Materials and Methods
1. Animals. Male Sprague-Dawley rats (n = 25) weighing180-200 g (6-7 weeks old) were purchased from Beijing Vital   River Laboratory Animal Technology Co., Ltd. and housed at the Experimental Animal Center of Hebei University of  Chinese Medicine. All rats were acclimatized and fed for  one week prior to the experiment and housed in a controlled  environment (12 h light/dark cycle, 22 ± 2 ° C, and 55 ± 5%  relative humidity) with free access to food and water. The environment and animals in this study were SPF grade and  were never used for other research procedures. All experimental procedures were performed in strict accordance with  the Guidance Suggestions for the Care and Use of Laboratory Animals (developed by the Ministry of Science and   Technology, China). The study protocol was obtained from
the Experimental Animal Management and Ethics Committee of Hebei University of Chinese Medicine (license number: DWLL202203131), and all animal handling procedures   were performed in accordance with the Guidelines for the    Protection and Use of Laboratory Animals of Hebei University of Chinese Medicine.
2. Experimental Grouping. After 1 week of acclimatization, the 25 SD rats were randomly divided into five groups: blank  control group (control), acne model group (acne), auricular  bloodletting therapy (ABT), auricular point sticking (APS),and auricular bloodletting therapy combined with auricular  point sticking (ABPS).

Mediators of InflammationFigure 1: Experimental roadmap and interventions. (a) The roadmap of this experiment. (b) The schematic diagram of the operation of
auricular bloodletting therapy (ABT). (c) The schematic diagram of the operation of auricular point sticking (APS).minced, and placed in a 50 ml test tube with a   stainless-steel screen. A single cell suspension was prepared  by grinding the spleen with a 5 ml rubber tip syringe while  adding ImunoSep cell sorting solution dropwise. Erythrocyte  lysis was performed using erythrocyte lysis solution (PBM,China). The cell suspensions were incubated with CD68-PE-Vio770 (Miltenyi, Germany), CD86-PE (Thermo Fisher,USA), and CD163-FITC (Bio-Rad, USA) for 30 min at room  temperature followed by flflow cytometry (ThermoFisherAttune NxT, USA) for detection. The experimental data were analyzed by Attune NxT software.at 4° C. PVDF membranes were washed fifive times with TBST at room temperature and incubated with a secondary anti body at room temperature for 1 h. PVDF membranes were rinsed again with TBST fifive times. The ECL kit (Sharebio,China) chemoflfluorescence method was used to cover thestrips, and a fully automated exposure machine was used

for exposure (GelView 6000Plus, China). Band densities were quantifified using ImageJ software (version 1.52a,National Institutes of Health, Bethesda, MD, USA). In this experiment, primary antibodies were used including NF-κB p65 (Cell Signaling Technology, USA), TLR2 (Abcam,UK), p-NF-κB p65 (Cell Signaling Technology, USA),β-actin (Abways, China), and HRP (Abways, China).

引用文獻：辛德維   炎癥介質    第2023卷，文章編號6627393，共9頁     https://doi.org/10.1155/2023/6627393