Acute kidney injury (AKI) is a multifunctional kidney disease characterized by a sudden kidney failure episode, increased serum creatinine levels, and a marked decrease in urine volume, with a significant risk of morbidity and mortality (Kellum et al., 2021). It has multiple etiology, such as renal ischemia, nephrotoxic drugs, sepsis, and urinary tract obstruction, and its pathogenesis is also related to various pathways (Bellomo et al., 2012). Nephrotoxic drugs include antibiotics, antipyretic analgesics, chemotherapy drugs, and antiepileptic drugs (Marley et al., 2022). Cisplatin was first approved as an anti-tumor drug in 1978, and it remains an essential and influential therapy for the treatment of various cancers. About 20% of chemotherapy regimens for malignant tumors contain cisplatin (Manohar and Leung, 2018). However, its clinical application is limited due to toxic side effects on normal tissues such as kidney, liver, heart, and nerves (Qi et al., 2019). In addition, the toxicity of cisplatin is positively related to the dose (Dasari and Bernard Tchounwou, 2014). Nephrotoxicity is the main complication, and AKI occurs in about 20%–30% of patients. The primary purpose of treating renal injury by cisplatin is its cause and symptoms. In clinical practice, hydration is often used before and after chemotherapy to promote the excretion of cisplatin to prevent nephrotoxicity. Amifostine is approved by the U.S. Food and Drug Administration for use in reducing progressive nephrotoxicity of repeated cisplatin dosing in patients with advanced ovarian cancer (el Arabey and Abd Allah, 2015). However, the final treatment chiefly relies on renal replacement therapy. Specific drugs are still lacking. Therefore, it is essential to discover effective medications to prevent and cure cisplatin-induced AKI. In ancient Chinese medicine books, AKI belongs to the categories of “edema” and “guange”. The onset is due to a lack of righteousness, exogenous damp-heat toxins, or drug poisoning. AKI caused by cisplatin is due to a lack of vital qi caused by drug poisoning (Li et al., 2019; Liu et al., 2021; Cai et al., 2022). Currently, clinical drugs are lacking for treating renal injury induced by cisplatin (Holditch et al., 2019).

San-Huang decoction (SHD) was the topic of this research. According to the theory of traditional Chinese medicine (TCM), it was found that astragalus, rhubarb, and Sanqi are used in the clinical treatment of AKI and CKD due to drug poisoning (Han et al., 2017; Tian et al., 2020; Chen et al., 2022). Astragalus, rhubarb, and sanqi are derived from Shen Nong’s Materia Medica. The three medicines complement each other. Among them, astragalus can replenish the kidney and qi, which is the foundation of nature; Rhubarb can boost blood circulation and eliminate blood stasis, dredge the fu organs and remove turbidity, clear heat, and detoxify; Sanqi promotes blood circulation, dissipates blood stasis and settles pain; All kinds of medicines play a role in strengthening the body, supplementing qi and deficiency, promoting blood circulation and removing blood stasis, clearing the five internal organs, removing turbidity and detoxification. Studies have shown that these three herbs have sound preventive effects against chronic renal insufficiency (Chen et al., 2022), diabetic nephropathy (Han et al., 2017), and AKI (Tian et al., 2020). Astragalus decoction regulates diabetic nephropathy engendered by streptozotocin in rats via TGF-β/MAPK/PPAR-γ signaling pathway (Han et al., 2017). A nationwide cohort study found that astragalus and rhubarb provide renal protection and survival benefits in patients with advanced chronic kidney disease (Chen et al., 2022). Sanqi oral liquid enhanced autophagy by reducing apoptosis and ameliorating renal ischemia/reperfusion injury by engaging the ERK/mTOR pathway (Tian et al., 2020). However, the role of SHD in the prevention and treatment of cisplatin-induced AKI and its specific molecular mechanism remain unclear.

Network pharmacology (Nogales et al., 2022) is a systems biology-based discipline that analyzes the targets of various drugs, components, and diseases. Molecular docking (Forli et al., 2016) is the docking of small molecules and protein binding sites. It is widely used to study biomolecular interactions and mechanisms and is applied to structure-based drug design. As a new drug research method, network pharmacology has the characteristics of high efficiency, multiple targets, and convenience. It is now extensively implemented in the research of molecular drug mechanisms and molecular docking. TCM is a great treasure house of the Chinese nation with a history of thousands of years. However, the single and compound components of TCM are complex, and the mechanism of their treatment of diseases is yet imprecise. Applying TCM research in network pharmacology helps explore the specific mechanism of TCM in treating conditions (Li and Zhang, 2013). Relevant studies (Qin et al., 2020; Tian et al., 2020) have used this research method to analyze the active components and explore the therapeutic mechanism of astragalus, rhubarb, and Sanqi. These studies offer a considerable possibility that, through network pharmacology and molecular docking analysis, the potential therapeutic targets of SHD can be further explored, which can help develop clinical strategies for cisplatin-induced AKI.

In this study, the combined methods of network pharmacology, molecular docking, and experimental verification were used to systematically study the bioactive components of SHD and the mechanism of prevention and treatment of AKI to provide a reference for further research in the future. The workflow of this study is shown in Figure 1.

AKI is a global disease with a poor prognosis. At present, cisplatin-induced AKI can neither be treated nor prevented. Renal replacement therapy becomes necessary as kidney function deteriorates and the disease progresses to its end stage. Consequently, effective treatment for cisplatin-induced nephrotoxicity is urgently needed.

SHD is refined from traditional Chinese prescriptions used for the clinical treatment of various kidney diseases. Astragalus, rhubarb, and Sanqi were recorded in “Shen Nong’s Materia Medica.” However, the complex chemical composition makes it difficult to elucidate the pharmacological mechanism of its underlying active compounds and treatment, despite its broad pharmacological activity.

Network pharmacology and molecular docking provide useful methods for the research of the “multi-component, multi-target, multi-biological regulatory function synergy” model of TCM, and have been widely utilized in TCM research and advanced drug findings. In this research, a combination of network pharmacology, molecular docking, and experimental validation was used to systematically investigate the bioactive components of SHD and its therapeutic mechanism for AKI.

Many overlapping targets were found in different compounds in SHD, indicating that SHD exerts anti-inflammatory and anti-apoptotic properties through the synergistic effect of its compounds. The primary active components in SHD may be the key to preventing and treating AKI. Among them, calycosin is the staple active component of astragalus. In studies, it has been shown to reduce renal ischemia-reperfusion injury by inhibiting NF-κB mediated inflammation through the PPARγ/EGR1 pathway (Zhang et al., 2022), and it is anti-inflammatory and promotes angiogenesis. Rhein is the main active component of rhubarb. Consequently, many AKI patients have progressed to chronic kidney disease. Rhein is able to curb the transdifferentiation of renal tubular epithelial cells to myofibroblasts. Studies (Ma et al., 2018) have found that emodin can inhibit the expression of transforming growth factor β1, block the TGF-β1/Smad signaling pathway, and inhibit the transdifferentiation of human renal tubular epithelial cells (HK-2), thereby reducing epithelial-myofibroblast transdifferentiation (TEMT). Rhein and its derivatives, analogs, and compound preparations protect against various kidney diseases, particularly diabetic nephropathy (DN) and drug-induced AKI (Zhu et al., 2022). In the research of Luo Yan et al., Rhein was found to restrain the SHH-Gli1-Snail signal pathway, extenuating renal fibrosis in rhein-treated UUO rats (Luo et al., 2022).

Ginsenoside Rh2 is the main active ingredient of Sanqi (Li et al., 2020). There is evidence that ginsenoside Rh2 has anticancer properties. Cisplatin is a clinically efficient broad-spectrum anticancer drug. Cisplatin nephrotoxicity affects nephrocyte apoptosis. Studies (Qi et al., 2019) have revealed that Rh2 has a protective effect on cisplatin-induced kidney dysfunction and can improve cisplatin-induced renal histopathological damage, inflammatory response, and renal tubular cell apoptosis. In the study of Kim Long Vu Huynh et al. (Vu-Huynh et al., 2019), from processed Vietnamese ginseng, they isolated six PPD-type saponins, including ginsenoside Rh2 and ocotillo genin, that displayed the potential to protect the kidney from cisplatin-induced toxicity. In addition, GO, and KEGG pathway analyses revealed that SHD mainly interferes with AKI occurrence and development through TNF and IL-17 signaling pathways. The abnormal activation of these targets can promote the biological process of AKI, such as inflammation, proliferation, and angiogenesis, further aggravating the condition. Treating tubular epithelial cells is a top priority in AKI. Inflammation, apoptosis, and vascular injury of renal tubular epithelial cells are all important potential targets. Therefore, treating inflammation and regulating apoptosis to reduce vascular injury are the focus of treating AKI. In the results of PPI analysis, we found that AKT, TNF-α, IL-6, IL-1β, caspase-3, and MMP9 were the core targets of SHD in preventing and treating cisplatin-induced AKI. The main compound bound to the core protein was further confirmed by molecular docking.

The predictive power of network pharmacology was further confirmed in vivo, suggesting that SHD decoction can prevent and treat cisplatin-induced AKI. It mainly exerts an anti-inflammatory effect mainly by regulating various pro-inflammatory and anti-inflammatory cytokines and exerts an anti-apoptotic effect mainly through the intrinsic and extrinsic apoptosis pathway.

SHD exerts significant anti-apoptosis and anti-inflammatory activities to attenuate cisplatin-induced kidney damage. Our study shows that SHD regulates the MMP9/PI3K/AKT anti-apoptosis pathway. MMPs family genes are abnormally expressed in the most common kidney diseases. In animal models of AKI induced by folic acid and ischemia/reperfusion (I/R), MMP9 activity was significantly downregulated. It could be activated through the soluble stem cell factor (SCF)/c-KIT pathway, and MMP9-mediated shedding of membrane SCF had a renoprotective effect in acute kidney injury. SCF binds to c-Kit, which induces anti-apoptotic signaling through the PI3K-AKT1 kinase (Arnould et al., 2009). MMP9 knockout mice have reduced SCFs, more severe kidney damage, and increased apoptosis than control mice, and renal function is not easy to recover (Bengatta et al., 2009). Interestingly, PI3K is dependently related to MMPs (Liang et al., 2021). PI3K activation can activate the downstream protein AKT, and the activated AKT can regulate a variety of downstream arnouapoptosis-related genes, among which Bad (Bcl-2-related cell death agonist) can positively regulate mitochondrial apoptosis. After AKT phosphorylates this protein, Bad activity is inhibited, and anti-apoptosis factor Bcl-2 activity is increased (Fulda, 2014). The experimental results showed that cisplatin-induced AKI inhibited the activity of MMP9 to inhibit the PI3K/AKT pathway, thereby promoting apoptosis, and SHD successfully reversed this phenomenon. However, in I/R-induced AKI mice, MMP9 deletion preserves tissue vascular endothelial growth factor levels and stabilizes microvascular density to protect against renal injury. (Lee et al., 2011). This study indicates that MMP9 is special in the pathology of AKI, which is worth exploring in further study.

There are two pathways of apoptosis in mammalian cells: the “extrinsic pathway” mediated by cell surface death receptors, and the other, the “intrinsic pathway” dominated by mitochondria. Caspase-3 is considered to be a common effector of exogenous and endogenous apoptosis. Studies have shown that inhibiting the activity of caspase-3 can inhibit cisplatin-induced apoptosis, which is in line with the results of network pharmacology (Em et al., 2015). Our experimental results indicated that the intrinsic and extrinsic apoptosis pathways are involved in cisplatin-induced AKI and play an essential role in its development. Because SHD exerts its anti-apoptosis through multi-targets and multi-ingredients. Mitochondria are the central organelle where AKT exerts its anti-apoptosis effect. To induce apoptosis, the mitochondrial outer membrane must become permeable so protein can escape. Bcl-2 is closely related to the release of mitochondrial intermembrane proteins. AKT participates in the regulation of different stages of mitochondria-mediated apoptosis by regulating Bcl-2. Cisplatin inhibits the PI3K/AKT pathway while activating the translocation of caspase-3, caspase-9, and Bax to mitochondria, resulting in cell expansion c, apoptosis-inducing factor (AIF), and endonuclease G release. Inhibition of caspase-3 and caspase-9 inhibits cisplatin-induced cell death. In contrast, cisplatin-treated Bax-deficient mice had reduced cytochrome c release and reduced apoptosis (Volarevic et al., 2019). We observed that SHD significantly attenuated the pathological damage of renal tubular cell apoptosis and that SHD significantly regulated the critical targets of the apoptosis pathway, Bcl2, caspase-3, caspase-9, and Bax. Furthermore, in cisplatin-AKI, TNF-α induced extrinsic apoptosis via caspase-8, which was also alleviated after SHD treatment.

Inflammation is an essential feature of the pathogenesis of AKI, in which the NF-κB signaling pathway is triggered by renal tubular epithelial cell injury by controlling the expression of pro-inflammatory factors, including IL-1β and IL-6. On the other hand, anti-inflammatory factors also play an important role in protecting renal function and inhibiting AKI. Anti-inflammatory factors, like IL10, can inhibit inflammation and tissue injury by suppressing migration, accumulation, and activation of monocytes and neutrophils. IL-10 can also significantly inhibit the cytotoxicity, inflammation, and apoptosis of renal injury (Zeinali et al., 2022). Other anti-inflammatory factors, such as IL13, activates M2 macrophages, which can promote the tissue repair and remodeling of acute nephritis by secreting various anti-inflammatory mediators, such as IL-4 and IL-10 (Mao et al., 2020). Studies have shown that cisplatin induces a cascade of inflammatory reactions with increased production of proinflammatory cytokines, particularly TNF-α, which has a central role in mediating cisplatin-induced inflammatory renal injury (EM et al., 2015). Inhibiting the expression of TNF-α can significantly attenuate renal injury induced by cisplatin. Meanwhile, TNF-α also plays a crucial role in renal tubular inflammation and is the upstream initiation stage of the inflammatory cascade.

Our previous findings (He et al., 2022) showed that inflammation and apoptosis are closely related to NF-kB signaling. Its’ activation is dependent on the phosphorylation of p65 and inhibitory IκB proteins. Data from our study suggest that SHD regulates the expression of NF-κB-related genes that are up or down-regulated in response to cisplatin induction. SHD can inhibit the secretion and emancipation of inflammatory factors such as TNF-α, IL-6, and IL-1β to protect renal tubules from damage, which is just opposite to the nephrotoxicity of cisplatin. Therefore, we confirmed the role of the NF-κB signaling pathway.

This study has certain limitations. First, public databases may not include some compounds and target genes due to the generality of network pharmacology research. Second, the clinical feasibility of SHD for cisplatin-induced AKI requires validation. Third, we found that MMP9 plays a special role in AKI pathology, and further in vitro and in vivo experiments are needed to determine this.

In conclusion, this study provided evidence that SHD plays a critical role in anti-inflammation and anti-apoptosis via inhibiting the NF-κB signaling pathway and activating PI3K/AKT anti-apoptosis pathway, indicating that SHD is a candidate herbal drug for further investigation in treating cisplatin-induced AKI. This is the first study to analyze the potential main compounds, targets comprehensively, and pathways of SHD in the prevention and treatment of AKI through network pharmacology, molecular docking, and experimental validation to provide supplementary therapy and additional support for AKI. Drug mechanism provides research ideas and foundations.