Renal fibrosis can lead to the progressive loss of renal function, with the characteristic of the proliferation of renal intrinsic cells, activation of renal interstitial fibroblasts, and deposition of extracellular matrix, which is the most common cause of kidney failure (Zhao et al., 2021). A large part of interstitial myofibroblasts, the main effector cells of renal fibrosis, are derived from tubular epithelial cells of the affected kidneys via epithelial–mesenchymal transition (EMT) (Hu et al., 2021). EMT, one of the main mechanisms of renal fibrosis, had been found to contribute to ongoing fibrosis in kidney disease through a variety of signal pathways, which ultimately led to renal function impairment, and then developed into chronic renal disease and end-stage renal disease. Despite extensive studies, the current treatment strategies of western medicine can hardly reverse the progression of renal fibrosis, underscoring the need to develop an alternative therapeutic approach to reverse or stop the progression. Traditional Chinese medicine (TCM), with the feature of multi-component, multi-target, and multi-channel, can enhance body functions and reduce drug toxicity through the synergistic actions of their main active ingredients, which could be a rich source for drug discovery. The experimental models and clinical studies have proved that TCM and its extracts have made great progress in the prevention or even treatment of renal fibrosis, and effectively inhibited the development of EMT in recent years (Xu et al., 2021).

Jian-Pi-Yi-Shen Formula (JPYSF) is derived from the addition and subtraction of “Yupingfeng San” and “Sijing Pills” (recorded in Shengji Zonglu). JPYSF has been prescribed for patients with chronic kidney disease in clinics, which consists of Astragalus mongholicus Bunge (A. mongholicus), Dioscorea oppositifolia L. (D. oppositifolia.), Salvia miltiorrhiza Bge. (S. miltiorrhiza), Atractylodes macrocephala Koidz. (A. macrocephala), Cistanche deserticola Ma (C. deserticola), Amomum kravanh Pierre ex Gagnep. (A. kravanh), Rheum palmatum L. (R. palmatum), and Glycyrrhiza uralensis Fisch. (G. uralensis). Adopting the two-complement five-way method, the two complement mainly consists of “tonifying the kidney” and “tonifying the spleen.” A. mongholicus, A. macrocephala., D. oppositifolia., and C. deserticola were used as spleen and kidney supplements (Zhong et al., 2016). A. mongholicus was used for nourishing the qi of the five viscera, and it was an important medicine for nourishing the qi of the spleen and kidney. “Five-way” means to make sure that feces, urine, sweat, breath, and blood are unobstructed. R. palmatum, A. macrocephala, and C. deserticola were used to pass bowel movements, having the functions of loosening the bowel to relieve constipation (Zhong et al., 2016). A. mongholicus and A. macrocephala were used for urination, having a diuretic effect (Fu et al., 2014). A. kravanh, which has the effect of warming middle energizer, dissipating hygrosis and regulating qi, and promoting sweat and breathing well, was used to drain sweat and breath. S. miltiorrhiza and R. palmatum were used to promote blood circulation, having the effect of promoting blood circulation and removing blood stasis (Mei et al., 2019; Pei et al., 2020). G. uralensis was used for invigorating the spleen and harmonizing herbs. Taken together, JPYSF, the combined use of all the above-mentioned herbs, has the effects of invigorating the spleen and kidney, promoting blood circulation, and removing turbidity. Previous pharmaceutical studies have revealed that JPYSF had effectiveness in the treatment of chronic kidney disease and renal fibrosis (Liu et al., 2018; Lu et al., 2018; Chen et al., 2019). However, the composition of JPYSF is complex and its mechanism of action is not clear enough, which limits its wide clinical application.

Network pharmacology is a theory based on systems biology and has been commonly used in the modern research of TCM in recent years. It emphasizes the multichannel regulation of signaling pathways, which is in accord with the characteristics of multi-components and multi-targets of Chinese medicine. Therefore, network pharmacology has become a new effective approach in TCM research at the molecular level (Zhang et al., 2021).

In this study, the integrated strategy of network pharmacology and verification in vivo, we first explored the main active ingredients, targets, and signal pathways of JPYSF in the treatment of EMT, and then the animal experiment was carried out to verify the effect of JPYSF against renal fibrosis by inhibiting EMT. The research may lay a good theoretical foundation for further study on developing new drugs for renal fibrosis.

JPYSF, a classic prescription of hospital preparations, has been used clinically to treat renal fibrosis as an effective and safe TCM. Previous studies confirmed that JPYSF significantly decreased fibrotic area to ameliorate interstitial fibrosis and down-regulated fibrosis-associated protein expression such as fibronectin and type IV collagen in 5/6 Nx rats (Liu et al., 2018; Lu et al., 2018; Chen et al., 2019). These results indicated that JPYSF could inhibit EMT, one of the main mechanisms of renal fibrosis. However, the bioactive components and multi-target mechanisms of JPYSF on EMT have not yet been fully studied. It is therefore of great importance to comprehensively investigate the pharmacological mechanisms of JPYSF on EMT to alleviate renal fibrosis.

First, network pharmacology was employed to explore the potential active ingredients and targets of JPYSF. Second, the common targets were the intersection targets between the potential targets of JPYSF and EMT-associated targets, and then they were identified to build a protein interaction network. According to the string database, the top 30 targets with action frequency are shown in Figure 1C. Some genes, such as HSP90AA1 and RELA have been associated with the pathogenesis of EMT. Studies have found that HSP90AA1 exhibited anti-hepatic fibrosis effects in LX-2 cells (Chen CH. et al., 2021), and EMT was reported to be suppressed by RELA/p65 through integrin-mediated signaling (Roupakia et al., 2021). Third, the GO functional and KEGG enrichment analyses were carried out to further understand the mechanism of JPYSF. The enrichment analysis results predicted that the therapeutic effects of JPYSF against EMT might be involved in anti-fibrosis (the Wnt signaling pathway, the JAK-STAT signaling pathway, the Hedgehog signaling pathway, and the TGF-beta signaling pathway), anti-inflammation (the PI3K-Akt signaling pathway, the NF-kappa B signaling pathway, and the Toll-like receptor signaling pathway), podocyte protection (the Autophagy–animal and the mTOR signaling pathway), and metabolism regulation (the AMPK signaling pathway). In accordance with previous studies, these results indicated that the above pathways had important effects on the progression and advancement of EMT. Among them, the Wnt/β-catenin pathway is the best researched anti-fibrosis pathway related to EMT (ChenQ et al., 2021; Gao, et al., 2020). Therefore, the Wnt/β-catenin pathway may play a crucial role in JPYSF against EMT. Other anti-fibrosis signal pathways were also reported in the EMT process. For instance, EMT and glomerulosclerosis in rats with chronic kidney disease were suppressed by JAK/STAT signaling pathway (Zhao et al., 2019), and cyclosporine-A-induced renal fibrosis was ameliorated by chrysin by the inhibition of TGF-beta-induced EMT (Nagavally et al., 2021). In addition, the PI3K-Akt signaling pathway, a major regulator of anti-inflammation, has been reported to prevent renal fibrosis by attenuating renal tubular epithelial cell–mesenchymal transition (Hu et al., 2021; Zhang Q et al., 2020; Wang et al., 2019). In line with this, JPYSF inhibited the inflammation via suppression of the NF–kappa B signaling pathway (Lu et al., 2018). Then, the network of CTS and core CTS were performed to comprehensively predict the core compounds, target genes, and mechanism of JPYSF on EMT, as shown in Figures 4, 5. Degree centrality is the most direct and important parameter of the node among the three topological characteristic parameters. The node with a greater degree is more important in the network. The closeness between a node and other nodes in the network was reflected by closeness centrality. The node that is closer to other nodes has greater proximity centrality. Between centrality means the number of shortest paths through a node. The results of the topological analysis of the C-T-S network showed that 17 core components and 23 core targets had higher values than the average degree centrality value, closeness centrality value, and between centrality value. All the compounds that might participate in the regulatory processes of JPYSF in EMT were considered as potential active ingredients. In support of this, several previous studies have shown that quercetin (Elumalai et al., 2021), kaempferol (Ji et al., 2020), luteolin (Cao et al., 2020), naringenin (Li et al., 2021), isorhamnetin, Tanshinone IIA (Fu et al., 2021), cryptotanshinone (Zhang Z et al., 2020), and aloe-emodin (Liu et al., 2021) had well-established roles in EMT. Taken together, our results demonstrate that the core components may be the effective ingredients of JPYSF for the EMT treatment.

Finally, the 5/6 Nx-induced renal fibrosis rat model was selected to evaluate the therapeutic effects of JPYSF on EMT to verify the network pharmacology prediction. As expected, JPYSF treatment significantly ameliorated interstitial fibrosis in 5/6 Nx rats by Masson staining analysis (Figure 6). In our previous studies, we have reported that JPYSF notably reduced the expression levels of fibronectin and type IV collagen by immunofluorescence analysis in renal fibrosis of 5/6 Nx rats (Liu et al., 2018). We, therefore, expect a similar outcome that the other markers of fibrosis can be identified. Furthermore, α-SMA, a mesenchymal cytoskeletal marker, and E-cadherin, an intercellular epithelial adhesion molecule, were selected to assess whether JPYFS had a modulating effect on EMT. The Wnt/β-catenin pathway, the best-enriched anti-fibrosis pathway related to EMT, was chosen to test the predicted molecular mechanisms and verify the accuracy of network pharmacology prediction. Consistently, the western blot analysis detected that JPYSF markedly up-regulated expressions of E-cadherin, Wnt3a, and β-catenin, and down-regulated expression of α-SMA in 5/6 Nx rats. In agreement with this, our previous studies have reported that JPYSF notably reduced the increased levels of fibronectin and type IV collagen in renal fibrosis of 5/6 Nx rats (Liu et al., 2018). Moreover, it has been reported that astragaloside IV up-regulates the decreased expressions of E-cadherin and occludin, whereas it down-regulates expressions of N-cadherin and vimentin in high glucose-induced EMT cells (Wang YN et al., 2020). Therefore, we assume that JPYSF could regulate other epithelial markers and mesenchymal markers. To support this notion, astragaloside IV, which is an active ingredient from A. mongholicus, is considered as the monarch drug, the ingredient that provides the principal curative action on the main syndrome in JPYS prescription and has also been identified within JPYSF by HPLC-MS analysis in the current study (Supplementary Figure S1). Since most of the EMT markers are cell- and drug-treatment-specific, checking the expression of EMT markers in the specific context by JPYSF would be the future potential work. Collectively, these results provide evidence that JPYSF could regulate EMT by inhibiting the Wnt3a/β-catenin pathway.

However, there were some limitations of research, such as the core compound with no effective dose, which still needed further experiments to identify. Moreover, the direct regulatory relationship of JPYSF in EMT and the Wnt3a/β-catenin pathway by gene knockout animal experiments and cell experiments would be our future potential work for a better understanding of this signaling pathway.

In this research, a combining approach of systematic network pharmacology and experimental verification were employed to determine therapeutic targets and pharmacological mechanisms of a complex herbal formulation, JPYSF, against EMT. The results suggested that 17 core compounds such as quercetin, kaempferol, luteolin, naringenin, and 23 core targets such as NCOA2, HSP90AA1, and PTGS1 might play an important role in JPYSF-treated renal fibrosis. The multi-target synergetic mechanisms of JPYSF in EMT mainly consist of four therapeutic aspects including anti-fibrosis, anti-inflammation, podocyte protection, and metabolism regulation. In vivo experiment data showed that JPYSF significantly ameliorated interstitial fibrosis by Masson staining and exerted the regulation of EMT by inhibiting the Wnt3a/β-catenin pathway (Figure 6). In summary, our study provides a basis and guidance for JPYSF research and its clinical application in the treatment of EMT, offering insights into the multi-target mechanisms by the system’s pharmacology approach (Figure 7).