Effects of SOCS 1/3 gene silencing on the expression of C/EBPα and PPARγ during differentiation and maturation of rat preadipocytes.

BACKGROUND: Suppressor of cytokine signaling-1 and -3 (SOCS-1 and SOCS-3) are two important negative regulators in the insulin-signaling pathway, and their overexpression may aggravate insulin resistance. Subjects with insulin resistance are often obese and have increased expressions of SOCS-1 and SOCS-3. We speculated that SOCS-1 and SOCS-3 may be involved in abnormal deposition of adipose tissues during insulin resistance. METHODS: A catch-up growth intrauterine growth retardation (CG-IUGR) rat model with insulin resistance was established; mRNA and protein expression of SOCS-1, SOCS-3, the CCAAT/enhancer binding protein (C/EBPα), and peroxisome proliferator-activated receptor (PPARγ) in adipose tissue were measured by real-time PCR and western blot; plasmids carrying small hairpin RNAs (shRNAs) targeting the SOCS-1 and SOCS-3 genes were constructed and transfected into preadipocytes, which were then induced to mature. At 72 h after differentiation was induced, the expressions of C/EBPα and PPARγ, two important molecules promoting the differentiation of preadipocytes, were detected. RESULTS: Expressions of SOCS-1, SOCS-3, C/EBPα, and PPARγ were markedly increased in adipose tissues of CG-IUGR rats, whereas the expressions of C/EBPα and PPARγ were significantly reduced after gene silencing of SOCS-1 or SOCS-3 in adipocytes. CONCLUSION: Overexpression of SOCS-1 and SOCS-3 may enhance the expression of C/EBPα and PPARγ, resulting in abnormal deposition of adipose tissues during insulin resistance.

Diagnosis of intellectual disability/global developmental delay via genetic analysis in a central region of China.

BACKGROUND: Advanced technology has become a valuable tool in etiological studies of intellectual disability/global developmental delay (ID/GDD). The present study investigated the role of genetic analysis to confirm the etiology in ID/GDD patients where the cause of the disease was uncertain in central China. METHODS: We evaluated 1051 ID/GDD children aged 6 months to 18 years from March 2009 to April 2017. Data concerning basic clinical manifestations were collected, and the method of etiology confirmation was recorded. Genome-wide copy number variations (CNVs) detection and high-throughput sequencing of exons in the targeted regions was performed to identify genetically-based etiologies. We compared the incidence of different methods used to confirm ID/GDD etiology among groups with differing degrees of ID/GDD using the Chi-square or Fisher exact probability test. RESULTS: We recruited 1051 children with mild (367, 34.9%), moderate (301, 28.6%), severe (310, 29.5%), and profoundly severe (73, 6.9%) ID/GDD. The main causes of ID/GDD in the children assessed were perinatal factors, such as acquired brain injury, as well as single gene imbalance and chromosomal gene mutation. We identified karyotype and/or CNVs variation in 46/96 (47.9%) of cases in severe ID/GDD patients, which was significantly higher than those with mild and moderate ID/GDD of 34/96 (35.4%) and 15/96 (15.6%), respectively. A total of 331/536 (61.8%) patients with clear etiology have undergone genetic analysis while 262/515 (50.9%) patients with unclear etiology have undergone genetic analysis (χ = 12.645, P < 0.001). Gene structure variation via karyotype analysis and CNV detection increased the proportion of children with confirmed etiology from 51.0% to 56.3%, and second-generation high-throughput sequencing dramatically increased this to 78.9%. Ten novel mutations were detected, recessive mutations in X-linked genes (ATPase copper transporting alpha and bromodomain and WD repeat domain containing 3) and dominant de novo heterozygous mutations in X-linked genes (cyclin-dependent kinase like 5, protocadherin 19, IQ motif and Sec7 domain 2, and methyl-CpG binding protein 2) were reported in the study. CONCLUSIONS: The present study indicates that genetic analysis is an effective method to increase the proportion of confirmed etiology in ID/GDD children and is highly recommended, especially in ID/GDD children with uncertain etiology.

Protective effects of activated protein C on neurovascular unit in a rat model of intrauterine infection-induced neonatal white matter injury.

Activated protein C (APC), a natural anticoagulant, has been reported to exert direct vasculoprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. Intraperitoneal injection of 300 µg/kg lipopolysaccharide (LPS) was administered consecutively to pregnant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infection- induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats immediately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eosin (H&E). Immunohistochemistry was used to evaluate myelin basic protein (MBP) expression in the periventricular white matter region. Blood-brain barrier (BBB) permeability and brain water content were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 (PAR1). Typical pathological changes of white matter injury were observed in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely activated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein expression levels were both down-regulated. Our results suggested that APC exerted neuroprotective effects on multiple components of the neurovascular unit in neonatal rats with intrauterine infection- induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

Neuroprotective effects of activated protein C on intrauterine inflammation-induced neonatal white matter injury are associated with the downregulation of fibrinogen-like protein 2/fibroleukin prothrombinase and the inhibition of pro-inflammatory cytokine expression.

Maternal intrauterine inflammation or infection is an important risk factor for neonatal cerebral white matter injury (WMI) and future neurological deficits. Activated protein C (APC), a natural anticoagulant, has been shown to exhibit anti-inflammatory, anti-apoptotic, profibrinolytic and cytoprotective activities. Recent studies have demonstrated that the novel prothrombinase, fibrinogen-like protein 2 (fgl2), contributes to the pathogenesis of a number of inflammatory diseases through the generation of fibrin. Thus, we hypothesized that APC may regulate coagulant and inflammatory processes and improve brain injury in an experimental rat model of intrauterine inflammation-induced WMI. The animal model was established by the administration of an intraperitoneal injection of lipopolysaccharide (LPS) to pregnant Sprague-Dawley rats on embryonic day (E)17 and E18. APC was administered intraperitoneally 30 min after the second LPS injection. The expression of fgl2 and the pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß expression in the placentas and fetal brains was determined on E19. Nerve cell death, the brain water content and protease-activated receptor 1 (PAR1) and nuclear factor κB (NF-κB) p65 expression was detected in the fetal brains. WMI in the neonatal rat brains was evaluated by hematoxylin and eosin (H&E) staining and immunohistochemistry for myelin basic protein (MBP). The results revealed that APC markedly reduced the LPS-induced increase in fgl2 expression and fibrin deposition, as well as the production of the pro-inflammatory cytokines, TNF-α, IL-6 and IL-1ß, in the placentas and fetal brains. In addition, APC attenuated cerebral apoptosis and brain edema, downregulated PAR1 and NF-κB p65 expression in the fetal brains, and improved hypomyelination and structural disturbances in the periventricular area of the neonatal rat brains. Our observations provide evidence that APC attenuates fetal neuroinflammation and the associated secondary WMI in the developing brain by inhibiting the expression of fgl2 and pro-inflammatory mediators, suggesting that APC may be a potential therapeutic approach for intrauterine inflammation-induced neonatal brain injury.