Objective To investigate the effects of extracellular vesicles(EVs)derived from synovial fluid of rheumatoid arthritis(RA)patients on angiogenesis of human umbilical vein endothelial cells,and to preliminarily explore the underlying mechanisms.Methods Synovial fluid samples of knee joint were collected from 20 patients with RA and 20 patients with osteoarthritis(OA)in this study.EVs were purified using ultracentrifugation.The morphology and size of EVs were observed by transmission electron microscopy and nanoparticle tracking analysis.CD9,CD63,cytochrome c(Cyt-c),vascular endothelial growth factor(VEGF),lysyl oxidase(LOX),matrix metalloproteinase 2(MMP2),tumour necrosis factor alpha(TNF-α),transforming growth factor beta1(TGF-β1)in EVs were detected using Western blot.Human umbilical vein endothelial cells(HUVEC)were treated with the EVs.The growth,migration and angiogenesis of HUVEC were observed by CCK8 assay,TranswellTM chamber assay,scratch test and matrigel angiogenesis assay,respectively.The effect of EVs on the PI3K/AKT pathway in HUVEC was assessed using Western blot.Results Both EVs from RA synovial fluid(RA-EVs)and OA synovial fluid(OA-EVs)were cup-shaped,mainly between 30-400 nm in diameter,expressing CD63 and CD9,but not Cyt-c.RA-EVs carried more VEGF,LOX,MMP2,TNF-α and TGF-β1 than OA-EVs.Compared with the OA-EVs intervention,RA-EVs significantly promoted the proliferation,migration,and angiogenesis of HUVECs,as well as upregulated PI3K/AKT phosphorylation.The inhibitor of PI3K suppressed angiogenesis induced by EVs.Conclusion EVs in synovial fluid of RA carried more cytokines and enzymes that related angiogenesis and inflammation.These EVs exert their pro-angiogenic effects by activating the PI3K/AKT pathway,then contributing to the pathological progression of RA.

Objective To investigate the effects of extracellular vesicles(EVs)derived from synovial fluid of rheumatoid arthritis(RA)patients on angiogenesis of human umbilical vein endothelial cells,and to preliminarily explore the underlying mechanisms.Methods Synovial fluid samples of knee joint were collected from 20 patients with RA and 20 patients with osteoarthritis(OA)in this study.EVs were purified using ultracentrifugation.The morphology and size of EVs were observed by transmission electron microscopy and nanoparticle tracking analysis.CD9,CD63,cytochrome c(Cyt-c),vascular endothelial growth factor(VEGF),lysyl oxidase(LOX),matrix metalloproteinase 2(MMP2),tumour necrosis factor alpha(TNF-α),transforming growth factor beta1(TGF-β1)in EVs were detected using Western blot.Human umbilical vein endothelial cells(HUVEC)were treated with the EVs.The growth,migration and angiogenesis of HUVEC were observed by CCK8 assay,TranswellTM chamber assay,scratch test and matrigel angiogenesis assay,respectively.The effect of EVs on the PI3K/AKT pathway in HUVEC was assessed using Western blot.Results Both EVs from RA synovial fluid(RA-EVs)and OA synovial fluid(OA-EVs)were cup-shaped,mainly between 30-400 nm in diameter,expressing CD63 and CD9,but not Cyt-c.RA-EVs carried more VEGF,LOX,MMP2,TNF-α and TGF-β1 than OA-EVs.Compared with the OA-EVs intervention,RA-EVs significantly promoted the proliferation,migration,and angiogenesis of HUVECs,as well as upregulated PI3K/AKT phosphorylation.The inhibitor of PI3K suppressed angiogenesis induced by EVs.Conclusion EVs in synovial fluid of RA carried more cytokines and enzymes that related angiogenesis and inflammation.These EVs exert their pro-angiogenic effects by activating the PI3K/AKT pathway,then contributing to the pathological progression of RA.

BACKGROUND:Indolepropionic acid has been shown to reduce diabetes-induced central nervous system inflammation.However,there is a lack of research on whether to inhibit microglia M1 polarization for the treatment of spinal cord injury. OBJECTIVE:To investigate the mechanism of indolepropionic acid inhibition of microglial cell M1 polarization for the treatment of spinal cord injury through cell and animal experiments. METHODS:(1)In vitro experiments:BV2 cell viability was assessed using the CCK-8 assay to determine optimal concentrations of indolepropionic acid.Subsequently,BV2 cells were categorized into control group,administration group(50 μmol/L indolepropionic acid),lipopolysaccharide group(100 ng/mL lipopolysaccharide),and treatment group(100 ng/mL lipopolysaccharide + 50 μmol/L indolepropionic acid).Nitric oxide content was quantified using the Griess method.Real-time quantitative PCR and western blot assay were employed to measure mRNA and protein levels of pro-inflammatory factors.Cell immunofluorescence staining was conducted to assess inducible nitric oxide synthase expression.The Seahorse assay was employed to assess glycolytic stress levels in BV2 cells.(2)In vivo experiments:30 SD rats were randomly divided into three groups:sham surgery group,spinal cord injury group,and indolepropionic acid group.Motor function recovery in rats after spinal cord injury was assessed using BBB scoring and the inclined plane test.Immunofluorescence staining of spinal cord tissue was conducted to evaluate the expression of inducible nitric oxide synthase in microglial cells.ELISA was employed to measure protein expression levels of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in spinal cord tissue. RESULTS AND CONCLUSION:(1)In vitro experiments:Indolepropionic acid exhibited significant suppression of BV2 cell viability when its concentration exceeded 50 μmol/L.Indolepropionic acid achieved this by inhibiting the activation of the nuclear factor κB signaling pathway,thereby suppressing the mRNA and protein expression levels of pro-inflammatory cytokines(interleukin-1β and tumor necrosis factor-α),as well as the M1 polarization marker,inducible nitric oxide synthase,in BV2 cells.Additionally,indolepropionic acid notably reduced the glycolytic level in BV2 cells induced by lipopolysaccharides.(2)In vivo experiments:Following indolepropionic acid intervention in spinal cord injury rats,there was a noticeable increase in BBB scores and the inclined plane test angle.There was also a significant decrease in the number of M1-polarized microglial cells in spinal cord tissue,accompanied by a marked reduction in the protein expression levels of pro-inflammatory cytokines(interleukin-1β and tumor necrosis factor-α).(3)These results conclude that indolepropionic acid promotes functional recovery after spinal cord injury by improving the inflammatory microenvironment through inhibition of microglia M1 polarization.

Objective To investigate the structure of deoxyhypusine synthase(DHS)in Saccharomyces cerevisiae(Dys1)and unravel the molecular mechanism of hypusine lysine modification,providing a theoretical basis for the treatment of highly proliferative diseases such as human immunodeficiency virus type 1(HIV-1)replication.Meth-ods Using the E.coli BL21 expression system,an in vitro expression vector was constructed and used to express the protein of Dys1.Dys1 protein samples were purified using methods such as affinity chromatography and molecu-lar sieving to achieve protein purification and isolation.The crystals of Dys1 were obtained using the crystallized so-lution containing 6％Polyethylene Glycol(PEG)8000,0.1 mol/L N-2-hydroxyethylpiperazine-N-ethane-sulphoni-cacid(Hepes)pH 6.5,and 8％ethylene glycol.The crystal structure of Dys1 was resolved at a resolution of 2.8 ? using X-ray crystallography.The structural analysis was performed with CCP4i and Coot software.Results The overall structure of Dys1 was a tetramer,each monomer containing a catalytic site and a cofactor NAD+binding site.The core region of the monomer adopted a Rossmann fold.The amino acid residues involved in the substrate binding sites were highly conserved among eukaryotes.Conclusion The crystal structure of Dys1 is being resolved for the first time.It reveals the binding mode of the cofactor NAD+to the enzyme and confirms that the enzyme functions as a tetramer,with the N-terminus serving as an essential modulator for its catalytic activity.

Objective:To explore the risk factors for nonunion or malunion after internal fixation of femoral neck fracture.Methods:A retrospective analysis was done of the 140 patients with femoral neck fracture who had been admitted to Department of Traumatology and Orthopedics, Weifang People's Hospital from January 2013 through June 2019. They were 61 males and 79 females, aged from 18 to 65 years (average, 51.3 years). All patients received "inverted triangle" internal fixation with 3 hollow nails after reduction. The univariate analysis was conducted of the 9 factors: age, gender, fracture side, Garden classification, injury-to-operation time, reduction quality, postoperative ambulation with crutches but no weight-bearing, preoperative bone density, and preoperative 25-hydroxyvitamin D3 concentration. Multivariate logistic regression analysis was used for the factors with P<0.05 to identify risk factors. P<0.05 was considered statistically significant. Results:This cohort of 140 patients obtained follow-up for 6 to 12 months (average, 10.8 months). Nonunion or malunion occurred in 25 cases of them (17.86%). The multivariate logistic regression analysis showed that Garden types Ⅲ&Ⅳ ( OR=2.873, 95% CI: 1.027~8.039, P=0.044), non-anatomic reduction ( OR=4.678, 95% CI: 1.133~19.310, P=0.033), postoperative ambulation time with crutches but no weight-bearing <3 months ( OR=0.230, 95% CI: 0.072~0.737, P=0.013), and preoperative 25-hydroxyvitamin D3 concentration<30 ng/mL ( OR=3.718, 95% CI: 1.337~10.341, P=0.012) were the risk factors for nonunion or malunion after internal fixation of femoral neck fracture. Conclusion:The risk factors for nonunion or malunion after internal fixation of femoral neck fracture may be the Garden types Ⅲ&Ⅳ, non-anatomic reduction, postoperative ambulation time with crutches but no weight-bearing <3 months, and preoperative 25-hydroxyvitamin D3 concentration < 30 ng/mL.