G protein-coupled receptor kinase 2 (GRK2) participates in the phosphorylation and desensitization of G protein-coupled receptor (GPCR), impacting various biological processes such as inflammation and cell proliferation. Dysregulated expression and activity of GRK2 have been reported in multiple cells in rheumatoid arthritis (RA). However, whether and how GRK2 regulates synovial hyperplasia and fibroblast-like synoviocytes (FLSs) proliferation is poorly understood. In this study, we investigated the regulation of GRK2 and its biological function in RA. We found that GRK2 transmembrane activity was increased in FLSs of RA patients and collagen-induced arthritis (CIA) rats. Additionally, we noted a positive correlation between high GRK2 expression on the cell membrane and serological markers associated with RA and CIA. Immunoprecipitation-mass spectrometry and pull-down analyses revealed tumor necrosis factor receptor-associated factor 2 (TRAF2) as a novel substrate of GRK2. Furthermore, surface plasmon resonance (SPR) and molecular docking assays determined that the C-terminus of GRK2 binds to the C-terminus of TRAF2 at the Gln340 residue. GRK2 knockdown and the GRK2 inhibitor CP-25 attenuated synovial hyperplasia and FLS proliferation in CIA both in vitro and in vivo by decreasing GRK2 membrane expression and activity. Mechanistically, increased GRK2 transmembrane activity contributed to the recruitment of TRAF2 on the cell membrane, promoting GRK2-TRAF2 interactions that facilitate the recruitment of the E3 ubiquitin ligase TRIM47 to TRAF2. This enhanced TRAF2 Lys63 polyubiquitylation and induced nuclear factor (NF)-κB activation, leading to synovial hyperplasia and abnormal proliferation of FLSs. Our study provides a mechanistic and preclinical rationale for further evaluation of GRK2 as a therapeutic target for RA.

Objective : To explore the effect and mechanism of CD151 on vascular permeability by regulating vesicle internalization and recycling. Methods: Wild-type mice and CD151 knockout mice were divided into WT-con group, WT-model group, KO-con group and KO-model group, with 6 mice in each group. WT-model group and KO-model group were intraperitoneally injected with LPS to prepare sepsis ALI model, and WT-con group and KO-con group were intraperitoneally injected with phosphate buffer saline(PBS) as a control. 24 h after modeling, pulmonary vascular permeability was measured by Miles test. The siRNA silencing CD151 expression(si-CD151) and negative control si-NC were transfected into EA.hy 926 cells. The permeability of endothelial cell layer to FITC-dextran at different time points was observed under basic conditions and vascular endothelial growth factor-A(VEGF-A) stimulation conditions. Transcriptome sequencing of endothelial cells in si-CD151 group and si-NC group; the distribution and internalization of CD151 in each group were measured using immunofluorescence. Western blot and real-time quantitative RT-qPCR were used to detect the expression of VE-cadherin in si-CD151 groupand other groups. The distribution and internalization of VE-cadherin in each group were measured using immunofluorescence. Results : Miles experiment results indicated that dye exudation in lung tissue of WT-model group was significantly higher than that of WT-con group(P<0.01). The dye exudation in the lung tissue of KO-model group increased compared with WT-model group(P<0.05). The results of endothelial cell layer permeability test showed that the permeability of FITC-dextran in si-CD151 group was significantly higher than that in control group after VEGF-A stimulation for 30, 60 and 120 min(P<0.05). Transcriptome sequencing results suggested that CD151 in endothelial cells was closely related to vesicle-mediated transport. Compared with other groups, protein and mRNA levels of VE-cadherin in CD151 knockdown endothelial cells was significantly lower(allP<0.01). The immunofluorescence assay demonstrated that after VEGF-A stimulation, the decrease of CD151 expression significantly impaired the expression of VE-cadherin at cell-cell contacts and reduced the CD151-VE-cadherin colocalization in the perinuclear region compared with other groups. Conclusion The absence of CD151 affects the internalization and recycling of endothelial cell vesicles, affects the expression and internalization of VE-cadherin, and then influences vascular permeability.

Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.

Objective To explore the effect and mechanism of Bajitianwan on preventing D-galactose (D-gal)-induced osteoblast bone loss. Methods Osteoblasts isolated from 24 h old Wistar rats were injured by D-gal and intervened with Bajitianwan extract. The osteoblastic proliferation and differentiation were determined by MTT and alkaline phosphatase (ALP), respectively. The cell reactive oxygen species (ROS) levels were detected by DCFH-DA fluorescent probes. The expression of cellular oxidation-related protein nuclear factor erythroid 2-related factor 2 (Nrf2), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase 1 (NQO1) were detected by Western blotting. The intranuclear expression of Nrf2 protein was measured by immunofluorescence. Results Bajitianwan extract had significantly increased the osteoblastic proliferation and differentiation, and significantly reduced the intracellular ROS level. Bajitianwan extract had activated the PI3K/AKT pathway via activating the phosphorylation of AKT in osteoblasts, and promoted NQO1 and HO-1 expression. In addition, Bajitianwan had significantly promoted the expression of Nrf2 in the nucleus of osteoblasts, activating Nrf2 signaling pathway, and further promoted bone formation. Conclusion This study confirmed that Bajitianwan could prevent D-gal injured osteoblastic bone loss for the first time. The mechanism might be related to the regulation of oxidative stress associated PI3K/AKT and Nrf2 signaling pathway.

Objective: To construct a human G protein-coupled receptor kinase 2 ( GRK2) eukaryotic expression system． Methods: The primers were designed ，and the His-GRK2 target gene was amplified by PCR using the Pans-EGFP-GrK2 (full-length) gene as the template．The His-GRK2 target gene was connected to the pcDNA3．1EGFP eukaryotic expression vector． The pcDNA3. 1-EGFP-His-GRK2 plasmid was transfected into HEK 293T cells．48 h later，the expression of GRK2 protein was detected by Western blot，and the GRK2 protein was purified by nickel chelated magnetic bead method．The purification of GRK2 protein was detected by Coomassie bright blue staining and Western blot，and the activity of GRK2 protein was detected by His pull down. Results : The results of double enzyme digestion and sequencing showed that pcDNA3. 1-EGFP-His-GRK2 eukaryotic expression plasmid was successfully constructed．Western blot analysis showed that the molecular weight of GRK2 protein was about 80 ku，indicating that GRK2 protein was successfully expressed in HEK 293T cells (t = 6. 433，P = 0. 003) ．GRK2 protein was purified by nickel chelated magnetic beads．His pull down experiment results showed that GRK2 was bound to prostaglandin E2 receptor 4 (EP4) ，suggesting that GRK2 protein had biological activity (t = 13. 5，P = 0. 000 2) ． Conclusion The pcDNA3．1-EGFP-His-GRK2 eukaryotic expression plasmid was correctly sequenced and the GRK2 recombinant plasmid was successfully constructed．The GRK2 recombinant plasmid was successfully expressed in eukaryotic cells HEK 293T and the protein expressed was biologically active.

BK polyomavirus-associated nephropathy (BKPyVAN) is a common cause of allograft failure. However, differentiation between BKPyVAN and type I T cell-mediated rejection (TCMR) is challenging when simian virus 40 (SV40) staining is negative, because of the similarities in histopathology. This study investigated whether donor-derived cell-free DNA (ddcfDNA) can be used to differentiate BKPyVAN. Target region capture sequencing was applied to detect the ddcfDNAs of 12 recipients with stable graft function, 22 with type I TCMR, 21 with proven BKPyVAN, and 5 with possible PyVAN. We found that urinary ddcfDNA levels were upregulated in recipients with graft injury, whereas plasma ddcfDNA levels were comparable for all groups. The median urinary concentrations and fractions of ddcfDNA in proven BKPyVAN recipients were significantly higher than those in type I TCMR recipients (10.4 vs. 6.1 ng/mL,

Objective To summarize the nursing for acute epididymitis within intermittent catheterization. Methods From August, 2013 to February, 2016, twelve patients suffering acute epididymitis within intermittent catheterization after spinal cord injury were reviewed. Re-sults Epididymitis was cured in all the patients after retention catheterization, psychological nuring, diet and drinking guide, reasonable drug use, etc. Conclusion Acute epididymitis within intermittent catheterization can be controlled by appropriate medication and nursing.

Objective To compare diagnostic values of the 2015 American Thyroid Association (ATA) Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer with the thyroid imaging reporting and data system (TI-RADS) for sonographic malignancy risk stratification of thyroid nodules.Methods From November 2011 to December 2015,485 thyroid nodules in 331 patients (mean age,42.9 years± 10.4)were included in this study.Characteristics includingsize,composition,shape(nonparallel or parallel),margin,echogenicity,calcifications and extrathyroidal extension of thyroid nodules were evaluated.Every nodule was stratificated by criteria set by TI-RADS and ATA guidelines,and malignant rate of each risk stratification were calculated and analysed.With pathology as the gold standard,different cutoff were taken to diagnose malignant nodules,and the sensitivity,specifity,positive predictive value,negativepredictive value and accuracy of the two methodologies were calculated at each cutoff.And the two methodologies were evaluated and measured by ROC curve.Finally their Kappa value were calculated at the best cutoff.Results Of the 485 thyroid nodules,96 were benign and 389 were malignant.The malignancy rates under TI-RADS category 2,3,4a,4b,4c,and 5 nodules were 0,12.0％ (3/25),22.2％ (10/45),29.8％ (14/47),99.2％ (261/363) and 100％ (101/101).Malignancy rates under ATA guidelines of benign,very low,low,intermediate,and high suspicion for malignancy were 0,12.5％ (1/8),16.1％ (10/62),27.7％ (13/47),and 99.2％ (365/368).There were significant differences inside each patterns (P ＜ 0.01) respectively and high correlation between risk stratification with TI-RADS (r=0.70) and ATA guidelines (r=0.83).Areas under the ROC curve of the TI-RADS and ATA guidelines classifications were 0.966 and 0.959.Best cut-off point for diagnosing malignant by TI-RADS and ATA guideline classifications were ≥ 4c and ≥ high suspicion,and at that point,diagnostic value of TI-RADS and ATA guidelines were nearly the same(sensitivity,93.1％vs 93.8％;specificity,97.9％ vs 96.9％;PPV,99.5％ vs 99.2％;NPV,75.7％vs 79.5％;and accuracy,94.0％vs94.4％),and there was no significant differences (P=0.50,P=0.50,P=0.50,P=0.53,P=0.55),Kappa=0.97.Conclusions Both TI-RADS and the ATA guidelinesprovide effective malignancy risk stratification for thyroid nodules.The diagnosticvalue of TI-RADS when considering ≥ 4c and ATA guidelines when considering ≥ high-suspicion nodules as malignant were nearly the same and both high.

Objective: To investigate the predictive value of an early inflammatory response factor, Rho kinase activity for left ventricle remodeling (LVR) in patients with acute ST-segment elevation myocardial infarction (STEMI).
Methods: A total of 120 acute STEMI patients treated in our hospital from 2010-10 to 2013-06 were studied, all patients were ifrst time received primary percutaneous coronary intervention (PCI) with stent implantation. Rho kinase activity and B-type natriuretic peptide (BNP) were measured before PCI, echocardiography was conducted at 24 hours and 12 months after STEMI respectively to clarify LVR diagnosis. The patients were divided into 2 groups as LVR group, n=97 and Non-LVR group, n=23, the above indexes were compared between 2 groups.
Results: The level of Rho kinase was higher in LVR group than that in Non-LVR group, P<0.001, after adjustment, Rho kinase was the independent predictor for LVR (OR 3.36, 95%CI 2.01–5.78, P<0.001). The ROC of Rho kinase was 0.88 (95%CI 0.82–0.94) and the ROC of BNP was 0.54 (95%CI 0.41–0.70).
Conclusion: High Rho kinase activity could predict LVR in acute STEMI patients with primary PCI and stent implantation.

Objective To evaluate the clinical value of ultrasonography guided percutaneous core needle biopsy in pancreatic lesions. Methods Thirty-four patients with 36 pancreatic lesions in Shanghai First People′s Hospital Afifliated to Shanghai Jiao Tong University from February 2012 to November 2013 underwent conventional ultrasound-guided percutaneous core needle biopsy using automatic gun and 18-gauge biopsy needles. The site, size, internal and surrounding vascularity, the sampling number of the lesions, and whether the specimens′ quality was satisfied were recorded. Then specimens were sent for pathological examination, and all above observations were compared with the ifnal diagnosis. Results The number of lesions with 2, 3 and 4 samplings was 32, 2 and 2, respectively. The average number of sampling was 2.2 (mean, 2.17;standard deviation, 0.51) and the acquisition rate of satisifed specimens was 89%(32/36). The pathological results of biopsy were malignant in 31 of 36 lesions including 27 cases of ductal adenocarcinoma, 2 cases of lymphoma, 1 case of small cell neuroendocrine carcinoma and 1 case of uterine leiomyosarcoma metastasis. The other 5 lesions were non-malignant including 3 cases of benign lesion, 1 cases of atypical hyperplasia and 1 cases of granulation tissue. The 36 lesions were ifnally diagnosed as 34 cases of pancreatic malignancy, 2 cases of non-malignant neoplasm. The sensitivity, speciifcity, accuracy, positive predictive value and negative predictive value of ultrasonography guided percutaneous core needle biopsy in pancreatic lesions were 91%(31/34), 100%(2/2), 92%(33/36), 100%(31/31) and 40%(2/5), respectively. Youden index was 0.91. Two patients had mild upper abdominal pain and 1 patient had transient elevated serum amylase. No pancreatitis, pancreatic fistula, peritonitis, bleeding or dispersion of malignant cells along the penetrating channel or other serious complications occurred. Conclusion Ultrasonography guided percutaneous core needle biopsy is a simple, rapid, safe and effective diagnostic method in pancreatic lesions with high clinical value.