SENP1 inhibits the IH-induced apoptosis and nitric oxide production in BV2 microglial cells.

To reveal SUMOylation and the roles of Sentrin-specific proteases (SENP)s in microglial cells under Intermittent hypoxia (IH) condition would provide more intensive view of understanding the mechanisms of IH-induced central nervous system (CNS) damage. Hence, in the present study, we detected the expression levels of SENPs in microglial cells under IH and normoxia conditions via RT-PCR assay. We found that SENP1 was significantly down-regulated in cells exposure to IH. Subsequently, the effect of IH for the activation of microglia and the potential roles of SENP1 in the SENP1-overexpressing cell lines were investigated via Western blotting, RT-PCR and Griess assay. The present study demonstrated the apoptosis-inducing and activating role of IH on microglia. In addition, we revealed that the effect of IH on BV-2 including apoptosis, nitric oxide synthase (iNOS) expression and nitric oxide (NO) induction can be attenuated by SENP1 overexpression. The results of the present study are of both theoretical and therapeutic significance to explore the potential roles of SENP1 under IH condition and elucidated the mechanisms underlying microglial survival and activation.

[The transcription levels of linker for activation of T cell and its upstream regulatory factors in T cells of asthmatic patients].

OBJECTIVE: To examine the mRNA expression of the linker for activation of T cell (LAT) and its upstream regulatory factors (Syk, Lck and ZAP-70) in the peripheral blood T cells of asthmatic patients. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of LAT and its upstream regulatory factors (Syk, Lck and ZAP-70) in 20 asthmatic patients and 20 nonallergic subjects. The results of LAT transcription level were confirmed by real-time RT-PCR Results were expressed as x +/- s. Differences between groups were assessed for significance by t test. RESULTS: Compared with nonallergic subjects, a significant decrease of mRNA expression of LAT gene was observed in T cells of asthmatic patients (0.54 +/- 0.14 vs 0.72 +/- 0.17, t = 3.11, P <0.01), which was verified by real-time RT-PCR (0.0065 +/- 0.0066 vs 0.0124 +/- 0.0045, t =0.0022, P <0.01). The Lck and ZAP-70 gene transcription levels were significantly up-regulated (Lck: 0.71 +/- 0.16 vs 0.53 +/- 0.17, t = 3.18, P<0.01; ZAP-70: 1.05 +/- 0.41 vs 0.82 +/- 0.27, t = 2.10, P < 0.05). CONCLUSION: A significant decrease of mRNA expression of LAT gene in T cells of asthmatic patients may be due to the up-regulation of its upstream regulatory factors (Lck and ZAP-70). The abnormal mRNA expression of LAT, Lck and ZAP-70 genes may be involved in the pathogenesis of asthma.

Propofol attenuates intermittent hypoxia induced up-regulation of proinflammatory cytokines in microglia through inhibiting the activation of NF-Bκ/p38 MAPK signalling.

As immune sentinels of the central nervous system (CNS), microglia is pivotal cellular mediator of neuroinflammatory processes. Activation of microglia might elicit the expression of proinflammatory cytokines involved in the progression of neuroinflammatory diseases. Numerous studies have demonstrated that propofol (2,6-diisopropylphenol) has an effective anti-inflammatory property. Intermittent hypoxia (IH), as a result of obstructive sleep apnoea (OSA), could lead to neuron damage and neuroinflammation in the CNS. Here, we determined the effects of propofol on the inflammatory response in microglia during IH. The levels of nuclear factor-Bκ (NF-κB) inhibitor (IκB) and activated p38 mitogen-activated protein kinase (MAPK) exposed to IH with or without propofol treatment were detected by Western blot. The viability of cells exposed to various concentrations of propofol was monitored with MTT assay. The production and mRNA levels of tumor necrosis factor- α(TNF-α) and interleukin-6 (IL-6) were evaluated by qRT-PCR and ELISA, respectively. As results, IH exposure obviously promoted the activation of NF-κB/p38 MAPK signalling and the secretion of TNF-α and IL-6. Propofol was not toxic to microglia. Compared with the control group, propofol attenuated the IH-induced activation of NF-Bκ and p38 MAPK, which accompanied with reduction of proinflammatory cytokine secretion. These data suggested that propofol down-regulated the IH-induced secretion of proinflammatory cytokine, and inhibit inflammatory responses in microglia, and might be involved in attenuation of the p38 MAPK and NF-κB signalling pathways. Overall, propofol could contribute to alleviating IH-induced CNS diseases in patients by inhibiting p38 MAPK and NF-κB mediated inflammation in microglia..

Linker for activation of T cells contributes to airway inflammation in an asthmatic mouse model.

BACKGROUND: Allergic asthma is associated with airway inflammation and hyperresponsiveness caused by dysregulated production of cytokines secreted by allergen-specific helper T-type 2 (Th2) cells. The linker for activation of T cells (LAT) is a membrane-associated adaptor protein, which has been shown to take part in regulating T cell receptor (TCR) signaling and T cell homeostasis. In this study, we established an asthmatic mouse model to examine the changes in LAT levels during allergic airway disease and the effects of LAT transgenic expression on airway inflammation. METHODS: T cells from mouse lung tissues were isolated from allergen challenged (ovalbumin (OVA)) and control mice, and the purity of these isolated T cells was examined by fluorescence-activated cell sorter (FACS). Semi-quantitative RT-PCR and Western blotting were used to detect the expression of the LAT gene and LAT protein, respectively. After an intranasally administered mixture of pCMV-HA-LAT plasmid and Lipofectamine 2000, 24 hours before and 72 hours after allergen challenge, the BALF cell count and the differential cytologies were studied. In addition, IL-4 and IFN-γ levels in the BALF were determined by ELISA, and pathological changes in lung tissues were observed. RESULTS: LAT protein and mRNA expression were decreased in lung T cells in a mouse model of allergen-induced airway disease. After intranasal administration of pCMV-HA-LAT, histopathological examination of the lungs showed that intervention with LAT overexpression prevented mice from developing airway inflammation, and the number of total cells, eosinophils, neutrophils, and lymphocytes in the BALF was reduced significantly compared with the OVA sensitized and challenged group. In addition, the Th2 cytokine IL-4 decreased, while the Th1 cytokine IFN-γ increased compared to the OVA sensitized and challenged group or the OVA sensitized group plus pCMV-HA treatment. CONCLUSION: This study demonstrates that LAT might effectively diminish Th2 cytokine responses, lung histopathological changes and lung inflammation to allergen challenge in a model of experimentally induced asthma.

Small interfering RNA-mediated knockdown of Notch1 in lung.

BACKGROUND: The immunologic response to allergens mediated by T lymphocytes is an incipient key element in the pathogenesis of asthma, and Th1/Th2 balance is regarded as the core of asthma pathogenesis. Notch is a single-pass transmembrane receptor protein that regulates differentiation, proliferation and apoptosis in a broad range of cells. It is considered that the Notch signal pathway works in every stage of T cell development and differentiation. Whether the pathway of asthma pathogenesis is related to Notch1 remains unknown. This study is aimed to investigate whether the pathway of asthma pathogenesis is related to Notch1 by examining the effect of knockdown of the Notch1 gene by small interfering RNA on T cell differentiation. METHODS: An OVA-induced asthma mouse model was established. The expression of Notch1 in the tissue and T cells of the lung from asthmatic mice was detected by RT-PCR and Western blotting. The expression of Notch1 and cytokine interleukin (IL)-4 and interferon (IFN)-gamma in activated lung T cells was detected by RT-PCR and enzyme-linked immunosorbent assay after blocking Notch1 by small interfering RNA. RESULTS: The mRNA and protein expression of Notch1 increased significantly both in the lung tissue and lung T cells of asthmatic mice (both P < 0.05). IL-4 decreased and IFN-gamma increased significantly in active lung T cells after Notch1 was blocked by Notch1-specific small interfering RNA (IL-4: (2.51 +/- 0.51) pg/ml vs 0.64 +/- 0.27) pg/ml protein; IFN-gamma: (21.72 +/- 4.24) pg/ml vs (39.79 +/- 4.09) pg/ml protein, P < 0.05). CONCLUSION: This study demonstrated that the Notch1 signal might play a role in the pathogenesis of asthma by its involvement in Th1/Th2 differentiation.