Modification of m6 A in human embryonic lung fibroblasts induced by neodymium oxide exposure and its mechanism / 环境与职业医学

Background Occupational and environmental particulate matter may cause fibrosis, accompanied by RNA m6A modification changes. Neodymium oxide (Nd2O3) can cause mouse lung fibrosis, which contains a large number of fibroblasts. Objective To investigate m6A modification of tumor necrosis factor receptor-associated protein 6/nuclear factor-κB (TRAF6/NF-κB) signaling pathway in fibrosis of human embryonic lung fibroblasts induced by Nd2O3, and identify the key m6A modification sites of TRAF6. Methods Designed concentrations of Nd2O3 (0, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg∙L−1) were infected with HELF cells for 24 and 48 h, and cell viability was detected to determine exposure time and dose. Measurements included indicators of fibrosis [hydroxyproline (HYP) and transforming growth factor-β1 (TGF-β1)], m6A methylation level, methyltransferases (METTL3 and METTL14), demethylases (FTO and ALKBH5), reading proteins (YTHDC2 and YTHDF2), fibrosis-associated genes (collagen-І, vimentin, and α-SMA), and proteins related to signaling pathway (TRAF6, NFKB1, P65, and P-P65). The enrichment of m6A in TRAF6 mRNA was measured by methylated RNA immunoprecipitation-quantitative real-time PCR (MeRIP-qPCR). Results The results of cell viability indicated that 6.25, 12.5, 25 mg∙L−1 Nd2O3 and 48 h exposure time were used for subsequent experiments. After 48 h exposure, compared with the control group, the HYP level in the 25 mg∙L−1 Nd2O3 group was increased, and the levels of TGF-β1 in the 6.25, 12.5, and 25 mg∙L−1 Nd2O3 groups were increased (P<0.05); the overall m6A methylation levels of HELF cells in the 12.5 and 25 mg∙L−1 Nd2O3 groups were increased (P<0.05). At mRNA level, compared with the control group, the mRNA expression levels of methyltransferases METTL3 and METTL14 (6.25, 12.5, and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the mRNA expression level of reading protein YTHDF2 (6.25, 12.5, and 25 mg∙L−1 Nd2O3) was increased (P<0.05), while the mRNA expression level of YTHDC2 (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the mRNA expression levels of demethylases FTO (12.5 and 25 mg∙L−1 Nd2O3) and ALKBH5 (25 mg∙L−1 Nd2O3) were decreased (P<0.05); the mRNA expression levels of fibrosis-related genes vimentin, α-SMA, and collagen-Ⅰ (6.25, 12.5, and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the mRNA expression levels of pathway-related genes TRAF6 (25 mg∙L−1 Nd2O3) and NFKB1 (12.5 and 25 mg∙L−1 Nd2O3) were increased (P<0.05). At protein level, compared with the control group, the expression levels of methyltransferases METTL3 (25 mg∙L−1 Nd2O3) and METTL14 (12.5 and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the expression level of reading protein YTHDF2 (12.5 and 25 mg∙L−1 Nd2O3) was increased, while the expression level of YTHDC2 (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the expression level of demethylase FTO (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the expression level of fibrosis-associated protein vimentin was increased at 25 mg∙L−1 Nd2O3, and the expression levels of α-SMA and collagen-Ⅰ were increased at 12.5 and 25 mg∙L−1 Nd2O3 (P<0.05); the expression levels of TRAF6 and P-P65 were increased at 25 mg∙L−1 Nd2O3 (P<0.05). The MeRIP-qPCR results showed that compared with the control group, the concentrations of m6A in all Nd2O3 groups were significantly increased (P<0.05). Conclusions Upon exposure of HELF cells to Nd2O3, the alterations in fibrosis-related indexes increase the expression of some m6A methylases and decrease the expression of demethylases, thereby increasing the m6A methylase level, and may promote the progression of fibrosis by activating the TRAF6/NF-κB signaling pathway.

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.

Change in expression of tumor necrosis factor receptor-associated factor 6 in lung tissues of rats with pulmonary hypertension / 中华麻醉学杂志

Objective:To determine the change in the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) in lung tissues of rats with pulmonary hypertension (PH).Methods:Sixteen SPF-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 200-220 g, were divided into 2 groups ( n=8 each) by the random number table method: control group (group C1) and PH group (group PH1). The model of PH was prepared by subcutaneous injection of monocrotaline. On day 28 after developing the model, the mean pulmonary arterial pressure (mPAP) was measured, and the Fulton index was calculated, and the percentage of media wall thickness of the small and medium pulmonary arteries and percentage of muscularized vessels were also calculated. The expression of TRAF6, transcription-3 (STAT3), phosphorylated STAT3 (p-STAT3) and Cyclin D1 in lung tissues was detected by Western blot, and p-STAT3/STAT3 ratio was calculated. The interaction between TRAF6 and STAT3 was determined by immunoprecipitation assay. Primarily cultured pulmonary artery smooth muscle cells of normal rats (group C2) and pulmonary artery smooth muscle cells of rats with PH (group PH2) were inoculated in 6-well plates ( n=3 each). The expression of TRAF6 mRNA was detected by quantitative polymerase chain reaction. The expression of TRAF6, STAT3, p-STAT3 and Cyclin D1 was detected by Western blot. Results:Compared with group C1, the mPAP, Fulton index, percentage of media wall thickness of the small and medium pulmonary arteries and percentage of muscularized vessels were significantly increased, the expression of TRAF6 and Cyclin D1 in lung tissues was up-regulated, the p-STAT3/STAT3 ratio was increased ( P<0.05 or 0.01), and the results of immunoprecipitation showed that TRAF6 interacted with STAT3 in group PH1. Compared with group C2, the expression of TRAF6 protein and mRNA and Cyclin D1 was significantly up-regulated, and the p-STAT3/STAT3 ratio was increased in group PH2 ( P<0.05 or 0.01). Conclusions:The expression of TRAF6 in the lung tissue is up-regulated in rats with PH, which may be related to pulmonary vascular remodeling by promoting the activation of STAT3.

Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells / 中国实验方剂学杂志

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.

Gummatous cutaneous tuberculosis associated with the use of infliximab for Crohn's disease

Abstract As the treatment of infectious and parasitic diseases improved, the prevalence of these conditions declined. However, with the expansion of the use of immunobiologicals, opportunistic infections have emerged, especially under atypical presentations. The present study reports the case of a patient treated with infliximab for Crohn's disease, who presented diarrhea, weight loss, abdominal pain, fever, and subcutaneous erythematous nodules that evolved with spontaneous fluctuation and ulceration. With the finding of alcohol-resistant bacilli and Mycobacterium tuberculosis DNA in a cutaneous fragment, through polymerase chain reaction, the diagnosis of gummatous tuberculosis was confirmed, probably secondary to hematogenous dissemination from an intestinal focus.

Effect of Danggui Niantongtang on Key Factors of Extrinsic and Intrinsic Apoptotic Pathway in Synovial Tissue of Adjuvant Arthritis Rats / 中国实验方剂学杂志

Objective:To observe the effect of Danggui Niantongtang on the protein and mRNA expression of key regulatory factors of the extrinsic and intrinsic apoptotic pathway in synovial tissue of adjuvant arthritis （AA） rats， and to further explore the mechanism of Danggui Niantongtang in the prevention and treatment of rheumatoid arthritis. Method:The general condition of AA rats， including its body weight， were observed. The changes of toe volume were detected by toe volume meter. Histopathological changes of synovium of knee joint were observed by hematoxylin-eosin （HE） staining. The mRNA and protein expression levels of tumor necrosis factor receptor super family 6 （Fas）， Fas-associating protein with a novel death domain（FADD）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax） and cysteinyl aspartate specific proteinase Caspase-3 （Caspase-3） were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Result:Compared with the normal group， the toe volume of the model group increased significantly （<italic>P</italic><0.01）， with significantly proliferated synovial cells， significantly reduced mRNA and protein expression levels of Fas， FADD， Bax and Caspase-3 in synovial tissues（<italic>P</italic><0.05，<italic> P</italic><0.01）， and significantly increased Bcl-2 level （<italic>P</italic><0.01）. Compared with the model group， the swelling degree of toes in Danggui Niantongtang group and Tripterygium group was significantly alleviated （<italic>P</italic><0.01）， with significantly improved synovial hyperplasia， significantly increased mRNA and protein expression levels of Fas， FADD， Bax and Caspase-3 （<italic>P</italic><0.05， <italic>P</italic><0.01）， and significantly decreased expression levels of bcl-2 mRNA and protein （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:Danggui Niantongtang can effectively reduce joint swelling and abnormal proliferation of synovial tissue in AA rats. Its mechanism may be related to regulating the expression of Fas， FADD， Bax， Bcl-2 and Caspase-3， and promoting the apoptosis of synovial cells.

Therapeutic Effect and Mechanism of Chaige Qinlian Tang on Young Mouse MRSA Pneumonia Model / 中国实验方剂学杂志

Objective::To explore the therapeutic effect and mechanism of Chaige Qinlian Tang on pneumonia in young mice. Method::The pneumonia model was duplicated by slowly dripping Staphylococcus aureus into the nasal cavity of mice.After successful modeling, the mice were randomly divided into model group, clindamycin group, and high and low-dose Chaige Qinlian Tang groups, with sham operation group as negative control group.The rats were given 200 mg·kg-1 high-dose Chaige Qinlian Tang, 100 mg·kg-1 low-dose Chaige Qinlian Tang and 120 mg·kg-1 clindamycin.The mice were observed every day.Colonies were counted in the lungs of each group five days later.The expression levels of interleukin(IL)-16, tumor necrosis factor (TNF)-α in lung lavage fluid of each group were determined by enzyme linked immunosorbent assay (ELISA). Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to measure the expression levels of IL-16, TNF-α in lung lavage fluid of each group.The expressions of tumor necrosis factor receptor (TNFR) 1, Caspase-3 and Caspase-7 in lung and the pathological changes of lung were observed. Result::Compared with the sham operation group, the respiratory state and the activity state of the model mice were worse, and the survival rate was higher in the high-dose Chaige Qinlian Tang group.Compared with the sham operation group, the pulmonary colony counts in the model group and treatment groups were increased, compared with the model group, the lung colony counts in clindamycin group and high-dose Chaige Qinlian Tang group were improved significantly (P<0.05, P<0.01). Compared with the control group, the expression levels of IL-16, TNF-α, TNFR1, Caspase-3, Caspase-7 mRNA and protein in the lung of model group and treatment groups were significantly increased (P<0.01). Compared with model group, the expression levels of IL-16, TNF-α and TNFR1, Caspase-3, Caspase-7 in the lung of clindamycin group and high and low-dose Chaige Qinlian Tang groups were significantly increased (P<0.01). The expression levels of protein and mRNA were significantly decreased (P<0.05, P<0.01), and the pathological changes of lung were improved, especially in clindamycin group and high-dose Chaige Qinlian Tang group. Conclusion::Chaige Qinlian Tang has a certain therapeutic effect on Staphylococcus aureus pneumonia in young mice.This effect may be related to regulating TNFR1, Caspase-3 and Caspase-7 pathways, reducing the secretion of IL-16 and TNF-alpha, and enhancing the clearance of staphylococcus aureus.

Establishment and validation of OX40/FcγR-humanized mice for the study of agonistic anti-OX40 antibody / 上海交通大学学报（医学版）

Objective • To establish a rapid method to evaluate the activity of agonistic antibody using OX40 (tumor necrosis factor receptor superfamily member 4)/FcγR (Fcγ receptor)-humanized mice. Methods • Bone marrow cells from OX40-humanized mice and FcγR-humanized mice were collected and mixed with equal ratio. Then the mixed bone marrow cells were administrated into irradiated wild-type mice through the tail veins. The reconstruction efficiency of the immune system was confirmed by detecting the expression of hOX40 and hFcγR in the immune cells of chimera mice. After the chimera mice were generated successfully, they were used to evaluate the immunostimulatory activity of anti-hOX40 antibodies to CD4+ or CD8+ T cells. The results of flow cytometry were statistically analyzed. The unpaired t-test was used to compare the means between the two groups, and oneway ANOVA was used to compare the means between multiple groups. Results • Flow cytometry analysis showed that wild-type recipient mice were efficiently reconstituted with hFcγR expressing cells and hOX40 expressing cells to generate OX40/FcγR-humanized bone marrow chimera mice. In these mice, B cells and myeloid cells expressed hFcγRs (P<0.05), and T cells expressed hOX40 upon in vitro stimulation (P<0.05). When these mice were used to evaluate the immunostimulatory activity of anti-hOX40 antibody, significant expressions of IFN-γ and hOX40 were observed (P<0.05). Conclusion • OX40/FcγR-humanized bone marrow chimera mice are generated based on hFcγR expressing cells and hOX40 expressing cells, suggesting a rapid method to build a mouse model with both hFcγR and hOX40 expression. These mice are suitable for evaluating the immunostimulatory activity of agonistic human anti-hOX40 antibodies.

Establishment and evaluation of a highly efficient and stable calcium phosphate transfection method for 293t cells / 吉林大学学报(医学版)

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.

The effects of silica dust on the expression of MyD88 and TRAF6 mRNA of lung macrophages in rats / 中华劳动卫生职业病杂志

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.

Clinical effect of recombinant human tumor necrosis factor-α receptor II: IgG Fc fusion protein combined with sodium hyaluronate intra-articular injection in the treatment of rheumatoid arthritis / 中国基层医药

Objective@#To investigate the clinical efficacy of recombinant human tumor necrosis factor-α receptor II: IgG Fc fusion protein combined with sodium hyaluronate in the treatment of rheumatoid arthritis (RA).@*Methods@#Forty adult RA patients (12 males, 28 females) aged from 36 to 79 were selected in Department of Rheumatology of the Second People′s hospital of Datong from February 2016 to February 2018.The patients were randomly divided into the observation group and the control group according to the digital table, with 20 cases in each group.The observation group was treated by intra-articular injection of recombinant human tumor necrosis factor-α receptor II: IgG Fc fusion protein and sodium hyaluronate, and the control group was only treated with recombinant human tumor necrosis factor-α receptor II: IgG Fc fusion protein.The joint swelling, joint pain, joint mobility, morning stiffness, X-ray grade and total effective rate of the two groups were statistically analyzed before and after treatment.@*Results@#Before treatment, there were no statistically significant differences in joint swelling, joint pain, joint mobility, morning stiffness, clinical score and X-ray grade between the two groups (all P>0.05). After treatment, the symptoms and signs of the two groups were obviously improved.The joint pain, joint pressure pain, joint activity, morning stiffness and clinical score in the observation group were (1.2±1.2)points, (0.8±0.7)points, (0.5±0.4)points, (0.7±0.7)points and (4.5±2.6)points, respetively, which were significantly better than those in the control group [(2.2±1.4)points, (1.5±0.9)points, (1.5±0.9)points, (1.5±0.6)points and (8.6±4.6)points, U=125, 111, 68, 89 and 97, all P<0.05]. The total effective rate of the observation group was 100% (20/20), while that of the control group was 75% (15/20), there was statistically significant difference between the two groups (χ2=14.10, P<0.01).@*Conclusion@#The injection of recombinant human tumor necrosis factor-α receptor II: IgG Fc fusion protein combined with sodium hyaluronate in the treatment of RA can effectively improve the clinical symptoms of RA patients, and it is more effective than single use.It is worthy of popularizing in clinic.

Effect of Xiao Chaihutang on NF-κB Signaling Pathway in Synovial Tissue of CFA Rats / 中国实验方剂学杂志

Objective:To observe the expression of tumor necrosis factor receptor-associated death domain (TARDD), nuclear transcription factor-κB inhibiting protein α(IκBα)IκB kinase-α (IKKα) and nuclear transcription factor (NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant (CFA) rats after treatment with Xiao Chaihutang (XCHT). Method:In animal experiments, SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table, the rats were randomly divided into the normal group, the model group, the low-dose XCHT group, the medium-dose XCHT group, the high-dose XCHT group, and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5.94 g·kg-1),medium-dose XCHT group(11.88 g·kg-1),high-dose XCHT group(23.76 g·kg-1),Tripterygium glucosides group(0.006 3 g·kg-1) were given corresponding drugs by gavage for three times a day, 2 mL/time. The histopathology of rat ankle joint was observed, and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot. Result:With the increase of the dosage of XCHT, the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats, compared with the model group, the statistical results of the low-dose XCHT group showed decreased protein expressions (PPPα, IκB α, NF-κB p65 in the NF-κB signaling pathway were significantly increased (PPα, IκBα, NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower (PPConclusion:This study shows that as the dose of Xiao Chaihutang increases, it could effectively improve synovitis, and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB, thereby preventing inflammation and suppressing bone erosion.

Possile Effect of Small Molecule in Hedysari Radix in Antagonizing Tumor Necrosis Factor Receptor 1 Based on Molecular Docking / 中国实验方剂学杂志

Objective: To study the effect of small molecule compounds of Hedysari Radix in ntagonizing tumor necrosis factor receptor type 1 (TNFR1) based on molecular docking. Method: The structure of small molecular compound of Hedysari Radix was downloaded from the chemical composition compound library of traditional Chinese medicine, and then optimized to obtain the composition compound library of Hedysari Radix. The three-dimensional structure of the inflammatory target TNFR1 (PDB ID:1TNR) was identified. After hydrotreating and anhydrating, the binding pocket residues were identified according to the literature. According to the defined target structure and binding pocket, the flexible molecular docking was conducted between the composition compound library and the target, and the score (Glide Score) was obtained. Based on the results of molecular docking, the first nine small molecular compounds of Glide Score were selected as candidate components. On this basis, the drug-likeness was analyzed, which involved small molecular compounds that meet the number of hydrogen-bonded receptors, the number of hydrogen-bonded donors, the formula weight, the number of rotatable key and the numerical range of lipo-hydro partition coefficient. Finally, the binding mode was analyzed according to pharmacokinetic parameters and complex structure of composition-target docking. Result: The residue set in the TNFR1 drug-binding pocket were identified as glutamic acid109 (Glu109), lysine 35(Lys35), alanine62 (Ala62), serine 74 (Ser74), lysine75 (Lys75), cysteine76 (Cys76), argnine 77(Arg77), glutamine82 (Gln82), threonine89 (Thr89), asparticacid91 (Asp91), argnine92 (Arg92), aspartic acid93 (Asp93), threonine 94(Thr94), valine95 (Val95), cysteine 96(Cys96), argnine104 (Arg104), tyrosine106 (Tyr106), asparagine110 (Asn110), leucine111 (Leu111), phenylalanine112(Phe112), glutamic acid 131(Glu131) and lysine132 (Lys132). Totally 43 small molecular compounds of Hedysari Radix were obtained. Five small molecular compounds, namely hedysari radix, quercetin, isoliquiritin, naringenin, calycosin and liquiritigenin, were screened by comprehensive factors, like docking scoring. Conclusion: Quercetin, isoliquiritin, naringenin, calycosin and liquiritigenin are the effective anti-inflammatory substances of Hedysari Radix, with a great possibility of becoming TNFR1 antagonists.

The role and mechanism of TRAF2 in the biological behavior of gastric cancer / 中华肿瘤杂志

Objective@#To clarify the effect of TRAF2 in the biological behavior of gastric cancer and explore the mechanism.@*Methods@#TRAF2 stably depleted AGS cell was established. Cell growth was monitored by x-CELLigence system. Cell proliferation was detected using cell viability assay. The apoptosis and cell cycle were detected by flow cytometry. The difference of migration and invasion abilities were measured by real-time xCELLigence system and Transwell. The expression and activity of NF-κB signaling pathway were measured by western blot and TransAM assay. The expression of TRAF2 in gastric cancer tissue and its clinical significance were detected by immunohistochemistry.@*Results@#The cell index of AGS-siTRAF2 cells was significantly lower than that of AGS-sictrl cells at 8 h. In the cell viability assay, the A values of AGS-siTRAF2 cells were 51 296.00±2 631.06, 68 389.25±6 703.21 and 65 559.50±6 339.22 at 24 h, 48 h and 72 h. The values of the viability of AGS-siTRAF2 cells were significantly lower than those of AGS-sictrl cells (P<0.001). The results of flow cytometry showed that the apoptosis rates of AGS-siTRAF2 cells were (1.42±0.07)%, (2.98±0.11)% and (1.56±0.03)% at 24 h, 48 h and 72 h, respectively, which were significantly higher than those of AGS-sictrl cells (all P<0.05). The distribution of S phase in AGS-siTRAF2 cells was (23.57±1.12)%, while that in the AGS-sictrl cells was (19.49±1.19)%. The difference was statistically significant (P=0.012). AGS-siTRAF2 cells migrated much slower than AGS-sictrl cells from 3 h and the number of migrated AGS-sictrl cells was 121.7±6.7 while that of AGS-siTRAF2 cells was 84.0±6.6 (P=0.002). The cell index of AGS-siTRAF2 cells was less than that of AGS-sictrl cells from 3 h. In Transwell assay, the number of invaded AGS-sictrl cells was 109.3±3.1 after 24 h of culture, significantly higher than 79.0±6.2 of AGS-siTRAF2 cells (P=0.002). Western blot analysis showed that the expression levels of RelA, RelB, p50 and p52 in AGS-siTRAF2 cells were significantly lower than those in AGS-sictrl cells. The activities of RelA, RelB, p50 and p52 in AGS-siTRAF2 cells were 0.01±0.00, 0.01±0.01, 0.92±0.01 and 0.53±0.03, respectively, significantly lower than those of AGS-sictrl cells (all P<0.001). High TRAF2 expression (TRAF2-high) was found in 53.0% of GC samples, while TRAF2-high was only observed in 38.0% of the paired adjacent tissues (P=0.033). The expression of TRAF2 was significantly higher in the tubular adenocarcinoma, poor differentiation advanced T, advanced N, and clinical staging (P<0.05). The median survival time were 17 months and 78 months in the TRAF2 high-expression and low-expression groups, respectively, and the difference was statistically significant (P=0.010).@*Conclusions@#Depletion of TRAF2 inhibits the AGS cell growth, migration and invasion. The expression of TRAF2 is increased in gastric tumor tissue. The expression of TRAF2 is associated with the prognosis of gastric cancer.

Study of tumor necrosis factor receptor superfamily 1B gene polymorphism in relation to the outcomes of HCV infection / 中华肝脏病杂志

Objective@#To investigate the tumor necrosis factor receptor superfamily 1B gene (TNFRSF1B) polymorphism in relation to the outcomes of hepatitis C virus (HCV) infection.@*Methods@#One thousand six hundred and forty-five cases without HCV infection, 545 cases with HCV clearance, and 783 cases with chronic HCV infection were enrolled. TaqMan probe method was used to investigate genotype rs1061622 (T > G) and rs1061624 (G > A). Two single nucleotide polymorphisms (SNPs) sites were genotyped and haplotypes were constructed to evaluate their relation with the outcome of HCV infection.@*Results@#Logistic regression analysis showed that there was no relation to the two SNPs with HCV infection susceptibility and chronicity (P > 0.05). Haplotype analysis showed that carrier TA had an increased susceptibility to HCV infection [adjusted odds ratio (OR) = 1.15, 95% confidence interval (CI): 1.01 to 1.30, P = 0.038)]. Carrier TA and GG haplotypes were conducive to chronic HCV infection (adjusted OR = 1.28, 95% CI: 1.08 to 1.53, P = 0.006; OR = 1.31, 95% CI: 1.03 to 1.66, P = 0.026).@*Conclusion@#The combinational effects of rs1061622 and rs1061624 in TNFRSF1B gene may increase the risk of HCV chronicity and infection.

Association between TNFRSF11A and TNFRSF11B gene polymorphisms and the outcome of hepatitis C virus infection / 中华流行病学杂志

Objective@#To explore the relationship between the tumor necrosis factor receptor superfamily members 11A (TNFRSF11A) and 11B (TNFRSF11B) gene polymorphisms and the outcome of hepatitis C virus (HCV) infection.@*Methods@#In this case-control study, 749 cases of persistent HCV infection, 494 cases of spontaneous clearance and 1 486 control subjects were included from 2008 to 2016. TaqMan-MGB probe method was used to detect the genotype of TNFRSF11A rs1805034 and TNFRSF11B rs2073617. The genotypes distribution of the two single nucleotide polymorphisms (SNP) were analyzed in different populations.@*Results@#Co-dominant model showed that individuals carrying the rs2073617 CC genotype were prone to have chronic HCV infection, compared with individuals carrying the rs2073617 TT genotype (OR=1.517, 95%CI: 1.055-2.181, P=0.024). Recessive model results showed that individuals carrying rs2073617 CC genotype were more likely to develop chronic HCV infection compared with individuals carrying rs2073617 TT or TC genotype (OR=1.435, 95%CI: 1.033-1.996, P=0.032). Additive model showed that the risk for chronic HCV infection increased with the increase of the number of rs2073617 C alleles (OR=1.204, 95%CI: 1.013-1.431, P=0.035).@*Conclusion@#The genetic polymorphism of TNFRSF11B rs2073617 might be related with the chronicity of HCV infection.

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.

Effect of Taiji Quan on Sleep Quality of Patients with Chronic Insomnia Disorder / 中国康复理论与实践

Objective:To explore the effect of Taiji Quan on the sleep quality of patients with chronic insomnia disorder (CID) and its mechanism. Methods:From January, 2015 to December, 2017, 31 patients with CID were enrolled in the sleep disorder clinic. Before and 24 weeks after Taiji Quan exercise, the Pittsburgh Sleep Quality Index (PSQI) was used to assess their sleep quality, the serum levels of tumor necrosis factor (TNF)-α, TNF-β, soluble tumor necrosis factor receptor (sTNF-R)1 and sTNF-R2 were detected with protein chip, and the correlation between the total score of PSQI and the serum levels of TNF-α, TNF-β, sTNF-R1 and sTNF-R2 were analyzed after exercise. Results:After Taiji Quan exercise, the scores of PSQI factors (subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, daytime dysfunction) and the total score of PSQI decreased (t > 4.080, P < 0.05). The serum levels of TNF-α and TNF-β decreased (t > 13.580, P < 0.01), however, the serum levels of sTNF-R1 and sTNF-R2 significantly increased (t > 160.189, P < 0.001). The serum levels of TNF-α and TNF-β were positively correlated with the total score of PSQI (r > 0.638, P < 0.001), while the serum levels of sTNF-R1 and sTNF-R2 were negatively correlated with the total score of PSQI (r > 0.532, P<0.001). Conclusion:Taiji Quan exercise could help to improve the sleep quality of patients with CID. The mechanism may be related to the decrease of the serum levels of TNF-α and TNF-β, and the increase of the serum levels of sTNF-R1 and sTNF-R2.

Role of spinal cord TRAF6/NF-κB signaling pathway in development of diabetic neuropathic pain in rats / 中华麻醉学杂志

Objective To evaluate the role of spinal cord tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa B (NF-κB)signaling pathway in the development of diabetic neuropathic pain (DNP)in rats.Methods Clean-grade healthy adult male Sprague-Dawley rats,aged 2 months,weighing 180-230 g,in which IT catheters were implanted,were used in this study.Streptozotocin 60 mg/kg was intraperitoneally injected after IT catheterization to establish the model of DNP.Twelve DNP rats were divided into 2 groups (n =6 each) by a random number table method:DNP group and DNP plus TRAF6 inhibitor group (group DTR).Another 6 age-matched Sprague-Dawley rats were used as normal control group (group NC).The rats in group DC and group DTR received IT injection of dimethyl sulfoxide 10 μl and TRAF6 inhibition 10 μg,respectively,once a day for 7 consecutive days starting from day 21 after establishing the model.The mechanical paw withdrawal threshold (MWT) was determined before establishing the model (T1),on 7,14 and 21 days after establishing the model (T2-4),and on 1,4 and 7 days after IT injection (T5-7).The rats were sacrificed after the last MWT measurement,and the L3-5 segments of the spinal cord were removed for determination of the expression of TRAF6 and NFκB p65 by Western blot.Results Compared with group NC,the MWT at T3-7 in group DC and at T3-6 in group DTR was significantly decreased,and the expression of spinal TRAF6 and NF-κB p65 was up-regulated in DC and DTR groups (P＜0.05).Compared with group DC,the MWT was significantly increased at T6-7,and the expression of spinal TRAF6 and NF-κB p65 was down-regulated in group DTR (P ＜ 0.05).Conclusion Spinal cord TRAF6/NF-κB signaling pathway is involved in the development of DNP in rats.

Anti-inflammatory mechanism of Scutellarlae Radix based on network pharmacology / 中草药

Objective: To explore the anti-inflammatory mechanism of Scutellarlae Radix (SR) by the network pharmacology. Methods: Firstly, the components in SR were searched through TCMSP database and screened with “Lipinski rule” and “Oral Bioavailability > 30%” rules. The targets of above components selected by PharmMapper web server and Cytoscape 3.4.0 was used to build a network between components and targets (component-target network, CTN). Secondly, “anti-inflammatory” targets was searched from Therapeutic Target Database (TTD) with keyword “anti-inflammatory”, and targets retrieved were used to build a protein-protein interaction (PPI) network based on the analysis by String database. To obtain anti-inflammatory targets of the active components in SR, the PPI network was fused with the CTN. Finally, the DAVID database was used to perform KEGG pathway enrichment analysis in order to explore the anti-inflammatory mechanism of SR. Results: Twenty-eight components in SR were obtained, including flavonoids such as baicalin, baicalein, wogonin, wogonoside, etc, alkaloids such as berberine, and epiberberine, and phenols such as dihydromyricetin, etc. Mitogen-activated protein kinase (MAPK14), tumor necrosis factor receptor superfamily 1A (TNFRSF1A), epidermal growth factor receptor (EGFR), and E-selectin (SELE) were the main targets of SR' anti-inflammatory effect. Salvigenin, epicatechin, and astragalusine mainly acted on MAPK14; Carthamidin acted on TNFRSF1A; Dihydromubutin A, 5,7,4’-trihydroxy-8-methoxyflavone, 5,7,4’-trihydroxy-8-methoxyflavanone, baicalin, and other components mainly acted on EGFR. There were 11 KEGG pathways, mainly related to TNF signaling pathways, MAPK signaling pathway, etc. Conclusion: There are three main anti-inflammatory mechanisms in SR, which can inhibit the production of inflammatory factors, inhibit the binding of inflammatory factors to their respective receptors, and block the initiation of inflammatory reactions.