Diabetic nephropathy (DN), also termed diabetic kidney disease (DKD), is a severe complication of diabetes and the leading cause of end-stage renal disease (ESRD). Its incidence is on the rise worldwide (Zhang et al., 2018a; Khan et al., 2020; Tang et al., 2021). In 2019, approximately 463 million individuals were affected by DN worldwide, and this figure is predicted to rise to 700 million by 2045. DN is characterized by a gradual increase in urine albumin, elevated blood pressure, and decreased glomerular filtration rate (Lu et al., 2021b). At present, the primary therapeutic strategy for DN focuses on the control of glucose and blood pressure (Mohsen et al., 2021). Blocking the renin-angiotensin-aldosterone system (RAAS) remains the predominant approach of managing DN (Block et al., 2022). Furthermore, some agents, such as sodium-glucose cotransporter type 2 (SGLT2) inhibitors (Shaffner et al., 2021) and dipeptidyl peptidase-4 (DPP-4) inhibitors (Trakarnvanich et al., 2021), reportedly have favorable therapeutic potential for DN. Despite improvements in the treatment of DN, current therapeutic approaches are insufficient to completely delay DN progression and numerous DN patients eventually progress into ESRD (Johnson and Spurney, 2015). Therefore, there is an urgent need to further understand the pathological mechanisms of DN, thereby developing novel and effective therapies to prevent its progression and occurrence.

Traditional Chinese medicine has attracted a lot of attention worldwide due to its good efficacy and safety in the treatment of various disorders, such as malignant tumors and inflammatory diseases. Some active ingredients of Chinese herbs, such as geniposide (Li et al., 2020), catalpol (Chen et al., 2020), tripterygium glycosides (Guo et al., 2021a), and quercetin (Hu et al., 2022), have exhibited their beneficial effects in the treatment of DN. Celastrol, also named tripterine, is a natural bioactive ingredient isolated from the root extract of Tripterygium wilfordii (a traditional Chinese herb) (Wagh et al., 2021; Guo et al., 2021b; Zhang et al., 2022). Furthermore, it has been reported that celastrol can also be synthesized (Lu et al., 2021a). Celastrol is well-known for its multiple pharmacological effects, including anti-inflammatory, anti-autoimmune, anti-cancer, anti-oxidative, and neuroprotective functions (Wagh et al., 2021). This has made celastrol an appealing therapeutic drug for treating cancer, inflammatory conditions, and autoimmune disorders. For instance, celastrol has been indicated to inhibit inflammation by suppressing the reactive oxygen species (ROS)/nuclear factor kappa B subunit 1(NF-κB)/NLR family pyrin domain containing 3 (NLRP3) axis to mitigate rheumatoid arthritis (Jing et al., 2021). Notably, a previous study revealed that celastrol could suppress inflammation and alleviate renal injury to protect the kidney of diabetic rats by blocking the mitogen-activated protein kinase (MAPK)/NF-κB pathway (Zhang et al., 2019). Further, celastrol has been recently demonstrated to mitigate renal injury, suppress glomerular basement membrane thickening, and assist podocyte homeostasis to delay the progression of early DN in rats (Nie et al., 2020). Another study revealed the inhibition of proteinuria and the kidney-protective effects of celastrol in the treatment of DN (Liu et al., 2021). Despite these findings, the specific mechanisms underlying the anti-DN role of celastrol have not yet been fully illustrated. This prompted us to further investigate the efficacy of celastrol against DN and its associated mechanisms.

In recent years, network pharmacology has been widely employed in the investigation and development of traditional Chinese medicines. Network pharmacology aims to systematically exhibit the mechanism of drug intervention in disease networks by integrating biology, computer science, and bioinformatics, to name a few (Lin et al., 2021).

In this study, we investigated the therapeutic effects of celastrol on DN and related mechanisms. We also conducted in vitro experiments to verify the efficacy of celastrol and its mechanisms in the treatment of DN. We aimed to uncover the pharmacological mechanisms of the anti-DN role of celastrol through network pharmacology and molecular docking, thereby providing novel targets for treating DN.

DN is one of the most common microvascular complications of diabetes mellitus, manifesting as thickened glomerular basement membrane, enhanced extracellular matrix formation, and podocyte loss (Han et al., 2017). DN tends to develop into ESRD which has high morbidity and mortality rates (Zhu et al., 2021; Noor et al., 2021). Natural bioactive compounds such as terpenoids have exhibited their potential to treat DN. For example, paeoniflorin could inhibit TLR4-mediated inflammation and ameliorate the clinical symptoms and the severity of DN (Shao et al., 2019). In another study, catalpol could mitigate endothelial dysfunction and inflammation to impede the progression of DN (Shu et al., 2021). Celastrol, a quinone methide triterpene, extracted from Tripterygium wilfordii has been shown to possess various pharmacological properties, including anti-inflammatory, anti-autoimmune, anti-cancer, and neuroprotective activities (Wagh et al., 2021; Lim et al., 2021; Gu et al., 2018). Further, celastrol has been extensively used in the treatment of autoimmune diseases, such as rheumatoid arthritis (Jing et al., 2021) and systemic lupus erythematosus (Song et al., 2020). An increasing number of studies indicated that celastrol could alleviate the pathological injury in DN (Nie et al., 2020; Zhan et al., 2018). Moreover, a recent study has elaborated that CN suppressed proteinuria, and showed anti-inflammatory and kidney-protective functions celastrol in DN (Liu et al., 2021). However, the mechanisms underlying the anti-DN effects of celastrol have not yet been fully elucidated. Therefore, it is essential to further explore the therapeutic effects of celastrol and its mechanisms in the treatment of DN.

In this study, sixty-six intersection targets of celastrol and DN were screened based on public databases. In the PPI network of 66 targets, IL6, JUN, CASP3, PTGS2, MMP9, STAT3, IL1B, MAPK3, TNF, and AKT1 were identified as the top 10 core targets for celastrol in the treatment of DN. The hub targets, such as IL6, IL1B, and TNF, were mainly associated with inflammatory and immune responses. Chronic low-grade inflammation has been a crucial characteristic of DN and a failure in addressing inflammation is a primary contributing factor in the constant development of DN (Wu et al., 2021). A previous study demonstrated that celastrol could reduce levels of IL6, IL1B, and TNF to attenuate oxidative damage of type 2 diabetes (Cascão et al., 2017). Further evidence indicated that down-regulation of IL6, IL1, and TNF can alleviate the pathological injury of DN (Wu et al., 2020; Sun et al., 2020). Another study showed that the inhibition of the STAT3 pathway-mediated inflammation can attenuate DN (Zhang et al., 2021). Further, celastrol was found to effectively inhibit the secretion of pro-inflammatory cytokines, including IL6, IL1B, and TNF-α, thus mitigating DN (Zhan et al., 2018). This accounts for the key role of IL6, IL1B, and TNF in treating DN as revealed in our study. Collectively, these results suggest that the anti-DN effects of celastrol can be partly attributed to its potent anti-inflammatory activity.

According to the GO functional enrichment analysis, the potential targets of celastrol against DN were mainly concentrated in BPs, such as response to lipopolysaccharide, response to molecules of bacterial origin, and oxidative stress. Relevant findings have been shown in other reports. For example the development of DN was associated with lipopolysaccharide-induced inflammation (Jiang et al., 2020), gut microbiota dysbiosis (Fernandes et al., 2019), and oxidative stress (Gerardo Yanowsky-Escatell et al., 2020), which were partially in line with our findings. Notably, a previous study indicated the crosstalk between inflammation and oxidative stress mechanisms in DN pathogenesis (Samsu, 2021). Another study found that celastrol could inhibit oxidative stress to ameliorate the pathological damage of the chronic complications of diabetes (Guan et al., 2016). The core targets related to GO functions included MAPK1, MAPK3, TNF, JAK2, FASLG, MAPK14, TGFB1, IL6, CCL3, and AKT1, which mainly participate in the regulation of inflammatory responses. For instance, MAPK signaling pathways are vital for the inflammatory process, including the secretion of pro-inflammatory cytokines induced by ROS (Galganska et al., 2021). CCL3 is a chemokine that triggers various pro-inflammatory reactions such as leukocyte chemotaxis and pushes T cells into the inflammatory tissue area from blood circulation (Zhang et al., 2018b). Consistent with our data, previous data has documented the involvement of CCL3 in DN development (Araújo et al., 2020). To summarize, our findings further indicate the complexity of the pathological mechanisms of DN, including inflammatory responses, response to lipopolysaccharide, response to molecules of bacterial origin, and oxidative stress.

KEGG pathway enrichment analysis showed that celastrol could address DN primarily through AGE-RAGE, TNF, IL-17, and MAPK signaling pathways. Evidence has indicated that the increase in end products of advanced glycosylation (AGEs) and its receptor (RAGE) can contribute to the onset and development of DN and nephron cellular injury (Barocio-Pantoja et al., 2021). Notably, a recent study has demonstrated that inhibiting AGE-RAGE-mediated inflammation signaling pathways could prevent the development and progression of DN (Chen et al., 2022). Further, accumulating evidence showed that the pro-inflammatory cytokines, TNF and IL-17, play a vital part in the development and progression of DN (Park et al., 2019; Kim et al., 2021). Additionally, blocking the TNF/IL-17 axis has been proven to alleviate pathological injury of DN (Ma et al., 2019). In terms of the MAPK signaling pathway, it was found to be associated with multiple pathological mechanisms of DN, including inflammation and oxidative stress (Song et al., 2018; Zhu et al., 2020; Ma et al., 2021). Interestingly, a previous study observed that celastrol-loaded nanomicelles could ameliorate inflammation, lipid accumulation, and insulin resistance in obese mice (Zhao et al., 2019). Thus, it is reasonable to hypothesize the positive effects of celastrol on inflammation, lipid homeostasis, and insulin resistance in preventing DN progression. Besides, our data showed that NFKB1, RELA, AKT1, IKBKB, MAPK8, MAPK3, MAPK1, TNF, MAPK14, and JUN were the key celastrol targets in the KEGG analysis. Similarly, these targets, such as MAPK8, MAPK3, MAPK1, TNF, and MAPK14, are mainly involved in inflammatory responses. Collectively, these data further demonstrate that celastrol inhibits DN progression mainly by regulating inflammatory responses through the AGE-RAGE, TNF, IL-17, and MAPK signaling pathways.

In the present study, we finally obtained three overlapping targets (MAPK3, TNF, and AKT1) from the PPI, GO, and KEGG networks of the hub targets. Molecular docking showed that celastrol exhibited good binding abilities with MAPK3, TNF, and AKT1.

An increasing number of studies demonstrates that cell apoptosis is one of the major mechanisms in the development and progression of DN. Elevated apoptosis has been found in the kidneys of DN patients and suppression of the cell apoptosis in DN mice could mitigate DN symptoms (Fan et al., 2022). It was previously revealed that celastrol pre-treatment reversed HG-induced cell apoptosis to ameliorate the pathological injury of DN (Zhan et al., 2018). Consistent with this report, our data also showed that celastrol significantly inhibited the apoptosis of HG-treated NRK-52E cells. Additionally, we found that the viability of HG-treated NRK-52E cells was suppressed compared with control cells, which was however reversed by celastrol treatment.

MAPK3, also known as ERK1, participates in a wide range of cellular processes including proliferation, inflammation, and cellular metabolism (Chen et al., 2019; Kassouf and Sumara, 2020). Further, MAPK3/MAPK1 signaling could enhance pancreatic β-cell mass and insulin production (Kassouf and Sumara, 2020). TNF, which is a common pro-inflammatory cytokine acts as a switch in immunity (Fischer et al., 2020). Therefore, TNF inhibitors have been developed and clinically applied to treat inflammatory and autoimmune disorders (Fischer et al., 2020). AKT1 is a key mediator of cellular survival and growth, which is involved in inflammatory responses (Zhang et al., 2021; Vergadi et al., 2017). An animal model-based investigation indicated that celastrol could suppress the expression of pro-inflammatory cytokines (e.g., TNF-α) to mitigate neuropathic pain in rats (Jin et al., 2022). Furthermore, a recent study has demonstrated that downregulation of the levels of MAPK3 and TNF by blocking AKT1 signaling could prevent the progression of DN (Li and Xu, 2022). We observed that celastrol effectively decreased the expression of MAPK3, TNF, and AKT1 in HG-treated NRK-52E cells. This was similar to that reported in research (Jin et al., 2022; Li and Xu, 2022). Collectively, these findings indicate that celastrol can be used to treat DN where MAPK3, TNF, and AKT1 are the pivotal therapeutic targets in its action.

Our study has some limitations. First, the data used in this study were from public databases that need to be updated in real-time. This makes it possible that some other vital targets or pathways associated with DN might have been overlooked. Second, we only verified the in vitro therapeutic effects of celastrol on DN without in vivo validation. Third, the crosstalk between the core mechanisms (e.g., inflammation and oxidative stress) revealed in this study remains to be further elucidated. These limitations will be perfected and addressed in our subsequent studies.

This study reveals that celastrol exerts therapeutic effects on DN through multiple pathways and mechanisms, including AGE-RAGE, TNF, IL-17, and MAPK signaling pathways, the inflammatory and immune response, and oxidative stress. MAPK3, TNF, and AKT1 are the foremost targets of celastrol against DN. Additionally, the anti-DN effects of celastrol and its therapeutic targets were confirmed in vitro. Our study provides a direction for follow-up animal and clinical studies, deepens our insights into the mechanisms related to DN, and furnishes a reference for developing celastrol as a novel drug for the treatment of DN.