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OBJECTIVE To explore the improvement effect and potential mechanism of total flavonoids from Alpinia zerumbet on gastric mucosa injury induced by absolute ethanol through microRNA-146a-5p （miR-146a-5p）. METHODS Using human gastric mucosa GES-1 cells as objects， the acute gastric ulcer model was established by absolute ethanol； based on the investigation of the effects of different concentrations of total flavonoids from A. zerumbet on cell activity and the selection of action concentration， the relative expression level of miR-146a-5p in GES-1 cells was detected， the protein expressions of tumor necrosis factor （TNF） receptor-associated factor 6（TRAF6）， nuclear factor-κB p65 （NF-κB p65） and TNF-α were detected， and the levels of interleukin- 1β （IL-1β）， IL-6 and prostaglandin E2 （PGE2） in cell supernatant were determined. The targeting relationship between miR-146a- 5p and TRAF6 was verified； the effects of overexpressed miR-146a-5p and TRAF6 knockdown on the levels of IL-1β， IL-6 and PEG2 in supernatant of model cells as well as the effects of miR-146a-5p knockdown on anti-gastric ulcer effect of total flavonoids from A. zerumbet were observed. RESULTS Compared with the blank group， the relative expression of miR-146a-5p in cells and the level of PGE2 in cell supernatant were decreased significantly in the model group （P＜0.01）， while the protein expressions of TRAF6， NF-κB p65 and TNF-α in cells and the levels of IL-1β and IL-6 in cell supernatant were increased significantly （P＜ 0.01）. Compared with the model group， the relative expression of miR-146a-5p in cells and the level of PGE2 in cell supernatant were increased significantly in model+A. zerumbet total flavonoids （60 mg/L） group （P＜0.01）， while the protein expressions of TRAF6， NF-κB p65 and TNF-α in cells and 82260767） the levels of IL-1β and IL-6 in cell supernatant were decreased significantly （P＜0.05 or P＜0.01）. There was a targeted relationship and a negative correlation between miR-146a-5p E-mail：3113836821@qq.com and TRAF6. After overexpression of miR-146a-5p or TRAF6 knockdown， the levels of IL-1β and IL-6 were decreased significantly in cell supernatant， while the level of PGE2 was increased significantly （P＜0.05）. After miR-146a-5p knockdown， the levels of IL-1β and IL-6 in cell supernatant and the protein expression of TRAF6 in cells administered with total flavonoids of A. zerumbet were increased significantly， while the level of PGE2 was decreased significantly （P＜0.05）. CONCLUSIONS Total flavonoids of A. zerumbet can improve the gastric mucosa injury induced by absolute ethanol. The mechanism may be related to up-regulating the expression of miR-146a-5p， inhibiting the expression of TRAF6， and further inhibiting the secretion of related inflammatory factors.
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Objective: To investigate the protective effect of human lipoxin A4 (LXA4) on N2a cell damage induced by β-amyloid protein 25-35 (Aβ25-35) and the underlying mechanism. Methods: Aβ25-35 was used to treat N2a cells to establish Alzheimer's disease (AD) cell injury model. Meanwhile, LXA4 was added to the experimental group at different concentrations (50, 100 and 200 nmol·L-1 ). MTT assay was used to detect the activity of N2a cells. The apoptosis was detected by Hoechst 33258-PI staining, the expression of P62 and TRAF6 mRNA was detected by RT-PCR, and the expression of P62 and TRAF6 protein was detected by Western blot. Results: Compared with that of the model group, the cell survival rate of LXA4 protective group (50,100 and 200 nmol·L-1 ) increased (P <0. 01) and the apoptosis of N2a cells induced by Aβ25-35 was reduced by LXA4 (100 and 200 nmol·L-1 ) . Compared with that of the model group, the expression of P62-mRNA and protein-P62 of N2a cells treated with Aβ25-35 increased (P <0. 05 or P <0. 01) and the expression of TRAF6-mRNA and protein-TRAF6 of N2a cells treated with Aβ25-35 were reduced (P <0. 05 or P <0. 01). Conclusion: LXA4 has protective effect on N2a cell damage induced by Aβ25-35 , and its mechanism may be related to the up-regulation of P62 gene and down-regulation of TRAF6 gene.
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Objective To study the correlation between single nucleotide polymorphism (SNP) of downstream mononucleotide in signal transduction of Toll-like receptors and predisposing genes of rheumatoid arthritis (RA).Methods One hundred and sixty-two RA patients were selected from northern part of Han people in China,and 188 healthy subjects were enrolled as normal controls.Rs7744 of Myd88,rs5030445 of TRAF6,rs11265618 and rs4845626 of IL-6R were analyzed.The data of genotypic frequency and allele genotypic frequency were statistical analyzed by x2 test between the two groups.Results The difference of allele frequency of TRAF6 rs5030445 between the two groups were remarkable (x2=5.716,P＜0.05),and so did the IL-6R rs11265618 (x2=5.704,P＜0.05).But the difference of genotypic frequency was not statistically significant for locus of Myd88 rs7744,TRAF6 rs5030445 and IL-6R rs11265618 (P＞0.05).And the differenceof allele frequency and genotypic frequency between the two groups had no statistical significance in locus of Myd88 rs7744 and IL-6R rs4845626 (P＞0.05).Conclusion There is significant correlation between single nucleotide polymorphism and predisposing genes of RA.The G allele of TRAF6 rs5030445 gene locus is a possible predisposing gene for RA.The T allele of IL-6R rs11265618 is a possible predisposing gene of RA.
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Objective To investigate the effects of curcumin on mRNA expression of cytokines related to Toll-like receptor (TLR) 4 signaling in THP-1 cells.Methods After pretreatment with different concentrations (50,25,12.5 mg/L) of curcumin or dexamethasone for 12 hours,THP-1 cells were stimulated by lipopolysaccharide (LPS.1 mg/L) for 4 hours followed by the collection of cells.Then total RNA was isolated from these cells and subjected to reverse transcription-polymerase chain reaction (RT-PCR) for the detection of mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),interleukin-1 receptorassociated kinase (IRAK1) and nuclear factor (NF)-κB.THP-1 cells without pretreatment or stimulation served as negative control,and those only stimulated with LPS served as LPS group.Results After stimulation with LPS (1 mg/L) for 4 hours,the mRNA expressions of TRAF6,IRAK1 and NF-κB were significantly upregulated in THP-1 cells compared with negative control cells (f=38.69,39.13,23.99,all P<0.01).Curcumin of 50 mg/L and 25 mg/L significantly inhibited the mRNA expressions of TRAF6.IRAK 1 and NF-κB upregulated by LPS with an inhibition rate of more than 50% (all P<0.0 1).Conclusions Certain concentrations of curcumin can inhibit the mRNA expressions of TRAF6.IRAK1 and NF-κB.which demonstrates the regulatory effect of curcumin on the mRNA expressions of TLR4 signaling pathway-associated cytokines.
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Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif 10PQENDE15 in STP-A11 reveals that Glu (E)12 residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.