Sodium valprovate suppresses autophagy in SH-SY5Y cells activating miR-34c-5p/ATG4B signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells.@*METHODS@#Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist.@*RESULTS@#VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells.@*CONCLUSIONS@#VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.

Effect of sulindac on improving autistic behaviors in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To test the effect of sulindac on autistic behaviors in a rat model and explore the possible mechanisms.</p><p><b>METHODS</b>Autistic rat models were established by a single intraperitoneal injection of sodium valproate (VPA) at 12.5 days of pregnancy. The pregnant rats were treated with oral sulindac at a daily dose of 80 mg/kg until weaning of the newborn rats (23 days after being born), which were divided into control, VPA treatment, sulindac treatment, and VPA+ sulindac treatment groups. The social interaction and neuroethology of the newborn rats were evaluated at 35 days, and the levels of β-catenin and phosphorylated Gsk3β in the brain tissues were investigated by Western blotting.</p><p><b>RESULTS</b>Compared with the control rats, the rats treated with VPA showed lower social interaction, longer moving time in central area, and reduced standing times. Treatment with sulindac alone resulted in no obvious changes in the social interaction or neuroethology of the newborn rats, but sulindac treatment corrected VPA-induced autistic-like behaviors. Sulindac also attenuated VPA-triggered p-Gsk3β downregulation and β-catenin upregulation in the prefrontal lobe, seahorse and cerebellum.</p><p><b>CONCLUSION</b>Sulindac can improve the behaviors of autistic rats possibly by suppressing Wnt signaling pathway.</p>

The effect of inhibition of MEK/ERK pathway on the behaviors of autistic rats / 重庆医学

Objective To explore the effect of inhibitor of MEK/ERK pathway on the behaviors of autistic rats .Methods Autistic rats were made by intraperitoneal injection of sodium valproate (VPA) after pregnancy for 12 .5 days .After VPA injec-tion ,pregnant rats were treated with U0126 via oral at 400 μg/kg dose per day until weaning .Young rats were divided to 4 groups :control group ,VPA group ,U0126 group ,VPA combined U0126 group .The social interaction and behaviors of young rats were e-valuated at 35 days after bornning .The levels of MEK and phosphorylated ERK in brain tissues were investigated by Western blot . Results The autistic rat mode was prepared successfully .Compared with control rats ,the rats treated with VPA showed low the social interaction ,long moving time in central area and reducing standing times .Treatment with U0126 alone didn′t change the so-cial interaction and behaviors of young rats ,but VPA combined U0126 group could improve VPA-induced autistic-like behaviors . Western blot results show that compared with the control group ,the rats treated with VPA could enhance the prefrontal cortex of rats ,the hippocampus and cerebellum in the organization of MEK and ERK phosphorylation level ;while VPA combined U0126 group could inhibit the brain tissue of MEK and ERK phosphorylation level .Conclusion U0126 can improve the model rats of au-tism disorders behavior ,the mechanism may be related to the inhibition of MEK/ERK signaling pathway in the brain .

Cloning and expression of human laminin alpha4 LG3-4 module / 第三军医大学学报

Objective To express and detect the antigenicity of human laminin alpha4 LG3-4 module (hLN?4LG3-4) protein by gene engineering techniques. Methods The cDNA encoding hLN?4LG3-4 was amplified by RT-PCR from human placenta, then inserted into pMD-18T vector by T/A cloning and sequenced. Prokaryotic expression vector pET-28a-LG3-4 was constructed by recombinant DNA technique. The hLN?4LG3-4 fusion protein expressed in BL21(DE3)/pET system was identified by SDS-PAGE, purified by Ni-NTA resin, and assayed by Western blotting. Results The cDNA fragment of hLN?4LG3-4 was cloned successfully. While BL21 (DE3)/pET28a-LG3-4 bacterium was induced with IPTG, a new protein band with a relative molecular weight of 44 000 was shown on SDS-PAGE profile. hLN?4LG3-4 fusion protein of high purity (95%) was obtained and specific protein band was detected by Western blotting. Conclusion The hLN?4LG3-4 fusion protein was successfully expressed.