Dioscin Mediated IgA Nephropathy Alleviation by Inhibiting B Cell Activation In Vivo and Decreasing Galactose-Deficient IgA1 Production In Vitro

The ethics committee of Guanganmen Hospital approved this experiment (animal experiment ethical approval number: IACUC-GAMH-2023-003).

1. Preparing mice for the experimental procedure

1. Raise 22 SPF-grade male Balb/c mice (6-7 weeks old, body weight 20-25 g) in the animal facility of the hospital/research center. Divide the animals into control (n = 8) and model (n = 14) groups using the random number table method.
2. After 1 week of adaptive rearing in the laboratory cage, feed the model group (IgAN group) with 0.1% bovine gamma globulin (BGG) solution in acidified water containing 6 mmol/L HCl for 9 weeks according to the modeling protocol of Zou et al.¹⁸.
3. Inject 0.1 mL of 0.1% BGG solution in saline into the tail vein for 3 consecutive days while continuing to drink BGG solution to prepare an IgAN experimental mouse model¹⁸.
4. Let the control group freely drink 6 mmol/L HCl acidified water without BGG for 9 weeks. Inject the corresponding volume of saline into the tail vein for consecutive 3 days.
   NOTE: The control and model groups were fed the same quality as normal feed.
5. After the tail vein injection, select 2 mice in the control group and 2 mice in the model group randomly and examine them by proteinuria, light microscopy, and immunofluorescence to determine whether the modeling was successful.
   NOTE: No food is provided to the animals, but they are not prohibited from water; record urine output.
6. Collect urine for 24 h by metabolic cages and centrifuge it at 400 x g for 5 min; discard urine sediment. After a 10-fold dilution of the supernatant, measure proteinuria concentration using a urine protein assay kit and then multiply by the dilution factor and urine volume to obtain 24 h of total urine protein.
   NOTE: Microscopy and immunofluorescence methods are shown in sections 3 and 4, respectively.
7. After successfully preparing the model, divide 12 mice in the model group into 6 mice, each in the model group (IgAN group) and DIO gavage group (DIO group), according to the random number table method.
8. Let the control group continue to drink 6 mmol/L HCl acidified water without BGG, and the model group 0.1% BGG solution composed of acidified water containing 6 mmol/L HCl. Calculate the dose of DIO group gavage administration according to the dose conversion formula of pharmacological experimental methodology (converted according to the human body mass of 70 kg)¹⁹. Gavage DIO tablets 0.06 g/kg once a day for 8 weeks.
9. After 8 weeks of gavage, anesthetize the mice intraperitoneally with 0.4% pentobarbital sodium (60 mg/kg), and after confirming proper anesthetization by toe pinch, isolate kidneys and Peyer's patch for subsequent light microscopy and immunohistochemistry analyses.
   NOTE: The scheme for the in vivo model is in Supplementary Figure 1.

2. Histological analysis

1. Paraffin sections for kidneys and Peyer's patch
   1. Fix 3 mm thick kidney tissues or 1 Peyer's patch with 4% paraformaldehyde for 24 h, dehydrate with gradient ethanol and xylene. Dip in wax for 2 h, seal, and freeze.
   2. Cut 2 µm thick kidney sections and 4 µm thick Peyer's patch sections and spread them in warm water. Scoop up the unfolded slices with a clean glass slide and bake them in a constant temperature oven at 40 °C for 1 h. Start staining after the pre-processing of the specimen.
      NOTE: Take the coronal surface of the hilar part of the kidney, with a thickness of 3 mm tissue block.
2. Dewax and stain the paraffin sections at room temperature (RT) for 10 min with periodic acid solution, avoiding light. Rinse with distilled water and wipe dry-stained with Schiff's staining solution for 20-30 min, avoiding light. Rinse with distilled water until the sections were red under the microscope.
3. Place the sections in hematoxylin staining solution, stain nuclei for 3 min (nuclei stained too profoundly can be divided by ethanol hydrochloride), and rinse with running water until the slides are colorless.
4. Perform routine dehydration with gradient concentrations of ethanol and xylene, seal the sections with neutral gum, and observe under a microscope. PAS-positive is red, and the nucleus is blue.

3. Immunohistochemical analysis of the Peyer's patch

1. Prepare paraffin sections of the Peyer's patch as described in step 2.1.
   NOTE: The thickness of the sections for Immunohistochemical and subsequent immunofluorescence is 4 µm.
2. Dewax the paraffin sections:
   1. Put the sections into xylene I for 5 min, xylene II for 5 min, and xylene III for 5 min.
   2. Further, rinse the slides in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min. Then rinse the slides in distilled water.
3. Antigen retrieval
   1. Prepare 50x stock solution of sodium citrate and dilute with distilled water to 1x for use. Heat it in an autoclave for 2 min, then place the slices in the autoclave, ensuring that the liquid level exceeds the level of the slices.
   2. Heat at high temperature for 5 min, then allow the slides to cool naturally. Wash the slices three times with PBS solution for 5 min each.
4. Blocking of endogenous peroxidase: Mark the borders of the tissue in a circle with an immunohistochemical pen. Incubate the sections in 3% hydrogen peroxide solution for 15 min at RT, protected from light, and wash the sections three times with PBS solution for 5 min each time.
5. Serum blocking: Block the sections by dropping 10% goat serum on the tissue sections marked for 30 min at RT. Ensure that the sections are evenly covered with the stain.
6. Primary antibody incubation: Gently shake off the blocking solution and add a proportion of the prepared primary antibody (CD20 [1:800]; CXCR5 [1:800]) to the section. Place the section flat in a wet box and incubate overnight at 4 °C.
   NOTE: Add a small amount of water to the wet box to prevent evaporation of the antibody.
7. Secondary antibody incubation: Wash the sections thrice with PBS solution for 5 min each time. Remove PBS by shaking the sections dry, cover the tissue with a drop of secondary antibody (HRP-label) of the related species of the primary antibody, and incubate at RT for 50 min.
8. 3,3'-diaminobenzidine (DAB) homogenization: Wash the sections with PBS solution thrice for 5 min each. After shaking the sections dry, drop the freshly prepared DAB chromogenic solution on the sections. Observe the color development time under the microscope; the positive is brownish-yellow. Rinse with tap water to terminate the color development.
9. Staining nuclei: Re-stain with hematoxylin for about 1 min, wash with tap water, and then rinse for 10 min with tap water to return to blue.
10. Dehydration and sealing:
    1. Place the sections in 75% alcohol for 5 min and 85% alcohol for 5 min. Place the sections in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and anhydrous ethanol III for 5 min.
    2. Wash the sections in xylene I for 5 min, take them out to dry slightly and seal the sections with neutral gum.
11. Image acquisition: Collect Images by microscopic examination and analyze by halo software for panoramic image analysis of the tissue.
    NOTE: Hematoxylin-stained nuclei are blue, and DAB positive expression is observed as brownish-yellow.

4. Renal IgA immunofluorescence

1. Prepare paraffin sections for kidneys as described in step 2.1.
2. Dewax paraffin sections:
   1. Put the sections into xylene I for 5 min, xylene II for 5 min, and xylene III for 5 min. Treat the sections in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and 50% ethanol, each for 5 min, and wash with distilled water.
3. Proteinase K retrieval: Shake the sections dry and draw a circle around the tissue section with a histochemical pen. Add the proteinase K working solution (1:9 ratio of stock solution and PBS) dropwise to cover the tissue and incubate at 37 °C for 30 min. Wash the sections three times with PBS for 5 min each.
4. Penetrating the cell membrane: Shake the sections dry slightly and then cover them with 0.1% Triton. Incubate for 20 min at RT, and wash the sections three times with PBS for 5 min each.
5. Blocking: Add 10% goat serum dropwise to cover the tissue evenly for blocking at RT for 30 min.
6. Primary antibody incubation: Add an appropriate amount of goat anti-mouse AF488-conjugate IgA antibody (1:500) dropwise to cover the tissue uniformly and incubate overnight at 4 °C.
7. Staining nuclei: Wash the slices thrice with PBS for 5 min each. After removing the PBS, add the 4′,6-diamidino-2-phenylindole (DAPI) stain dropwise on the sections and incubate for 15 min at RT, protected from light.
8. Wash and seal the sections: Wash the sections thrice with PBS for 5 min, then seal them with an antifade mounting medium.
9. Microscopy and photography: Observe the sections under a fluorescent microscope and take images.
   NOTE: For DAPI, ultraviolet excitation is wavelength 330-380 nm, and the emission wavelength is 420 nm, blue light. Fluorescein isothiocyanate (FITC) excitation wavelength is 465-495 nm, and the emission wavelength is 515-555 nm, green light.

5. Cell culture

1. Obtain human B lymphocyte line DAKIKI from ATCC, USA. Culture DAKIKI cells in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin.
2. Culture the cells in a 37 °C, 5% CO₂ incubator and sub-culture them every 2-3 days. Use cells at the logarithmic growth phase for all experiments.
3. At 70%-80% confluency, collect the cells with a sterile pipette and centrifuge the cells at 140 x g for 5 min. Discard the supernatant, resuspend with the serum-free medium, and after 24 h, leave all cells in a quiescent period for subsequent treatment.

6. LDH cytotoxicity assays for screening safe concentrations of DIO on normal DAKIKI cells

1. Seed DAKIKI cells in 96-well plates at a density of 4×10⁵ cells/well and set up a Low control group, a High control group, and different concentrations of DIO (0.25, 0.5, 1.0, 2.0, 4.0, 8.0 µg/mL). Incubate in 5% CO₂, 37 °C incubator for 24 h after corresponding treatment according to the grouping method.
2. According to the instructions of the cytotoxicity detection kit, add 5 µL of lysate per well in the high control group, and then place the plate in a 5% CO₂, 37 °C incubator for 15 min.
3. Take out the plate, add 100 µL of the reaction mixture to each well, incubate in the dark for 10 min at RT, and then add 50 µL of the stop reaction solution. Measure the OD value at 490 nm on the microplate reader as soon as possible.
4. Calculate the LDH release rate of different concentrations of DIO and ≤10% as the maximum dose administered according to the formula: LDH release rate = (experimental well LDH – low control LDH) / (high control LDH – low control LDH) x 100%.

7. CCK-8 assay for detecting the effect of DIO on DAKIKI cell proliferation

1. Based on the results of our previous experiments²⁰, establish an IgAN model using LPS 40 µg/mL to induce DAKIKI cells.
2. Then, seed 4×10⁵ cells/well in 96-well plates and divide into the control, model, and DIO low, medium, and high concentration groups (0.25 µg/mL, 0.5 µg/mL, and 1.0 µg/mL). Incubate the plates in a 5% CO₂, 37 °C incubator for 24 h.
3. Then, add 20 µL of CCK-8 reagent to each well and place the plates back in the incubator (5% CO₂, 37 °C) for 2 h. After the incubation, detect the OD at a wavelength of 450 nm on the microplate reader as soon as possible.

8. ELISA for detecting the effect of DIO on the secretion of IgA and Gd-IgA1 by DAKIKI cells

1. Seed DAKIKI cells in 6-well plates at a density of 6×10⁶ cells/well, and group and treat the cells according to step 7.2. Culture the cells for 24 h, then centrifuge at 850 x g for 10 min at 4 °C to obtain the supernatant.
2. Detect IgA and Gd-IgA1 concentrations according to the kit instructions.

9.qRT-PCR for detecting the effect of DIO on C1GALT1 and Cosmc mRNA levels in DAKIKI cells

1. Seed the DAKIKI cells at a density of 6×10⁶ cells/well in a 6-well plate, group and treat the cells as CCK8 mentioned in step 7.2, and incubate for 24 h. Extract total RNA from DAKIKI cells according to the instructions of the total RNA extraction kit.
2. After taking 1 µL of extracted RNA from each group of samples and measuring its concentration, transcribe reversely 1 µg of total RNA from each sample into cDNA according to the kit instructions.
3. Then, perform RT-PCR amplification to detect the expression of each gene (95 °C for 15 min, 95 °C for 10 s, and 60 °C for 30 s). Calculate the expression level of each gene using the 2^-ΔΔCT method with β-actin as the internal reference.
   NOTE: The primer sequences were as follows:
   C1GALT1: 5'-AAGGTTGACACCCAGCCTAA-3', 5'-CTTTGACGTGTTTGGCCTTT-3';
   Cosmc: 5'-GCTCCTTTTTGAAGGGTGTG-3', 5'-TACTGCAGCCCAAAGACTCA-3';
   β-actin: 5'-TCACCCACACTGTGCCCATCTACGA-3', 5'-CAGCGGAACCGCTCATTGCCAATGG-3'.

10. Western blotting for examining the effect of DIO on the expression of C1GALT1 and Cosmc proteins in DAKIKI cells

1. Seed the DAKIKI cells at a density of 6×10⁶ cells/well in a 6-well plate. Group and treat them as mentioned in step 7.2. After 24 h of incubation, collect each group of cells.
2. Add an appropriate amount of cell lysis solution (PMSF: phosphatase inhibitor: RIPA lysis solution = 1:1:100) and incubate on ice for 30 min. Then centrifuge at 13,500 x g for 10 min at 4 °C and collect the supernatant.
3. Determine the protein concentration using the BCA protein concentration assay kit.
4. Mix the protein samples with 5x SDS-PAGE loading buffer at 4:1 by vortexing, and heat the mixed samples at 100 °C for 5 min to denature the protein.
5. To detect proteins with different molecular weights, add the protein marker (5 µL/well) and samples (20 µg/well) into different lanes of a 12% SDS-PAGE gel, run SDS-PAGE electrophoresis, and transfer the gel to PVDF membranes.
6. Block the membranes with 5% nonfat milk for 2 h at RT and incubate with the corresponding primary antibodies (C1GALT1 [1:1000], Cosmc [1:2000]) for 24 h. Use β-actin antibody (1:100000) as an internal control.
7. Wash the polyvinylidene fluoride (PVDF) membrane with 1x Tris-Buffered Saline, 0.1% Tween 20 detergent (TBST) three times (10 min/time), then incubate with the corresponding secondary goat anti-rabbit IgG antibody (1: 10000) at RT for 2 h.
8. Wash the membrane with TBST again (three times for 10 min each), and treat it with an appropriate amount of enhanced chemiluminescence (ECL) working solution (per the manufacturer's instructions) for protein band detection.
9. Capture the images using the chemiluminescence imaging system and perform a semi-quantitative analysis of the gray values of proteins using the Image J image analysis system.

11. Statistical analysis

1. Use an appropriate software application for analyzing the data. Express all data as the means ± SD (standard deviation) and evaluate multiple samples by a one-way ANOVA test for comparison between groups.
   NOTE: SPSS statistical software 26.0 was used for statistical analysis. The LSD method was used for two-way comparisons between groups when the variances were equal, and the Dunnett T3 method was used for two-way comparisons between groups when the variances were not equal. P<0.05 was considered to indicate a statistically significant difference.