Based on Network Pharmacology Tools to Investigate the Mechanism of Tripterygium wilfordii Against IgA Nephropathy

Abstract

Background: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerular disease and poses a global major public health burden. The preparation of Tripterygium wilfordii Hook F (TwHF) is widely applied for treating patients with Immunoglobulin A nephropathy in China, while the molecular mechanisms remain unclear. This study aimed to verify the therapeutic mechanism of TwHF on IgAN by undertaking a holistic network pharmacology strategy in combination with in vitro and in vivo experiments. Methods: TwHF active ingredients and their targets were obtained via the Traditional Chinese Medicine Systems Pharmacology Database. The collection of IgAN-related target genes was collected from GeneCards and OMIM. TwHF-IgAN common targets were integrated and visualized by Cytoscape. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine the predominant molecular mechanisms and pathways of TwHF on the treatment of IgAN. The protein-protein interaction network was constructed by the STRING online search tool, and hub genes were identified using R software. The expression of hub gene and related signaling were evaluated in TwHF-treated mice through immunohistochemistry and western blot and further validated in human mesangial cells (HMCs). In addition, Cell counting kit 8 (CCK8) and flow cytometry were used to detect the effects of TwHF on cell proliferation and cell cycle of mesangial cells. Results: A total of 51 active ingredients were screened from TwHF and 61 overlapping targets related to IgAN were considered potential therapeutic targets, GO functions and KEGG analyses demonstrated that these genes were primarily associated with DNA-binding transcription factor binding, lipid and atherosclerosis pathway. Genes with higher degrees including AKT1, CXCL8, MMP9, PTGS2, CASP3, JUN are hub genes of TwHF against IgAN. Verification of hub gene JUN both in vitro and in vivo showed that TwHF significantly attenuated JUN phosphorylation in the kidneys of IgAN mice and aIgA1-activated HMCs, meanwhile suppressing HMCs proliferation and arresting G1-S cell cycle progression. Conclusion: Our research strengthened the mechanisms of TwHF in treating IgAN, inhibition of JUN activation may play a pivotal role in TwHF in alleviating IgAN renal injury.

Keywords: IgA nephropathy; JUN; Tripterygium wilfordii Hook F; mesangial cell; network pharmacology.

Figure 1

A flow chart exploring TwHF against IgAN based on network pharmacology.

Figure 2

Network of potential targets between IgAN and TwHF ingredients. (A) Overlapping target genes between IgAN and TwHF. (B) Network of putative targets. The yellow and purple nodes stand for IgAN and TwHF, respectively. The blue nodes represent the active components of TwHF, and the red nodes represent the corresponding targets. The lines stand for interactions.

Figure 3

Functional enrichment analysis of the IgAN-TwHF ingredients common targets. (A) Top 20 GO terms and (B) Top 20 enriched KEGG pathways of targets. The color depth of the nodes refers to the adj p-value. The size of the nodes refers to the number of genes.

Figure 4

PPI network and the key genes of the IgAN-TwHF ingredients common targets. (A) The PPI network of targets was constructed using string. (B) The Top 30 key genes were obtained from the PPI network. The numbers on the bar represent the number of related nodes for each gene.

Figure 5

TGT downregulated JUN signaling in the kidney of IgAN mice. (A) Representative images and quantitative analysis of glomerular immunohistostaining with JUN and p-JUN in TGT treated mice or control mice. The average optical density was analyzed by ImageJ software. (B) Western blot analysis for JUN, p-JUN expressions in IgAN. Data are presented as mean ± SD of three independent experiments. ^bp < 0.01 vs. control group, ^dp < 0.01 vs. IgAN group.

Figure 6

Triptolide inhibited HMCs proliferation and reduced phosphorylation of JUN signaling. (A) The proliferation of HMCs with different concentrations of triptolide intervention was assessed by CCK8 assay. (B) The cell cycle of HMCs treated with 25 μg/ml aIgA1 and 50 nM triptolide was detected by flow cytometry. (C) Immunoblot analyses and quantitative determination of the protein levels of JUN, p-JUN in HMCs. Data are the mean ± SD of three independent experiments. ^ap < 0.05, ^bp < 0.01 vs. no treatment group, ^dp < 0.01 vs. only aIgA1-treated group.