In vivo and in vitro procedures

One day after model establishment, total RNA was extracted from brain tissue of each group using the Trizol method and TNF-α and IL-6 mRNAs were detected. At 3, 12, 24 and 48 hours after model establishment, total RNA was extracted in each group, and let-7a and MKP1 mRNAs were detected. To determine the effects of let-7a on MKP1 mRNA levels in PC12 cells, cells were collected after transfection with miRNAs, and total RNA was extracted using the Trizol method. cDNA was synthesized using EasyScript First-Strand cDNA Synthesis SuperMix. 2.5 μL of cDNA and 1 μL of specific primer were added to TransStart™ SYBR Green qPCR Supermix. qPCR was conducted with a ABI 7500 PCR system (ABI, Bedford, MA, USA). 5S ribosomal RNA served as an internal reference for miRNA, and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA was used as the internal reference for mRNA. The concentrations of products were quantified and results are expressed as 2^–ΔΔCt (Song et al., 2015b).

Primer information is as follows:

Total protein was extracted from cells or brain tissue using a radio-immune precipitation assay (Cell Signaling Technology, Boston, MA, USA). Total protein concentrations were measured using the bicinchoninic acid assay. Twenty micrograms of total protein of each sample were mixed with loading buffer. After denaturation in boiling water for 5 minutes, each sample underwent sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% discontinuous gels (110 V; 90 minutes), and was then wet transferred onto a membrane (200 mA; 60–90 minutes). Membranes were blocked with skimmed milk for 2 hours, washed six times in Tris-buffered saline containing Tween-20 (10 minutes each wash), and then incubated with rabbit anti-rat caspase 3, MKP1, p-p38 MAPK, p-JNK or GAPDH IgG polyclonal antibodies (1:1,000; Abcam, Cambridge, MA, USA) at 4°C overnight. On the next day, membranes were rinsed six times in Tris-buffered saline containing Tween-20 (10 minutes each wash), incubated with horseradish peroxidase-labeled goat anti-rabbit polyclonal antibody (1:5,000; Abcam) at room temperature for 2 hours, followed by enhanced chemiluminescence. The ratio of the target protein gray value to that of GAPDH was calculated.