Interleukin 6 (il 6)

Mactra chinenesis was purchased from a seafood supermarket (Lianyungang, China). Commercial kits for the determination of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), total cholesterol (TC), triacylglyceride (TG), alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), superoxide dismutase (SOD), and malondialdehyde (MDA) were purchased from Grace Biotechnology Co., Ltd. (Suzhou, China). Detection kits for quantification of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were obtained from Xinyu Biotechnology Co., Ltd. (Shanghai, China). Antibodies against phospho-adenosine 5′-monophosphate (AMP)-activated protein kinase (p-AMPK), toll-like receptor 4 (TLR4), cytochrome P450 2E1 (CYP2E1), myeloid differential protein-88 (MyD88), peroxisome proliferator-activated receptor-α (PPAR-α), carnitine palmitoyltransferase 1a (Cpt1a), sterol regulatory element binding protein 1c (SREBP-1c), and interleukin-6 (IL-6) were bought from Servicebio Technology Co., Ltd. (Wuhan, China). A protein content determination kit was offered by Beyotime Biotechnology Co., Ltd. (Shanghai, China). Chinese spirits (alcohol content of 56%) were provided by Red Star Co., Ltd. (Beijing, China). Other chemical reagents were of analytical grade.

For immunohistochemistry (Kusakawa et al., 2015 (link)), the slides were incubated with antibodies against caspase-3 (1:750, rabbit source, Servicebio, Wuhan, China), TNF-α (1:500, rabbit source, Servicebio, Wuhan, China), and IL-6 (1:1000, rabbit source, Servicebio, Wuhan, China) overnight at 4°C. Subsequently, paraffin sections of the tissues were washed with PBS, and the secondary antibody was added and incubated for 1 h.

For immunohistochemical analysis, the dewaxed sections were boiled in 10 mmol/L citrate buffer for 20 min for epitope retrieval following washing three times in PBS. After incubating in 3% hydrogen peroxide, the sections were blocked with 1% BSA for 1 hour. The slices were incubated with anti-rat antibodies against IL-10 (1:200, Servicebio, China), TGF-β3 (1:200, Servicebio, China), IL-6 (1:400, Servicebio, China), TNF-α (1:300, Servicebio, China), IFN-γ (1:150, Servicebio, China), α-SMA (1:1000, Servicebio, China), MCP-1 (1:500, Servicebio, China), CCR2 (1:800, Servicebio, China) and CCRL2 (1:200, Servicebio, China) at 4°C overnight and then incubated with HRP labeled Goat anti-rabbit IgG general secondary antibody (DAKO, Denmark). Finally, the antibody binding was visualized by using a DAB kit (Thermo Scientific). All sections were imaged using an automatic slide scanning system (AxioScan.Z1, Zeiss, Germany) at 20X magnification.

Conforming to the standard protocol, immunofluorescence staining was executed using the following antibodies and dilutions: TNF-α (1:500; Servicebio, Wuhan, Hubei, China), IL-6 (1:500; Servicebio, Wuhan, Hubei, China), and F4/80 (1: 1,000; Servicebio, Wuhan, Hubei, China). After being deparaffinized and rehydrated, the samples were subjected to epitope retrieval by microwaving the slides in an EDTA antigen retrieval buffer with a pH value of 8.0 (Servicebio, Wuhan, Hubei, China). The samples were first permeabilized with BioDewax and Clear Solution (Servicebio, Wuhan, China) and blocked with 3% BSA. Only one antigen was found in each stage, which included primary and secondary antibody incubation, as well as tyramine signal amplification (TSA) visualization. After protein blocking and epitope retrieval as mentioned prior, the next antibody was labeled. To finally stain the nuclei of the cells, the samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The immunofluorescent images were visualized and captured using the PANNORAMIC MIDI (3DHISTECH™, Budapest, Hungary) at 200× magnification. Semi-quantitative image analysis was performed with ImageJ software. The data were presented as relative fluorescence intensity to the Con group.

The histological observation methods were similar to those previously reported (Jiao et al., 2022a (link)). Briefly, after fixation, embedding and section, Hematoxylin-eosin (HE) and Masson tri-chrome staining were performed. The method of evaluating fiber alignment was described in the previous studies (Adeoye et al., 2022 (link)) (Ozlu et al., 2019 (link)) (Erisken et al., 2013 (link)). For immunohistochemical staining, we incubated sections overnight with different antibodies (COL I, COX-2, IL-6; Servicebio; China). On the next day, the sections were incubated with the secondary antibody (HRP-anti-rabbit IgG, Servicebio, China). After that, they were observed and captured.

After dewaxing and antigen retrieval, the sections were washed 3 times with phosphate buffer solution (PBS, pH = 7.4). Then, they were soaked in 0.3% hydrogen peroxide for 25 min to block endogenous peroxidase at room temperature and washed on a decolorizing shaker three times. After 30 min incubation with 3% Bovine Serum Albumin (BSA), the primary antibody against MMP13 (Abcam, 1:100) or IL-6 (Servicebio, 1:100) in PBS was incubated overnight at 4 °C. The secondary antibody was incubated for 50 mins followed by diaminobenzidine solution (DAB). Nuclei were counterstained with hematoxylin for 3–8 min. Then, the sections were dehydrated and sealed and analyzed using ImageJ.

Immunohistochemistry was performed as previously described (Tu et al., 2017 (link)). The renal tissue slides were incubated with primary antibodies against IL6 (1:500), LC3 (1:500), and BECN1 (1:500) (Servicebio), as well as the primary antibodies CD206 (1:500, sc-58986) and ARG1 (1:500, sc-271430) (Santa Cruz Biotechnology) overnight at 4°C. The slices were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:500) (Servicebio). Changes in the kidneys and the positively stained areas were observed using light microscopy (Olympus, Tokyo, Japan). The percentages of the positive areas were calculated by Image-Pro Plus 5.0 software.