This study provides experimental data for treating immunoglobulin A nephropathy (IgAN) with Dioscin (DIO), the active ingredient of Dioscoreae Nipponicae Rhizoma (DNR), and a paradigm for studying herbal medicine’s effects and underlying mechanisms in vivo and in vitro.
The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal immune responses, which is a critical link in the pathogenesis of IgA nephropathy (IgAN). Peyer's patch, the prominent place where B lymphocytes are transformed into IgA-secreting plasma cells, is the primary source of IgA. In addition, the lower expression of core 1β-1,3-galactosyltransferase (C1GalT1) and its molecular chaperone, C1GalT1-specific molecular chaperone (Cosmc), is related to abnormal glycosylation of IgA1 in IgAN patients. Our clinical experience shows that Dioscoreae Nipponicae Rhizoma's (DNR) herbal medicine can relieve proteinuria and hematuria and improve renal function in IgAN patients. Dioscin (DIO) is one of the main active ingredients of DNR, which has various pharmacological activities. This study explores DIO's possible mechanism in treating IgAN.The IgAN model mouse was established by mucosal immune induction. The mice were divided into the control, model, and DIO gavage groups. The glomerular IgA deposition in mice, renal pathological changes, and B cell markers CD20 and CXCR5 expression in Peyer's patch were detected by immunofluorescence and immunohistochemistry. After lipopolysaccharide (LPS) stimulation, DIO's effects on DAKIKI cells proliferation, IgA and Gd-IgA1 secretion, C1GalT1, and Cosmc expression were studied by cell counting kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA) test, quantitative real-time polymerase chain reaction (QRT-PCR), and western blotting (WB). In in vivo studies, IgA deposition accompanied by glomerular mesangial hyperplasia and increased expression of CD20 and CXCR5 in Peyer's patch in the IgAN model mouse was alleviated by DIO. In vitro studies showed 0.25 µg/mL to 1.0 µg/mL DIO inhibited LPS-induced DAKIKI cell proliferation, IgA and Gd-IgA1 secretion, and up-regulated the mRNA and protein expression of C1GalT1 and Cosmc. This study demonstrates that DIO may reduce Gd-IgA1 production by inhibiting excessive activation of IgA-secreting cells and up-regulating C1GALT1/Cosmc expression.
IgA Nephropathy (IgAN) is the most common type of primary glomerulonephritis, for which there is no specific treatment, and it remains a significant cause of end-stage renal disease1. Although the pathogenesis of IgAN is still not fully understood, the "multi-hit hypothesis" is generally accepted and supported by a large body of clinical and experimental research evidence2. The pathogenesis of IgAN involves activating B cells and producing Galactose-deficient IgA1 (Gd-IgA1)3. The increase in circulating Gd-IgA1 due to the excessive proliferation and activation of IgA-secreting cells during the mucosal immune response is a critical link in the pathogenesis of IgAN4,5,6. As the central place for the proliferation and activation of B lymphocyte phenotype conversion to IgA-secreting cells, Peyer's patch is the primary source of IgA secretion, closely related to the occurrence and development of IgAN7,8. In addition, the proliferation of IgA1-secreting cells, as well as expression of Core 1β-1,3-galactosyltransferase(C1GalT1) and C1GalT1-specific molecular chaperone (Cosmc), were associated with abnormal glycosylation of IgA1, which causes GD-IgA1 production in IgAN patients6,9.
Clinical study on IgAN treatment with herbal medicine has progressed in recent years. Yiqi Qingjie Formula is an essential formula for treating IgAN by the Department of Nephrology of Guang'anmen Hospital. The previous study of our group found that Gd-IgA1 decreased in the serum of IgAN patients after treatment with Yiqi Qingjie Formula. As one of the most used herbs in the Yiqi Qingjie Formula, Dioscoreae Nipponicae Rhizoma (DNR) is the dried rhizome of Dioscorea Nipponica Makino, which has various functions such as regulating immunity, suppressing inflammation, relieving cough and asthma10,11. Several scholars treated IgAN with DNR and achieved good results12,13,14. As the main active ingredient in DNR15, Dioscin (DIO) lowers uric acid, inhibits fibrosis, inhibits inflammatory response, and anti-oxidative stress16,17. Therefore, DIO may have a novel action mechanism to inhibit the cellular secretion of excessive Gd-IgA1 and exert specific kidney protection effects. Still, no study has been reported on DIO's action mechanism for treating IgAN.
To explore the potential therapeutic mechanism of DIO on IgAN and provide a new method for the treatment of IgAN, we carried out experiments for the therapeutic effects of DIO on IgAN in vivo and 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
2. Histological analysis
3. Immunohistochemical analysis of the Peyer's patch
4. Renal IgA immunofluorescence
5. Cell culture
6. LDH cytotoxicity assays for screening safe concentrations of DIO on normal DAKIKI cells
7. CCK-8 assay for detecting the effect of DIO on DAKIKI cell proliferation
8. ELISA for detecting the effect of DIO on the secretion of IgA and Gd-IgA1 by DAKIKI cells
9.qRT-PCR for detecting the effect of DIO on C1GALT1 and Cosmc mRNA levels in DAKIKI cells
10. Western blotting for examining the effect of DIO on the expression of C1GALT1 and Cosmc proteins in DAKIKI cells
11. Statistical analysis
Effect of DIO on kidney tissue in IgAN mice model
Compared with the control group, the mucosal immune-induced IgAN mice model (model group) had a significant increase in proteinuria (Supplementary Figure 2), IgA deposition was visible in the mesangial region, fluorescence was uniformly distributed in clusters throughout the entire mesangial region (Figure 1A), PAS staining of the renal tissue showed mesangial cells proliferation and stromal hyperplasia (Figure 1B), which was reduced in the DIO gavage group (DIO group).
Effect of DIO on B lymphocytes in Peyer's patch
Peyer's patch is the leading site of the conversion of B lymphocytes into IgA-secreting cells. We took Peyer's patch as the research object to observe the effect of DIO on B lymphocytes by detecting the expression of B cell markers CD20 and CXCR5. Immunohistochemical results showed that the expression of CD20 and CXCR5 was significantly higher in the model group compared with the control group. DIO could inhibit the expression of the above molecular markers (Figure 2A,B).
The safe concentration range of DIO on DAKIKI cells
LDH is a marker of plasma membrane integrity and an indicator of cell death, with higher LDH release rates indicating more severe cell damage. The LDH release assay was used to determine the safe concentration range of DIO. The maximum safe concentration of DIO was determined by an LDH release rate below 10%. The results (Figure 3) showed no significant cytotoxicity induced by DIO at concentrations of 0.25 to 1.0 µg/mL. Therefore, the following study used 0.25, 0.5, and 1.0 µg/mL DIO as the dosing level.
Effects of DIO on DAKIKI cell proliferation
The experimental results (Figure 4) showed that compared with the model group (LPS-stimulated group), DIO inhibited LPS-induced DAKIKI cell proliferation in a concentration-dependent manner. DIO at 0.5 and 1.0 µg/mL concentrations significantly inhibited LPS-induced DAKIKI cell proliferation (P < 0.01).
Effects of DIO on the secretory function of DAKIKI cells
Gd-IgA1 levels are closely related to the pathological process of IgAN, and total IgA is tested together as an indicator of cellular secretory function. An ELISA assay was used to detect IgA and Gd-IgA1 content in the supernatant of the DAKIKI cell culture. The results showed (Figure 5A,B) that DAKIKI cells stimulated by LPS secreted more IgA compared with the control group (P < 0.01). In comparison, DIO significantly inhibited DAKIKI cells from secreting IgA (P < 0.01) in a concentration-dependent manner. Compared with the control group, DAKIKI cells stimulated by LPS secreted more Gd-IgA1 with a statistical tendency (P < 0.10), and DIO inhibited Gd-IgA1 secretion from LPS-stimulated DAKIKI cells in a concentration-dependent manner (P < 0.05 and P < 0.01), among which DIO at 1.0 µg/mL significantly inhibited the secretion of Gd-IgA1 with the inhibited rate of 25%.
The mechanism of DIO inhibits Gd-IgA1 secretion by DAKIKI cells
To further investigate the possible mechanism of DIO inhibiting excessive Gd-IgA1 secretion by DAKIKI cells, the levels of glycosylated transferase C1GALT1 and chaperone protein Cosmc mRNA in DAKIKI cells were detected by qRT-PCR, and the results showed (Figure 6A,B) that the relative mRNA expression of C1GALT1 and Cosmc was down-regulated in DAKIKI cells in the model group compared with the control group (P < 0.01). DIO up-regulated the relative mRNA expression of C1GALT1 and Cosmc to different degrees compared with the model group, with DIO 1.0 µg /mL significantly up-regulated the relative mRNA expression of C1GALT1 and Cosmc (P < 0.05).
At the same time, the WB method was used to detect the effect of DIO on the protein expression of C1GALT1 and Cosmc in DAKIKI cells. Compared with the control group, the protein expression of C1GALT1 and Cosmc in DAKIKI cells in the model group decreased obviously (P < 0.05). Compared with the model group, the protein expression of C1GALT1 and Cosmc after DIO intervention was up-regulated. The protein expression of C1GALT1 and Cosmc was significantly up-regulated by DIO at a concentration of 1.0 µg/mL (P < 0.05) (Figure 7A–C).
Figure 1: Histopathology of the kidneys. (A) Immunofluorescence microscope. Kidney sections of mice in each group were stained with anti-IgA (green) and DAPI (blue). The above picture scale bar = 200 μm. The below picture scale bar = 50 μm. n = 6 per group. (B) Representative pictures of PAS staining of kidney tissue from mice in the Control, Model, and DIO groups. Scale bar = 30 μm. The downward arrow shows the mesangial cells and the upward arrow shows the stroma. Scale bar = 30 μm. n = 6 per group. Please click here to view a larger version of this figure.
Figure 2: Effect of DIO on B lymphocyte markers. (A) The expression of CD20 in the Peyer's patch. Scale bar = 200 μm. n = 6 per group. (B) The expression of CXCR5 in the Peyer's patch. The scale bars are in the lower right corner of the image. Scale bar = 200 μm. n = 6 per group. Please click here to view a larger version of this figure.
Figure 3. Screening of the safe concentration of DIO on DAKIKI cells. Statistical values are expressed as the mean ± SD from three independent experiments. Please click here to view a larger version of this figure.
Figure 4. Different concentrations of DIO affect the proliferation of DAKIKI cells. The data were expressed as mean ±SD. Compared with the control group, **P < 0.01; compared with the model group, #P < 0.05‚ ##P < 0.01; The results of all experiments were repeated three times. Please click here to view a larger version of this figure.
Figure 5. DIO inhibits IgA and Gd-IgA1 secretion by DAKIKI cells. (A) ELISA method detected the expression of IgA in each group. (B) ELISA method detected the expression of Gd-IgA1 in each group. The data were expressed as mean ± SD. Compared with the control group, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01; all experimental results were repeated three times. Please click here to view a larger version of this figure.
Figure 6. The mechanism of DIO inhibits excessive Gd-IgA1 secretion by DAKIKI cells. (A) QRT-PCR detected the mRNA expression of C1GALT1. (B) QRT-PCR detected the mRNA expression of Cosmc. The data were expressed as mean ± SD. Compared with the control group, **P<0.01; compared with the model group, #P < 0.05, ##P < 0.01; all experimental results were repeated three times. Please click here to view a larger version of this figure.
Figure 7. DIO affects protein expression of C1GALT1 and Cosmc in DAKIKI cells. (A) WB verified the up-regulation of protein expression of C1GALT1 and Cosmc by DIO. (B) Semi-quantitative analysis of C1GALT1 expression was performed using Image J. (C) Semi-quantitative analysis of Cosmc expression using Image J. The data were expressed as mean ±SD. Compared with the control group,*P < 0.05; compared with the model group, #P < 0.05, ##P < 0.01, all experimental results were repeated three times. Please click here to view a larger version of this figure.
Supplementary Figure 1. The schema for the in vivo model. Please click here to download this figure.
Supplementary Figure 2. Changes in proteinuria. The data were expressed as mean ± SD; n = 6 per group. Please click here to download this figure.
The characteristic pathological feature of IgAN is the deposition of IgA1 and GD-IgA1-containing immune complexes in the mesangial region of the glomerulus21,22. Reducing the formation of immune complexes can reduce renal injury and alleviate the clinical symptoms of IgAN. In an in vivo experiment, we studied the therapeutic effects of DIO on IgAN, and we found that DIO can reduce IgA deposition in the kidney of IgAN model mice. It is demonstrated that the accumulation of IgA-secreting cells in the kidney is related to the pathogenesis of IgAN23. As an important site of proliferation and activation of B lymphocytes, Peyer’s patch is an important source of IgA-secreting cells, so we examined the expression of B lymphocyte markers (CD20, CXCR5) in Peyer’s patch and found that DIO could inhibit the expression of B lymphocytes in the Peyer’s patch of IgAN mice model. These experimental results could provide a basis for applying DIO in treating IgAN.
We performed the following experiments In vitro to further investigate the action mechanism of DIO on IgAN. Firstly it has been demonstrated earlier that DAKIKI, an EBV-immortalized B cell line that secretes IgA1, part of which is GD-IgA124, is ideal for in vitro research of the drug’s mechanism of action on IgAN. We chose DAKIKI cells to investigate the molecular mechanism of DIO in the treatment of IgAN. In addition, the mucosal inflammatory immune response plays an integral role in the pathogenesis of IgAN. As mentioned above, we use LPS to stimulate DAKIKI cells, which can release pro-inflammatory factors and mediate inflammatory responses, which can better mimic the mechanism of mucosal immune responses in IgAN. The in vitro cellular model may help investigate the possibility and mechanism of other drugs for treating IgAN. The results showed that DIO inhibited the proliferation of DAKIKI cells stimulated by LPS in a concentration-dependent manner. DIO could inhibit the secretion of IgA and Gd-IgA1 in DAKIKI cells caused by LPS stimulation and up-regulate the expression of mRNA and protein of C1GalT1 and its chaperone Cosmc in DAKIKI cells, suggesting that DIO could reduce the secretion of Gd-IgA1 by up-regulating C1GALT1/Cosmc expression and thus inhibit the excessive activation of DAKIKI cells.
Key steps should be noted during the experimental procedures. The concentration of Gd-IgA1 in the DAKIKI cell supernatant is not within the detection range of the ELISA kit (1.56~100 ng/mL), and the collected supernatant must be centrifuged by an ultrafiltration tube to obtain the concentrated Gd-IgA1. Also, ensure the volume of supernatant starting from each group is the same and the final volume of concentrate obtained after ultrafiltration is the same.
In this study, we used both in vitro and in vivo methods simultaneously, which can mutually support each other in pharmacological effects and provide an example for studying the effects and their mechanisms of herbal medicine. Some things could be improved in this protocol. Firstly, We did not detect blood concentrations in the mice’s DIO gavage group; therefore, the concentration of DIO equivalent to blood concentrations is not used in vitro experiments. Secondly, only the DIO monomer, the active component of DNR, was investigated; the effects of other components of DNR on IgAN still need further study.
In conclusion, this study provides an experimental basis for treating IgAN with DIO, the active ingredient of DNR. This study established a cellular pathological model of IgAN by mimicking the mucosal immune response of IgAN both in vitro and in vivo. It gives a new idea for studying traditional Chinese medicine to prevent and treat IgAN.
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (81973675).
Anti-CD20/MS4A1 Antibody | Boster Biotechnology Company | A03780-3 | |
Antifade mounting medium | Beyotime, Shanghai, China | P0128S | |
Balb/c mice | Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. | 110322220101424000 | |
blocking serum | Solarbio, Beijing, China | SL038 | |
Bovine gamma globulin | ShangHai YuanYe Biotechnology Company | S12031 | |
C1GALT1 polyclonal antibody | Proteintech Group, Inc,USA | 27569-1-AP | |
Citrate antigen retrieval solution(50×) | Phygene Biotechnology Company | PH0422 | |
COSMC polyclonal antibody | Proteintech Group, Inc,USA | 19254-1-AP | |
Cytotoxiciy detection kit | Roche Company | 4744926001 | |
Dako REAL EnVision detection system, Peroxidase/DAB+ | Dako | K5007 | |
DAPI | Invitrogen | D1306 | |
Dioscin | National Institute For Food and Drug Control | 111707-201703 | |
DIO tablets | Chengdu No 1 Pharmaceutical Co. Ltd. | H51023866 | |
ECL working solution | Merck Biotechnology, Inc | WBKLS0100 | |
Enhanced cell counting kit-8 | Beyotime, Shanghai, China | C0043 | |
Fasking one-step removal of gene cDNA first-strand synthesis premix | TIANGEN,Beijing, China | KR118-02 | |
Glycogen Periodic acid Schiff (PAS) stain kit | BaSO Biotechnology Company | BA4080A | |
Goat anti-mouse IgA-AF488 | SouthernBiotech | 1040-30 | |
Goat anti-rabbit IgG antibody (H+L), HRP conjugated | BeiJing Bioss Biotechnology Company | BS-0295G-HRP | |
Human Gd-IgA1 ELISA kit | IBL | 27600 | |
Human IgA ELISA kit | MultiSciences (LiankeBio) | 70-EK174-96 | |
Pierce BCA protein assay kit | Thermo Scientific | 23227 | |
PMSF solution | Beyotime, Shanghai, China | ST507 | |
Proteinase K | Phygene Biotechnology Company | PH1521 | |
Rabbit anti-CXCR5 polyclonal antibody | BeiJing Bioss Biotechnology Company | bs-23570R | |
RIPA lysis buffer | Beyotime, Shanghai, China | P0013B | |
RNAsimple total RNA extraction kit | TIANGEN,Beijing, China | DP419 | |
RPMI Medium 1640 | Solarbio, Beijing, China | 31800 | |
Super-Bradford protein assay kit | CWBIO, Beijing, China | CW0013 | |
Triton X-100 | Beyotime, Shanghai, China | ST795 | |
β-Actin Rabbit mAb | Abclonal, Wuhan, China | AC026 |
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