Il 10

Frozen cerebral cortex samples were firstly homogenized in cold-PBS supplied with protease inhibitors (Sigma–Aldrich, St Louis, MO, U.S.A.). The sample weight to PBS ratio was 1 g: 9 ml. To further break the cells, the samples were sonicated with an ultrasonic cell disrupter. Then the homogenates were contrifugated for 5 min at 5000 × g to get the supernatants. The concentration of the total proteins was determined by the Bradford method. The levels of IL-1, IL-1β, IL-6, IL-10, TNFα, TGFβ, and KL-6 were detected via an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Shanghai Renjie Biotech LTD). Each sample was detected in triplicate. The proteins expression levels of IL-1β, IL-6 (Abcam, U.S.A.), IL-10 (Sangon, Shanghai, China), and TGFβ (R&D Systems, U.S.A.) were also examined by Western blotting with corresponding primary antibodies and secondary antibodies. The optical densities of the immunoreactive bands were assessed using Image J software. The protein levels were normalized to Tubulin and expressed as ratios.

Total RNA was extracted with an Axyprep multisource RNA miniprep kit (Axygen, America), and cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing). Quantitative real-time PCR was performed using a SYBR Green kit (TransGen Biotech, Beijing) with a StepOnePlus real-time PCR system (ABI), and the primer sets used for MTTP, ApoA1, ApoB, ApoC2, CD31, TGFβ, TSP1, VEGFR1, IL-1β, TNFα, IL-6, IL-10, CCL2, PPARα, SIRT1, and β-actin (Sangon Biotech, Shanghai) are listed in Table 1. Relative gene expression was measured with triplicates for each sample and normalized to β-actin.