Lipopolysaccharide lps o55 b5

Shanghai Yuan ye Bio-tech Co., Ltd. (Shanghai, China) supplied us with silibinin (≥98%, suspended in 0.5% carboxymethylcellulose sodium solution) and D-galactosamine (D-GalN). Lipopolysaccharide (LPS) (O55:B5), purchased from Sigma (Sigma, USA) coupled with D-GalN, was dissolved in saline.
The antibodies used (and their concentrations) were listed below: p-PI3K (CAT#4228, 1 : 1000, CST, USA), PI3K (CAT#4292, 1 : 1000), p-EGFR (CAT# ab40815, 1 : 1000), EGFR (CAT# ab52894, 1 : 1000), p-AKT (CAT# ab38449, 1 : 1000), AKT (CAT#4691, 1 : 1000), p-mTOR (CAT#5536, 1 : 1000), mTOR (CAT#2972, 1 : 1000), p-ERK1/2 (CAT#4370T, 1 : 1000, CST, USA), ERK1/2 (CAT#9102S, 1 : 1000), Nrf2 (CAT# ab137550, 1 : 1000), HO-1 (CAT# ab52947, 1 : 1000), GAPDH (CAT# 60004-1-Ig, 1 : 1000).

Male C57BL/6 mice (6–8 weeks) were housed 3–5 per cage and had free access to water and food throughout the experiment; the padding was replaced at least three times a week. The protocol complied with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and was approved by the Animal Ethics Committee of the Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University, and was reported in accordance with the ARRIVE guidelines.
The mice were randomly separated into four experimental groups: control, DEX, sepsis, and sepsis + DEX group (Figure 1). The control and DEX groups received an intraperitoneal (i.p.) injection of saline solution, and the other groups received lipopolysaccharide (LPS) (O55:B5, Sigma Aldrich, St. Louis, MO, United States) injection (i.p.) at a dose of 10 mg/kg (14 (link)–17 (link)). After 24 h, mice with signs of lethargy, piloerection, and tachypnoea were diagnosed with sepsis (6 (link)). Dexamethasone (Sigma Aldrich, St. Louis, MO, United States), 1 mg/kg, was injected intraperitoneally into mice in the DEX group and sepsis + DEX group. The control and sepsis groups were injected with saline solution (i.p.).

The role of TLRs in inducing inflammation in oligodendrocytes, in response to B. burgdorferi exposure was determined by gene silencing using small interfering RNA (siRNA) technology. Transfection complexes were generated using 2 μL HiPerfect transfection reagent (Qiagen, Valencia, CA) and 12.5–25 nM siRNA (Santa Cruz Biotechnology, Dallas, TX) in experimental medium. The complexes were incubated at room temperature for 30 min, and 200 μL of the complex was added to cells, after replacing the old medium in the 6-well plates. Immediately after the addition of transfection complexes, 800 μL of experimental medium was added to prevent drying of cell layer. After a 24-h incubation at 37 °C, 5% CO₂, B. burgdorferi (MOI 10:1) was added and further incubated for 48 h, at the end of which, supernatants were collected as before and analyzed for chemokine and cytokine expression. A non-specific control siRNA was used as negative control for all experiments. Pam3CSK4 (Imgenex, San Diego, CA; TLR2), lipopolysaccharide (LPS) O55:B5 (Sigma Aldrich, St. Louis, MO; TLR4) were used as TLR-specific positive controls when required.