L, the expression level of HBV DNA in cellular supernatant was 0.32±0.09, which was statistically lower than that in the control (1.00±0.05, t=-0.93, P=0.020), and the expression level of 3.5 kb HBV RNA was 0.49±0.11, which was statistically lower than that in the control (1.00±0.05, t=-0.68, P=0.040), while the mRNA levels of IFN-β and downstream effector molecules ( ISG56, MxA and OAS) were all significantly increased compared with the control ( t=4.66, 9.38, 7.29 and 7.01, respectively, all P<0.01). With poly (dAT: dAT) treatment and vesicular stomatitis virus (VSV) stimulation, the levels of IFN-β in HepG2 cells with knockdown of NEDD4 L were (776.41±115.49) ng/L and (961.21±130.19) ng/L, respectively, which were both statistically higher than those of the control group ((320.15± 56.05) ng/L, t=2.43, P=0.020; (440.17±67.82) ng/L, t=2.85, P=0.030, respectively). With poly (dAT: dAT) treatment and VSV stimulation, the levels of IFN-β in HepG2 cells with overexpression of NEDD4 L were (156.18±26.47) ng/L and (176.67±34.51) ng/L, respectively, which were both statistically lower than those of the control group ((320.38±49.39) ng/L, t=-2.03, P=0.040; (440.59±68.83) ng/L, t=-1.93, P=0.030, respectively). Western blot showed that the replication of HBV reduced the protein level of melanoma differentiation-associated protein 5 (MDA5), a key molecule in upstream of IFN-β, but the down-regulation was not obvious in cells with the knockdown of NEDD4 L. Conclusion:The replication of HBV could promote the up-regulation of NEDD4L protein and subsequently reduce the protein level of MDA5, thereby inhibiting the production of IFN-β, which facilitates HBV to escape the innate immune response.
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ABSTRACT:Objective To explore the role of HIF‐1αin the pathogenesis of hepatopulmonary syndrome (HPS) and its relationship with GRP78 .Methods The HPS model in rats was induced by multiple pathogenic factor .The samples were assessed by using Western blotting analysis for HIF‐lα, GRP78 and VEGF164 . The expressions of VEGFR‐2 and CD105 were observed by using immunohistochemical staining .Results The protein level of HIF‐1αwas significantly increased in HPS group at week 8 compared with that at week 4 and 6 groups and corresponding normal control groups .With the development of HPS ,protein level of GRP78 was gradually increased at each time point significantly and reached the highest level at week 8 ;protein level of VEGF164 showed a similar change with GRP78 ,but the peak was at week 6 .Immunohistochemical results showed that the protein expressions of VEGFR‐2 and CD105 were gradually increased in lung tissue as HPS progressed .The protein level of GRP78 was positively correlated with HIF‐1α,VEGF164 ,VEGFR‐2 and CD105 ,respectively (P<0 .05) .Conclusion HIF‐1αis most likely together with GRP78 to play a critical role in promoting pulmonary microvascular remodeling in the pathogenesis of HPS in rats .
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AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α~7 subunit-containing nicotinic receptor (α~7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α~7nAch receptor expression.
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0.05).Both HMGB1 mRNA expression and HMGB1 protein level were remarkably higher in LPS treatment group than that in control group(P0.05).CONCLUSION:RPM inhibits HMGB1 expression not only by directly suppressing STAT3 activation,but also by indirectly reducing TNF-? level.