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

Our main research area is how Chinese herbal medicine works to treat chronic kidney disease like IgA nephropathy. In this study, based on our clinical experience and treatment of IgA nephropathy using Dioscoreae Nipponicae Rhizoma, abbreviation, DNR, and our research result of the main active substance of DNR, we try to confirm the potential value of dioscin as one of the main active substance of DNR, and discover its possible mechanism of action in the treatment of IgA nephropathy through in vivo and in vitro experiments. Abnormal IgA1 production, that is, the increased lactose deficient IgA1, is important in the pathogenesis of IgA nephropathy, and there is strong evidence that gut-associated lymphoid tissue plays a significant role in the production of galactose-deficient IgA1.

In vitro experiments, we found that dioscin has a very distinct inhibitory effect on the proliferation of IgA-secreting cells, DAKIKI cell line, at relatively low concentration, as well as an inhibitory effect on the secretion of Gd-IgA1 by DAKIKI cells. These findings indicate that dioscin may have a potential therapeutic value for IgA nephropathy, as well as disease linked to excessive B cell activation, such as systemic lupus erythematosus. Due to the complexity of Chinese herbal ingredients, the study of the therapeutic mechanism of Chinese herbal medicine faces considerable difficulties, especially in cell experiments in which mechanism discussion like ours, using the main active ingredient of a non-herbal medicine as the representative substance of the drug may be commonly adopted research method, and we hope these kinds of works may provide the foundation for the discovery of new drugs for natural medicine.

Begin preparing the immunoglobulin A or IgA nephropathy and control models after subjecting the mice to one week of adaptive rearing in a laboratory cage. Then, for nine weeks, feed the model mice with 0.1%bovine gamma globulin or BGG solution in 6 millimolar aqueous hydrochloric acid. Next, inject 0.1 milliliters of 0.1%BGG solution in saline into the model animal's tail vein for three consecutive days while continuing the feeding as before.

After successfully preparing the IgA neuropathy model, on week nine, begin the dioscin or DIO intervention to half of the mice from the model group selected as the DIO gavage group. Feed appropriate drinks to the groups as demonstrated for eight weeks before proceeding with the subsequent analysis. To begin preparing the paraffin sections of the kidney and Peyer's patch isolated from properly anesthetized IgA neuropathy model mouse, first, fix one Peyer's patch or 3-millimeter-thick kidney tissues with 4%paraformaldehyde for 24 hours.

Then dehydrate them, using a gradient of ethanol and xylene before washing them with water. Submerge the tissues in wax for two hours before sealing and freezing them. Next, cut 4-micron-thick Peyer's patch sections or 2-micron-thick kidney sections, and place them in warm water to unfold.

Using a clean glass slide, gently pick up the unfolded slices and incubate them in a constant temperature oven at 40 degrees Celsius, for one hour, before staining. After preparing the paraffin sections of the kidney and Peyer's patch isolated from immunoglobulin A or IgA neuropathy model mouse, proceed to dewax and stain the desired paraffin section using periodic acid solution at room temperature for 10 minutes, while avoiding exposure to light. Rinse the section with distilled water before blotting it dry.

Then stain the dry section with Schiff's staining solution for 20 to 30 minutes, while avoiding exposure to light. Rinse the section with distilled water until it appears red under the microscope. Immerse the rinsed section in hematoxylin staining solution for three minutes to stain the nuclei, and then rinse it under running water until the section becomes colorless.

After performing routine dehydration with xylene and ethanol gradients, seal the section with neutral gum to examine it under a microscope. Compared with the control group, the mucosal immune-induced IgA neuropathy mice model has visible IgA deposition in the entire mesangial region. PAS staining of the renal tissue showed mesangial cell proliferation and stromal hyperplasia in the model group, which was reduced in the dioscin gavage group.

To begin dewaxing previously prepared paraffin sections of the Peyer's patch isolated from immunoglobulin A or IgA neuropathy model mouse, submerge each section sequentially in xylene and ethanol gradients for five minutes. Then rinse the section in distilled water. For antigen retrieval, heat sodium citrate solution for two minutes in an autoclave.

Then place the tissue slice in the solution and heat it at a high temperature for five minutes. Once cooled down, incubate the section in a 3%hydrogen peroxide solution for 15 minutes, at room temperature, away from light. Then apply 10%goat serum evenly on the tissue section and incubate for 30 minutes at room temperature to block the section.

After shaking off the blocking solution, apply an appropriate amount of the prepared primary antibody to each section. Incubate the sections laid flat in a wet box overnight at 4 degrees Celsius. On the next day, cover the tissue with a drop of the HRP labeled secondary antibody, and incubate it at room temperature for 50 minutes.

Then apply freshly prepared DAB chromogenic solution to each section and wait till color development. Restain the section with hematoxylin for approximately one minute. Subsequently, rinse the sections for 10 minutes with tap water until they return to a blue color.

For dehydration, place each section sequentially in gradients of ethanol and xylene for five minutes each. Once all the sections are dried slightly, seal each section with neutral gum. Immunohistochemical results showed that B cell markers CD20 and CXCR5 expression were significantly higher in the IgA neuropathy model group compared with the control group.

However, dioscin could inhibit the expression of the molecular markers. To begin, seed DAKIKI cells in 96 well plates at a density of 0.4 million cells per well. Set up the different experimental groups, and after the corresponding treatments based on the grouping method, incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide.

For the lactate dehydrogenase cytotoxicity assay, add 5 microliters of lysate to each well in the high control group, and incubate the plate at 5%carbon dioxide and 37 degrees Celsius for 15 minutes. Then add 100 microliters of the reaction mixture to each well. To quench the reaction, add 50 microliters of the stop reaction solution per well.

For the Cell Counting Kit-8 or CCK-8 assay, after the initial 24 hour incubation, add 20 microliters of CCK-8 reagent to each well and return the plates to the incubator at 5%carbon dioxide and 37 degrees Celsius for two hours. The results of the lactate dehydrogenase cytotoxicity assay showed insignificant cytotoxicity induced by dioscin at concentrations of 0.25 to 1.0 micrograms per milliliter, which had lactate dehydrogenase release rates below 10%CCK-8 assay showed that compared with the model group, dioscin inhibited LPS-induced DAKIKI cell proliferation in a concentration-dependent manner with the concentrations of 0.5 and 1.0 micrograms per milliliter showing significance inhibition. To begin, seed DAKIKI cells in a six-well plate at a density of 6 million cells per well.

Set up the different experimental groups, and after the corresponding treatment based on the grouping method, incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Extract total RNA from the DAKIKI cells, following the instructions in the total RNA extraction kit. Take one microliter of the extracted RNA from each group of samples to measure its concentration.

Reverse transcribe 1 microgram of total RNA from each sample into complimentary DNA following the kit instructions. Perform reverse transcription polymerase chain reaction or RT-PCR amplification to detect the expression of each gene. For western blotting after the initial 24-hour incubation, collect the cells from each group.

Add an appropriate volume of cell lysis solution to the collected cells before incubating them on ice for 30 minutes. Centrifuge the samples at 13, 500 G for 10 minutes at 4 degrees Celsius and collect the supernatant for further analysis. Vortex the protein samples with 5X SDS-PAGE loading buffer at a ratio of 4 to 1, and heat the mixed samples at 100 degrees Celsius for five minutes to denature the protein before electrophoresis.

Once the run is complete, transfer the gel onto the PVDF membranes. To block the membrane, incubate it in 5%non-fat milk for two hours, at room temperature. Then incubate the membrane with the corresponding primary antibodies for 24 hours while using beta actin antibody as an internal control.

Subsequently, incubate the membranes with the corresponding secondary goat anti-rabbit IgG antibody at room temperature, for two hours. Finally, treat the membranes with an appropriate amount of enhanced chemiluminescence working solution for protein band detection. The quantitative RT-PCR results showed that the relative mRNA expression of C1GalT1 and Cosmc was downregulated in DAKIKI cells in the model group compared with the control group.

Dioscin upregulated the relative mRNA expressions of both to different degrees compared with the model group. Western blotting results also showed that compared with the control group, the protein expression of C1GalT1 and Cosmc in DAKIKI cells in the model group decreased. The protein expressions were upregulated after dioscin intervention.