Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells / 中华肿瘤杂志

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Andrographolide Inhibits Proliferation and Promotes Apoptosis in Bladder Cancer Cells by Interfering with NF- κ B and PI3K/AKT Signaling In Vitro and In Vivo / 中国结合医学杂志

OBJECTIVE@#To explore the influences of andrographolide (Andro) on bladder cancer cell lines and a tumor xenograft mouse model bearing 5637 cells.@*METHODS@#For in vitro experiments, T24 cells were stimulated with Andro (0-40 µmol/L) and 5637 cells were stimulated with Andro (0 to 80 µmol/L). Cell growth, migration, and infiltration were assessed using cell counting kit-8, colony formation, wound healing, and transwell assays. Apoptosis rate was examined using flow cytometry. In in vivo study, the antitumor effect of Andro (10 mg/kg) was evaluated by 5637 tumor-bearing mice, and levels of nuclear factor κ B (NF- κ B) and phosphoinositide 3-kinase/AKT related-proteins were determined by immunoblotting.@*RESULTS@#Andro suppressed growth, migration, and infiltraion of bladder cancer cells (P⩽0.05 or P⩽0.01). Additionally, Andro induced intrinsic mitochondria-dependent apoptosis in bladder cancer cell lines. Furthermore, Andro inhibited bladder cancer growth in mice (P⩽0.01). The expression of p65, p-AKT were suppressed by Andro treatment in vitro and in vivo (P⩽0.05 or P⩽0.01).@*CONCLUSIONS@#Andrographolide inhibits proliferation and promotes apoptosis in bladder cancer cells by interfering with NF- κ B and PI3K/AKT signaling in vitro and in vivo.

Role of TRPV1/NF-κB signaling pathway in dexmedetomidine-induced alleviation of VILI in rats / 中华麻醉学杂志

Objective:To evaluate the role of transient receptor potential vanilloid receptor 1 (TRPV1)/nuclear factor-κB (NF-κB) signaling pathway in dexmedetomidine-induced alleviation of ventilator-induced lung injury (VILI) in rats.Methods:One hundred clean-grade healthy male Sprague-Dawley rats, weighing 270-320 g, aged 4-5 months, were divided into 5 groups ( n=20 each) using a random number table method: control group (group C), VILI group (group V), AMG9810 group (group A), dexmedetomidine group (group D), and dexmedetomidine + RTX group (group DR). VILI model was prepared by mechanical ventilation with a tidal volume of 40 ml/kg for 4 h. In group A, TRPV1 inhibitor AMG9810 30 mg/kg was intraperitoneally injected at 1 h before mechanical ventilation.Dexmedetomidine 5.0 μg/kg was intravenously infused at 20 min before mechanical ventilation, and dexmedetomidine was intravenously infused at the rate of 5.0 μ g·kg -1·h -1 during ventilation in group D and group DR.In group DR, RTX 70 μ g/kg was intraperitoneally injected for 3 consecutive days before mechanical ventilation.At 4 h of mechanical ventilation, the concentrations of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-6 in bronchoalveolar lavage fluid (BALF) were detected, oxygenation index (OI) and wet/dry lung weight (W/D) ratio were measured, the histopathological changes of lung tissues were observed, and lung injury was assessed and scored.The expression of TRPV1 and NF-κB in lung tissues was detected by Western blot, and real-time polymerase chain reaction was used to detect the expression of TRPV1 and NF-κB mRNA. Results:Compared with group C, the concentrations of IL-1β, TNF-α and IL-6 in BALF were significantly increased, OI was decreased, the W/D ratio and lung injury scores were increased, and the expression of TRPV1 and NF-κB protein and mRNA was up-regulated in group V ( P<0.05). Compared with group V, the concentrations of IL-1β, TNF-α and IL-6 in BALF were significantly decreased, OI was increased, the W/D ratio and lung injury scores were decreased, and the expression of TRPV1 and NF-κB protein and mRNA was down-regulated in A, D and DR groups ( P<0.05). Compared with group D, the concentrations of IL-1β, TNF-α and IL-6 in BALF were significantly increased, OI was decreased, the W/D ratio and lung injury scores were increased, and the expression of TRPV1 and NF-κB protein and mRNA was up-regulated in group DR ( P<0.05). Conclusions:The mechanism by which dexmedetomidine alleviates VILI is partially related to inhibition of the activation of TRPV1/NF-κB signaling pathway and inhibition of the inflammatory responses in lung tissues of rats.

Expression of mammalian target of rapamycin (mTOR), nuclear factor-κ B (NF-κ B) and sterol regulatory element binding protein 2 (SREBP2) in serum and placentas among gravidas with preeclampsia / 中华围产医学杂志

Objective To investigate the level of mammalian target of rapamycin (mTOR) in serum and the expression of mTOR,nuclear factor-κ B (NF-κ B) and sterol regulatory element binding protein 2 (SREBP2) in placenta among gravidas with preeclampsia.Methods From August 2015 to August 2017,60 gravidas including 40 with severe preeclampsia (SPE) and 20 with mild preeclampsia (MPE) who underwent regular prenatal care and delivered by caesarean section were selected from the Second Xiangya Hospital of Central South University.According to the ratio of 2:1,30 gravidas who delivered through caesarean section due to cephalopelvic disproportion,abnormal fetal position or social factors during the same period were enrolled as the control group.Peripheral blood samples were obtained to determine the concentrations of serum mTOR,high density lipoprotein-cholesterol (HDL-C),low density lipoprotein-cholesterol (LDL-C),triglyceride (TG) and total cholesterol (TC) by enzyme linked immunosorbent assay (ELISA).The expression of mTOR,phospho-mTOR (p-mTOR),NF-κ B and SREBP2 in placenta were measured by Western blot.Clinical datas were statistically analyzed using one-way ANOVA,Bonferroni or Dunnett's T3 test,and Pearson's correlation analysis.Results (1) The serum levels of mTOR and LDL-C in the SPE and MPE group were both higher than that in the control group [mTOR:(11 765.56± 1 698.95) and (8 278.56±1 106.59) vs (4 366.19±716.43) pg/ml;LDL-C:(7.81 ±1.90) and (4.11 ±0.75) vs (2.42±0.45) mmol/L,all P＜0.05].Furthermore the serum levels of mTOR and LDL-C in the SPE group were both higher than those in the MPE group (both P＜0.05).The serum level of HDL-C in the SPE and MPE group were lower than that in the control group [(0.36±0.12) and (0.85±0.11) vs (1.33± 0.16) mmol/L,both P＜0.05],and that in the SPE group was lower than that in the MPE group (P＜0.05).Women in the SPE group showed higher TG level when comparing with the MPE and control group [(46.19± 18.92)vs (35.55±6.54) and (33.24±9.78) nmol/L,both P＜0.05],while the TC levels in the SPE and MPE group were higher than that in the control group[(24.72±7.17) and (21.83±4.19) vs (16.32±3.88) nmol/L,both P＜0.05].(2) The placental expressions of mTOR,p-mTOR,NF-κ B and SREBP2 protein in the SPE and MPE group were higher compared with that in the control group [mTOR:(0.52±0.09) and (0.38±0.08) vs (0.24±0.05);p-mTOR:(0.42±0.08) and (0.26±0.05) vs (0.14±0.03);NF-κ B:(0.58±0.10) and (0.36±0.05) vs (0.21 ± 0.03);SREBP2:(0.52 ± 0.08) and (0.33 ± 0.05) vs (0.20 ± 0.05);all P＜0.05],and those expressions of the SPE group also higher comparing with the MPE group.Otherwise the p-mTOR/mTOR ratios in the SPE group and MPE group were higher than that in the control group [(0.75±0.10) and (0.69±0.14) vs (0.59 ±0.13),both P＜0.05].(3) Pearson's correlation analysis showed that serum level of mTOR and placental expressions of mTOR and p-mTOR in the SPE group were positively correlated with serum LDL-C (r=0.682,0.584 and 0.504,all P＜0.05),TG (r=0.612,0.658 and 0.422,all P＜0.05),while serum level of mTOR and placental expressions of mTOR in the SPE group were positively correlated with TC (r=0.598 and 0.452,all P＜0.05),but were negatively correlated with serum HDL-C (r=-0.375,-0.442 and-0.390,all P＜0.05).The NF-κ B expression in placenta of the SPE group was significantly positively correlated with the mTOR expression in placenta and serum LDL-C (r=0.375 and 0.391,both P＜0.05).Moreover,in the SPE group,the SREBP2 level in placenta was significantly positively correlated with placental expression of mTOR and serum TC level (r=0.364 and 0.392,both P＜0.05).(4) In the MPE group,mTOR level in serum and levels of mTOR and p-mTOR in placenta were significantly positively correlated with serum LDL-C (r=0.813,0.641 and 0.465,all P＜0.05),TG (r=0.646,0.529 and 0.502,all P＜0.05) and TC (r=0.558,0.482 and 0.483,all P＜0.05),while the level of serum mTOR was negatively correlated with the level of serum HDL-C (r=-0.606,P＜0.05).The NF-κ B level in placenta in MPE group was positively correlated with the mTOR in placenta and the serum LDL-C (r=0.458 and 0.595,both P＜0.05),while the SREBP2 level in placenta was significantly positively correlated with mTOR in placenta and serum TC (r=0.580,0.560,respectively;both P＜0.05) in the MPE group.Conclusions mTOR,NF-κ B and SREBP2 may play important roles in the onset and development of preeclampsia by interfering lipid metabolism.

Effect of Tripterygium glycosides combined with Recombinant human tumor necrosis factor receptorⅡ antibody fusion protein for injection on serum vascular endothelial growth factor, rheumatoid factor, receptor activator of nuclear factor κ B ligand levels in patients with rheumatoid arthritis / 中国综合临床

Objective To investigate the effect of tripterygium glycosides combined with recombinant human tumor necrosis factor receptor Ⅱ antibody fusion protein for injection on serum vascular endothelial growth factor (VEGF),rheumatoid factor (RF),receptor activator of nuclear factor κ B ligand (RANKL) levels in patients with rheumatoid arthritis.To provide reference for rational clinical application.Methods From December 2014 to January 2016,132 patients with rheumatoid arthritis were divided into observation group and control group by random number table method,,with 66 cases in each group,The control group was treated with tripterygium glycosides alone (10 mg each time,3 times daily,orally) for 3 months.The observation group was treated with a combination of tripterygium glycosides and recombinant human tumor necrosis factor receptor Ⅱ (0.4 mg/kg,once weekly,hypodermic injection) for 3 months.The clinical efficacy,and serum VEGF,RF and RANKL levels were compared between 2 groups.Results The effective rate of the observation group was 92.4％ (61/66),which was significantly higher than that in the control group (80.3％,53/66).There was a significant difference between the two groups (x2=4.117,P＜0.05).There was no significant difference in the levels of serum VEGF,RF and RANKL between the two groups (t =0.174,0.103,0.359,all P＞0.05).After treatment,the levels of serum VEGF,RF and RANKL in the observation group and the control group were (20.8± 11.5) ng/L and (27.3 ±13.1) ng/L,(258.4±54.5) U/L and (298.1 ±49.5) U/L,(0.083±0.021) pmol/L and (0.197 ± ±0.064),respectively.There were significant differences between the two groups (t =3.029,4.381,13.750,all P＜0.05).The incidence rate of adverse reactions in the control group was 10.5％ (7/66),which was significantly higher than that in the observation group (1.5％,1/66).There was a significant difference between the two groups(x2 =4.790,P＜0.05).The one-year recurrence rate was 25.0％ (13/52) in the control group and that was 6.7％ (4/60) in the observation group,respectively,and there was a significant difference between the two groups (x2 =7.272,P＜ 0.05).Conclusion Tripterygium glycosidescombined with recombinant human tumor necrosis factor receptor Ⅱ antibody fusion protein for injection is effective in the treatment of rheumatoid arthritis,which reduces the levels of serum VEGF,RF and RANKL,and has a low incidence of adverse reactions and recurrence.

Extract of Fructus Schisandrae chinensis Inhibits Neuroinflammation Mediator Production from Microglia via NF-κ B and MAPK Pathways / 中国结合医学杂志

OBJECTIVE@#To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis (EFSC) on lipopolysaccharide (LPS)-induced BV-2 cells and the possible involved mechanisms.@*METHODS@#Primary cortical neurons were isolated from embryonic (E17-18) cortices of Institute of Cancer Research (ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group (treated with 1 μg/mL LPS only) and EFSC groups (treated with 1 μg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide (MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide (NO) and interleukin-6 (IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence.@*RESULTS@#EFSC (200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression in LPS-induced BV-2 cells (P<0.01 or P<0.05). EFSC (200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia (P<0.01). In addition, EFSC alleviated cell apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium (P<0.01). The mechanistic studies indicated EFSC could suppress nuclear factor (NF)-?B phosphorylation and its nuclear translocation (P<0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase (MAPK) pathway (P<0.01 or P<0.05).@*CONCLUSION@#EFSC acted as an anti-inflammatory agent in LPS-induced glia cells. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.

Autophagy plays a protective role in advanced glycation end products- induced apoptosis of chondrocytes via regulation of tumor necrosis factor-α , nuclear factor-κ B and reactive oxygen species

Objective:To study the adverse effects of advanced glycation end products (AGEs) on chondrocytes and the role of autophagy in this process.Methods:Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of tumor necrosis factor- α (TNF- a ) and nuclear factor- κ B (NF- κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species (ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively.Results:The chondrocytes viability was significantly reduced after 12 h incubation with AGEs (P<0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF- α and NF- κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis.Conclusions:Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF- α and NF- κ B expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEs- induced adverse effects.

Protective effects of hydroxyethyl starch 130/0.4 against myocardial ischemia-reperfusion injury in rats / 第二军医大学学报

Objective: To explore the influence of hydroxyethyl starch(HES) 130/0.4 on myocardial ischemia-reperfusion (I/R) injury in rats and the possible mechanism. Methods: Twenty-four SD rats were evenly randomized into four groups(n = 6): the sham operation group, the I/R(IR) group, albumin + I/R(A-IR) group, and HES+I/R(H-IR) group; rats in the latter three groups were made into I/R models and were treated respectively with 7.5 ml/kg saline, 5% albumin and HES 130/0.4 through femoral vein at 25 min of ischemia. At 180 min of reperfusion, animals were sacrificed and the pathological changes of myocardium were observed. Serum concentrations of TNF-α and IL-1β and the myocardial NF-κB activity were also measured. Results: Histological examination showed that the injury in H-IR group was ameliorated compared with those in IR and A-IR groups. NF-κB activity and TNF-α, IL-1β concentrations in the sham operation group were significantly lower than those of the other 3 groups (P<0.05); and the increases of the above parameters in H-IR group were smaller than those of the IR and A-IR groups (P<0.05). Conclusion: HES 130/0.4 can improve myocardial function and attenuate ischemia-reperfusion injury, and the mechanism might be related to the inhibition of myocardial NF-κB activity and reduction of proinflammatory factors.