Diabetic nephropathy (DN), a serious microvascular complication of type 1 and type 2 diabetes, is characterized by proteinuria and persistent renal function injury [1]. Proteinuria, an independent risk factor of disease progression, is the most important clinical characteristic of DN, and is also the leading cause of end-stage renal disease [2, 3]. Without early intervention, 50% of patients with microalbuminuria will progress to macroalbuminuria [4]. There are many risk factors for the development of DN including increased inflammatory factors and oxidative stress, changes in fat and protein metabolism, and overexpression of the renin-angiotensin-aldosterone system. These lead to the apoptosis and loss of renal podocytes and decreased filtration capacity of glomeruli, thus aggravating DN [5, 6]. Although several recent studies have confirmed that angiotensin-converting enzyme inhibitors/angiotensin receptor blockers can reduce DN proteinuria and play a role in delaying disease progression, they are ineffective in DN patients with normal blood pressure [7]. Due to the limitations of traditional Western medical approaches, some DN patients have turned to alternative treatments such as traditional Chinese medicine (TCM).

Triptolide (TP) is a component of the following traditional Chinese herbal medicines: Tripterygium wilfordii Hook. F., Tripterygium hypoglaucum Levl. Hutch, Tripterygium regerii Sprague et Takeda, and Tripterygium forretii Dicls [8]. It is also a major active component of the colquhounia root tablet and tripterygium glycoside tablet, which have long been used in China to treat DN due to their marked anti‑inflammatory, anti-proteinuria, and podocyte‑protective effects [9, 10, 11]. Several randomized controlled clinical trials have indicated that TP possibly imparts nephroprotective effects by decreasing proteinuria, serum creatinine levels, and blood urea nitrogen levels [12, 13, 14]. Although TP is effective for improving renal function and reducing proteinuria in DN, the exact mechanism is still unclear.

Network pharmacology is a research method based on virtual computing technology, high-throughput data, and public database, which combines system computing with experiments, introducing a new field of pharmacology [15, 16]. Network pharmacology constructs a multi-level network of disease-phenotype-gene-drug through multi-target interaction [17]. Through analysis of the overall network, we can better predict drug targets to provide help for the research and design of new drugs [18]. For TCM, each component in its prescription has its target, and the effect of the drug is often the result of the synergistic effects of multiple component targets [19]. Network pharmacology can reveal the role of various components at the molecular level so that people can make better use of TCM [20].

In this study, we used network pharmacology to identify the potential targets and signaling pathways of the TCM component TP for the treatment of DN and revealed its possible mechanism. Fig. 1 shows a flowchart of the online pharmacological processes of this study.

Fig. 1.

Network pharmacological workflow to determine the potential mechanisms and targets of triptolide in the treatment of diabetic nephropathy.

DN is a chronic kidney disease and the leading cause of end-stage renal disease in most developed countries [5]. The causes of DN are complex, but inflammation and oxidative stress are known to be involved in its progression [5, 33]. As a new approach, network pharmacology can well analyze the overall relationship between drugs and diseases, including how to participate in the therapeutic process [34]. TP markedly attenuates albuminuria and podocyte injury, regulating the T helper cell balance and macrophage infiltration in an animal model of DN [35, 36]. To elucidate the possible mechanism and potential targets of TP in the treatment of DN, we constructed and analyzed the targets through network pharmacology and molecular docking.

Among the 113 TP- and DN-related targets, 7 targets were found to play an essential role through network analysis including ALB, AKT1, CASP3, EGFR, STAT3, TNF, and TP53. Individually, ALB functions as an intravascular transporter, which not only binds a variety of ions, hormones, and drugs but also stabilizes osmotic pressure, anti-inflammation, and antioxidation [37]. When the concentration of glucose is too high, glycosylation of ALB occurs [38]. After additional events, glycosylated ALB further forms AGEs and stimulates cells to produce oxidative stress, thus damaging cells [39]. CASP3 belongs to the family of cysteine proteases and is an essential factor in regulating apoptosis [40]. High glucose can stimulate mitochondria, release cytochrome C, and increase the expression of CASP9 and CASP3 [41]. At the same time, activation of CASP12 and CASP3 through endoplasmic reticulum stress can also be independent of mitochondria, resulting in apoptosis [42]. EGFR is an important receptor tyrosine kinase, which is closely related to the development of DN and is widely distributed in glomeruli and renal tubules [43]. EGFR can be activated by high glucose and Src kinase, mediating the phosphorylation of Akt, stimulating a large number of reactive oxygen species (ROS), and inducing the MAPK signal pathway all of which leads to the release of inflammatory factors and reduces insulin secretion in islet cells, resulting in insulin resistance [44]. The Janus kinase/STAT pathway can be activated by high glucose and ROS, and is involved in the pathogenesis of DN [45]. After being phosphorylated, STAT3 enters the nucleus to stimulate the transcription of target genes, increasing the expression of inflammatory and fibrosis factors [46]. Inhibiting the activity of STAT3 can decrease TNF-$\alpha$ and interleukin beta 1 (IL-b1) levels, ameliorating renal fibrosis [47]. In diabetic patients, the levels of inflammatory factors are significantly elevated [48]. As an inflammatory factor, TNF can greatly promote the development of DN and damage the glomerular filtration barrier [49]. Moreover, it can bind to insulin-like growth factor binding protein-3 to induce the apoptosis of mesangial cells [50]. TP53 is a tumor suppressor, which regulates the apoptosis of podocytes [51]. After phosphorylation, AKT1 activates the MAPK signaling pathway, releasing a large number of inflammatory factors and causing renal fibrosis [52].

A total of 113 common targets of TP and DN were identified by using network pharmacology, and the binding ability of TP to these targets was verified by molecular docking experiments. After KEGG enrichment analysis, six pathways were found to play a key role in the therapeutic effect of TP on DN (Fig. 10). The MAPK signaling pathway is a classic inflammatory pathway, which is composed of p38-MAPK, c-Jun N-terminal kinase 1 (JNK1), and extracellular signal-regulated kinase 2 (ERK2) [53]. When stimulated by ROS, p38-MAPK, JNK, and ERK release signaling factors such as ATF1/2 and c-Jun that mediate the transcription of transforming growth factor beta (TGF-$\beta$), IL-1$\beta$, fibronectin, and type IV collagen, leading to nephritis, renal fibrosis, podocyte apoptosis, and proteinuria [54]. A high glucose environment can stimulate the glycosylation of serum ALB and gradually transform it into AGEs [38]. AGEs continuously accumulate and activate RAGE, leading to oxidative stress, activation of the MAPK pathway, chronic inflammation, and eventually renal injury [55, 56, 57]. The PI3K/Akt pathway can activate the nuclear factor kappa B (NF-$\kappa$B) pathway, increasing the expression of IL-6 and leading to glomerular basement membrane thickening and mesangial expansion [52]. Meanwhile, Akt can phosphorylate FOXO3a in the FOXO signaling pathway, causing extracellular matrix hyperplasia [58]. Relaxin, a member of the insulin family, has vasodilatory and antifibrotic effects. Activation of the relaxin pathway inhibits SMAD2 activation and TGF-$\beta$ production, reducing synthesis of the extracellular matrix (ECM) [59]. When FOXO3a is phosphorylated by Akt, the expression of bisindolylmaleimide and manganese superoxide dismutase decreases, resulting in ECM accumulation and accelerating the occurrence of DN [60]. Meanwhile, activation of the TNF pathway will elevate the expression of ROS, leading to the altered permeability of the capillary wall and triggering proteinuria [61]. Generally, the results showed that TP could improve DN via its anti-inflammatory, anti-renal fibrosis, anti-oxidant, and podocyte-protective effects.

Fig. 10.

Schematic diagram of the underlying mechanism of TP in the treatment of DN. High glucose stimulates the EGFR receptor, which activates the PI3K / Akt signaling pathway, which in turn mediates the transcription of genes via the MAPK pathway, leading to TGF-$\beta$, Collagen IV, fibronectin as well as TNF-$\alpha$, IL-1$\beta$ of the levels rise. The inflammatory factors will trigger ECM and EMT, causing nephritis, renal fibrosis, proteinuria, meanwhile, inflammatory factors will also bind to the corresponding receptors to stimulate cells to release more inflammatory factors. All events ultimately initiate DN.

The data used to support the findings of this study are available from the corresponding author upon request.

ACEIs, Angiotensin-converting enzyme inhibitors; AGEs, Advanced glycation end products; ALB, Albumin; ASPL, Average shortest path length; ARBs, Angiotensin receptor blockers; BC, Betweenness centrality; CASP3, Caspase-3; CC, Closeness centrality; DN, Diabetic nephropathy; EGFR, Epidermal growth factor receptor; EMT, Epithelial mesenchymal transition; ER, Endoplasmic reticulum; ERK2, Extracellular signal-regulated kinase; ESR1, Estrogen receptor; HG, High glucose; HO-1, Heme oxygenase-1; JAK, Janus kinase; MAPK, Mitogen-activated protein kinase; NF-$\kappa$B, Nuclear factor kappa B; RAGE, Receptor for advanced glycation end products; ROS, Reactive oxygen species; STAT3, Signal transducer and activator of transcription 3; SRC, SRC proto-oncogene, non-receptor tyrosine kinase; TP53, Tumor protein p53; TGF-$\beta$, Transforming growth factor-$\beta$; TNF, Tumor necrosis factor; VEGF, Vascular endothelial growth factor.

MY conceived the study, MY and DF designed research; XA, DF, YZ, RT, and JZ performed research; DF, ZY performed data analysis; MY and DF prepared all figures and wrote the manuscript; MY edited the final and prepared the revised manuscript. All authors read and approved the final manuscript.

Not applicable.

We thank Darren Chu from Rutgers University for his linguistic assistance in proofreading this manuscript.

This work was supported by the National Natural Science Foundation of China (81774248, 82074359), the National Integrative Medicine Key Training Project (2019-44), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Open Projects of the Discipline of Chinese Medicine of the Nanjing University of Chinese Medicine; the Subject of Academic priority discipline of Jiangsu Higher Education Institutions (ZYX03KF027).

The authors declare no conflict of interest.