Preparation of Chinese medicine monomers

The 10 selected traditional Chinese medicine monomers were purchased from the China National Standard Material Network (http://www.bzwz-china.com/) and dissolved in methanol in cell experiments. EA for use in the animal and cell research was purchased from Herb Substance Biotechnology Co., Ltd. (Chengdu, China). In animal experiments, EA was dissolved in olive oil (80 mg/kg/d, concentration was selected according to reference (You et al. 2015)). In the cell experiment, EA was dissolved in methanol.

Cell culture and stimulation - Selecting experiment of traditional Chinese medicine monomers

HK-2 cells were starved in low-glucose DMEM (2% FBS) for 24 h, then changed to high-glucose (30 mM) medium (10% FBS), and added to the same concentration of traditional Chinese medicine monomers (ecliptasaponin A, martynoside, astragalus polysaccharides, emodin, rhein, aucubin, betaine, ursolic acid, icariin, geniposide) for 48 h.

TGFβ1 stimulation

HK-2 cells were starved in low-glucose DMEM (2% FBS) for 24 h and then changed to low-glucose DMEM (10% FBS), and TGFβ1 (10 ng/mL) was added to each well for 48 h.

EA treatment

After the HK-2 cells were starved for 24 h, the medium was changed to low-glucose DMEM (10% FBS), and TGFβ1 (10 ng/mL) and EA were added to each well of the plate.

Animals and drug administration

Six-week-old C57BL/6 J mice (n = 24) were purchased from Southern Medical University Laboratory Animal Science and Technology Development Co., Ltd. (Guangzhou, China). Throughout the whole experiment, the mice were provided with sufficient food and water. After 1 wk of adaptive feeding, all mice were anesthetized by intraperitoneal injection of 1% pelltobarbitalum natricum (50 mg/kg). The ureters were separated below the left kidney with the animals in the prone position. In the operation group (unilateral ureteral obstruction, UUO), the left ureters were separated and ligated with No. 4 silk thread to complete the obstruction of the left ureters. In the sham group, the left ureter was separated without ligation, and the other operations were the same as those in the UUO group. The mice were divided into three groups: (1) control group: the mice received sham operation treatment and were given solvent (olive oil) by gavage on the second day after the operation; (2) UUO group: the mice underwent the UUO operation, and the solvent (olive oil) was given by gavage on the second day after the operation; (3) UUO + EA group: the mice underwent the UUO operation. On the second day after the operation, the mice were given EA (80 mg/kg/d) by gavage for 10 consecutive d. Ten days after the operation, the mice were killed. The renal capsule was stripped with curved tweezers, and the kidneys were removed on both sides. Filter paper was used to absorb the surface water, and the kidneys were weighed. The kidney tissue was preserved in a − 80℃ refrigerator.

Histological analysis

The left kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, cut into 6-μm-thick sections, stained with HE, PAS, and Masson’s trichrome and observed under a microscope (BX51, Olympus Corporation, Tokyo, Japan). The area positive for Masson staining was calculated with ImageJ.

Cell counting Kit-8 assay

HK-2 cells were seeded into 96-well plates. After starving the cells for 24 h, the medium was replaced with low-glucose DMEM (10% FBS) and subjected to the following conditions: (1) blank group: cell-free group containing only medium; (2) 0 dosing group: a group containing cells but without EA; (3) EA group: the cells were subjected to different concentrations of EA (5, 10, 15, 20, 25, 30 nM) for 48 h, and three wells were taken from each group to calculate the average value and draw the proliferation curve. After 48 h, the solution was changed to low-glucose DMEM (10% FBS), and 10 μL of CCK-8 solution was added to each well; the plates were incubated at 37°C for 1–4 h, and the absorbance at 450 nm was measured with a microplate reader.

RNA extraction library construction and sequencing

HK-2 cells in the control group, TGFβ1 (10 ng/mL) group, and TGFβ1 (10 ng/mL) + EA (20 nM) group were cultured for 48 h, and total RNA was extracted with TRIzol reagent (Beijing Dingguo Biotechnology Co., Ltd, Beijing, China.) according to the manufacturer’s instructions. After total RNA was extracted, magnetic beads with oligo (DT) and Poly A were used for A-T base pairing, and mRNA was isolated from total RNA. Then, the mRNA was randomly broken into small fragments of approximately 300 bp by adding a fragment buffer. The cut RNA fragment was then reverse transcribed by reverse transcriptase to obtain cDNA. Then, a base was added to the 3′ end of each chain and connected to the index adapter. The ligation products were amplified by PCR. Finally, we performed 2 × 150-bp paired-end sequencing (PE150) on the Illumina NovaSeq™ 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China) according to the manufacturer’s protocol.

Bioinformatics analysis - Quality control, comparison, expression analysis, and expression difference analysis of original sequencing data

Conduct sequencing–related quality evaluation on the raw sequencing data of each sample, including (1) base error rate distribution statistics; (2) base content distribution statistics.

Using software: fastp.

1.

Base error rate distribution statistics

The sequencing error rate increases with the length of sequenced reads, which is caused by the consumption of chemical reagents during the sequencing process, and is a common feature of Illumina’s high-throughput sequencing platform. In addition, there is also a high sequencing error rate in the positions of the first six bases, and this length is exactly equal to the length of random primers required for reverse transcription during the RNA seq library construction process. The high sequencing error rate of these bases may be due to incomplete binding between random primers and RNA templates.

2.

Base content distribution statistics

The distribution of base content is generally used to detect the presence of AT and GC separation phenomena. For RNA seq, in view of the randomness of sequence interruption and the principle of equal G/C and A/T contents, theoretically, the GC-content and AT content in each sequencing cycle are equal (if it is chain specific database building, AT separation and/or GC separation may occur), and the whole sequencing process is basically stable, showing a horizontal line. However, in existing high-throughput sequencing techniques, the 6-bp random primers used for reverse transcription synthesis of cDNA can cause certain preferences in the nucleotide composition of the first few positions, which is a normal fluctuation.

After obtaining clean data (reads), RSEM (http://deweylab.github.io/RSEM) was used for quantitative analysis of genes and transcripts. RSEM can calculate the expression level of genes/transcripts using single- or double-ended sequencing data. RSEM builds a maximum likelihood abundance estimation model based on the maximum expectation algorithm, and considers paired end reads, the length of reads, the length distribution of fragments, quality values, etc., to distinguish which transcripts are different subtypes of the same gene. Raw read counts of gene expression differences among samples (≥ 2) items were analyzed by using the R package DESeq2. If the conditions (FDR < 0.05 and |log2fc|≥ 1.0) were met, the genes were regarded as differentially expressed genes (DEGs).

Venn diagrams

From the above differentially expressed genes, the genes that were upregulated in the TGFβ1 group, downregulated in the TGFβ1 + EA group, and had significant differences were used as targets to explore the possible potential mechanism of EA. The specific screening conditions were as follows: (1) Genes were upregulated in the TGFβ1 group compared with the control group and downregulated in the TGFβ1 + EA group compared with the TGFβ1 group; (2) the difference in expression was | log2fc |≥ 1.5; (3) the difference was statistically significant, that is, padjust < 0.05.

A Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to screen out the genes that met the above conditions involved in the possible mechanism of action of EA.

Protein‒protein interaction (PPI)

A PPI protein network (https://cn.string-db.org/) was used to analyze the mutual intersection genes screened by a Venn diagram and evaluate the interaction relationship between the proteins encoded by these genes.

Gene ontology (GO) categories and Kyoto encyclopedia of genes and genomes (KEGG)

Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to analyze and predict the biological functions of target genes. GO (http://www.geneontology.org/) analysis clarified the main biological processes in three aspects: cell composition, molecular function, and biological process. The results are expressed as p values. The lower the p value is, the more significant the enrichment result.

KEGG (https://www.genome.jp/kegg/) is a database resource that analyzes differentially expressed mRNAs through genetic biology to explore important pathways related to the target genes. The lower the p value is, the more significant the enrichment result.

Real-time qPCR

The gene expression levels of ECM proteins (CTGF, Col-1, Col-3), α-SMA, E-cadherin, vimentin, MMP10, and MMP13 were evaluated by RT-qPCR. Total RNA was extracted from mouse kidney or HK-2 cells using TRIzol reagent (Dingguo, Beijing, China). Primescript RT Master Mix (Yeasen, Shanghai, China) was used to reverse transcribe RNA into cDNA. Quantitative analysis was performed with a qPCR instrument (LC480) using SYBR ® Green Master Mix (Takara, Kyoto, Japan). β-Actin was used as the internal standard for mRNA. The relative expression of each gene was quantified using the 2−∆∆Ct (∆∆Ct = ( Cttarget gene-Ctreference gene)-mean Ctreference gene) method. The primer sequences are shown in Supplementary Table 1.

Western blot

The kidney tissue of mice or HK-2 cells was lysed with RIPA lysis buffer (Beyotime, Beijing, China) containing 0.1% protease inhibitor and 1% phosphatase inhibitor for 10 min and centrifuged at 12,000 rpm for 15 min, and the supernatant was transferred to a new test tube. A BCA Protein Assay Kit (Takara, Kyoto, Japan) was used to assess the protein concentration. A Gel Preparation Kit (Yisheng, Shanghai, China) was used for SDS-PAGE. The isolated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and blocked with primary dilution antibodies overnight at 4°C. Anti-Hsp90 primary antibody (1:1000, mouse, TA-12, ZSGB-BIO, Beijing, China), anti-FN (1:1000; rabbit; A16678; Abclonal, Boston, MA), anti-Col-1 (1:1000, rabbit, A1352, Abclonal, Boston, Massachusetts), anti-Vimentin (1:1000, rabbit, 10,366–1-AP, Proteintech, Rosemont, IL), anti-TGFβ (1:1000, rabbit, A18692, Abclonal), anti-Col-3 (1:1000, rabbit, WL03186, WanleiBio, Shenyang, China), anti-MMP10 (1:1000, rabbit, MAB9101, R&D, Minneapolis, MN), and anti-MMP13 (1:1000, rabbit, A11755, Abclonal) were used as the primary antibodies following incubation with the membranes for 12 h at 4°C. After sufficiently washing the membranes with Tris-buffered saline + 0.1% [v/v] Tween-20 (TBST), the membranes were incubated with the corresponding secondary antibody for 1 h at room temperature and then washed three times with TBST. The protein level was determined by BLT GelView 6000 Pro enhanced chemiluminescence (FDbio, Hangzhou, China).

Immunofluorescence

HK-2 cells induced by TGFβ1 and EA treatment for 48 h were washed with PBS 3 times. The cells were fixed with 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.1% Triton X-100 for 20 min, washed with PBS, and blocked with 5% BSA at room temperature for 2 h. Then, the cells were incubated with anti-MMP13 antibody (1:100, rabbit, A11755, Abclonal) overnight at 4°C. The next day, the slides were washed in PBS and incubated with the appropriate secondary antibodies (Proteintech, Rosemont, IL, sa00013-4, 1:200) (Proteintech sa00013-3, 1:200) at room temperature for 1 h. In the last step, the cover glass was washed with a fade-resistant mounting medium with DAPI and mounted on the slide. Images were captured using a fluorescence inverted microscope (Imager D2, Zeiss, Oberkochen, Germany).

Immunohistochemistry

The preparation of slides was the same as that for histological analysis. EDTA antigen repair solution was used for antigen repair and then cooled at room temperature. After the samples were washed with PBS, 3% hydrogen peroxide solution was used to block the cells for 10 min. MMP13 primary antibody was incubated at 4°C for 12–18 h. The next day, the slides were washed in PBS and incubated with the appropriate secondary antibody at room temperature for 30 min. After washing, DAB was added to the slides and incubated for 1 min. After coloring, the slides were washed, stained with hematoxylin for 3 min and 1% hydrochloric acid alcohol solution for 2 s, and baked in an oven at 60℃ for 10 min. Images were captured using a microscope (BX51, Olympus Corporation, Tokyo, Japan).

Plasmid construction and cell transfection

The MMP13 overexpression plasmid was constructed by WZBio (Shandong, China). According to the manufacturer’s protocol, 2500-ng plasmid and 6 μL Lipofectamine® 3000 transfection reagent (Invitrogen, Carlsbad, CA) were used to transfect cells. RNA and protein expression levels were detected 48 h after transfection, and each experiment was repeated at least three times. The overexpression efficiency of MMP13 was verified by RT-qPCR.

Statistical analysis

GraphPad Prism 8.0.1 was used for data analysis and mapping, and the data are expressed as the mean ± standard deviation (‾χ ± SD). A two independent-sample t test was used to compare two groups of measurement data that conformed to a normal distribution, and one-way ANOVA was used to compare the mean between multiple groups. When p < 0.05, the difference was considered statistically significant.