Experimental animals

Twenty-seven clean-grade Sprague-Dawley (SD) male rats with 180 ±20 g body weight were purchased from Beijing HFK Bioscience Co., Ltd. (lot No.: SCXK (Beijing) 2009-0004). All rats were kept in the animal room of the laboratory and were routinely fed. They were given free access to water. This study was supported by the Ethics Committee of Chongqing Hospital of Traditional Chinese Medicine.

Drugs and reagents

Streptozotocin (STZ, Sigma-Aldrich, USA); HSYA (mass fraction ≥ 98%; National Institute for the Control of Pharmaceutical and Biological Products, China); foetal bovine serum (FBS) and DMEMF12 medium (Gibco, USA); SuperReal PreMix Kit (Tiangen Biotech (Beijing) Co., Ltd, China); RNAiso Plus Kit, TRIzol Kit and M-PER Kit (Life Technologies, USA); Lipofectamine 2000 Transfection Reagent and Lipofectamine RNAiMAX Kit (Thermo Fisher Scientific, USA); BCA Protein Quantification Kit (Beyotime Biotechnology, Jiangsu, China); miRNA-140-5p mimic and inhibitor (RiboBio, Guangdong, China); rabbit anti-TLR4 polyclonal antibody (ProteinTech, USA); rabbit anti-NLRP3 polyclonal antibody (Bioss Antibodies, China); rabbit anti-Notch2 polyclonal antibody (MDL, China); anti-collagen type IV (Col-IV) protein monoclonal antibody (Sigma-Aldrich, USA); anti-β-actin antibody (Boster, Wuhan, China); Total Anti-oxidant Capacity (T-AOC) and Malondialdehyde (MDA) Assay Kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); Immunohistochemical Streptavidin-Peroxidase (SP) Two-Step Assay Kit, diaminobenzidine (DAB) substrate solution (ZsBio, Beijing, China); Enzyme-Linked Immunosorbent Assay (ELISA) Kit (NEOBIOSCIENCE, Beijing, China); and Dual-Luciferase Reporter Gene Assay Kit (Promega, USA) were used.

Diabetic animal model establishment and animal grouping

With free access to water, the animals fasted for 4–6 h prior to modelling. Then, STZ (55 mg/kg) was administered via a single tail vein injection to establish the diabetic rat model. After 72 h, the animals fasted for 4 h, and then blood was collected from the tail vein tip for the measurement of fasting blood glucose. Blood glucose ≥ 16.7 mmol/l and a positive result of the urine glucose test indicate the successful establishment of the diabetes model [7]. The animals were randomly divided into the diabetic (DM) and HSYA groups 2 weeks after the establishment of eight model rats in each group. Another eight rats were assigned to the normal control (NC) group. The HSYA group was intragastrically administered a dose of 10 mg/kg daily for 6 consecutive weeks. The blank control and DM groups were administered the same volume of normal saline. Insulin was not utilised in the experiment. All animals were free to eat and drink, and no deaths were reported.

Cell culture and high glucose-induced renal tubular epithelial cells

The human immortalised renal tubular epithelial (HK-2) cells were purchased from the American Type Culture Collection. In the Thermo 3111 cell culture incubator set at a constant temperature of 37°C, the cells were cultured in a DMEM-F12 culture system containing 10% foetal bovine serum at 5% CO₂ and 95% relative humidity. The HK-2 cells were processed with 30 mmol/L D-glucose for 48 h to construct the DN cell model.

The cells in the HSYA group were administered 10 mg/ml HSYA.

Transient transfection assay

The HK-2 cells were inoculated into the 35-mm dish and then left to stand for cell adhesion. Lipofectamine RNAiMAX was used to transfect the blank plasmids and inhibitor with the scrambled RNA fragment as the control. DMEM-F12 medium containing 10% FBS was replaced after 12 h, followed by high sugar stimulation at an appropriate time. The specific steps were as follows: 250 µl of Opti-MEM medium was first added with the fragment at the corresponding concentration, followed by 5 µl of Lipofectamine RNAiMAX. The diluted Lipofectamine RNAiMAX and miRNA-NC or miRNA-140-5p inhibitor were placed at room temperature for 5 min; the mixture was added dropwise to the transfection dish (medium volume, approximately 2.5 ml), thus making the volume of the final culture system 3 ml. miR-140-5p inhibitor (antisense 5’-CUA CCA UAG GGU AAA A CCA CUG-3’), NC empty vector (cat. no. v855-20; Invitrogen; Thermo Fisher Scientific, Inc.).

Specimen collection and indicator determination

The 24-h urine volume of rats was collected at the end of the eighth week of experiment, and the urine protein (UP) concentration was measured using the Biuret method. The 24-h total UP was defined as the product of UP concentration and 24-h urine volume. Subsequently, blood was collected from the common femoral artery under ether anaesthesia. The blood glucose (BG) level was detected using the glucose oxidase method, whereas the enzyme assay was employed to detect total cholesterol (TC) and triglyceride (TG) in the blood and culture supernatants. The normal saline pre-cooled at 4°C was injected via the left ventricle to repeatedly lavage the kidneys, which were then dissociated, fixed with 4% paraformaldehyde, and sectioned for immunohistochemical staining. For homogenisation, 0.1 g of kidney cortex was added to 500 µl of tissue protein lysate. The protein concentrations in the serum and culture supernatants were measured using the BCA method based on the T-AOC and MDA Kit instructions. The levels of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in renal tissues and culture supernatants were determined using the ELISA Kit.

Immunohistochemical staining of the kidneys using he and masson stains

The fixed rat kidneys were cut into 3-µm thick paraffin sections for pathological observation via HE and Masson staining. The kidney cortex was stained using the immunohistochemical SP two-step assay to detect Notch2, NLRP3, and TLR4 (1 : 50), followed by dewaxing, hydration, microwave heating for antigen retrieval, antibody incubation, and DAB colour development. The expression sites were observed under light microscope and then photographed. ImageJ software was used for the analysis. The collagen volume fraction (CVF, %) was calculated using the equation CVF = collagen area/measured field area × 100%.

RT-qPCR assay

For the tissues, an appropriate amount of sample tissues was placed into 1 ml of RNAiso Plus and homogenised. The clarified liquid was transferred to a 1.5-ml centrifuge tube and then left to stand at room temperature for 5 min. Then, the total RNA was extracted according to the instructions. The cells in the logarithmic phase were inoculated into a six-well plate at a density of 2 × 10⁵ cells/well and then treated accordingly after adhesion. The supernatants were removed at the end of the treatment, and 1 ml of TRIzol Reagent was added to each well to extract cellular RNA according to the instructions. After determining the total RNA concentration using a Qubit 2.0 Fluorometer, 1 µg of total RNA was obtained for reverse transcription reaction, where the PCR parameters were set at 42°C for 60 min and then 70°C for 10 min to terminate the reaction. cDNA was treated according to the instructions in the SuperReal PreMix Kit and then submitted for real-time quantitative PCR experiment together with the primers. The primer sequences of miRNA-140-5p were as follows: miR-140-5p, F 5’-CAG TGG TTT TAC CCT ATG GTA G-3’ and R 5’-TGG TGT CGT GGA GTC G-3’; U6, F 5’-CTC GCT TCG GCA GCA CA-3’ and R 5’AAC GCT TCA CGA ATT TGC GT-3’. PCR parameters were set at 95°C for 10 min and 40 cycles of 95°C for 10 s, 62°C for 30 s and 72°C for 30 s, followed by slow warming to 95°C. After the completion of the reaction, the relative expression level of miRNA-140-5p was calculated according to the 2^–ΔΔCt method.

Detection of protein expression using the western blot method

HK-2 cells were inoculated into the 35-mm culture dish. After the corresponding treatment, the supernatants were removed from all groups and then gently rinsed with pre-cooled PBS thrice. Approximately 200 µl of cell lysate, the concentration of which was determined using the BCA Kit, was added to each well. After the protein was quantified to the same concentration, 20 µg of the protein sample was placed in a 5 × SDS loading buffer and then heated in boiling water for 5 min to completely denature the protein. Then, separation was performed via sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to the PVDF membrane using the wet method. The sample was blocked with 5% skimmed milk powder at room temperature for 1 h and then incubated with the diluted primary antibody (antibody diluent, 5% BSA) overnight at 4°C. It was then rinsed with TBST thrice (5 min each) the next day, followed by incubation with the corresponding secondary antibody at room temperature for 1 h, TBST rinsing three times, and colour development by chemiluminescence and photographing.

Cellular immunofluorescence assay

The cells of the different groups were inoculated into 24-well plates, respectively, placed in a CO₂ cell incubator for 24 h, and then fixed with 95% ethanol for 1 h. Subsequently, the flowing were perfomred: permeation by 0.05% Triton X-100 for 20 min, PBS rinsing, the addition of the primary antibody, dilution at 1 : 100 and incubation overnight at 4°C, PBS rinsing, incubation with goat anti-rabbit fluorescent-labelled secondary antibody for 1 h, 3× PBS rinsing, incubation with DAPI for 15 min in the dark, 3× PBS rinsing, observation, and photographing under a fluorescent inverted microscope. The expression sites and levels of TLR4, NLRP3, Notch2, and Col-IV proteins were detected using ImageJ software, and the translocation of NF-κB (p65) into nuclei was also analysed.

Dual-luciferase reporter assay

The TLR4 gene 3’-UTR was cloned into plasmid pGL3 to construct the pGL3-TLR4 wild- and mutant-type vectors. The HK-2 cells were inoculated into 12-well plates; after adhesion, Lipofectamine 2000 was utilised for the co-transfection of pGL3-TLR4 wild- or mutant-type vector and miRNA-140-5p mimic. Firefly luciferase activity was detected after 24 h using a Synergy H1 hybrid multi-mode microplate reader according to the instructions of the Dual-Luciferase Reporter Assay Kit, where Renilla luciferase activity was adopted as the internal reference.