Qingre huazhuo tang regulates IgA nephropathy through immune checkpoints and ferroptosis

Abstract

Background

Qingre huazhuo tang (QRHZT) is a traditional Chinese medicine decoction that has been used clinically by traditional Chinese medicine masters for more than 50 years with good results. However, its mechanism is unknown, and further elucidation is necessary.

Aim of the study

To verify the mechanism by which QRHZT regulates IgA nephropathy through immune checkpoints and ferroptosis by combining network pharmacology, single-cell sequencing and experimental studies.

Materials and methods

The single-cell sequencing data obtained from podocytes of immunoglobulin A (IgA) nephropathy (IgAN) patients were screened, and the QRHZT target information was obtained from the ETCM database. Moreover, the KEGG, GSEA, immune checkpoint and ferroptosis data were analyzed and plotted using R language. Finally, the relevant immune checkpoint and ferroptosis targets were validated experimentally.

Results

QRHZT can regulate IgAN through the immune checkpoints FIT, FTH1, AKR1C3, and IL6 and the ferroptosis-related genes PVR and IFNG.

Conclusion

QRHZT has the potential to regulate IgAN, and omics strategies combined with network pharmacology is a feasible method for exploring the mechanisms of traditional Chinese medicines.

Graphical abstract

Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerular disease among Chinese children, with hematuria and/or proteinuria being the primary clinical manifestations.¹ In addition, 25 %–40 % of IgAN cases progress to end-stage renal disease within 20–25 years.² The main mechanism of IgAN pathogenesis is currently believed to the deposition of IgA immune complexes in the mesangial glomerulus caused by mucosal infection on the basis of genetic factors, and infection control, hormones and immunosuppressants are the main treatment methods. However, these treatment modalities also come with adverse effects, such as immunosuppression and liver damage.³ As the main branch of complementary and alternative medicine, traditional Chinese medicine (TCM) has long been considered to have the unique advantages of excellent curative effects, good economic benefits, high safety and few adverse reactions. In recent decades, TCM has been widely used in the prevention and treatment of kidney diseases, and a series of evidence-based medicines have been developed.⁴ The multitarget, multimechanism, and multipathway approach of TCMs to restore the human body to its normal state can often achieve exceptional results. For example, TCM can reduce protein in the urine of individuals with IgAN by modulating miRNA-223 and NLRP3⁵ and regulate sphingosine-1-phosphate to attenuate the expression of renal fibrosis markers, inflammation and oxidative stress in IgAN rats.⁶ Moreover, artemisinin can protect against IgAN by inhibiting the NF-κB pathway and the NLRP3 inflammasome.⁷ However, the multicomponent, multitarget, and multimechanism characteristics of certain TCMs have not been fully characterized, and most studies have focused only on a single mechanism and a single target. These shortcomings necessitate the use of new methods to conduct in-depth explorations of the integrated regulatory mechanisms of TCMs to provide additional evidence to aid in the further development of TCMs.

To further explore how TCM can be applied for IgAN treatment, in this study, the effects of Qingre huazhuo Tang (QRHZT), which has been used clinically for 50 years by Chinese masters of TCM, were evaluated. Moreover, network pharmacology, single-cell sequencing and experimental studies were combined to verify the combined mechanism of action of the multiple QRHZT pathways.

1. Materials and methods

1.1. Composition and targets of QRHZT

The components of QRHZT include unprocessed rehmannia root, yerbadetajo herb, India madder root, baical skullcap root, field thistle herb, and licorice root. The targets and components were obtained from the recognized TCM database ETCM database (http://www.tcmip.cn/ETCM/); thus, the resulting drug data are relatively comprehensive and reliable.⁸ The components and targets of QRHZT are shown in Supplementary Material 1, and the main 10 components of each herb are also listed in Table 1.

Table 1.

Main components of QRHZT.

Herb	Ingredient	Herb	Ingredient
unprocessed rehmannia root	Stachyose	baical skullcap root	Sitosterol,Î′-Sitosterol
unprocessed rehmannia root	D-Mannitol,Cordycepic Acid	baical skullcap root	Stigmasterol
unprocessed rehmannia root	Alexandrin, Daucosterol,Caproic Acid, Eleutheroside A,Sitogluside, Strumaroside,Î′-Sitosterol-Î′-D-Glucoside	baical skullcap root	Cetylic Acid, Hexadecanoic Acid, Palmitic Acid
unprocessed rehmannia root	8-Epiloganic Acid	baical skullcap root	Sucrose
unprocessed rehmannia root	Fructose	baical skullcap root	Campesterol,M-Cresol
unprocessed rehmannia root	Geniposide	baical skullcap root	4′-Hydroxywogonin,5,7-Dihydroxy-8-Methoxylflavone
unprocessed rehmannia root	Aucuboside, Aucubin	baical skullcap root	Scutellarin
unprocessed rehmannia root	Ajugol	baical skullcap root	Wogonin
unprocessed rehmannia root	D-Glucose,Glucose	baical skullcap root	5-Hydroxy-6,7,8,4′-Tetramethoxyflavone,Skullcapflavone I,Skullcapflavone II
unprocessed rehmannia root	Raffinose	baical skullcap root	Oroxylin A
licorice root	Sitosterol,Î′-Sitosterol	yerbadetajo	2-(Buta-1,3-diynyl)-5-(4-chloro-3-hydroxybut-1-ynyl) thiophene
licorice root	Rutin, Rutoside,Vitamin P	yerbadetajo	alpha-Terithenyl acetate
licorice root	Isoquercitrin, Isoquercetrin,Kuwanon H	yerbadetajo	alpha-Terthienyl methanol
licorice root	Nicotiflorin	yerbadetajo	Butein
licorice root	Corylifolinin, Isobavachalcone	yerbadetajo	Butin
licorice root	(S)-5,7-Dihydroxy-2-Phenylchroman-4-One,Pinocembrin	yerbadetajo	Chloromaloside
licorice root	Lupiwighteone	yerbadetajo	Demethylwedelolactone-7-glucoside
licorice root	3,3′-Dimethylquercetin	yerbadetajo	Demissine
licorice root	3-O-Acetyl-Glycyrrhetinic Acid	yerbadetajo	Ecliptasaponin B
licorice root	6,8-Bis(C-Î′-Glucosyl)-Apigenin,Vicenin-2	yerbadetajo	Edulinine
India madder root	2-Methylanthraquinone	field thistle	Sitosterol,Î′-Sitosterol
India madder root	1-Hydroxy-2-Methoxyanthraquinone,Alizarin-2-Methylether	field thistle	Guercetol, Quercetin,Quercetin, Sophoretin,Meletin, Xanthaurine
India madder root	3Î′-Acetoxyolean-12-En-28-Oic Acid	field thistle	Caffeic Acid
India madder root	Nordamnacanthal	field thistle	Rutin, Rutoside,Vitamin P
India madder root	Alizarin	field thistle	Stigmasterol
India madder root	2-Carboxymethyl-3-Phenyl-2,3-Epoxy-1,4-Naphthoquinone	field thistle	3′-Caffeoylquinic Acid,5′-Caffeoylquinic Acid, Chlorogenic Acid, Heriguard
India madder root	Digiferrugineol	field thistle	3,4-Dihydroxybenzoic Acid, Protocatechuic Acid
India madder root	4-Hydroxy-2-Carboxyanthraquinone	field thistle	Tyramine
India madder root	1-Hydroxy-2-Carboxy-3-Methoxyanthraquinone	field thistle	Ψ-Taraxasteyl Acetate
India madder root	1-Hydroxy-2-Methyl-6-Methoxyanthraquinone	field thistle	Triacontanol, Thea Alcohol A

1.2. Single-cell sequencing and enrichment analysis

The single-cell sequencing data were obtained from the publicly available GEO dataset GSE127136, which includes information from 3620 samples, and the text of the source study was reviewed in detail.⁹ To improve the stability and interpretability of the expression estimates, the original data were imported into R software (version 4.2.1), and then the DESeq2 package was used for analysis. The obtained total cell cluster was the same as the original author's analysis. Podocytes from the normal and IgAN groups were selected for differential expression analysis, and the ggplot package was used to visualize the results. After intersection analysis of the differentially expressed genes and QRHZT targets was conducted, expression analysis of factors related to immune checkpoints and ferroptosis was performed. Thirty-nine immune checkpoint genes were identified in previous studies.^10,11

1.3. Enrichment analysis

KEGG analysis was performed using the clusterProfiler package in R, volcano plots were drawn in ggplot, heatmaps were constructed using pheatmap, and GSEA was performed using the gseahallmarks gene set, applying the enrichplot package for data visualization. The ferroptosis genes were obtained from the FerrDb database (http://www.zhounan.org/ferrdb/).

1.4. Cell experiments

1.4.1. Drugs and materials

The ingredients of QRHZT (unprocessed rehmannia root, yerbadetajo herb, India madder root, baical skullcap root, field thistle herb, and licorice root) were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. The high-performance liquid chromatography–tandem mass spectrometry (HPLC‒MS/MS) results for all the drugs are shown in Supplementary Material 2. The components of QRHZT were mixed in a ratio of 15:10:10:10:10:6 and dissolved and diluted in dimethyl sulfoxide (DMSO) to generate solutions with concentrations of 4 μg/ml, 8 μg/ml, and 16 μg/ml for use. RIPA lysis buffer (catalog number: MD912016), a BCA protein concentration determination kit (catalog number: MD913053), a SDS‒PAGE precast gel kit (catalog number: MD911919), the PVDF membranes (Millipore, USA; ISEQ00010), and a medium protein molecular weight marker (26617) were purchased from Thermo, USA, and the AKR1C3 antibody (ab192865) and IgA1 protein (ab91020) were purchased from Abcam, USA. Sialidase (11080725001) and β-galactosidase (G5635) were purchased from Sigma‒Aldrich, USA. Ferritin heavy chain 1 (FTH1; #3998) was purchased from Cell Signaling Technology, whereas ferritin light chain (FTL; CSB-PA11409A0Rb) was purchased from Wuhan Huamei Bioengineering Co., Ltd. Moreover, a SDS‒PAGE system (U.S. Bio-Rad Company, model Mini-PROTEAN), wet protein transfer instrument (U.S. Bio-Rad Company, model Mini Trans-Blot), and Western blot imaging system (U.S. Bio-Rad Company, model 170–8280) were used.

1.4.2. Cells and models

Human kidney podocytes were purchased from Shanghai Xuanke Biotechnology Co., Ltd. and cultured in RPMI 1640 supplemented with 10 % fetal calf serum (FCS) at 37 °C with 5 % CO2. The medium was changed every 2 days. When cell confluency was greater than 80 %, 0.25 % trypsin was added for digestion and passage. After the cells were passaged to obtain a sufficient number, they were grouped as follows. According to previous studies,^12,13 sialidase and β-galactosidase (Sigma, G5635) were added to IgA1 for incubation at 37 °C for 6 h to yield desialylated and degalactosylated IgA1. Then, 50 μl of IgA1 (1 μg/ml) was added to the human kidney podocytes in the model group, which were incubated for 6 h. Additionally, 4 μg/ml, 8 μg/ml or 16 μg/ml QRHZT was added to the QRHZT groups, and the changes in various indicators were detected.

1.4.3. Detection methods

After the cells in each group were centrifuged, RIPA lysis buffer was added, and the cells were shaken for 5 min and centrifuged at 12000 rpm for 15 min. Then, the protein concentration in the supernatant was measured via a BCA assay. After the addition of loading buffer with mixing, the samples were boiled in a water bath for 5 min and subjected to SDS‒PAGE. The proteins were transferred to PVDF membranes via the wet transfer method, and 5 % skim milk powder was added for blocking at room temperature for 2 h. The membranes were incubated with primary antibody overnight at 4 °C. After the membrane was washed with TBST, the secondary antibody was added, and the mixture was incubated at 37 °C for 1 h. After the membrane was washed with TBST again, color development was performed. The enzyme-linked immunosorbent assay (ELISA) kits for IL-1β, IL-6, and INF-γ, purchased from Thermo Fisher Technology Co., Ltd. (China) were used according to the manufacturer's instructions.

1.5. Statistical analysis

The data are expressed as the mean ± standard deviation (SD) (n > 3) and were analyzed with GraphPad Prism 8.0 (GraphPad Software) via a t-test and one-way ANOVA followed by Tukey's post hoc multiple comparison test. ∗∗, p < 0.05.

2. Results

2.1. Basic analysis of the podocytes in the normal and IgAN groups

After reanalysis of the original podocyte data, in total, information from 4 normal samples and 22 IgAN samples were obtained. Principal component analysis (PCA) dimensionality reduction analysis was performed using the plotPCA function of the DESeq2 package in R, as shown in Fig. 1A. Four groups of podocytes were found to separate the normal group from the IgAN group. To exclude nonexpressed and invalid genes, logFC correction was performed. Fig. 1B shows the gene data before correction, and Fig. 1C shows the corrected gene data. Notably, the corrected data are more concentrated, and the errors due to abnormal expression are excluded.

Fig. 1.

PCA dimensionality reduction of the healthy and IgAN groups and subsequent data correction. A PCA dimensionality reduction analysis performed with the plotPCA function of the DESeq2 package in R software. Green represents the normal group, and red represents the IgAN group. B and C Gene correction results of both groups. B Before correction and C after correction.

2.2. Analysis of the differentially expressed genes between the podocytes in the normal group and those in the IgAN group

A heatmap was constructed to further analyze the differentially expressed genes between the normal group and the IgAN group. As shown in Fig. 2A, the normal group and the IgAN group could be clearly distinguished. Similarly, the volcano plots in Fig. 2B show the significantly differentially expressed genes. The KEGG enrichment results (Fig. 2C) indicated that the degradation of valine, leucine and isoleucine; carbon metabolism; and glutathione metabolism were the top three pathways in which the differentially expressed genes were enriched. Leucine, isoleucine, and valine are all branched chain amino acids, which are the main energy-supplying amino acids in the body.¹⁴ Carbon metabolism, the most basic aspect of life,¹⁵ is involved in major biological pathways, including the hydroxypropionate-hydroxybutyrate cycle and the dicarboxylic acid-hydroxybutyrate cycle. Moreover, glutathione is a tripeptide composed of glutamic acid, cysteine and glycine that contains amide bonds and sulfhydryl groups and is present in almost every cell of the body. Glutathione helps maintain normal immune system function and has endogenous antioxidant and detoxification effects.¹⁶ The GSEA enrichment results (Fig. 2D) show that protein secretion was the main function of these cells. Cotransporters are present on the apical surface of epithelial cells and transport glucose, amino acids, phosphate, lactate, citrate and sodium ions into epithelial cells. In addition, sodium can enter epithelial cells through Na+/H+ exchangers on the apical surface of epithelial cells. Epithelial–mesenchymal transition (EMT) is the process by which polar epithelial cells transform into active mesenchymal cells and acquire the abilities to invade and migrate. EMT is involved in many physiological and pathological processes in the human body.¹⁷ The induction of EMT involves multiple signal transduction pathways and complex molecular mechanisms related to calcein, growth factors, transcription factors, and the microenvironment. EMT is also closely related to the invasion and metastasis of tumor cells.

Fig. 2.

Differentially expressed gene expression and functional enrichment between the normal group and IgAN group. A Heatmap, B volcano map, C KEGG enrichment results, and D GSEA enrichment results.

2.3. Analysis of ferroptosis and immune checkpoints in the normal and IgAN groups

The differentially expressed genes between the normal group and the IgAN group were further analyzed. As shown in Fig. 3A, a significant difference in “suppressor in ferroptosis” was detected, implying that ferroptosis was significantly suppressed in the IgAN group. To determine whether QRHZT has a role in regulating these suppressor genes, we intersected the QRHZT targets with the differentially expressed suppressor genes. After manual proofreading, three closely related genes were significantly differentially expressed, FTL, FTH1 and ACSL3, as shown in Fig. 3B. Fig. 3C shows the differentially expressed genes that intersect with immune checkpoints, while the two genes in Fig. 3D intersect with the QRHZT targets on the basis of Fig. 3C. The above results confirmed that the targets of QRHZT are related to both ferroptosis suppression and immune checkpoints. To verify the role of QRHZT in ferroptosis and immune checkpoints, further experimental verification was conducted.

Fig. 3.

Analysis of ferroptosis and immune checkpoints in the healthy and IgAN groups. A Level of ferroptosis. B Genes whose expression significantly differed after intersection of the QRHZT targets and suppressor genes and manual proofreading. C Immune checkpoints that intersect with the differentially expressed genes. D Immune checkpoints that intersect with the QRHZT targets on the basis of the data in C.

2.4. Experimental verification

To mimic the pathogenesis of IgAN, IgA1 was used to stimulate renal podocytes, after which the levels of the inflammatory factors IL-1β, IL-6 and INF-γ were significantly increased, as shown in Fig. 4A–C. However, QRHZT treatment caused the levels of these factors to decrease significantly. The levels of the ferroptosis- and immune checkpoint-related proteins AKR1C3, FTL and FTH1 also increased significantly after IgA1 stimulation, but these changes were reversed by intervention with QRHZT, as shown in Fig. 4D.

Fig. 4.

QRHZT regulates podocytes through the ferroptosis pathway and immune checkpoints. A, B, C, D, and E indicate the control, model, QRHZT 4 μl, QRHZT 8 μl, and QRHZT 16 μl groups, respectively.

3. Discussion

3.1. QRHZT regulates IgAN through ferroptosis and immune checkpoints

In this study, QRHZT was found to affect the degradation of FTH1/FTL (the ferritin component) through autophagy, ultimately increasing the level of iron.¹⁸ Moreover, the levels of AKR1C3 and IL6 were also altered. The ligand PVR can bind to the receptor TIGIT, which is expressed on T and NK immune cells, and inhibit the killing function of immune cells. Interferon-γ (IFNG) can enhance immune function, but it also promotes T-cell exhaustion through PD-L1. Experimental studies have shown that the expression of interferon-γ is reduced after QRHZT intervention. These findings indicate that QRHZT can regulate IgAN disease progression through ferroptosis and immune checkpoints. The mechanism of ferroptosis, a newly identified type of iron-dependent regulated cell death, is dependent on iron and involves the peroxidation of the unsaturated fatty acids that are highly expressed on the cell membrane, which induces cell death. Immune checkpoints are receptors that bind to their respective antibodies and immune cell surface receptors to further activate or inhibit immune cells. Although these two mechanisms seem unrelated, they are actually closely related. CD8⁺ T cells activated by immunotherapy release IFN-γ, which downregulates SLC3A2 and SLC7A11, resulting in a decrease in antioxidant capacity and increased ferroptosis.¹⁹ PD-L1 is also positively correlated with the expression of most ferroptosis regulatory factors (such as ACSL4, CARS, and NCOA4) but negatively correlated with the expression of HSPB1, MT1G, RPL8, and GPX4. After damage-associated molecular patterns, lipid metabolites, cytokines, and chemokines are released during ferroptosis to induce immunogenic death in tumor cells, these antigens can be directly presented to effector T cells to promote their activation and infiltration.^20,21 Notably, IFN-γ is the "intersection" of ferroptosis and immune checkpoints. PD-L1 inhibitors can promote the release of IFN-γ, and IFN-γ can inhibit the expression of SLC7A11, thereby promoting ferroptosis.²² In this study, QRHZT first increased ferroptosis by inhibiting the ferroptosis-related proteins FTH1, FTL, and AKR1C3, accelerating abnormal cell death. Moreover, QRHZT targets the immune checkpoint receptor PVR to regulate the immune response. Notably, the single-cell sequencing data in this study showed that IFNG (also known as IFN-γ) expression was reduced in the IgAN group, whereas in the experimental group (Fig. 4C), IFN-γ expression increased, possibly because cells undergoing ferroptosis can activate interferon-related signals to release more IFN-γ. In summary, the results of this study revealed that QRHZT has multiple effects in IgAN, including promoting ferroptosis, regulating immune checkpoints, and reducing the release of inflammatory factors.

3.2. Combining omics strategies and network pharmacology is a novel way to explore drug mechanisms or discover new drugs

Omics technology is based on systems biology, among which genomics, transcriptomics, proteomics, and metabolomics are widely used. Collectively, these technologies are referred to as the four omics technologies. As research progresses, these technologies will become more refined. As a novel method, single-cell sequencing overcomes the limitation of traditional sequencing methods that measure the average expression of genes in a cell population, as the traditional methods do not obtain information on heterogeneity. Single-cell sequencing has been widely used in biomedical research fields, such as those that study tumors, microbiology, neurobiology, genetic diseases, immune diseases, COVID-19, and epigenetics. The findings here show that the continuous development of omics technology has played an important role in the promotion of medical progress. Network pharmacology is another research method that is widely used to explore new drugs and new pharmacological mechanisms, especially TCMs. The combination of these two methods can elucidate the specific mechanism of a TCM at the cellular level, which is the ultimate goal during TCM development. From a data perspective, omics data such as those from genomics, transcriptomics, proteomics, metabolomics, and single-cell sequencing, can reveal the molecular changes that occur during both the development of a diseases and the responses after drug intervention. These data can be integrated with network pharmacology analysis to construct a more accurate and comprehensive drug‒target‒disease network. Network construction analysis, node analysis, and module analysis of this network can help researchers extract key information from complex multiomics data and identify potential drug targets and biomarkers. In addition, network pharmacology can also provide guidance and verification for multiomics research by predicting the potential mechanisms of action and side effects of drugs.

The combination of omics strategies and network pharmacology can also be used to determine the active ingredients and targets of TCMs by analyzing the effects of the TCM on metabolites in organisms; clarifying the mechanism of action of the TCM in multiple biological systems and molecular pathways; and revealing the scientific connotations of the TCM more deeply and improving the clinical efficacy and safety of the TCM. After combining the functions of predicting the interactions between drugs and targets via network pharmacology and integrating the multiomics data, the potential mechanisms of action of new drugs can be explored, guidance for the development of new drugs and the design and optimization of existing drugs can be provided, and the efficiency of new drug development can be improved.

3.3. Further research on QRHZT is needed

The most well-known Chinese medicine practitioners in China, known as the masters, have at least 50 years of medical experience. Their methods and logic of using TCM to treat diseases are excellent, and their experience is highly respected by the Chinese government. QRHZT is a clinically effective prescription for IgAN that has been used for more than 50 years. Although this study focused on immune checkpoints and ferroptosis for IgAN treatment, shortcomings remain. First, the most notable limitation is the possible differences in the single-cell sequencing results and the actual measurement data of the ferroptosis-related proteins NQO1, ACSL3 and IL6. The most critical reason for these differences may be that the sample size of the normal group was too small, leading to certain errors in the omics results. Therefore, further research on the regulation of IgAN by QRHZT is still needed, which would help to elucidate the modern scientific basis of well-known, old TCM prescriptions.

4. Summary

On the basis of the combination of single-cell sequencing data and network pharmacology analysis, in this study, the potential mechanism of QRHZT, a Chinese traditional medicine master's empirical formula, in the treatment of IgAN was explored. Certain ferroptosis suppressor and immune checkpoint genes, such as PVR and IFNG, seem to be affected by QRHZT. In conclusion, single-cell sequencing combined with network pharmacology is a feasible method for exploring the mechanisms of TCMs.

Authors' contributions

Yan Xu and Shanshan Han designed the experiments. Ting Guo, Yudi Li and Donglin Li performed the experiments and collected the data. Yan Xu and Ying Ding wrote the manuscript. All the authors read and approved the final manuscript.

Funding

This work was supported by the Henan Province Postdoctoral Project (HN2022096), the National Natural Science Foundation of China (82205190), the China Postdoctoral Science Foundation General Project (2023M731027), Special Grant from China Postdoctoral Science Foundation (2024T170253), and the Henan Provincial Health Commission National Traditional Chinese Medicine Inheritance and Innovation Center Scientific Research Special Project (2023ZXZX1073).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Material 1: Components and targets of QRHZT.

Supplementary Material 2: HPLC‒MS/MS analysis of QRHZT.