Lps from escherichia coli 055 b5

All rodent experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee at the University of California, Irvine (UCI). LPS treatment: LPS (from Escherichia coli 055:B5; Sigma) was dissolved in phosphate buffered saline (PBS) at a concentration of 0.1 mg/ml and administered intraperitoneally (IP) at a dose of 0.5 mg/kg body weight. Following any treatments, mice were sacrificed and brains isolated. One-half of the brain was fixed in 4 % paraformaldehyde and the other half was snap frozen on dry ice and stored at −80 °C until analysis.

Recombinant rat macrophage-colony stimulating factor (M-CSF), interferon-γ (IFN-γ), and interleukin-4 (IL-4) were purchased from PeproTech (Rocky Hill, NJ, http://www.peprotech.com). LPS from Escherichia coli 055:B5 was obtained from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com). Antibodies included FITC anti-rat CD11b/c and PE anti-rat CD80 (BioLegend, San Diego, CA, http://www.biolegend.com), Alexa Fluor® 647 anti-rat CD163 (Bio-Rad, US, http://www.bio-rad.com/), Annexin V-Alexa Fluor647/PI Apoptosis Assay Kit (FMSAV647-100, FcMAS, Nanjing, China, http://www.fcmacs.com/), inducible nitric oxide synthase (iNOS) (R&D, MN, https://www.bio-techne.com/), and Arginase 1 (Arg-1) (CST, https://www.cellsignal.com/).
Cell culture-related reagents included fetal bovine serum (FBS) and DMEM low sugar medium (glucose content 1 g/ml; Gibco, US, http://www.thermofisher.com/cn), as well as penicillin/streptomycin and PBS (Hyclone, US, http://www.thermofisher.com/cn).

The experimental procedures and animal management in this study were approved by the Institutional Animal Care and Use Committee at the Henan Institute of Science and Technology. Forty commercial ross 308 eggs were obtained from Henan Doyoo Industrial Co., Ltd. (Hebi, China) and incubated in an electric forced-draft incubator at 37.5 ± 0.5 °C and 65% relative humidity. At the day of hatching, 30 chicks with similar body weights (BW) were randomly divided into three groups—the control group, LPS group and Betaine + LPS group—with an equal number in each group. The chickens were kept in one room at a standard temperature, moisture, light, and ventilation. Water and a corn-soybean meal diet were provided ad libitum. Betaine at a dose of 1000 mg/L was added to the drinking water of the Betaine+LPS group. After two weeks of feeding, the chickens in the LPS and Betaine+LPS groups were intraperitoneally injected with LPS from Escherichia coli 055:B5 (Sigma-Aldrich, Darmstadt, Germany) at a dose of 1 mg/kg BW. The chickens in the control group were intraperitoneally injected with the same volume of PBS. Two hours after the treatment, six chickens in each group were randomly chosen and sacrificed, and the leg muscles were collected, snap-frozen in liquid nitrogen, and stored at −70 °C.

C57Bl6/J mice were euthanized using CO₂. Both femurs were collected; the diaphysis removed and rinsed though a 40-μm cell strainer (Falcon) with ice-cold PBS w/o calcium and magnesium (Lonza). These bone marrow cells were centrifuged with 300g for 5min at 4°C, re-suspended in macrophage differentiation medium containing 500ml DMEM (Gibco), 10% FCS (PAN Biotech), 1% penicillin-streptomycin (P/S) (Bio Whittaker), and 30ng/ml M-CSF (PeproTech) and plated in an incubator at 37°C and 5% CO₂. Non-adherent cells were removed after 3 days by replacing the macrophage differentiation medium. The macrophage differentiation medium was removed after 7 days, and adherent cells were washed twice with PBS with calcium and magnesium (Lonza). Macrophage differentiation and cell culture purity was verified via flow cytometric analysis. For M1 differentiation, BMDMs were incubated 5h at 37°C and 5% CO₂ with DMEM + 10% FCS + 1% P/S with 10ng/ml IFNγ (PeproTech) and 100ng/ml LPS from Escherichia coli 055:B5 (Sigma Aldrich). For M2 differentiation, BMDMs were incubated 5h at 37°C and 5% CO₂ with DMEM + 10% FCS + 1% P/S with 20ng/ml IL-4 (PeproTech). Non-stimulated M0 macrophages only received DMEM + 10% FCS + 1% P/S for 5h at 37°C and 5% CO₂.

Oridonin (HPLC ≥ 98 %) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). The purity of Ori was determined by HPLC. The assay was performed on an EChrom2000 DAD Data System (Elite, Dalian, China). Chromatography was performed using a Hyper ODS-2 C18 column (5μm, 250×4.6 mm, Dikma Technology, California, USA). Elution was performed with acetonitrile/water (30:70), and the flow rate was 1.0 mL/min with DAD detection at 242 nm (Figure 1B). LPS (from Escherichia coli 055: B5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The indicated primary antibodies and actin were obtained from Cell Signalling Technology (Beverly, MA, USA). qPCR was carried out using the SYBR Green Plus Reagent kit (Roche Applied Science, Mannheim, Germany). All of the other chemicals and reagents were of the highest commercial grade available.