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ObjectiveTo observe the repair effect of Dahuanglingxian prescription （DHLX） on bile duct epithelial cells of rats. To explore whether its mechanism of action is to adjust the mutual binding of transforming growth factor -β （TGF-β） activated kinase 1（TAK1） and tumor necrosis factor receptor-associated factor 6 （TRAF6）， and regulate the activation of the nuclear transcription factor -κB （NF-κB）/mitogen-activated protein kinase （MAPK） signaling pathway. MethodThe 20 SD rats were randomly divided into normal group and DHLX group， 10 rats in each group， were given saline and DHLX （320 mg·kg-1·d-1） for 8 days， to prepare normal serum and DHLX serum. Biliary epithelial cells were extracted from normal SD rats and divided into 9 groups： Normal group， model group （20 mg·L-1）， LPS+DHLX group （20 mg·L-1+10% DHLX）， LPS+PDTC group （20 mg·L-1+200 μmol·L-1）， LPS+SB203580 group （20 mg·L-1+0.5 μmol·L-1）， LPS+PDTC+SB203580 group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1）， LPS+PDTC+DHLX group （20 mg·L-1+200 μmol·L-1+10% DHLX serum）， LPS+SB203580+DHLX group （20 mg·L-1+0.5 μmol·L-1+10% DHLX serum）， LPS+PDTC+SB203580 +DHLX group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1+10% DHLX serum）. The microscopic observation of morphological changes in each group of cells after drug intervention. Enzyme-linked immunosorbent assay（ELISA） was used to detect the expression of （IL）-1β and IL-6 in each group of cells. Western blot detected the expression levels of TAK1 and TRAF6 proteins in each group of cells， Co-IP detected the interaction between TAK1 and TRAF6， and further observed the distribution and co-localization of TAK1 and TRAF6 using Laser confocal microscope. ResultAfter the action of LPS， the cell synapses are reduced， the cell body becomes significantly rounded and smaller， but the cell morphology of each group tends to be normal after medication. Compared with normal group， the expression levels of IL-1β and IL-6 in model group were significantly increased （P<0.05）， while the expression level of TAK1 was decreased while the expression level of TRAF6 was increased （P<0.05）. The content of TAK1-TRAF6 protein complex showed a decreasing trend， and the two proteins co-located in the cytoplasm. Compared with model group， the expression levels of IL-1β and IL-6 in LPS+DHLX group were significantly decreased （P<0.05）， the expression level of TAK1 was increased and the expression level of TRAF6 was decreased （P<0.05）， the content of TAK1-TRAF6 protein complex was significantly increased （P<0.01）， and the two proteins were significantly co-located in cytoplasm. Compared with LPS+DHLX group， the expression levels of IL-1β and IL-6 in other groups were significantly decreased （P<0.05，P<0.01）. TAK1-TRAF6 protein complex content in each group was significantly decreased after pathway blocker intervention （P<0.05）， while TAK1-TRAF6 protein complex content in each group was significantly increased after pathway blocker combined with DHLX intervention （P<0.05）. Co-localization of the TAK1-TRAF6 in cytoplasm was not obvious. ConclusionIn the LPS-induced inflammatory response of bile duct cells， the binding of TAK1 and TRAF6 showed a weakening trend， but DHLX could reverse the phenomenon， we think the mechanism of action may be related to promoting the mutual binding of TAK1 and TARF6 to inhibit the activation of the NF-κB/MAPK signaling pathway.
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Objective@#To investigate the effects of silica dust on the expression of Myeloid differentiation factor 88 (MyD88) mRNA and tumor necrosis factor receptor-associated factor (TRAF6) mRNA of lung macrophages in rats.@*Methods@#Selecting 40 SPF-class Wistar rats with average weight (200±20) g randomly divided into control group and 30 d, 60 d, 120 d experimental groups with 10 rats in each group according to body weight. The experimental groups rats were injected with 1 ml of SiO2 (100 mg/ml) suspension through the trachea into lung only once, then they were respectively killed after 30, 60, 120 days. The control group rats were injected with 1 ml of saline into lung, and killed after 120 days. The lungs of the rats were taken for pathological observation. Lung macrophages were extracted and counted, and their activity was detected by MTT. RT-qPCR was used to assess the relative contents of MyD88 mRNA and TRAF6 mRNA.@*Results@#Silica dust inhalation led to infiltration of lung tissue cells, thickening the alveolar wall and destruction of alveolar structure. The longer the exposure to dust, the more obvious the results were. The number of macrophages in all experimental groups and activity in the 30 d, 60 d groups were significantly higher than that in the control group (P<0.05) . Among them, 30 d group had the largest number and the highest activity. Compared with the control group, the expression of MyD88 mRNA and TRAF6 mRNA of lung macrophages in rats increased in the experimental groups (P<0.05) , especially in the 60 d group.@*Conclusion@#Silica dust inhalation can increase the expression of MyD88 and TRAF6 in macrophages, suggesting that silica dust can induce silicosis fibrosis by activating TLR/NF-κB signal pathway.
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Objective: To explore the effects of two kinds of calcium phosphate transfection methods in the 293T cells, and to establish the method of achieving high-efficiency and stable calcium phosphate transfection in the 293T cells. Methods: Fluorescence microscope was used to observe the transfection efficiencies of transfection of pCDH-GFP-3xflag-TRAF6 plasmid into the 293T cells by two kinds of calcium phosphate transfection methods (traditional calcium phosphate transfection method and improved calcium phosphate transfection method). Real-time PCR and Western blotting method were used to detect the expression levels of TRAF6 mRNA and flag protein in the 293T cells after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by two kinds of calcium phosphate transfection methods. Results: Under fluorescence microscope, compared with traditional calcium phosphate transfection method, the transfection efficiencies of improved calcium phosphate transfection method 24 an 48 h after transfection of pCDH-GFP-3xflag-TRAF6 plasmid into the 293T cells were significantly increased (P<0. 01). The Real-time PCR and Western blotting results showed that compared with traditional transfection method, the expression levels of TRAF6 mRNA and flag protein in the 293T cells 24 and 48 h after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by calcicum phosphate transfection method were significantly increased (P<0. 01). Conclusion: The improved calcium phosphate transfection method established in this reseach is a highly efficient and stable DNA transfection method.
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Background and purpose: Abnormal expression and amplification of transforming growth factor beta 1 (TGF-β1) and Notch3 in ovarian carcinoma tissues are associated with metastasis and low survival rate, respectively. The crosstalk between TGF-β1 and Notch3 signaling pathway promotes invasion and metastasis in various cancers. However, the mechanism is still under debate. Therefore, this study was designed, using in vitro cytological assays, to investigate the effects of TGF-β1 and Notch3 signaling pathway on ovarian cancer cell biological behavior and the potential mechanisms in terms of the crosstalk between TGF-β1 and Notch3 signaling pathway. Methods: Hey A8 and Hey cell lines were used as models in the study. The levels of TGF-β1 in supernatants from culture media were measured by ELISA. Both cell lines were treated with 500 ng/mL TGF-β1 neutralizing antibody (control group), 10 ng/mL TGF-β1, 50 μmol/L DAPT, 10 ng/mL TGF-β1 and 50 μmol/L DAPT, 50 μmol/L tumor necrosis factor receptor-associated factor 6 (TRAF6) peptide inhibitor, 10 ng/mL TGF-β1 and 50 μmol/L TRAF6 peptide inhibitor, respectively. The protein expression levels of TGF-β1 and Notch3 signaling pathway molecules as well as TRAF6 from cell lines with different treatments were detected by Western blot. Cell proliferation, migration and invasion were tested by cell counting kit-8 (CCK-8), scratch and Transwell assays, respectively. Results: The levels of TGF-β1 were timedependently increased in supernatants of culture media from Hey A8 and Hey cell lines. Compared with control group, TGF-β1 treatment increased the expression levels of Notch3-ICD and Hes1, while no obvious change was observed in the group treated with DAPT and TGF-β1. Moreover, TGF-β1 promoted cell proliferation, migration and invasion while DAPT decreased the proliferation, migration and invasion in cell lines treated with TGF-β1. These results indicated that TGF-β1 might promote proliferation, invasion and migration of ovarian epithelial cancer cells through activating the Notch3 signaling pathway. Further study showed that TGF-β1 up-regulated TRAF6 and activated the Notch3 signaling pathway. The activation of the Notch3 signaling pathway by TGF-β1 was inhibited in cells treated with the TRAF6 specific inhibitor. Conclusions: TGF-β1 may promote the proliferation, invasion and migration of ovarian epithelial carcinoma cells through TRAF6-mediated activation of the Notch3 signaling pathway.
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Objective:To explore expression of miR-146b in peripheral blood serum and aortic wall tissues in patients with acute Stanford type A aortic dissection (TAAD),and to discuss the significance and underlying mechanisms.Methods:The subjects were divided into a control group (excluded relative aortic diseases) (n=23) and a TAAD group (n=27).The miR-146b levels of serum and aortic wall tissues were detected by quantitative real-time PCR (qRT-PCR).Serum miR-146b and aortic wall tissues miR-146b were compared among different risk TAAD groups.The correlations between miR-146b and severity of aortic dissection were analyzed.MiR-146b related target genes were predicted by the DIANA LAB-TarBase 6.0 and TargetScan.Results:The expression levels of miR-146b in the serum and aortic wall tissues in the TAAD group were significantly elevated compared with those in the control group (P＜0.001).Compared with the mild risk group,the miR-146b levels of serum and aortic wall tissues were significantly higher in the moderate risk and severe risk groups (P＜0.05).The expression of miR-146b was positively correlated with the risk severity of TAAD patients (r=0.862,0.872;P＜0.05).Nuclear factor kappa B1 (NF-κB1),tumor necrosis factor receptor-associated factor 6 (TRAF6),matrix metalloproteinase 16 (MMP16) and actin alpha 2 (ACTA2) were miR-146b related target genes.Conclusion:The upregulation of miR-146b in peripheral blood serum and aortic wall tissues may contribute to the pathogenesis of TAAD and the severity of this disease.
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Objective:To investigate the changes of expression levels of TLR9 and IL-6 in the periodontal tissue during the experimental tooth movement of the rats, and the effecst of the MT01 on the expression of TLR9,TRAF6 and IL-6 in periodontal tissue,and to clarify its related mechanisms.Methods:Thirty Wistar rats were randomly divided into MT01 intervention group(n=6) and non-MT01 group(n=24).Force of 0.49 N was applied to move the upper first molars mesially. The rats in Non-MT01 intervention group were sacrificed on the days 3,7,14 and 21, and the rats in MT01 intervention group were all sacrificed on the day 7. RT-qPCR was used to detect the expression levels of TLR9,TRAF6 and IL-6 mRNA in maxillary first molar alveolar bone in each group.Results:The expression levels of IL-6 and TLR9 mRNA in loaded side were higher than those in control side(P<0.05), and reached the maximum levels on the day 7(P<0.01);with the interpose of MT01, the expression levels of TLR9 and TRAF6 mRNA were lower than control side(P<0.01).Conclusion: MT01 could down-regulate the expression levels of TLR9 and TRAF6 during orthodontic tooth movement and eventually resists the inflammation during the tooth movement.
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Objective To detect the effects of bifidobacterium or bifidobacterium cultured supernatant on the mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),glycogen synthase kinase-3β (GSK-3β) and the miRNA-146a in rat small intestinal epithelial cell(IEC-6) induced by lipopolysaccharide (LPS).Methods IEC-6 in logarithmic phase were randomly divided into LPS group,cultured supernatant group and inactivated bacteria group.All the 3 groups were exposed to 5 mg/L LPS for 5 hours,and then 1 mL sterile saline was added in LPS group and culturing continued for 24 hours ; 1 mL bifidobacterium cultured supernatant was added in cultured supernatant group and culturing continued for 24 hours;1 mL inactivated bifidobacterium 1 x 1010 CFU/L added in inactivated bacteria group and continued culturing for 24 hours.The mRNA expressions of TRAF6,GSK-3 β and miRNA-146a were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results The level of TRAF6,GSK-3 β of culture supematant group (0.72 ± 0.05,0.46 ± 0.14) were all lower than LPS group (1.01 ± 0.14,1.02 ± 0.25),but the level of miRNA-146a(3.05 ± 0.40) was higher than that in LPS group(1.01 ± 0.12),and there were significant differences between them (t =5.278,6.316,13.218,P =0.000).The level of GSK-3 β of inactivated bacteria group(0.59 ±0.13) was significantly lower than that in LPS group(t =4.837,P =0.000).The levels of TRAF6 and miRNA-146a of inactivated bacteria group(1.05 ±0.11,0.78 ±0.22) had no significant differences with LPS group (t =0.732,1.463,P ＞ 0.05).The level of TRAF6 of cultured supernatant group was lower than that in inactivated bacteria group,and the level of miRNA-146a was higher than that in inactivated bacteria group,and there were significant differences between 2 groups (t =6.009,14.687,P =0.000).Conclusions Bifidobacterium cultured supernatant and inactivated bacteria both have certain protective effect on the IEC-6 induced by LPS.One of the protective mechanisms of bifidobacterium cultured supernatant may be achieved by elevating the expression of miRNA-146a,and decreasing the levels of inflammation related factor TRAF6 and damage related factor GSK-3β.The protective effects of inactivated bifidobacterium may be achieved by decreasing the level of damage related factor GSK-3β.
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Objeciive To investigative the expressoin of TNF receptor-associated factor 6 (TRAF6) in the peripheral blood mononuclear cells (PBMCs) of primary Sjögren syndrome (pSS) patients and its clinical significance. Methods The expression of TRAF6 mRNA was determined by quantified reverse transceipsional PCR in the PBMCs of 23 pSS patients and 23 heaithy controls. The correlation 0f TRAF6 mRNA level with the laboratory findings, including serum rheumatoid factor (RF), globulin, anli-SSA and anli-SSA antibody, were analyzed by Spearman test. Nine of the pSS patients were followed up and their TRAF6 mRNA expression was detected after glucocorlicoid treatment for 1-2 months. Results The TRAF6 mRNA level in pSS patients was 2. 77 times higher than that in the healthy controls (P< 0. 01). The TRAF6 mRNA level in pSS patients was positively correlated with serum RF (r=0. 45, P = 0. 03) and globidln (r=0. 43, P = 0, 04), but was not assosiated the antibody. TRAF6 mRNA level was decreased by aboijt 50% n the palients who were treated with glucocorlicoids for 1-2 mcmths (P<0. 05). Conclusion TRAF6 n PBMCs is involved in the pathogenesiss of pSS and it may be a potential biomarker for pSS.
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This study investigated the influence of silencing TRAF6 with shRNA on lipopolysac-charide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA 1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-α, IL-1β and TGF-β1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA 1, 2 could significantly reduce the production of pro-inflammatory cyto-kines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibit NF-κB nuclear transloca-tion. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-β1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflam-matory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.
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Objective To study the effect of prolactin(PRL) on the activation of T lymphocytes stmiulated by concanavalin A(ConA),and to explore the action of PRL in the activation of T lymphocytes. Methods After CD4 +T cell line JurkatE6-1 cells were respectively stmiulated by 5 mg/L ConA,25 ?g/L PRL and 500 ?g/L bromocriptine(Brc).The blank control group,the ConA group,the PRL and ConA group(PRL group),the Brc and ConA group(Brc group),the PRL and Brc group(PRL-Brc group) were set in the experiment.The total RNA was extracted by Trizol after 48 hours and was reversed transcription immediately.The expression of tumor necrosis factor receptor associated factor 6(TRAF6) mRNA of T lymphocytes was checked by PCR.The expressions of tumor necrosis factor(ligand) super family 4(TNFSF4) and Killer specific secretory protein of 37 000(KSP37) mRNA of T lymphocytes were detected by real-time polymerase chain reaction. Results The PRL group and the Brc group could inhibit the expressions of TRAF6,TNFSF4,and KSP37 mRNA of the activated T lymphocyte compared with the blank control group and the ConA group(P a0.05).The PRL-Brc group could inhibit significantly the expressions of TRAF6,TNFSF4,and KSP37 mRNA of the activated T lymphocyte compared with the ConA group(P a