miR-20a-5p inhibits proliferation of lung cancer A549 cells by down-regulating HOXB13 / 南方医科大学学报

OBJECTIVE@#To investigate the molecular mechanism by which miR-20a-5p regulates HOXB13 gene expression and inhibits lung cancer cell proliferation.@*METHODS@#The expression levels of HOXB13 mRNA and protein in lung cancer A549 cells transfected with HOXB13 overexpression plasmid or HOXB13 siRNA were detected with real-time fluorescence quantitative PCR (qRT-PCR) and Western blotting. CCK-8 and EdU assays were used to examine the effect of modulation of HOXB13 expression on cell proliferation. We screened possible binding miRNAs of HOXB13 by bioinformatics analysis. In A549 cells transfected with miR-20a-5p mimic or miR-20a-5p inhibitor, the expression level of miR-20a-5p was detected by qRT-PCR and the protein expression of HOXB13 was determined with Western blotting. CCK-8 and EdU assays were used to assess the effect of miR-20a-5p overexpression on the proliferation of A549 cells. miR-20a-5p mimic and HOXB13 overexpression plasmids were co-transfected into A549 cells, and the changes in cell proliferation were evaluated with CCK-8 and EdU assays.@*RESULTS@#HOXB13 overexpression obviously promoted the proliferation of A549 cells (P < 0.05). miR-20a-5p was identified as the potential binding miRNA of HOXB13. Overexpression of miR-20a-5p in A549 cells significantly decreased the expression of HOXB13 protein (P < 0.05), while interference of miR-20a-5p obviously increased HOXB13 expression (P < 0.05). The results of cell proliferation experiment showed that miR-20a-5p and HOXB13 had opposite effects on cell proliferation, and the cells overexpressing both miR-20a-5p and HOXB13 showed a lower proliferation activity than the cells overexpressing HOXB13 but higher than the cells overexpressing miR-20a-5p alone (P < 0.05).@*CONCLUSION@#miR-20a-5p inhibits proliferation of lung cancer cells by down-regulating the expression of HOXB13.

Primary culture of neurons under low density conditions by immunomagnetic beads method combined with conditioned medium / 中国药理学与毒理学杂志

OBJECTIVE To establish a primary culture of neurons under low density conditions by immunomagnetic beads (1MB) based cell separation technology combined with astrocyte conditioned medium (A-CM). METHODS The cerebral hemispheres of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension for primary culture. After 10 d of culture, the microglia and other irrelevant cells were removed by constant temperature oscillation, while the purity of astrocytes was identified by immunofluorescence staining with anti-glial fibrillary acidic protein (GFAP) antibody. A-CM was prepared using cell cultures that met purity requirements. The cerebral cortex and hippocampus of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension. High-purity neurons were obtained by immunomagnetic beads based cell separation technology, and A-CM was applied to primary culture of neurons under low density conditions. Morphological parameters of neurons during culture were observed, and neurons were identified by immunofluorescence staining with anti-160 ku neurofilament antibody. RESULTS The purity of astrocytes was over 95% by cellular immunofluorescence staining identification, which met the cell purity requirements of subsequent experiments. Morphological observation and cellular immunofluorescence staining of the mouse cortex and hippocampal neurons showed that the neurons grew well under low density culture conditions with high purity, and the morphological characteristics were distinct at different developmental stages. CONCLUSION The primary culture of the mouse cerebral cortex and hippocampal neurons under low density conditions is successfully established.

Gαi and Gβγ subunits have opposite effects on dexmedetomidine-induced sedation / 中国药理学与毒理学杂志

OBJECTIVE To explore the mechanism of Gαiand Gβγsubunits on dexmedetomidine (DMED)-induced sedation.METHODS Kunming mice were randomly placed into three groups(DMED group, DMED+dbcAMP/rolipram/gallein/M119 group, dbcAMP/rolipram/gallein/M119 group) to explore the regulation of dbcAMP/rolipram/gallein/M119 on DMED-induced sedation by establishing loss of righting reflex (LORR) model. DbcAMP/rolipram was intracerebroventricular injected and gallein/M119 was intraperitoneal injected 15 min before DMED intravenous injection. In CHO-α2A-AR cells, after administration of DMED/gallein/M119, the regulation on the cAMP accumulation stimulated by Forskolin (FSK) was detected, so was the intracellular calcium ion concentration ([Ca2+]i. The levels of pERK/pCREB were detected by Western Blot to explore the key signal molecules involved in DMED-induced sedation. RESULTS The ED50of DMED-induced LORR (200.0 nmol·kg-1) was increased to 375.0 or 433.3 nmol·kg-1by pre-treatment with cAMP analog dbcAMP(50 nmol/5μl per mouse)or phosphodies-terase 4 inhibitor rolipram(100 nmol/5μl per mouse).In addition,the ED50of DMED-induced LORR was decreased to 113.6 or 136.5 nmol·kg-1when pre-treated with Gβγsubunits inhibitor M119(100 mg·kg-1) or gallein(100 mg·kg-1)respectively.Administration of dbcAMP,rolipram,gallein or M119 alone had little effect on LORR of mice.Gallein(10 μmol·L-1)significantly inhibited forskolin-stimulated cAMP accumu-lation in CHO-α2A-AR cells.Compared with Gβγsubunits inhibitors or DMED alone,[Ca2+]iand pERK1/2 significantly increased after co-administration of Gβγsubunits inhibitors with DMED.DbcAMP(5 μmol·L-1) or rolipram (5 μmol·L- 1) alone had little effect on ERK1/2 phosphorylation, but decreased DMED-induced ERK1/2 phosphorylation after co-administration with DMED. Gβγsubunit inhibitors treatment increased DMED-induced phosphorylation of CREB, whereas dbcAMP or rolipram had little effect on pCREB induced by DMED.CONCLUSION Gβγsubunits might inhibit DMED-induced sedation through cAMP and pERK1/2 pathway,which was opposite to Gαisubuint.

Mechanism and impact of a novel allosteric AMPA receptor modulator on protection against respiratory depression / 中国药理学与毒理学杂志

OBJECTIVE Respiratory depression hinders the use of anaesthetics and sedative hyp-notics.To explore the mechanism of LCX001 on protection against respiratory depression,a novel AMPA receptor modulator LCX001,synthesized by our Institute of Medicinal Chemistry,is expected to relieve suppressed respiration. METHODS LCX001 was tested to alleviate respiratory depression triggered by opioid(fentanyl and TH-030418),propofol and pentobarbital in the plethysmography recording.The acetic acid writhing and hot-plate tests were conducted to evaluate analgesic effect of LCX001.Binding assay and whole-cell recording were used to analyze the property of LCX001 on positive modulation. The function of AMPA receptors were determined by location of receptors in the membrane and state of channel opening, and both processes were impressed by AMPA receptor regulatory proteins. Ac-cording to the theory,the effect of LCX001 on the expression of stargazin was measured firstly by west-ern blotting. The variation of receptor surface location was observed by live cell imaging. The regula-tion on neuronal Ca2+and cell function was investigated intensively by Ca2+imaging to clarify mecha-nism of LCX001. RESULTS LCX001 effectively rescued and prevented opioid (fentanyl and TH-030418), propofol, and pentobarbital-induced respiratory depression by strengthening respiratory fre-quency and minute ventilation in rats. The acetic acid writhing test and hot-plate test revealed potent anti-nociceptive efficacy of LCX001,in contrast to some ampakines that did not affect analgesia. Fur-thermore,LCX001 potentiated[3H]AMPA and L-glutamate binding affinity to AMPA receptors,and facili-tated glutamate-evoked inward currents in HEK293 cells stably expressing GluA2(R).Importantly,appli-cation of LCX001 generated a significant increase in GluA2(R) surface expression in a mechanism of stargazin up-regulation,and restrained opioid-induced abnormal intracellular Ca2+load,which might par-ticipate in breathing modulation. CONCLUSION The novel pharmacological effect and potential new mechanism of LCX001 might promote ampakines to be a therapeutic option for protection against respi-ratory depression.

Stable co-expression of human kappa opioid receptor and PKAcat-EGFP in CHO cells and functional identification / 中国药理学通报

Aim To establish a cell model which stably co-ex-press human kappa opioid receptor (hKOR) and enhanced green fluorescent protein ( EGFP) labeled catalytic domain of cAMP-dependent protein kinase A(PKAcat) fusion protein (PKAcat-EGFP) in Chinese hamster ovary(CHO) cells, laying the foun-dation for the high-throughput screening of hKOR drugs and drug molecular mechanisms in vitro. Methods Hygromycin B resist-ant hKOR recombinant plasmid [ pcDNA3.1/Hygro ( + ) -hKOR] was transfected into CHO cells stably expressing PKA-cat-EGFP by a lipofectin based method. Transfected cells were selected in culture medium containing hygromycin B. The posi-tive clones were selected by PKA redistribution assay. Z’ factor was used for evaluation and validation the reliability of the cell model. PKA redistribution assay and LANCE cAMP 384 Kit were used to test the function of the receptors in selected clone. Results CHO-PKAcat-EGFP/hKOR-13 cell model exhibited stable response in PKA redistribution assay and LANCE cAMP 384 Kit. Treated with 100 nmol·L-1U-50488 for 30 min, the average value of Z’ factor was 0.596, proving the reliability of the cell model. The hKOR expression in cell model remained stable after a few generations. Conclusion The CHO-PKAcat-EGFP/hKOR-13 cell model with stable co-expression of hKOR and PKAcat-EGFP has been successfully established.

Primary investigation on heterodimerization of kappa-opioid receptor and ORL1 receptor / 药学学报

This study investigates whether kappa-opioid receptor and ORL1 receptor may interact to form a heterodimer. In immunofluorescence and co-immunoprecipitation experiments, differentially epitope-tagged receptors, colocalization and heterodimerization of kappa-opioid receptor and ORL1 receptor were used and examined in primary culturing rat neurons, Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. The results show that fluorescence of both kappa-opioid receptor and ORL1 receptor were overlapping in primary culturing hippocampal and cortical neurons. Similarly in co-expressing CHO or HEK293 cells, HA-KOR and Myc-ORL1 were almost exclusively confined to the membranes, revealing extensive colocalization. When Flag-KOR and Myc-ORL1 were co-expressing in CHO cells, heterodimerization was identified to have the ability to co-immunoprecipitate ORL1-receptors with kappa-opioid receptor and vice versa. In the current study, further evidence was provided for the direct interaction of two subtypes of opioid receptors, kappa-opioid receptor and ORL1-receptor, to form the heterodimerization. The finding represents the novel pharmacological mechanism for modulation of opioid receptor function as well as diversity of G protein-coupled receptors.

Effect of spinal glutamate transporter 1 on chronic constriction injury of sciatic nerve and morphine tolerance of rats / 药学学报

In order to investigate the role of spinal glutamate transporter 1 (GLT-1) in the neuropathic pain and morphine tolerance, rat chronic constriction injury (CCI) of sciatic nerve was performed, and the mechanical allodynia was evaluated by mechanical withdrawal threshold (MWT), the expression of GLT-1 was measured by real-time PCR and Western blotting analysis. The results showed that compared to sham group, the MWT of CCI group had decreased approximately 80%. Administration of morphine alone could develop tolerance rapidly in initial two days, and then had no significant difference with CCI group, the expression of GLT-1 was down-regulated. Ceftriaxone sodium alone could improve mechanical allodynia. Co-administration of ceftriaxone sodium with morphine attenuated morphine tolerance and up-regulated GLT-1 expression, and the MWT remained at high level after 6 days. In conclusion, change of spinal GLT-1 expression and function has close correlation with the development of neuropathic pain and morphine tolerance.