*2.6. Cell Experiment*

Raw 264.7 cells were a gift from Professor Hu Fuliang, college of Animal Science, Zhejiang University. Raw 264.7 cells were cultured with DMEM high glucose medium containing 10% heat-inactivated fetal bovine serum and double antibodies (streptomycin (100 μg/mL) and penicillin (100 IU/mL)) at 37 ◦C Incubation was in a 5% CO2 incubator [37], to maintain the stable growth of the cells.

### 2.6.1. ECH Measurement on Cell Relative Survival Rate

Raw 264.7 cells in the logarithmic growth phase were inoculated into 96-well plates with 1 × 10<sup>5</sup> cells/mL, 100 μL per well, incubated overnight in a 5% CO2 incubator at 37 ◦C, until the cell adherence reached 70–80%. The cells were incubated with 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, or 20 μg/mL ECH for 24 h, and the culture medium was aspirated. The cells were washed with new cell culture medium for two to three times, and a blank control group was set. We added 10 μL CCK-8 reagen<sup>t</sup> and cell culture medium into each well, incubated for 3 h, and then the absorbance at 450 nm was determined [38,39].

2.6.2. Determination of the Nitric Oxide (NO) Concentration in LPS-Induced Cells Treatment with ECH

The NO concentration was measured with a Griess reagent. We referred to the method of Lee et al. and adjusted [40]. Raw 264.7 cells in the logarithmic growth phase were inoculated into 24-well plates with 1 × 10<sup>5</sup> cells/mL, 500 μL per well, incubated in a 37 ◦C, 5% CO2 incubator, until the cell adherence reached 70–80%, and we set the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group, we successively added 2.5 μg/mL and 5 μg/mL ECH for 1 h, followed by LPS (1 μg/mL) for 24 h. The cell culture fluid was collected, centrifuged at 5000 r/min for 10 min, and the supernatant was collected. We mixed 100 μL of each sample with an equal volume of Griess reagen<sup>t</sup> and added these samples to a 96-well plate, and incubated at room temperature for 10 min. We measured the absorbance at 540 nm.

2.6.3. ECH Detection of the mRNA Expression Related to Inflammation and Oxidation in LPS-Induced Cells

Raw 264.7 cells in the logarithmic growth phase were inoculated into 24-well plates with 1 × 10<sup>5</sup> cells/mL, 500 μL per well, incubated in a 37 ◦C, 5% CO2 incubator, until the cell adherence reached 70–80%, and we set the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group, we successively added 2.5 μg/mL and 5 μg/mL ECH and incubated for 1 h, and then induced with LPS (1 μg/mL) for 6 h. The cells were collected, and then the total cell RNA was extracted using a CarryHelix RNA extraction kit, and its concentration and purity were determined with a NanoDrop 2000 ultramicro spectrophotometer. Using 1 μg of extracted total RNA as a template, the PrimeScript® TM RT Master Mix reverse transcription kit was used to perform reverse transcription of the cDNA, and the product was placed in a −20 ◦C refrigerator for use. Real-time quantitative PCR was performed with a TB Green® Premix Ex TaqTM kit. The total volume of the reaction system was 10 μL: TB Green® Premix Ex TaqTM 5.0 μL, cDNA template 0.2 μL, RNase Free dH2O 4.4 μL, upstream and downstream primers 0.2 μL each, related to use. The primer sequences are shown in Table 2.



2.6.4. ECH Detection of Related Protein Expression in LPS-Induced Cells

Raw 264.7 cells in the logarithmic growth phase were inoculated in 6-well plates with 1 × 10<sup>5</sup> cells/mL, 1 mL per well, and incubated in a 37 ◦C, 5% CO2 incubator, so that the cell adherence reached 90%, and we set the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group we successively added 2.5 μg/mL and 5 μg/mL ECH and incubated for 1 h, and then induced with LPS (1 μg/mL) for 0.5 h. The collected cells were washed twice with PBS, and inhibited with *NP-40* protein lysate Cellular protein was extracted from the reagent, and the concentration of the extracted protein was measured by BCA. The protein samples of each group of cells were detected by immunoblotting. The total sample loading was 20 μg of total protein, with β-actin as a reference, through 12% SDS-PAGE gel electrophoresis, transfer, hybridization, alkaline phosphatase color development and other steps to obtain the hybridization bands of IκBα, P-IκBα, Nrf-2 and HO-1 [42].

#### 2.6.5. E ffect of ECH on LPS-Induced Nuclear Localization of p65 (NF-κB)

Raw 264.7 cells in the logarithmic growth phase were inoculated into the slide confocal small dish with 1 × 10<sup>5</sup> cells/mL, incubated in a 37 ◦C, 5% CO2 incubator overnight, and we set up the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group, we added 5 μg/mL of ECH and incubated for 1 h. Then, we induced with LPS (1 μg/mL) for 0.5 h. A methanol-acetone mixture (1:1, *v*/*v*) was used as the fixing solution for 30 min. Permeabilization was performed with 0.5% Triton X-100 for 30 min, blocked with 10% serum blocking solution for 30 min at room temperature. We added primary antibody (NF-κB-p65) (1:50 dilution) and secondary antibody goa<sup>t</sup> anti-rabbit (IgG) (1:500 dilution) and incubated for 1 h, with DAPI (4,6-diamidino-2-phenylindole) (1:2000 dilution) stained nuclear processing coverslips, observed with a laser confocal scanning microscope, and analyzed the results [42].
