To investigate the effect of DIS on renal interstitial fibrosis, the present study used network pharmacology to predict the targets of DIS. PubChem (https://pubchem.ncbi.nlm.nih.gov/) was used to get the structured and SMILE files of DIS. The SMILE files were imported into SwissTargetPrediction (http://www.swisstargetprediction.ch/) and TargetNet (http://targetnet.scbdd.com/) to search the potential targets. The keywords ‘renal interstitial fibrosis’ were entered into DrugBank (https://go.drugbank.com), CTD (http://ctdbase.org), Genecards (http://www.genecards.org), DisGeNET (https://www.disgenet.org) and OMIM databases (http://omim.org) to collect the targets of disease. Then, the process of DIS on renal fibrosis was analyzed using Kyoto Encyclopedia of Genes and Genomes pathway analysis in Metascape database (http://www.metascape.org/). Significant pathways with P<0.05 were chosen.

To determine the sample size for each group of mice, the present study reviewed studies reporting on UUO mice and noted the high mortality rate of mice when UUO surgery was performed on the mice. The kidneys of the mice were intended for western blotting, reverse transcription-quantitative (RT-q) PCR and histopathological staining. Due to the amount of mouse kidney tissue required for these analyses, it decided to set the sample size at 10 mice per group, based on the research paper by Cao et al (18) and Gu et al (19).

Male C57BL/6 mice from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. weighing 17–22 g. The mice were kept in a cage with a 12-h light/dark cycle, a standard feed and unlimited access to water at a temperature of 22±2°C and humidity of 40±5%. The bedding of the cages was changed daily and the condition of the mice was checked daily. The animal study was reviewed and approved by the Ethics Committee of the China-Japan Friendship Institute of Clinical Medical Sciences (approval no. zryhyy21-22-01-09; affiliated with the China-Japan Friendship Hospital, Beijing, China).

A total of 80 mice were randomly assigned to one of four groups (n=20/group), which included the sham-operated group, the UUO group, the UUO + 50 mg/ kg DIS group and the UUO + 100 mg/kg DIS group. After three days of adaptive feeding, a model of interstitial renal fibrosis was prepared in mice by using UUO surgery. Pentobarbital (1%; 40 mg/kg) was used 1% to anesthetize mice by intraperitoneal injection before the surgery. The unilateral ureteral obstruction surgery was described previously (20). The sham-operated group underwent a sham operation. The mice were fasted for 12 h but were given free access to drinking water before the operation. The DIS group was administered from the first day after surgery and gavaged with DIS 50 mg/ kg or 100 mg/kg every 24 h for 7 days. DIS was purchased from Herbpurify company (CAS: 19057-60-4, purity >98.0%, ID: S-048, Chengdu Herbpurify Co., Ltd.). The sham-operated and the UUO groups were orally administered physiological saline of equal volumes every 24 h for 7 days. Half of the mice were sacrificed on the 3rd and 7th day of treatment to obtain kidney tissue, respectively. The mice were fasted for 12 h before sacrifice. Pentobarbital sodium (1%; 50 mg/kg) by intraperitoneal injection was used, followed by intracardiac exsanguination. Animal mortality was verified by the absence of a heartbeat and respiration for >3 min.

At the end of the experiment, urine in mice was collected. Urine protein was detected by mouse albumin ELISA kit (Bethyl Laboratories, Inc.). Urine creatinine was detected by an automatic biochemical analyzer. Then, the ratio of urine protein to urine creatinine was calculated.

Human renal tubular epithelial cells (HK-2) were obtained from Professor HY Lan (Chinese University of Hong Kong) and placed in a cell incubator at 37°C and 5% CO₂. The cells were cultured with DMEM/F-12 medium (Corning, Inc.), 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and 1X double antibiotics (penicillin and streptomycin). TGF-β1 (2 ng/ml) was used to induce renal fibrosis model in HK-2 cells. Then, the cells were respectively treated with DIS (3.125, 6.25, 12.5 µM) and Bay11-7082 (1 µM; cat. no. B5556; MilliporeSigma) for another 24 h. DIS (CAS: 19057-60-4; purity >98.0%; cat. no. SD8350) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

CCK-8 assay (Mei5 Biotechnology, Co., Ltd.) was used to evaluate DIS on HK-2 viability. HK-2 cells were seeded and grown in a 96-well plate for 24 h at 37°C. Subsequently, the cells were treated with DIS for indicated periods (24, 48 and 72 h), respectively. Then the cells were incubated for 1 h with 10 µl of CCK-8 solution (5 mg/ml). An enzyme marker was used for analyzing cell absorbance at 450 nm.

Kidney tissue specimens from mice were fixed with 10% formalin solution overnight at 4°C and were dehydrated by ethanol. These specimens were then embedded in paraffin and were cut into 2–3-µm sections by a routine procedure (21). The sections were stained with hematoxylin and eosin (HE) and Masson's trichrome as described previously (22). Briefly, for HE staining, the sections were stained with hematoxylin for 5 min followed by eosin for 3 min, both at room temperature. For Masson's trichrome staining, the sections were stained with hematoxylin for 5 min, lichon red acidic magenta solution for 10 min, 1% phosphomolybdic acid solution for 5 min and aniline blue solution for 5 min. All Masson's trichrome staining steps were conducted at room temperature. All stained sections were viewed by a light microscope (Olympus BX53; Olympus Corporation).

The sections were routinely dewaxed and hydrated with 3% H₂O₂ solution for 10 min and rinsed three times with distilled water. Subsequently, the antigen was retrieved using a microwave oven. The sections were heated in a microwave oven on high with 0.01 M sodium citrate buffer for 10 min, cooled and rinsed once with PBS buffer. The blocking solution was added dropwise for 20 min. Primary antibodies against TNF-α (1:50; cat. no. sc-52746; Santa Cruz Biotechnology, Inc.), alpha-smooth muscle actin (α-SMA; 1:50; cat. no. 19245; Cell Signaling Technology, Inc.) and fibronectin (FN; 1:50; cat. no. ab2413; Abcam) were added dropwise, incubated for 2 h at room temperature and then rinsed 5 times for 2 min each using PBS buffer. The secondary bio-goat anti-mouse IgG and bio-goat anti-rabbit IgG antibodies (1:50 dilution; both from the immunohistochemistry staining kit; cat. no. MF501-01; Mei5 Biotechnology Co., Ltd.) were added dropwise, incubated for 30 min at room temperature and then rinsed 5 times with PBS buffer. Streptavidin-POD working solution was added according to the immunohistochemistry kit instructions, kept for 30 min and rinsed 5 times with PBS buffer. The reaction was terminated by dropwise addition of ready-to-use DAB, color development for 30 min and rinsing with distilled water. Finally, after light re-staining with hematoxylin and dehydration, neutral gum was used to seal the sections.

The HK-2 cells on coverslips were fixed with 4% paraformaldehyde for 10 min at 4°C. The cells were stained with primary antibodies against NOD-like receptor thermal protein domain associated protein 3 (NLRP3; 1:100; cat. no. ab260017; Abcam), p-NF-κB p65 (1:100; cat. no. sc-136548; Santa Cruz Biotechnology, Inc.) and secondary antibody goat anti-mouse IgG/Alexa Fluor 488 (1:1,000; cat. no. K0031G-AF488; Solarbio Technology Company, Beijing, China), respectively. Subsequently, the cells were incubated with DAPI (1 µg/ml; cat. no. C0060; Beijing Solarbio Science & Technology Co., Ltd.) for 5 min protected from light and sealed with 80% glycerol. A fluorescent inverted microscope was used to capture final fluorescence images.

Radioimmunoprecipitation lysis buffer was used to extract proteins from the kidney tissue and cultured cells. The concentration of protein was determined using a BCA Protein Assay Kit (cat. no. MF071-01; Mei5 Biotechnology, Co., Ltd.). Each lane was loaded with 100–120 µg of protein. Electrophoresis was performed with 10 or 15% SDS-PAGE gels. The proteins were transferred from the gels onto PVDF membranes (cat. no. IPFL00010; Merck KGaA) and blocked with a BSA-based western blot blocking solution (cat. no. MF432-01; Mei5 Biotechnology, Co., Ltd.) for 1 h at room temperature. After blocking, the PVDF membranes were incubated with primary antibodies for 2 h at room temperature and secondary antibodies for 1 h at room temperature (23). In the present study, primary antibodies against β-actin (1:10,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.), IL-1β (1:1,000; cat. no. sc-52012; Santa Cruz Biotechnology, Inc.), IL-6 (1:1,000; cat. no. sc-130326; Santa Cruz Biotechnology, Inc.), TNF-α (1:1,000; cat. no. sc-52746; Santa Cruz Biotechnology, Inc.), NF-κB p65 (1:1,000; cat. no. sc-8008; Santa Cruz Biotechnology, Inc.), phosphorylated (p-)NF-κB p65 (1:1,000; cat. no. sc-136548; Santa Cruz Biotechnology, Inc.), NLRP3 (1:1,000; cat. no. ab260017; Abcam) and monocyte chemotactic protein 1 (MCP-1; 1:1,000; cat. no. sc-52701; Santa Cruz Biotechnology, Inc.) and secondary antibodies against goat anti-rabbit IgG (1:10,000; cat. no. MF094-01; Mei5 Biotechnology, Co., Ltd.), goat anti-mouse IgG (1:10,000; cat. no. MF093-01; Mei5 Biotechnology, Co., Ltd.) and goat anti-rat IgG (1:10,000; cat. no. MF756-01; Mei5 Biotechnology, Co., Ltd.) were used for incubation. An iBright CL1000 gel imaging system program (Thermo Fisher Scientific, Inc.) was used for capturing signals, which were quantified by ImageJ software (ImageJ bundled with 64-bit Java; version 1.8.0_172; National Institutes of Health).

To extract RNA, HK-2 cells were seeded in 6-well culture plates at a density of 2×10⁶ cells/well. The total RNA from HK-2 and renal tissues was isolated (Universal RNA Mini kit; Mei5 Biotechnology, Co., Ltd.) and reversed-transcribed into first strand cDNA (RevertAid First Strand cDNA Synthesis kit; Mei5 Biotechnology, Co., Ltd.), both according to the manufacturer's protocol. Subsequently, the UltraSYBR Green Mixture qPCR kit (Mei5 Biotechnology, Co., Ltd.) was used to perform RT-qPCR according to the manufacturer's protocol. The thermocycler conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 65°C for 15 sec and 72°C for 45 sec, ending with 1 cycle of 72°C for 10 min. All experiments were repeated three times. The results were analyzed by the 2^−ΔΔCq method and normalized to the expression levels of the internal control gene, β-actin (24). All primer sequences used in this study are shown in Table I.

A total of 100 µl each of cell culture medium and kidney protein suspension after drug treatment was added to a 96-well plate. The concentrations of IL-1β (HiPer Human IL-1β ELISA Kit; cat. no. MFH-02-01; Mei5 Biotechnology Co., Ltd.) and IL-18 (HiPer Human IL-18 ELISA Kit; cat. no. MFH-22-01; Mei5 Biotechnology Co., Ltd.) were detected with Elisa kit, according to the manufacturer's instructions. Then, the absorbance was measured in an enzyme marker instrument (Bioteke Corporation). The concentrations were calculated using a standard curve.

All results were evaluated using the GraphPad Prism version 8.0 (Dotmatics) and quantitative data are expressed as the mean ± standard error of the mean. One-way ANOVA and Dunnett's test for multiple comparisons were used for analysis. P<0.05 was considered to indicate a statistically significant difference.

According to a network pharmacology analysis (Fig. S1), it was demonstrated that the NF-κB signaling pathway is an important pathway for DIS in improving renal interstitial fibrosis.

The sham-operated group demonstrated no abnormal pathological changes in morphological structure (Fig. 1). In the UUO group, the renal tubular structure was disorganized, some of the tubular epithelial cells were detached from the brush border, cellular debris was occasionally seen in the tubular lumen and the renal interstitium was infiltrated with inflammatory cells and collagen was deposited. Although the ratio of urine protein to urine creatinine in mice did not show a statistically significant difference (Fig. S2), the degree of kidney tissue damage in the DIS group at each time point was less than that in the UUO group. In brief, DIS attenuated renal injury in UUO mice.

To further clarify the mechanism of DIS, the kidney tissue in mice was analyzed. Immunohistochemistry staining (Fig. 2) showed that the expression of TNF-α, fibronectin (FN) and α-SMA increased in the UUO group. However, the expression of TNF-α, FN and α-SMA decreased in the UUO + DIS group. Western blotting (Fig. 3A) and RT-qPCR (Fig. 3B) indicated that the expression of MCP-1, TNF-α, the ratio of p-NF-κB p65 and NF-κB p65, NLRP3, IL-1β and IL-6 increased in the UUO group compared to the sham-operated group. After being treated with DIS, the expressions of NLRP3, MCP-1, TNF-α, the ratio of p-NF-κB p65 and NF-κB p65, IL-1β and IL-6 were significantly decreased in the DIS group. Therefore, it was hypothesized that DIS alleviated renal injury in UUO mice by inhibiting the secretion of inflammatory factors linked to the NF-κB pathway.

To further clarify the mechanism of DIS, TGF-β1 was used to induce a renal fibrosis model in vitro. First, the cells were treated with NF-κB inhibitor Bay11-7802. The expressions of NLRP3, IL-6, TNF-α, the ratio of p-NF-κB p65 and NF-κB p65, MCP-1, IL-1β and IL-18 were reduced (Fig. 4), which indicated that the inflammatory response was attenuated and the NF-κB pathway was inhibited. Subsequently, the cells were treated with DIS. The expressions of NLRP3, MCP-1, IL-6, TNF-α, IL-1β, IL-18 and the ratio of p-NF-κB p65 and NF-κB p65 were also reduced (Fig. 5), showing a similar effect to Bay11-7082, which indicated that DIS attenuated the inflammatory response by suppressing the NF-κB pathway.

To clarify the impact of DIS on renal interstitial fibrosis, the toxicity of DIS on cells was tested by CCK-8 assay. The result showed no significant toxicity to cells when the concentration of DIS was in the range of 0–20 µM and significant toxicity to cells when its concentration was in the range of 40–80 µM (Fig. 6A). The concentrations of 3.125, 6.25 and 12.5 µM were selected for further experiments and did not find that DIS trigger inflammation (Fig. 6B).