Objective : To generate heterozygous TRAF2 knockout mice, the CRISPR/Cas9 technology was successfully employed. These mice were served as a valuable model to explore the pathological mechanisms underlying inflammatory and immune disorders mediated by abnormal TNF-α-TRAF2 signaling and to develop new therapeutic targets. Methods : A vector targeting the knockout of the TRAF2 gene was constructed. Lead RNA and Cas9 Mrna were introduced into the fertilized eggs of C57BL/6JGpt mice through microinjection to mediate the TRAF2 gene mutation in mice. The mouse tail protein was extracted and the genotype of the F0 generation was determined by PCR and Western blot. TRAF2+/- mice were successfully obtained. F0 generation mice were backcrossed with C57BL/6JGpt wild-type mice to obtain stable TRAF2+/- mice for propagation and subsequent experiments. The body weight of TRAF2+/- mice was detected; Western blot was used to detect the expression of TRAF2 in the spleen, liver and kidney tissues of TRAF2+/- mice. The development of spleen, liver and kidney tissues in TRAF2+/- mice was detected by HE staining. Results : PCR identification using specific primers demonstrated that TRAF2+/- mice exhibited a target band at 679 bp. Western blot analysis results indicated that, compared with the WT group, the expression of TRAF2 in the tail protein of TRAF2+/- mice was significantly reduced(P+/- mice had a lower body weight compared to their littermate WT mice(P+/- mice was decreased(P+/- mice and WT mice. Conclusion The successful construction of TRAF2+/- mice has provided an important animal model for exploring the role of TRAF2 in developmental regulation, revealing the mechanism of inflammatory immune diseases mediated by abnormal TNF-α-TRAF2 signaling, and screening related drug targets.

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.

BACKGROUND:Compensatory changes in sagittal parameters of the cervicothoracic spine after orthopedic surgery in patients with lumbar degenerative scoliosis and their intrinsic relationship,as well as the impact of these changes on quality of life,are still lacking.OBJECTIVE:To evaluate the compensatory alignment of cervical and thoracic vertebrae after correction of lumbar degenerative scoliosis.METHODS:103 patients who underwent surgical correction of lumbar degenerative scoliosis were included in this study.Patients'demographic characteristics and spinal sagittal parameters were assessed,and prediction equations between changes in cervical sagittal parameters and lumbar deformity correction were attempted.Simultaneously,the SRS-22 scale was used to assess the quality of life of patients and to explore the relationship between the compensatory changes of the cervical and thoracic spine after correction and the patients'health-related quality of life.RESULTS AND CONCLUSION:(1)At 3 months and 2 years after surgery,all indicators of the cervical spine and thoracic spine were significantly improved compared with those before surgery(P＜0.05),but there was no significant change at 3 months after surgery compared with 2 years after surgery(P＞0.05).At 3 months and 2 years after surgery,the lumbar spine parameters including lumbar lordosis,C7-S1 sagittal vertical axis,and pelvic incident-lumbar lordosis had significant changes compared with those before surgery(P＜0.05),but the change was not significant at 3 months after surgery compared with 2 years after surgery(P＞0.05).(2)Correlation analysis showed that the lumbar lordosis was highly correlated with the C3-C7 cervical lordosis,C1-C7 cervical lordosis,C2-7 sagittal vertical axis,thoracic inlet angle,and C7-S1 sagittal vertical axis(|r|≥ 0.5,P＜0.000 1).The lumbar lordosis was correlated with the thoracic kyphosis(r=-0.280).(3)Two prediction formulas were established for compensatory changes in cervical spine:cervical lordosis=0.524x,lumbar lordosis=-6.612,C2-7 sagittal vertical axis=-0.263x,and lumbar lordosis=-5.436(P＜0.05,R2＞0.6).(4)When postoperative C2-7 sagittal vertical axis was between 14.4 and 26.8 mm;cervical lordosis was between 9° and 41°,lumbar lordosis was between 42.7° and 68.7°,and sagittal vertical axis was between-40 and 40 mm,patients had better quality of life recovery.(5)It is indicated that significant compensatory changes in the sagittal plane of the cervical spine can be observed after correction of lumbar degenerative scoliosis.We found that each 1° increase in lumbar lordosis was associated with a corresponding increase of about 0.5° in cervical lordosis and a corresponding decrease of about 0.3 mm in the vertical axis of the C2-7 sagittal plane.Patient satisfaction was higher if compensatory changes were closer to normal sagittal plane.

BACKGROUND:Compensatory changes in sagittal parameters of the cervicothoracic spine after orthopedic surgery in patients with lumbar degenerative scoliosis and their intrinsic relationship,as well as the impact of these changes on quality of life,are still lacking.OBJECTIVE:To evaluate the compensatory alignment of cervical and thoracic vertebrae after correction of lumbar degenerative scoliosis.METHODS:103 patients who underwent surgical correction of lumbar degenerative scoliosis were included in this study.Patients'demographic characteristics and spinal sagittal parameters were assessed,and prediction equations between changes in cervical sagittal parameters and lumbar deformity correction were attempted.Simultaneously,the SRS-22 scale was used to assess the quality of life of patients and to explore the relationship between the compensatory changes of the cervical and thoracic spine after correction and the patients'health-related quality of life.RESULTS AND CONCLUSION:(1)At 3 months and 2 years after surgery,all indicators of the cervical spine and thoracic spine were significantly improved compared with those before surgery(P＜0.05),but there was no significant change at 3 months after surgery compared with 2 years after surgery(P＞0.05).At 3 months and 2 years after surgery,the lumbar spine parameters including lumbar lordosis,C7-S1 sagittal vertical axis,and pelvic incident-lumbar lordosis had significant changes compared with those before surgery(P＜0.05),but the change was not significant at 3 months after surgery compared with 2 years after surgery(P＞0.05).(2)Correlation analysis showed that the lumbar lordosis was highly correlated with the C3-C7 cervical lordosis,C1-C7 cervical lordosis,C2-7 sagittal vertical axis,thoracic inlet angle,and C7-S1 sagittal vertical axis(|r|≥ 0.5,P＜0.000 1).The lumbar lordosis was correlated with the thoracic kyphosis(r=-0.280).(3)Two prediction formulas were established for compensatory changes in cervical spine:cervical lordosis=0.524x,lumbar lordosis=-6.612,C2-7 sagittal vertical axis=-0.263x,and lumbar lordosis=-5.436(P＜0.05,R2＞0.6).(4)When postoperative C2-7 sagittal vertical axis was between 14.4 and 26.8 mm;cervical lordosis was between 9° and 41°,lumbar lordosis was between 42.7° and 68.7°,and sagittal vertical axis was between-40 and 40 mm,patients had better quality of life recovery.(5)It is indicated that significant compensatory changes in the sagittal plane of the cervical spine can be observed after correction of lumbar degenerative scoliosis.We found that each 1° increase in lumbar lordosis was associated with a corresponding increase of about 0.5° in cervical lordosis and a corresponding decrease of about 0.3 mm in the vertical axis of the C2-7 sagittal plane.Patient satisfaction was higher if compensatory changes were closer to normal sagittal plane.

Objective:To investigate the association between plasma anti-Müllerian hormone(AMH) levels and ischemic stroke.Methods:In this case-control study, 93 ischemic stroke patients were randomly selected as the case group from a study on the prevention and treatment of metabolic syndrome, which was conducted in 2018-2019 in Changshu, Jiangsu Province, while 372 nonischemic stroke patients were selected as the control group according to the principle of 1∶4 matching.An enzyme-linked immunosorbent assay was used to measure plasma AMH levels.The conditional logistic regression model and restricted cubic spline were used to analyze the relationship between AMH levels and ischemic stroke.Results:A total of 465 subjects with an average age of (68.7±7.4)years were included in this study, of whom 215(46.2%)were men and 250(53.8%)were women.According to our conditional Logistic regression analysis, the risk of ischemic stroke was reduced by 44% for every unit increase in the log-AMH level( OR=0.56, 95% CI: 0.37-0.85)in the overall population after multivariate adjustment.Compared with the tertile with the lowest AMH level, the risk of ischemic stroke in the tertile with the highest AMH level decreased significantly( OR=0.37, 95% CI: 0.19-0.69). When subgrouped by sex, the tertiles with the highest AMH levels were associated with a 66% lower risk of ischemic stroke in men( OR=0.34, 95% CI: 0.13-0.88)and a 64% lower risk of ischemic stroke in women( OR=0.36, 95% CI: 0.15-0.87), compared with the tertiles with the lowest AMH levels.The results of restricted cubic spline analysis showed that there was a linear dose-response relationship between plasma AMH levels and ischemic stroke both in the general population and in male or female population( Pvalues for linear trends were 0.0002, 0.008 and 0.007, respectively). Conclusions:Higher plasma AMH levels decrease the risk of ischemic stroke with a dose-response pattern.

With the rapid development of nanotechnology, the research and development of nanomedicine has become one of the current development directions of drug innovation. The pharmacokinetic characteristics of nanomedicine are significantly different from general drugs because of the scale effect based on nanostructures, and pharmacokinetics studies of nanomedicine may be different from the general drugs. This article focuses on the research strategies and considerations on non-clinical pharmacokinetics of nanomedicine, including test agents, in vivo/in vitro assays, biological sample analysis, data evaluation and analysis etc., providing references for developers.

Tree shrew is a novel and high-quality experimental animal model. In this study, the real-time polymerase chain reaction methods were established to detect infection-related cytokines interleukin-6 (IL-6), IL-8, IL-10, IL-17A, interferon-γ (IFN-γ) and housekeeping gene glyceraldehyde-phosphate dehydrogenase ( ) of tree shrew. The results indicated that the establised methods had good specificity. The high point of the linear range of these reagents reached 1 × 10 copies, and the low points ranged from 10 copies (IL-6, IL-17A), 100 copies (IL-10, ) to 1 000 copies (IL-8, IFN-γ). In this interval, the linear correlation coefficient of each reagent was greater than 0.99. The lowest detectable values of IL-6, IL-8, IL-10, IL-17A, IFN-γ and were 8, 8, 4, 8, 128 and 4 copies, respectively. The results showed that the established detection methods had good specificity, sensitivity and wide linear range. The methods were suitable for detection of multiple concentration range samples, and could be used for the subsequent studies of tree shrew cytokines.
Animals ; Cytokines ; analysis ; Real-Time Polymerase Chain Reaction ; Shrews

Objective To investigate the association between ten single nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptors and pulse pressure (PP) as well as the relationships between gene-gene interaction between PPARα/δ/γ genes and PP.Methods A total of 820 subjects,with 550 females and 270 males,were recruited from a cohort study of “Prevention of Metabolic Syndrome and Multi-metabolic Disorders in Jiangsu Province of China Study (PMMJS)”.Ten SNPs of PPARα/δ/γ genes were selected.GMDR software (version 1.0.1) was used to evaluate the gene-gene interactions among PPARs SNPs associated with PP.Results The mean levels of PP in people with mutant genotype of rs1805192 in PPARγ genes (PA+AA) showed a significant increase by 1.341 mmHg (95％CI:0.431-2.252 mmHg) when compared to the persons with wild genotype (PP).In the subgroup of subjects with more than 30 mmHg levels of PP,a six-locus model comprised rs135539 of PPARα,rs2016520 of PPARδ,rs10865710,rs1805192,rs709158 and rs3856806 of PPARγshowed a highest level of prediction accuracy (0.577) and displayed a better cross-validation consistency (10/10).In the subgroup of subjects with less than 40 mmHg levels of PP,a two-locus model was statistically associated with PP with 0.628 of prediction accuracy and 10/10 of cross-validation consistency.Conclusion PPARγrs1805192 was associated with the occurrence of PP.Gene-gene interactions among rs135539 of PPARα,rs2016520 of PPARδ,rs10865710,rs1805192,rs709158 and rs3856806 of PPARγ were all significantly related to PP.

OBJECTIVETo investigate the therapeutical effects of visfatin and metformin on insulin resistance and reproductive endocrine disorder in rats with polycystic ovary syndrome (PCOS).

METHODSForty female Wistar rats were divided into 4 equal groups, and in groups A, B and C, the rats were injected subcutaneously with dehydroepiandrosterone (DHEA) for PCOS modeling, with group D as the blank control injected with soybean oil. Vaginal smears and serological testing were taken to assess the modeling. After the modeling, the rats in group A received 10 µg reorganized visfatin injection and those in group B were treated with metformin (14 mg/100 g) on a daily basis for 15 days. Serum levels of T, LH, FSH, FINS and blood glucose levels during OGTT were measured before and after the treatments, and HOMA-IR and LH to FSH ratio were calculated. The ovaries were then dissected for pathological examination.

RESULTSIn groups A and B, FINS, FPG, T, HOMA-IR and blood glucose levels during OGTT were significantly decreased after the treatments (P<0.05), which resulted in recovery of regular menses in 8 (80%) rats in group A and 7 (77.8%) rats in group B with the development of normal follicles. Visfatin and metformin produced equivalent therapeutic effects in improving the insulin resistance and hyperandrogenism in PCOS rats.

CONCLUSIONVisfatin and metformin have equivalent therapeutic effects in improving insulin resistance and hyperandrogenism and in promoting the recovery of regular menses and development of normal follicles in PCOS rats.

Animals ; Female ; Humans ; Insulin Resistance ; Metformin ; pharmacology ; Nicotinamide Phosphoribosyltransferase ; pharmacology ; Polycystic Ovary Syndrome ; complications ; drug therapy ; Rats ; Rats, Wistar

Objective To explore the correlation between the amount of residual undifferentiated embryonic stem cells (ESCs) in embryoid bodies and its tumorigenicity.Methods Mouse R1 ESCs were cultured in suspension to form embryoid bodies (EBs).Ten days later,EBs were digested into single cells and then re-plated in standard ESCs culture condition.The residual undifferentiated embryonic stem cells surface maker SSEA-1 was examined by flow cytometry in EBs.The morphology of residual undifferentiated cells in EBs were observed,meanwhile the surface marker SSEA-1 was examined by immunofluorescent staining.EBs were digested into single cells and grouped into 104,105,106,2×106,and then injected into limb muscle of nude mice.The correlation of the amount of cells and its tumorigenicity was observed.Results Residual undifferentiated ESCs were observed after EBs differentiated for 10 days,which displayed clonal morphology and expressed undifferentiated ceil markers of ESCs,such as SSEA-1.The expression rate of undifferentiated cells surface marker SSEA-1 was (13.5±0.75)％ in EBs differentiated for 10 days.Only two millions single cells harvested from EBs were able to form teratoma after being injected into muscle of nude mice for 6 weeks.Mature endoderm,mesoderm and ectoderm tissues could be found in teratoma.No teratoma formed in other groups.Conclusion A certain amount residual undifferentiated ESCs still exist after differentiation of ESCs into EBs.About 2.7× 105 undifferentiated cells are able to form teratoma by iniecting into muscle of nude mice.