Bidirectional regulation of Pam3CSK4?induced inflammatory response by ATP?binding cassette transporter A1 knockdown in mouse mononuclear macrophages in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the regulatory effect of ATP?binding cassette transporter A1 (ABCA1) knockdown on inflammatory response induced by Pam3CSK4 in mouse mononuclear macrophage RAW264.7 cell line.</p><p><b>METHODS</b>A mouse mononuclear macrophage RAW264.7 cell line with stable ABCA1 knockdown was constructed and stimulated with Toll?like receptor 2 (TLR2) ligand Pam3CSK4, and the changes in the transcriptional levels of the proinflammatory and anti-inflammatory cytokines were analyzed in this cell model.</p><p><b>RESULTS</b>In RAW264.7 cells, ABCA1 knockdown significantly up-regulated Pam3CSK4 stimulation?induced expressions of IL?1β, TNF?α and IL?6 and also enhanced the expression of transcription factor cAMP?dependent transcription factor 3 (ATF3) without obviously affecting the expressions of the transcription factors ATF1, ATF2, ATF4 or ATF5.</p><p><b>CONCLUSION</b>ABCA1 knockdown in macrophages may have both proinflammatory and anti?inflammatory effects. ABCA1 knockdown up?regulates the transcription of ATF3 possibly through a mechanism that is different from that for the other members of the ATF protein family.</p>

The pharmacological mechanism of gastrodin on calcitonin gene-related peptide of cultured rat trigeminal ganglion / 药学学报

The Chinese herbal medicine Tianma (Gastrodia elata) has been used for treating and preventing primary headache over thousands of years, but the exact pharmacological mechanism of the main bioactive ingredient gastrodin remains unclear. In present study, the effects of gastrodin on calcitonin gene-related peptide (CGRP) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) expression were observed in rat trigeminal ganglion (TG) after in vitro organ culture to explore the underlying intracellular mechanism of gastrodin on primary vascular-associated headache. CGRP-immunoreactivity (CGRP-ir) positive neurons count, positive area, mean optical density and integrated optical density by means of immunohistochemistry stain were compared at different concentrations of gastrodin, which was separately co-incubated with DMEM in SD rat TG for 24 hours. Only at 5 or 10 mmol L(-1) concentration, gastrodin demonstrated significantly concentration-dependent reduction of CGRP-ir (+) expression and its action closed to 1.2 mmol L(-1) sumatriptan succinate. While at 2.5, 20, and 40 mmol L(-1) concentration, gastrodin did not show remarkable effects on CGRP-ir (+) expression. The optimal concentration of gastrodin (5 and 10 mmol L(-1)) similarly inhibited CGRP-mRNA expression level separately compared with 1.2 mmol L(-1) sumatriptan succinate and 10 micromol L(-1) flunarizine hydrochloride, which was quantitatively analyzed by real-time PCR (RT-PCR). pERK1/2 level was examined by Western blotting after co-cultured with optimal concentration of gastrodin and effective specific ERK1/2 pathway inhibitors PD98059, U0126. The result indicated that gastrodin significantly reduced pERK1/2 protein actions similarly to ERK1/2 pathway specific blockade. It suggests ERK1/2 signaling transduction pathway may be involved in gastrodin intracellular mechanism. This study indicates gastrodin (5 and 10 mmol L(-1)) can remarkably reduce CGRP-ir (+) neuron, CGRP-mRNA and pERK1/2 expression level in cultured rat TG, with its actions similar to the effective concentration of sumatriptan succinate, flunarizine hydrochloride and specific ERK1/2 pathway blocker. The intracellular signaling transduction ERK1/2 pathway may be involved in the gastrodin reducing CGRP up-regulation in rat TG after organ culture.

Expression of protein arginine N-methyltransferases in E3 rat models of acute asthma / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the expression of protein arginine N-methyltransferase (PRMT) genes in the lung and spleen of E3 rats with acute asthma.</p><p><b>METHODS</b>E3 rats with ovalbumin-induced pulmonary inflammation were divided into two groups (n=10), and the validity of the acute asthma model was evaluated by histological observation with HE and PAS staining and by measurement of NO production. Semi-quantitative RT-PCR was employed to detect the expressions of PRMT1-PRMT6 genes in the lung and spleen tissues of the rats.</p><p><b>RESULTS</b>In the lung tissue of the asthmatic rats, the gene expressions of PRMT1 (P<0.01), PRMT2 (P<0.01), PRMT3 (P<0.05) and PRMT5 (P<0.05) were significantly increased, but the expression of PRMT4 gene (P<0.05) was significantly decreased as compared with those in the control tissue. In the spleen tissue of the asthmatic rats, the expressions of PRMT2 (P<0.05) and PRMT5 genes (P<0.05) showed a significant increase as compared with those in the control rat tissue.</p><p><b>CONCLUSION</b>The gene expressions of PRMTs vary significantly between asthmatic rats and control rats, suggesting that PRMTs play an important role in the post-translational modification process of asthma-related genes.</p>

Identification of differentially expressed genes in rat asthma model by suppression subtractive hybridization technology / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To identify differentially expressed genes related to asthma by using a rat model.</p><p><b>METHODS</b>Total RNA extracted from the asthmatic rats was taken as the tester and the total RNA from the control rats as the driver. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGEM-T Easy vector to establish the subtractive library. The library was amplified through E.coli transformation and positive clones of the transformants were screened. The white clones in selective medium from cDNA library were isolated and digested by EcoR I restriction endonuclease. Thirty-six positive clones were chosen randomly and sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank (NCBI).</p><p><b>RESULTS</b>There were more than 300 white clones in the cDNA library. The clones were sequenced and similarity search (http://www.ncbi.hlm.nih.gov/BLAST) revealed 4 known genes, 2 ESTs without homologous genes and 3 potential new gene fragments.</p><p><b>CONCLUSION</b>The forward-subtracted cDNA library for differentially expressed in the lung of asthmatic rats has been successfully constructed and the interesting candidate genes related to asthma have been identified.</p>

KCNE2 protein S98 phosphorylation in heart of old SHR rats detected by point mutagenesis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the phosphorylation of KCNE2 protein in heart of old SHR rats.</p><p><b>METHODS</b>The membrane proteins from ventricular myocardium of old SHR were extracted, treated with or without alkaline phosphatase and tested binding with Ab2 (an anti-KCNE2 polyclonal antibody) by Western blot. A KCNE2 fusion protein with c-myc was obtained from in vitro translation system and treated with or without alkaline phosphatase. A series of mono- and double-point mutated fusion KCNE2 proteins with c-myc were obtained from an in vitro translation system, and Western blots with Ab2 or anti-myc antibody were performed.</p><p><b>RESULTS</b>After alkaline phosphatase treatment, Ab2 significantly attenuated its binding with KCNE2. In vitro translation system confirmed that after alkaline phosphatase treatment, Ab2 weakened binding ability to KCNE2 while binding to c-myc was not changed. Point mutation experiments showed that serine residue in position 98 of KCNE2 proteins might be phosphorylated.</p><p><b>CONCLUSION</b>KCNE2 protein in heart of old SHR rats is phosphorylated and this phosphorylation takes place in serine residue of position 98.</p>