Healthy male Sprague-Dawley (SD) rats (body weight, 220–240 g; n = 30) were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. (Changchun, Jilin). Before starting the experiment, mice were adaptively fed for 1 week under specific pathogen-free (SPF) conditions (12/12 h light/dark cycle) at a controlled temperature (22 ± 1 °C) and humidity (50 ± 1 °C). %), and all mice had free access to drinking water and food. All animal experiments were approved by the Animal Ethics Committee of the Jilin Academy of Agricultural Sciences (approval number: SCXK2020-0001, Jilin, China).
After one week of adaptation, 30 rats were randomly divided into three groups (n = 10 per group): the control group, the HFD group and the HFD + S9 group. Mice in the control group were fed a normal diet containing 10% fat, while the HFD group and the HFD + S9 group were fed a HFD containing 60% fat for 8 weeks.40. Subsequently, the HFD + S9 group was administered orally 2 ml L. Plantae S9, control group, and HFD group were given the same dose of normal saline (once daily for 6 weeks). The nutritional composition of the diet was presented in Table 1. The body weight and blood glucose level of mice in each group were recorded every week. After administration, all rats were euthanized for 12 h and anesthetized by intraperitoneal injection of 5% sodium pentobarbital, blood samples were collected through cardiac puncture and serum was obtained after centrifugation at 4000g for 10 minutes at 4 °C. Then, mice were sacrificed to cervical dislocation, and serum and liver were collected. Furthermore, a portion of the liver was placed in 10% paraformaldehyde for pathological staining, and the remaining liver tissue was frozen in liquid nitrogen and stored at −80 °C for further analysis.41.
Define relevant parameters
The final weight and height of the mice were recorded one day before sacrifice, and weight gain was calculated for each group according to the established formula [final weight (g) − initial body weight (g)]Lee’s index was calculated using the following formula: Lee’s index = cube root of final weight (g) / anal length (cm). When rats were sacrificed, the livers were immediately removed, the weight of the liver was recorded, and the liver index was calculated using the following formula: Liver index = (liver weight/final body weight) × 100. Body weight and body length The average number of rats in each Collection.
Analysis of biochemical parameters in serum
According to the manufacturers’ protocols, serum levels of insulin, total cholesterol (TC), TG, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were detected using a kit. Purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), the contents of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by ELISA kit (Shanghai Jianglai Biotechnology Co., Ltd.). Ltd., Shanghai, China). Therefore, the fasting blood glucose level of rats was measured using an ACCU-CHEK active blood glucose meter (Roche Diabetes Care GmbH, Mannheim, Germany), the homeostasis model index–insulin resistance (HOMA-IR) and beta cell function ratio (HOMA index) were calculated. -B as follows: HOMA-IR = fasting blood glucose (mmol/L) x fasting insulin concentration (mU/L)/22.5; HOMA-B = 20 x fasting insulin concentration (mIU/L)/[fasting blood glucose (mmol/L) − 3.5].
According to a previous study42, liver tissues were fixed in 10% neutral paraformaldehyde (pH = 7.0), then embedded in paraffin, and 4 μm paraffin-embedded sections were prepared and placed on clean glass microscope slides for hematoxylin and eosin (H&E) staining and oil-red staining. Then, images were analyzed visually with an optical microscope (Eclipse E100; Nikon, Tokyo, Japan), and the pathological changes of the liver were observed in each group via Image J software (National Institutes of Health, Bethesda, MD, USA).
Western blot analysis
Rat liver total protein was extracted by the RIPA kit (ComWin Biotech Co., Ltd., Beijing, China) according to the method of Wang et al.43The liver extract was centrifuged at 10,000 .g for 10 min at 4 °C and separated to harvest the protein extract. Then, the protein content of the supernatant was detected using a BCA kit (Wanleibio Co., Ltd., Shenyang, China), adjusted according to the regular concentration, and thoroughly mixed with the buffer solution. [60 mM Tris–HCl, 2% sodium dodecyl sulfate (SDS), and 2% β-mercaptoethanol, pH = 7.2], and stir them for 10 minutes in boiling water. Then, protein samples were separated by 10% SDS-polyacrylamide electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Protein samples were sealed with Tris-buffered saline 0.05% Tween-20 (TBST), containing 3% bovine serum albumin (BSA) at room temperature for 60 min. Finally, incubation was performed with rabbit anti-TLR4 (1:1000, bs-20379R, Bioss, China), anti-rabbit p38 (1:1000, bs-0637R, Bioss, China), anti-rabbit p-p38 (Thr180, 1:1). 1000, bs-5476R, Bioss, China), rabbit anti-IκBα (1:1500, bs-1287R, Bioss, China), rabbit anti-p-IκBα (Ser 36, 1:1000, bs-18129R, Bioss, China) ), rabbit anti-NFκB p65 (1:1500, bsm-52305R, Bioss, China), rabbit anti-p-p65 (Ser 281, 1:1500, bs-17502R, Bioss, China), and rabbit anti-β-actin (1:1000, bs-0061R, Bioss, China) at 4 °C for 12 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at 37 °C for 60 min. β-actin served as a loading control, and the optical density of the target band was measured using Image Quant LAS 4000 (Fujifilm, Tokyo, Japan) (Supplementary Figures).
All experimental results were expressed as mean ± standard deviation. SPSS 20.0 software (IBM, Armonk, NY, USA) was used to analyze differences between groups using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. s< 0.05 was considered statistically significant.
All animal experiments were approved and directed by the Animal Welfare Committee of the Jilin Academy of Agricultural Sciences (approval number: SCXK2020-0001).
All animal studies (including euthanasia of rats) were performed according to AAALAC and IACUC guidelines.
All research methods given in the manuscript are performed in accordance with the requirements of ARRIVE.
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