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Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2f/fLyz2-Cre+/- mice. Synovial inflammation and M1 polarization were improved in GRK2f/fLyz2-Cre+/- mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1+ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.
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Objective@#To establish an inducible macrophage-specific knockout G protein-coupled receptor kinase 2 ( GRK2) gene ( GRK2flox / flox Lyz2-CreERT + ) mice model.@*Methods@#GRK2flox / flox Lyz2-CreERT + mice were constructed based on Cre / LoxP system.The genotypes of GRK2flox / floxLyz2-CreERT + mice were identified by PCR amplification and agarose gel electrophoresis.After the mice were sacrificed by carbon dioxide method,the expression of GRK2 in bone marrow-derived macrophages ( BMDMs) and peritoneal macrophages ( PMs) was detected by Western blot.Immunofluorescence was used to detect GRK2 expression in mouse brain,heart and spleen macrophages.The M1 / M2 ratio in PMs induced by prostaglandin E2 (PGE2) was analyzed by flow cytometry. @*Results@#The results of genotype identification showed that the mice with a band at 355 bp in the length of the flox amplification product and a band at 355 bp in the length of the Cre amplification product were GRK2flox / floxLyz2-CreERT + mice.Western blot results showed that GRK2 expression in BMDMs and PMs of GRK2flox / floxLyz2-CreERT + mice decreased compared with GRK2flox / flox mice(P<0. 01) .Immunofluorescence results showed that GRK2 expression decreased in the brain,heart and spleen of GRK2flox / floxLyz2-CreERT + mice compared with GRK2flox / flox mice (P < 0. 01) .Flow cytometry showed that compared with GRK2flox / flox mice,there was no significant difference in the proportion of CD86 / CD206 in the PMs of GRK2flox / floxLyz2-CreERT + mice.Under PGE2 ( 10 μmol / L) stimulation, the proportion of CD86 / CD206 in GRK2flox / floxLyz2-CreERT + mice PMs increased (P <0. 01) .The proportion of CD86 / CD206 in the PMs of GRK2flox / flox mice was higher than that of GRK2flox / floxLyz2-CreERT + mice(P<0. 01) . @*Conclusion @#In this study,GRK2flox / floxLyz2-CreERT + mice model was successfully constructed,and the mice promoted PGE2-induced polarization of PMs to M2-type macrophages compared with control mice.
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Previous studies have shown that the sciatic nerve has neurotrophic activity, and nerve regeneration, differentiation, and axon outgrowth can be modulated by different sciatic nerve preparations. However, numerous animals may have to be sacrificed to obtain enough sciatic nerves to make a sciatic nerve preparation. Some studies have demonstrated that the role of sciatic nerve preparations in neural differentiation depends on the neurotrophins that Schwann cells secrete, and these factors are highly conserved among different species. To reduce the use of experimental animals, in this study, we made a leachate by using the sciatic nerve of cattle and explored its effect on neuronal differentiation of rat PC12 cells (a useful model for studying neuronal differentiation). Results showed the neurite outgrowth of PC12 cells treated with the cattle sciatic nerve leachate for 3, 6, and 9 days was significantly improved, and the expressions of β3-tubulin and microtubule-associated protein 2 (two neuron-specific proteins) were increased. Moreover, the ERK1/2 signaling pathway was activated after PC12 cells were incubated with cattle sciatic nerve leachate for 9 days. Thus, a sciatic nerve leachate obtained from cattle can effectively induce neuronal differentiation of rat PC12 cells via ERK1/2 signaling pathway.
Asunto(s)
Animales , Bovinos , Ratas , Axones , Proteínas Asociadas a Microtúbulos , Factores de Crecimiento Nervioso , Regeneración Nerviosa , Neuritas , Neuronas , Células PC12 , Células de Schwann , Nervio CiáticoRESUMEN
ObjectiveTo establish the autophagy model of normal human liver cell line 7702 induced by hypoxia and starvation, and to lay a foundation for further studies on the influence of autophagy on liver function. MethodsThe 7702 cells were selected and incubated with 95% air and 5% CO2 at a temperature of 37 ℃(normal control group). The Binder three-gas incubator was used, with a temperature of 37 ℃, a CO2 concentration of 5%, and an O2 concentration of 0.3% to provide a hypoxic environment, and the serum-free DMEM was used to induce starvation. These cells were divided into 6-, 12-, 18-, and 24-hour hypoxia-starvation groups. Western blot was used to measure the protein expression of Beclin 1, Atg5, and LC3 in the normal control group and experimental groups, RT-qPCR was used to measure the mRNA expression of Beclin 1 and Atg5 in each group, and after transfection of LC3 plasmid, immunofluorescence assay was used to observe autophagy in each group. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for further comparison between any two groups; the chi-square test was used for comparison of categorical data between groups. ResultsThe 6-hour hypoxia-starvation groups had higher protein expression of Beclin 1, Atg5, and LC3 than the normal control group or other treated groups. Compared with all the other groups, the 6-hour hypoxia-starvation group showed significantly increased mRNA expression of Beclin 1 and Atg5, as well as significantly greater increases in the mRNA expression of Beclin 1 and Atg5 (all P<0.05). The hypoxia-starvation groups had significantly lower numbers of autophagosomes than the normal control group, and the 6-hour hypoxia-starvation group had the highest number of autophagosomes (all P<0.05). ConclusionHypoxia and starvation established by physical methods can successfully induce hepatocyte autophagy, which is the most remarkable at 6 hours of hypoxia and starvation.
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Objective Aortic dissection is accute in occurrence along with fast change , which is inclined to to induce psy-chotic symptoms and affect the lives of patients .The study was to investigate the effects of healthcare cooperative intervention on pa-tients with psychiatric symptoms secondary to aortic dissection . Methods We established medical cooperation organization structure to clarify the division of responsibility along with a quick assessment form , a process chart and a green channel for emergencey treat-ment.The above method was applied to intervene 32 patients with psychiatric symptoms secondary to aortic dissection and observe the diagnosis time , the time from diagnosis to surgery , the remission time of psychiatric symptoms , the incidence of adverse events and the length of stay. Results 32 patients were diagnosed within 30min followed by emergency surgery within 30min after diagnosis, which was 1-2 hours shorter than the previous diagnosis and surgery preparation time .5-21 d after surgery , psychiatric symptoms have been effectively controlled.Extubation, falling out of bed, self-injury or injury and other adverse events did not occur .No patient died, and the average length of stay was 13.5d, which was 1.75d shorter than the length of stay of previous similar patients . Conclusion The healthcare cooperation method can quickly identify patients with psychiatric symptoms secondary to aortic dissection , prevent adverse events and shorten the length of stay .
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Objective Because of the major trauma from thoracotomy , postoperative pain may arouse patients'psychological and physiological stress response and hence affected the treatment outcome and functional recovery seriously .We retrospectively studied the correlation between the staging of pain and nursing interference to investigate the effect of nursing interference on the pain intensity after thoracic surgery . Methods Five hundred and eighty cases of patients surviving thoracotomy between December 2013 and March 2014 in Nanjing Jingling Hospital were reviewed .Correlations between comfortable nursing measures such as effective analgesia , postural care , catheter care , environmental interventions , psychological intervention and the standard assessment of pain were analysed according to postoperative pain stage . Results With comprehensive nursing interference , the highest pain score occurred in the first 24 hour (2.89 ±0.39).The score was reduced gradually to 2.25 ±0.90 in stage Ⅱ, 1.58 ±0.57 in stage Ⅲ and 1.06 ±0.24 in stage Ⅳrespectively . Conclusion Comprehensive nursing interference according to pain staging may relieve pain effectively after thoracotomy .
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To develop the awareness of in student nurses during their clinical nursing practice for the prevention of legal disputes and protection of patients’ safety. Enough efforts should be made to strengthen legal education at the beginning, to enrich legal knowledge in the middle, and to stress nursing safety and related countermeasures towards the end of the clinical nursing practice. It is necessary to have student nurses under the supervision of the teachers make sure that they are aware of and abide by the law all the time.