P jnk

The RAW264.7 cells (TCM13) were purchased from the American Type Culture Collection (Rockville, MD, USA). LPS was obtained from Beijing Solarbio Co., Ltd. (Beijing, China). CCK-8 kit was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (Jiangsu, China). NO was obtained from Jiancheng Biotechnology Co., Ltd. (Nanjing, China). TNF-α, IL-6 and PGE₂ enzyme linked immunosorbent assay (ELISA) kits were provided by Shanghai Youchu Trading Co., Ltd. (Shanghai, China). Nuclear transcription factor E2 related factor 2 (Nrf2), p65 and β-tubulin antibodies were obtained from Proteintech Group, Inc. (USA). P-p65 and p-p38antibodies were obtained from Affinity Biosciences (USA). ERK1/2, p-ERK1/2, p38, p-JNK, JNK antibodies and DCFH-DA kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). β-actin, heme oxygenase-1 (HO-1) and IgG antibodies were purchased from Boster Biological Engineering Co., Ltd. (Wuhan, China). GAPDH, iNOS and COX-2 primers were obtained from Servicebio Biotechnology Co., Ltd. (Wuhan, China), etc.

The total protein was extracted from the corpus callosum of rats for western blot analysis. Briefly, rats were deeply anesthetized with pentobarbital and perfused transcardially with 4°C saline (50 ml). The corpus callosum was rapidly separated and removed on ice. After adding the lysis buffer, the brain tissue was fragmented using a tissue grinder. Protein concentrations of all extracted samples were measured using Bio-Rad Protein Assay (BioRad, Hercules, CA, United States) and bovine serum albumin (BSA) standards. A 30 μg protein sample was loaded and separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then incubated overnight at 4°C with the following primary antibody: ERK (1:1,000, Cell Signaling Technology, Boston, MA, United States), P-ERK (1:1,000, Beyotime, China), JNK (1:1,000, Cell Signaling Technology, Boston, MA, United States), P-JNK (1:1,000, Beyotime, China), P38 (1:1,000, Cell Signaling Technology, Boston, MA, United States), P-P38 (1:1,000, Beyotime, China) and GAPDH (1:1,000, Servicebio, Wuhan, China). Next, the membrane was washed and incubated with horse-radish peroxidase-labeled goat anti-rabbit and goat anti-mouse secondary antibody (1:10,000, Boster, China) for 1 h at room temperature. Finally, the enhanced chemiluminescence system was used to observe protein bands.

Tetrahydrocurcumin was purchased from Yuanye Biotechnology Co. Ltd (Shanghai,China). E-cadherin, ZEB, Snail, Twist, HIF-1α, p-mTOR, mTOR, p-ERK, ERK, GAPDH and β-actin antibodies were purchased from Santa cruz Biotechnology (Santa cruz, CA, USA). Vimentin, N-cadherin, VEGF, MMP-2, MMP-9, LaminA/C, Atg5, Atg7 and Beclin-1 antibodies were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). LY294002, SB203580, Rapamycin, β-catenin, p-Akt, Akt, p-JNK, JNK, p-p38, p38, IκB-α, p65, Hsp90 and GSK-3β antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). Antibody against LC3B was purchased from Abcam (Cambridge, UK). All other reagents were from common commercial sources.