Il 17

The serum samples were collected, and the supernatant was subjected to 10 min centrifugation at 3000 rpm. The levels of infammatory factors in serum were evaluated by using ELISA kits, including IL-4 (No. 220805012R, Jiangsu Meimian Industrial Co., Ltd, Yancheng, Jiangsu, China), IL-6 (MM-0190R1, Jiangsu Meimian Industrial Co., Ltd, Yancheng, Jiangsu, China), and IL-17 (No. MM-0088R1, Jiangsu Meimian Industrial Co., Ltd, Yancheng, Jiangsu, China).

BAS the Affiliated Hospital of Hebei Engineering University provided the 3% BAS. IL-17, IL-22, IL-23 Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Jiangsu Meimian Industrial Co., Ltd. IL-17, IL-22, and IL-23 rabbit anti-mouse polyclonal antibodies were purchased from Beijing Biosynthesis Biotechnology Co., Ltd.

Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify levels of netrin-1 (MEIMIAN, China), IL-17 (MEIMIAN, China) and IL-10 (RayBiotech, United States) in duplicate wells containing serum and CSF samples according to the manufacturers’ protocols. Blood was collected via a right ventricular puncture and about 1–2 ml serum per rat was obtained following centrifugation at 3000 rpm for 5 min. Approximately 100 μl of CSF was collected from each rat via foramen magnum puncture. Briefly, for detection of IL-10, samples were incubated for 2.5 h in a microtiter plate pre-coated with monoclonal antibody, and then for 60 min with biotinylated antibody at room temperature, followed by the addition of streptavidin solution for 45 min and one-step substrate reagent for 30 min as performed in the dark at 37°C. For netrin-1 and IL-17, each sample and its control were incubated in the 96-well microtiter plate for 0.5 h at 37°C, followed by HRP-conjugated reagent and chromogen solution at 37°C. All optical densities were measured at 450 nm. Each sample was detected in duplicated wells and the results were averaged.