Puerarin ameliorates apoptosis and inflammation induced by kidney stones through the PI3K/AKT pathway: Network pharmacology and experimental validation

Puerarin ameliorates apoptosis and inflammation induced by kidney stones through the PI3K/AKT pathway: Network pharmacology and experimental validation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Puerarin ameliorates apoptosis and inflammation induced by kidney stones through the PI3K/AKT pathway: Network pharmacology and experimental validation Yuexian Xu, Hu Liang, Xike Mao, Zhenyu Song, Xudong Shen, Defeng Ge, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4509583/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Puerarin(PUE), an isoflavonoid extracted from Pueraria root, has anti-apoptotic effects. The objective of this research is to examine the impact of PUE on renal apoptosis and inflammation resulting from renal calculi and to elucidate its mechanism.The approach of network pharmacology and molecular docking was employed to discover potential targets and pathways of PUE. An animal model of calcium oxalate crystal deposition by intraperitoneal injection of glyoxylate and a model of COM-induced human renal tubular epithelial cells (HK2) were used to investigate the pharmacological mechanisms of PUE against apoptosis and inflammation. We used hematoxylin-eosin (H&E) and Periodic Acid-Schiff staining (PAS) to assess the effect of PUE on crystal deposition and damage. The mechanism of PUE was elucidated and validated using Western blotting, histology, and immunohistochemical staining.Network pharmacology findings indicated that the PI3K/AKT pathway plays a crucial role in PUE. We experimentally demonstrate that PUE alleviated COM-induced changes in apoptotic proteins, increased inflammatory indicators and changes in oxidative stress-related indicators in HK2 cells by activating the PI3K/AKT pathway, reduced serum creatinine and urea nitrogen levels in mice caused by CaOx, alleviated crystal deposition and damage, and alleviated apoptosis, oxidative stress and inflammation.Puerarin attenuates renal apoptosis and inflammation caused by kidney stones through the PI3K/AKT pathway. Puerarin Kidney stone Apoptosis Network pharmacology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The prevalence of kidney stones, a frequent urological condition, has been on the rise for the last twenty years[1-3]. Despite notable advancements in less invasive surgical methods, the rate of recurrence within a span of five years following the initial operation remains at 50%[4], resulting in substantial socioeconomic and medical burdens[5]. The main component of kidney stones is calcium oxalate (CaOx) crystals, which account for up to 80% of all kidney stones[6]. CaOx causes kidney damage mainly because of apoptosis, autophagy, inflammation, fibrosis and so on[7-10]. Considering this, the discovery of novel therapeutic targets holds immense theoretical importance and practical value in the treatment and prevention of kidney stones. The method of treating kidney stones with herbal medicine has a long history and profound cultural heritage. With the deepening of scientific research, more and more studies have confirmed that herbal medicine has significant efficacy in the treatment of kidney stones. Its unique advantage lies in its smaller side effects and high safety, providing a more gentle and effective treatment approach for kidney stone patients[11-13]. PUE, derived from Pueraria Mirifica, is a bioactive compound abundant in flavonoids, peptides, and other bioactive substances[14]. An increasing amount of pharmacological research has shown that PUE exhibits a range of biopharmacological properties. PUE has been shown to have anti-inflammatory, anti-injury, and anti-apoptotic effects in vitro in a large number of experiments[15-19]. Not only in vitro experiments, but also in vivo experiments have fully verified its efficacy. PUE attenuates neurological deficits apoptotic pathways in subarachnoid hemorrhage mice[20]. PUE suppresses inflammation and alleviates pain symptom in osteoarthritic mice[21]. PUE protects against myocardial ischemia in rats[22]. The effect of PUE on the kidney has also been demonstrated by a large number of in vivo experiments. The renoprotective effect of PUE has been demonstrated in diabetic nephropathy, renal fibrosis, and cisplatin-induced acute kidney injury[23-26]. Nonetheless, limited research has been conducted on the safeguarding impact of PUE against CaOx-induced renal damage. Network pharmacology, as a new research methodology, has been successful in revealing multiple targets and complex mechanisms of drugs in various diseases.Using a network pharmacology method, we examined the possible targets and pathways through which PUE can reduce renal injury caused by CaOx.To validate our hypothesis, we conducted experiments both in vivo and in vitro. 2. Materials and methods 2.1.Network pharmacology GeneCards (Giftes≥5) was used to search through the Swiss target prediction database (www.swisstargetprediction.ch) for potential targets associated with kidney stones. Venny tool draw the Venn diagram, kidney stones and PUE-related targets. Protein-protein interaction(PPI) between potential targets were evaluated using the STRING database (https://cn.string-db.org/). Using Cytoscape 3.7, we built targets and kidney stones of PUE potential targets for shared between PPI, through the CytoHubba plug-in filtered on key targets. The DAVID database (david.abcc.ncifcrf.gov) was used for enrichment analysis of the intersecting genes. Molecular docking was performed using AutoDock. 2.2.Chemicals and reagents PUE and LY294002 (PI3K/AKT inhibitor) were purchased from TargetMol (Shanghai, China). Glyoxylate and calcium oxalate monohydrate (COM) were obtained from Sigma (Germany). TNF (26405-1-AP), IL1(16765-1-AP), IL6(21865-1-AP), Akt(10176-2-AP), p-Akt(28731-1-AP), Pi3k(20584-1-AP), Bax(50599-2-Ig), Bcl-2(26593-1-AP) and Caspase3(19677-1-AP) were obtained from Peprotech (Wuhan, China). p-PI3K(#AF3242) was obtained from Affinity Biosciences(Jiangsu, China). Using the Cell Counting Kit-8 (CCK-8) sourced from Beyotime (Nanjing, China) and ELISA kits from Solarbio (China), we assayed various oxidative stress markers—specifically, MDA, SOD, LDH, and GSH—in the cell homogenates derived from different treatment cohorts. 2.3.Cell culture and treatment The human kidney cell line HK-2 was obtained from the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences. These cells were cultured in an environment containing 10% fetal bovine serum and a DMEM/F12 mixed medium, maintained at 5% carbon dioxide and 95% humidity for routine cultivation. Before stimulating with COM (1000 mg ml-1), we exposed the HK-2 cells to different concentrations of PUE (1, 2, 4, 8, 16, 32, 64, 128 μM) or LY294002 (10 μM) for 30 minutes. Afterwards, they were transferred to a new environment containing 0.5% fetal bovine serum and a DMEM/F12 mixed medium for overnight cultivation. 2.4.Cell viability determination CCK-8 was utilized to assess cell viability.In summary, HK2 cells were seeded in 96-well dishes and prior to stimulation with COM (1,000 mg L-1), they were treated with varying amounts of PUE for 24 hours.Next, 10 microliters of CCK-8 solution was introduced into every well and incubated for a period of 60 to 90 minutes.The manufacturer's instructions (Multiskan MK3, Thermo, United States) were followed to analyze the optical density at 492 nm. 2.5.Animals and experimental design We conducted rigorous and cautious animal experiments. In this experiment, we carefully selected male C57BL/6J mice aged between 6 and 8 weeks as the subjects to ensure the accuracy and reliability of the experimental results. It's worth mentioning that we strictly adhere to animal ethics norms, and this experiment has been officially authorized by the Animal Ethics Committee of Anhui Medical University (numbered LLSC20232250). This committee is committed to ensuring that all scientific research activities involving animals follow the highest ethical standards, fully respecting and protecting the rights and interests of animals. Their authorization provides important guarantees for the compliance and morality of our experiment. To create a mouse model for CaOx renal calcium deposition, a daily intraperitoneal injection of 100 mg/kg glyoxylate was administered, and the mice were euthanized after 7 days of injection.Mice received PUE(10, 50, and 100mg/kg) via oral administration, 12 hours prior to glyoxylate injection, followed by daily administration. We anesthetized the mice with Sodium barbiturate (50 mg/kg) (Sigma, 11715) intraperitoneally, collected blood samples from their orbits for analysis, and killed them after they had been fasted and water deprived for 12 hours. The blood that was acquired underwent testing for Cr and BUN in accordance with the instructions provided by the manufacturer, while the renal tissue was regularly embedded in paraffin and examined for molecular objectives. ImageJ software was used to determine the percentage of CaOx crystal deposition in each kidney section. 2.6. Tubular injury determination In order to assess the damage to tubules, we conducted PAS staining using a PAS staining kit according to the established protocol. Subsequently, we invited three experienced pathologists to collaboratively determine the tubule damage score based on the degree of loss of brush border, tubule dilation and atrophy, as well as the formation of intraluminal casts. Regarding tubular lesions, we have established the following grading criteria[27]: 0 (indicating normal), 1 (≤10%), 2 (11–25%), 3 (26–50%), 4 (51–75%), and 5 (≥76%). 2.7.Histology and immunohistochemical staining Paraffin-embedded sections of mouse kidneys were prepared using standard histopathology techniques, including 4% paraformaldehyde fixation, dehydration, waxing, embedding, and sectioning (4 μm). Kidney sections were then stained with HE and PAS reagent kits using standard protocols (Solarbio, Beijing, China). First, Immunohistochemistry was performed by renal biopsy antigen repair, and then endogenous peroxidase activity was blocked. The antibodies were incubated for 30 minutes at a temperature of 4℃. Tissue samples were stained using the DAB (3,3'-diaminobenzidine) staining method. After staining was completed, all observed images were captured and recorded using an Olympus IX83 microscope (made in Japan). 2.8.Western blot analysis Using 10% SDS-PAGE technology, separate 30 micrograms of tissue lysate and cell samples. The separated samples of cells mixed with tissue lysate are then transferred to a nitrocellulose (NC) membrane using electrophoretic transfer. Block the NC membrane for 2 hours using tris-buffered saline containing 0.05% Tween 20 and 5% non-fat milk. Cells are incubated with a specific primary antibody for 8 hours. Subsequently, the cells are co-incubated with a secondary antibody at 37 degrees Celsius for 90 minutes. Images are captured and analyzed using the Licor/Odyssey infrared imaging system. Finally, the gray values of each band are quantified using ImageJ software. 2.9. TUNEL staining Following the guidelines provided by Solarbio (Beijing, China), we conducted the TUNEL assay on 5µm tissue sections and HK2 cells. After completing the assay, we first rinsed the slides twice with phosphate-buffered saline, and then stained them with DAPI (Bioworld). Finally, we carefully examined the slides under a fluorescence microscope (Olympus C-5050, made in Japan). 2.10. Cell apoptosis assay The rate of programmed cell death was measured by utilizing a kit for detecting apoptosis, which involved annexin V/PI double staining (Solarbio, Beijing, China).The cells from every group were gathered, rinsed two times with chilled PBS at 4°C, and suspended again in 1×Annexin V solution at a concentration of 1×106cells/mL. Next, 5 microliters of both Annexin V-FITC and PI staining solution were introduced into 100 microliters of the cell suspension.The cells were thoroughly blended and dyed for 15 minutes at room temperature, ensuring they were kept away from any source of light.Flow cytometry was conducted within one hour. 2.11. Enzyme-linked immunosorbent assay (ELISA) We used an ELISA kit (provided by Solarbio, Beijing, China) to detect the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and lactate dehydrogenase (LDH). The entire experimental process strictly followed the operating guidelines of the cellular ELISA kit. 2.12.Statistical analysis Statistical analyses were conducted using GraphPad Prism 8.3.0 (GraphPad Software, San Diego, CA, USA). The presented data represent the mean with standard deviation. Variations among distinct groups were analyzed via one-way ANOVA. 3. Results 3.1.Network pharmacology analysis After cross-referencing kidney stone targets with PUE targets, we successfully identified 39 common drug-disease targets (Fig.1A). Leveraging the STRING online platform, we constructed an interaction network for these shared targets (Fig.1B). Subsequently, we imported the network diagram and data information obtained from the platform into Cytoscape 3.7.0 software for in-depth analysis, and based on this, we built a PPI network (Fig1C). To determine the key nodes in the network, we used the cytoHubba plug-in to screen for hub genes (Fig1D). Detailed information on these core regulatory genes is listed in Table 1. We performed enrichment analysis using 39 shared drug-disease targets of the GO and KEGG pathways. The top of each part is shown in the bubble graph. Based on the analysis of biological processes (BP), the identified targets are linked to the inflammatory response, promotion of apoptotic process, inhibition of apoptotic process, and apoptosis of epithelial cells (Fig.1E). Cellular component(CC) analysis showed that the target distribution in the nucleus, plasma membrane, cytoplasm, cytosol and nucleoplasm(Fig.1F). Molecular function (MF) show the connections and functions between these targets, such as protein binding, cysteine−type endopeptidase activity involved in apoptotic signaling pathway, cysteine−type endopeptidase activity involved in apoptotic process(Fig.1G). The KEGG enrichment analysis results indicated a strong association between the shared targets and the PI3K/AKT signaling pathway (Fig. 1H). Table1 :Basic topological properties of core regulatory genes. Geen Symbol Average Shortest PathLength Betweenness Centrality Closeness Centrality Neighborhood Connectivity IL1B 1.13157895 0.02327975 0.88372093 24.24242424 STAT3 1.07894737 0.03130638 0.92682927 23.6 CASP3 1.10526316 0.02567058 0.9047619 24.20588235 PTGS2 1.13157895 0.02462452 0.88372093 24.12121212 MMP9 1.10526316 0.03072048 0.9047619 23.79411765 TLR4 1.23684211 0.01227401 0.80851064 25.86206897 HIF1A 1.05263158 0.04019006 0.95 23.19444444 AKT1 1.02631579 0.04827868 0.97435897 22.7027027 TNF 1.05263158 0.04119749 0.95 23.02777778 BCL2 1.15789474 0.02057588 0.86363636 24.5625 3.2. Molecular docking study Based on the selected HUB gene, we used molecular docking technology to predict the binding affinity between PUE and its target protein (Fig2). Affinity is shown in Table 2. We found that PUE has strong binding affinity with the target proteins of HUB genes. Table2 : Docking score of PUE with each core target. Gene Name Dockingscore (kcal mol −1 ) HIF1 -7.3 IL1 -7.8 CAS3 -7.4 SAT3 -8.3 TNF -6.4 TLR4 -7.2 BCL2 -8.1 PTGS2 -8.5 MMP9 -8.0 AKT1 -7.8 3.3.PUE attenuated COM crystal-induced HK2 cell injury, inflammation and oxidative stress The molecular formula of PUE is shown in Fig.3A. Initially, the impact of PUE on HK2 cells was assessed for its cytotoxicity, and the outcomes from CCK-8 tests indicated that HK2 cell viability remained largely unaffected by PUE concentrations lower than 64 uM. Furthermore, we conducted additional research on the impact of PUE on the damage caused to HK2 cells by COM crystals(Fig.3B). The CCK-8 experiments indicated that exposure to COM crystals resulted in a notable reduction in the viability of HK2 cells, which was alleviated by PUE(Fig.3C)，PUE concentrations of 8, 16 and 32 µM were more effective in improving HK2 cell viability, so we used these three concentrations in subsequent cell experiments. We then found that COM crystals induced a significant increase in IL1, IL6, and TNFα, while PUE significantly reduced the inflammatory response(Fig.3D-G). To determine whether PUE causes oxidative stress in HK-2 cells by inhibiting COM, we further examined oxidative stress-related indicators. By measuring COM crystal-induced ROS generation by fluorescence and confocal microscopy, we found that PUE treatment partially attenuated ROS(Fig.3H,I). By assaying MDA, SOD, LDH and GSH we further confirmed that PUE significantly reduced the level of COM-induced oxidative stress as the dose increased(Fig.3J-M). 3.4. In vitro, PUE counteracts the impact of COM on the PI3K/AKT signaling pathway and apoptosis. PUE has been repeatedly shown to have anti-apoptotic effects[28-30]. In this study, we investigated whether PUE can reduce apoptosis in HK2 cells through the PI3K/AKT signaling pathway in vitro using COM.Initially, it was observed that p-PI3K and p-AKT exhibited a notable decrease in the COM group when compared to the control group. However, this decrease was counteracted by PUE. Additionally, the COM group displayed significant alterations in Cleaved-caspase3, Bax, and Bcl2 levels, which were also reversed by PUE in a dose-dependent manner(Fig.4A-F). This suggests that PUE can reverse the apoptosis produced by HK2 cells caused by COM. We then used TUNEL staining to show that treatment with PUE ameliorated COM-induced apoptosis in HK-2 cells(Fig.4G). We also stained the cells with PI/Annexin V and analyzed them by flow cytometry and found that PUE significantly reduced the number of HK-2 cells that showed COM-induced apoptosis(Fig.4H). We fully demonstrated that PUE alleviated COM-induced apoptosis in HK2 cells. 3.5. PUE treatment for renal calcification is a CaOx disease mouse model has a protective effect on inflammation and damage Subsequently, we examined the defensive impact of PUE on in vivo renal calcinosis caused by CaOx.Mice were given PUE at doses of 10, 50, and 100 mg/kg/d after being pretreated with gavage 12 hours prior to the injection of glyoxylate.The findings indicated that PUE mitigated the decrease in body weight in a mouse model of CaOx renal calcinosis(Fig.5A) and significantly decreased elevated levels of serum Cr and BUN in a mouse model of CaOx renal calcinosis(Fig.5B,C).Additionally, there was a decline in the formation of CaOx crystals in the renal system, as depicted in Figure.5D, E.PAS staining showed that PUE treatment also partially reversed renal tubular injury(Fig.5F, G). Western blot analysis demonstrated that PUE effectively decreased the protein levels of IL1, IL6, and TNFα induced by CaOx, as depicted in Figure.5H-K. 3.6. In vivo, PUE counteracts the impact of CaOx on the PI3K/AKT signaling pathway and apoptosis. We then observed in vivo the changes in p-Pi3K, p-Akt and apoptosis-related proteins induced by PUE on CaOx.By Western blotting and IHC we found that PUE attenuated the inhibitory effect of CaOx on the renal Pi3K/Akt signaling pathway and alleviated the CaOx-induced changes in cleaved-caspase3, Bax and Bcl2 levels(Fig.6A-F). Immunohistochemistry yielded consistent results(Fig.6G). The results of TUNEL staining also showed that PUE ameliorated CaOx-induced renal apoptosis(Fig.6H). 3.7. PI3K inhibitor reverses the effects of PUE to alleviate COM damage, inflammation and oxidative stress in HK2 cells To provide additional confirmation of PUE's ability to mitigate the impact of COM on inflammation, apoptosis, and oxidative stress in HK2 cells through modulation of the PI3K/Akt signaling pathway, we employed PI3K inhibitor(LY294002). For the concentration of PUE we used 32uM, which was the most effective in the in vitro experiments described above. Western blot analysis revealed that LY294002 counteracted the anti-inflammatory and anti-apoptotic impacts of PUE(Fig.7A-I). Subsequently by TUNEL staining we found that LY294002 reversed the anti-apoptotic effect of PUE(Fig.7J). Then for further proof, we further examined oxidative stress-related indicators and found that LY294002 reversed PUE to reduce COM-induced superoxide anion and ROS levels in HK2 cells(Fig.7K,L). Finally, cells were stained with PI/Annexin V and analyzed by flow cytometry to further confirm that LY294002 reversed the apoptotic effect of PUE on COM leading to HK2 cells(Fig.7M). 4. Discussion Network pharmacology has been extensively employed to comprehend the intricate mechanisms of medication treatment. Network pharmacology allows rapid screening of drug targets, prediction of pathways of action and systematic analysis of drug-disease interactions[31]. PUE is a major isoflavonoid extracted from the Chinese herb Pueraria Mirifica.Pueraria Mirifica, originating from Southeast Asia, has been utilized for countless centuries as sustenance, remedy, and animal feed. Moreover, it is among the earliest plants employed in ancient China[32]. Through the utilization of network pharmacology, we anticipated the capability of PUE in addressing kidney stones and subsequently validated its efficacy via both in vitro and in vivo experiments. This study revealed a strong correlation between apoptosis and the potential target of PUE in the context of kidney stones. The PI3K/AKT pathway may be associated with the anti-apoptotic mechanism of PUE, as indicated by enrichment analysis of the constructed PPI network. Phosphoinositide 3-kinase (PI3K), a lipid kinase, has a crucial function in both normal and abnormal cellular processes.PI3K controls the regulation of proliferation, differentiation, programmed cell death, and migration by activating protein kinase B (PKB or AKT) [33,34]. The role of Pi3k/Akt pathway in inflammation, oxidative stress and apoptosis in kidney disease has been demonstrated by a large number of studies and plays an important role in kidney disease[35,36]. The PI3K/AKT pathway has been confirmed to be closely related to kidney stones[37,38]. PUE has also been shown to exert its pharmacological effects through the Pi3k/Akt pathway[39,40]. Later we also predicted the affinity of PUE and pathway proteins using molecular docking. Therefore, we believe that PUE can alleviate CaOx-induced renal apoptosis through the PI3K/AKT pathway and proved our hypothesis through in vitro and in vivo experiments. Reactive oxygen species (ROS), collectively referred to as free radicals, consist of atoms or molecules possessing unpaired electrons. These highly reactive species are crucial in the modulation of signaling molecules[41]. In addition, they have the ability to chemically alter and breakdown proteins, lipids, carbohydrates, and nucleotides.Prior research has indicated the existence of ROS buildup in renal tissues containing calcium oxalate crystal deposits, indicating that ROS might play a role in the advancement and growth of calcium oxalate kidney stone disorder[42]. In addition, clinical studies additionally validated that the serum of patients with stones exhibited a reduced level of antioxidant enzymes in comparison to the normal group. This indicates that individuals with stones have a diminished antioxidant capacity in their bodies, and their levels of antioxidant enzymes are lower compared to the normal group[43]. The levels of SOD and CAT activity, as well as the levels of MDA and GSH in tissues, are representative of the oxidative status of the tissues, lipid peroxidation, and cellular injury[44-46]. Therefore, we responded to the level of oxidative stress by measuring MDA, GSH,SOD and LDH in cells. Studies have reported differential proteins associated with inflammatory cells and processes in CaOx stone patients and controls[47]. We found that PUE significantly reduced the level of CaOx-induced oxidative stress and inflammatory responses in vivo and in vitro by a series of methods. PUE not only successfully alleviated the renal function of mice with a reduction in serum creatinine and urea but also attenuated the deposition and damage of calcium oxalate crystals. Since the PI3K/AKT pathway is a key hub where PUE acts, we found by western blotting and HIC that PUE activated the PI3K/Akt pathway and restored the reduction in p-PI3K and p-AKT by CaOx. The involvement of apoptosis in kidney disease is substantiated by human studies that reveal the activation of pro-apoptotic pathways within kidney tissue. Additionally, preclinical data suggest that the protective effect is achieved through the disruption of genuine pro-apoptotic proteins[48]. The ability of CaOx to cause apoptosis in the kidney has been well documented[49-51]. In our study, we confirmed our idea by testing the anti-apoptotic effect of PUE by various means in vivo and in vitro. To further confirm that PUE acts through the PI3K/AKT pathway, we applied an inhibitor of PI3K (LY294002)[52]. We found that the anti-inflammatory, antioxidative stress and antiapoptotic effects of PUE were greatly reduced after inhibiting the PI3K/AKT pathway. This further proves our idea. 5. Conclusion Using the method of network pharmacology, we initially examined the mechanism of action of PUE in treating kidney stones.The PI3K/AKT pathway was identified by network pharmacology as the mechanism through which PUE exerts its anti-apoptotic effects.Following that, the in vivo and in vitro experiments confirmed the preventive and therapeutic capabilities of PUE in treating kidney stones.The scientific foundation for the impact of PUE on kidney stones and the practical use of PUE is established. Declarations Funding This study was supported by the National Natural Science Foundation of China (82070724) , (82370768). Ethics approval and consent to participate The animal study was carried out in compliance with the ARRIVE guidelines. All animal procedures were approved and conducted according to the Animal Experimentation Ethics Committee guidelines. The animal study was reviewed and approved by The Animal Experimentation Ethics Committee of Anhui Medical University (No: LLSC20232250). Author Contributions: YX, HL and XM: Data curation, Writing original draft. XS,ZS and DG:Conceptualization, Methodology, Software. ZH, BH and YC: Writing-review & editing. Declaration of Competing Interest The authors declare that they have no competing interests. Consent for publication All authors supported the publication of the manuscript. Data Availability Statement All data generated or analyzed during this study are included in this published article and its supplementary information files. References Hesse, A.; Brändle, E.; Wilbert, D.; Köhrmann, K.U.; Alken, P. Study on the prevalence and incidence of urolithiasis in Germany comparing the years 1979 vs. 2000. Eur. Urol. 2003 , 44 , 709-713. Scales, C.D. Jr; Smith, A.C.; Hanley, J.M.; Saigal, C.S. Prevalence of kidney stones in the United States. Eur. Urol. 2012 , 62 , 160-165. 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Puerarin alleviates cisplatin-induced acute renal damage and upregulates microRNA-31-related signaling. Exp Ther Med 2020 , 20 , 3122-3129. Zhou, X.; Bai, C.; Sun, X.; Gong, X.; Yang, Y.; Chen, C.; Shan, G.; Yao, Q. Puerarin attenuates renal fibrosis by reducing oxidative stress induced-epithelial cell apoptosis via MAPK signal pathways in vivo and in vitro. Ren Fail 2017 , 39 , 423-431. Zhu, Q.; Yang, S.; Wei, C.; Lu, G.; Lee, K.; He, J.C.; Liu, R.; Zhong, Y. Puerarin attenuates diabetic kidney injury through interaction with Guanidine nucleotide-binding protein Gi subunit alpha-1 (Gnai1) subunit. J. Cell. Mol. Med. 2022 , 26 , 3816-3827. Liu, H.; Ye, T.; Yang, X.; Liu, J.; Jiang, K.; Lu, H.; Xia, D.; Peng, E.; Chen, Z.; Sun, F.; et al. H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway. EBioMedicine 2019 , 50 , 366-378. Liu, L.J.; Liu, L.Q.; Bo, T.; Li, S.J.; Zhu, Z.; Cui, R.R.; Mao, D.A. Puerarin Suppress Apoptosis of Human Osteoblasts via ERK Signaling Pathway. Int J Endocrinol 2013 , 2013 , 786574. Shukla, R.; Banerjee, S.; Tripathi, Y.B. Antioxidant and Antiapoptotic effect of aqueous extract of Pueraria tuberosa (Roxb. Ex Willd.) DC. On streptozotocin-induced diabetic nephropathy in rats. BMC Complement Altern Med 2018 , 18 , 156. Xu, B.; Li, J.; Chen, X.; Kou, M. Puerarin attenuates cisplatin-induced apoptosis of hair cells through the mitochondrial apoptotic pathway. Biochim Biophys Acta Mol Cell Res 2022 , 1869 , 119208. Nogales, C.; Mamdouh, Z.M.; List, M.; Kiel, C.; Casas, A.I.; Schmidt, H. Network pharmacology: curing causal mechanisms instead of treating symptoms. Trends Pharmacol. Sci. 2022 , 43 , 136-150. Prasain, J.K.; Peng, N.; Rajbhandari, R.; Wyss, J.M. The Chinese Pueraria root extract (Pueraria lobata) ameliorates impaired glucose and lipid metabolism in obese mice. Phytomedicine 2012 , 20 , 17-23. Davis, W.J.; Lehmann, P.Z.; Li, W. Nuclear PI3K signaling in cell growth and tumorigenesis. Front Cell Dev Biol 2015 , 3 , 24. Samakova, A.; Gazova, A.; Sabova, N.; Valaskova, S.; Jurikova, M.; Kyselovic, J. The PI3k/Akt pathway is associated with angiogenesis, oxidative stress and survival of mesenchymal stem cells in pathophysiologic condition in ischemia. Physiol Res 2019 , 68 , S131-S138. Wang, X.; Jiang, L.; Liu, X.Q.; Huang, Y.B.; Wang, A.L.; Zeng, H.X.; Gao, L.; Zhu, Q.J.; Xia, L.L.; Wu, Y.G. Paeoniflorin binds to VEGFR2 to restore autophagy and inhibit apoptosis for podocyte protection in diabetic kidney disease through PI3K-AKT signaling pathway. Phytomedicine 2022 , 106 , 154400. Zhang, B.; Zeng, M.; Li, B.; Kan, Y.; Wang, S.; Cao, B.; Huang, Y.; Zheng, X.; Feng, W. Arbutin attenuates LPS-induced acute kidney injury by inhibiting inflammation and apoptosis via the PI3K/Akt/Nrf2 pathway. Phytomedicine 2021 , 82 , 153466. Chen, X.; Zhang, X.B.; Li, D.J.; Qi, G.N.; Dai, Y.Q.; Gu, J.; Chen, M.Q.; Hu, S.; Liu, Z.Y.; Yang, Z.M. miR-155 facilitates calcium oxalate crystal-induced HK-2 cell injury via targeting PI3K associated autophagy. Exp. Mol. Pathol. 2020 , 115 , 104450. Yuan, H.; Zhang, J.; Yin, X.; Liu, T.; Yue, X.; Li, C.; Wang, Y.; Li, D.; Wang, Q. The protective role of corilagin on renal calcium oxalate crystal-induced oxidative stress, inflammatory response, and apoptosis via PPAR-γ and PI3K/Akt pathway in rats. Biotechnol. Appl. Biochem. 2021 , 68 , 1323-1331. Chen, F.; Chen, Z.Q.; Wang, H.; Zhu, J.J. Puerarin pretreatment inhibits myocardial apoptosis and improves cardiac function in rats after acute myocardial infarction through the PI3K/Akt signaling pathway. Adv Clin Exp Med 2021 , 30 , 255-261. Wang, L.; Jiang, W.; Wang, J.; Xie, Y.; Wang, W. Puerarin inhibits FUNDC1-mediated mitochondrial autophagy and CSE-induced apoptosis of human bronchial epithelial cells by activating the PI3K/AKT/mTOR signaling pathway. Aging 2022 , 14 , 1253-1264. Dröge, W. Free radicals in the physiological control of cell function. Physiol. Rev. 2002 , 82 , 47-95. Khan, S.R. Reactive oxygen species as the molecular modulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations. J. Urol. 2013 , 189 , 803-811. Ceban, E.; Banov, P.; Galescu, A.; Botnari, V. Oxidative stress and antioxidant status in patients with complicated urolithiasis. Journal of medicine and life 2016 , 9 , 259-262. Goc, Z.; Szaroma, W.; Kapusta, E.; Dziubek, K. Protective effects of melatonin on the activity of SOD, CAT, GSH-Px and GSH content in organs of mice after administration of SNP. Chin J Physiol 2017 , 60 , 1-10. Mehdi, M.; Menon, M.; Seyoum, N.; Bekele, M.; Tigeneh, W.; Seifu, D. Blood and Tissue Enzymatic Activities of GDH and LDH, Index of Glutathione, and Oxidative Stress among Breast Cancer Patients Attending Referral Hospitals of Addis Ababa, Ethiopia: Hospital-Based Comparative Cross-Sectional Study. Oxid Med Cell Longev 2018 , 2018 , 6039453. Xu, J.; Lin, C.; Wang, T.; Zhang, P.; Liu, Z.; Lu, C. Ergosterol Attenuates LPS-Induced Myocardial Injury by Modulating Oxidative Stress and Apoptosis in Rats. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2018 , 48 , 583-592. Canales, B.K.; Anderson, L.; Higgins, L.; Ensrud-Bowlin, K.; Roberts, K.P.; Wu, B.; Kim, I.W.; Monga, M. Proteome of human calcium kidney stones. Urology 2010 , 76 , 1017.e13-20. Sanz, A.B.; Santamaría, B.; Ruiz-Ortega, M.; Egido, J.; Ortiz, A. Mechanisms of renal apoptosis in health and disease. J. Am. Soc. Nephrol. 2008 , 19 , 1634-1642. Khan, S.R.; Byer, K.J.; Thamilselvan, S.; Hackett, R.L.; McCormack, W.T.; Benson, N.A.; Vaughn, K.L.; Erdos, G.W. Crystal-cell interaction and apoptosis in oxalate-associated injury of renal epithelial cells. J. Am. Soc. Nephrol. 1999 , 10 Suppl 14 , S457-463. Yan, L.; Chen, J.; Fang, W. Exosomes derived from calcium oxalate-treated macrophages promote apoptosis of HK-2 cells by promoting autophagy. Bioengineered 2022 , 13 , 2442-2450. Ye, Q.L.; Wang, D.M.; Wang, X.; Zhang, Z.Q.; Tian, Q.X.; Feng, S.Y.; Zhang, Z.H.; Yu, D.X.; Ding, D.M.; Xie, D.D. Sirt1 inhibits kidney stones formation by attenuating calcium oxalate-induced cell injury. Chem. Biol. Interact. 2021 , 347 , 109605. Xiao, Q. Cinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155. Ren Fail 2022 , 44 , 601-614. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4509583","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309363509,"identity":"f4b87b1b-57a0-4124-84d7-fcf09ba42dd4","order_by":0,"name":"Yuexian Xu","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuexian","middleName":"","lastName":"Xu","suffix":""},{"id":309363510,"identity":"7a32cf63-0773-42f7-a26c-da2813f08d90","order_by":1,"name":"Hu Liang","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hu","middleName":"","lastName":"Liang","suffix":""},{"id":309363511,"identity":"be6bfd9a-5e35-4b60-bb73-4e78dabd9869","order_by":2,"name":"Xike Mao","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xike","middleName":"","lastName":"Mao","suffix":""},{"id":309363512,"identity":"0eb501a0-f3e7-4a5c-b25b-eb5814a82933","order_by":3,"name":"Zhenyu Song","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Song","suffix":""},{"id":309363513,"identity":"0e7e15fb-beab-454c-b7c2-f481270489ff","order_by":4,"name":"Xudong Shen","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xudong","middleName":"","lastName":"Shen","suffix":""},{"id":309363514,"identity":"69a64352-80b6-478c-a5b1-1db956e63ca7","order_by":5,"name":"Defeng Ge","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Defeng","middleName":"","lastName":"Ge","suffix":""},{"id":309363515,"identity":"054322b3-8594-4c5c-8e54-c9974d884d81","order_by":6,"name":"Yang Chen","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Chen","suffix":""},{"id":309363516,"identity":"18129221-2524-4f1c-8116-c27894eca97a","order_by":7,"name":"Bingbing Hou","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bingbing","middleName":"","lastName":"Hou","suffix":""},{"id":309363517,"identity":"2e0fc771-0e03-42b0-a85d-c359aaa3dffa","order_by":8,"name":"Zongyao Hao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACAwST+cCBDz9I08KWeHBmD2laeIwPc7ARocWc/fDhDx931Cb2t/d8OMzAwyDPL3YAvxbLnrQEw5lnjifOOHN2w+ECCwbDmbMTCDjsQI5BMm/bscSGG7kbDs/gYUgwuE1Iy/k3BodBWubfyHlwmIeNGC03cgybedtqEjfcyGEgVsuzZMaZbQeMN545ZgAMZAki/HI+GRhibXWy8443P/7w4YeNPL80AS1QcNixAcKQIEo5CNTZE610FIyCUTAKRh4AAKzRT2wbmSAGAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zongyao","middleName":"","lastName":"Hao","suffix":""}],"badges":[],"createdAt":"2024-05-31 14:21:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4509583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4509583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58319605,"identity":"1f5436ae-8064-4ee9-9796-b23d224df045","added_by":"auto","created_at":"2024-06-13 22:54:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25561381,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram and PPI network and Enrichment analysis. (A) Venn diagram of the intersection target between PUE and Kidney stone. (B) PPI network of targets generated using STRING 11.0.(C) PPI network of targets genetrated useing Cytoscape 3.7. (D) Top 10 targets of PPI network. (E) Top 20 Gene Ontology cellular components. (F) Top 20 Gene Ontology biological processes. (G) Top 20 Gene Ontology molecular functions. (H) Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Abscissa represents gene proportion, ordinate represents pathway name, the size of the bubble represents the number of targets in the pathway and the color represents P-value.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/d12befcc0f714a9302342111.jpg"},{"id":58319609,"identity":"c16324ce-e5a3-4e1c-90f5-4378fca01871","added_by":"auto","created_at":"2024-06-13 22:54:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24903132,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking. Molecular models of the binding of PUE with MMP9, STAT3, CASP3, IL1B, HIF1A, AKT1, PTGS2, BCL2, TLR4, TNF.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/70abf854418626979d9daaf1.jpg"},{"id":58319603,"identity":"86e1fdc1-9186-409d-a250-34122afed203","added_by":"auto","created_at":"2024-06-13 22:54:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16557896,"visible":true,"origin":"","legend":"\u003cp\u003ePUE prevents COM crystal-induced cell injury and inflammation.(A) The molecular formula of PUE. (B) We tested the cytotoxicity of PUE on HK2 cells at different concentrations using the CCK-8 assay. (C) The PUE pretreatment of HK2 cells exposed to COM crystals restored their viability.(D-G) Western blot and quantitative analysis of IL1, IL6 and TNFα. (H,I) The levels of superoxide anion and reactive oxygen species in HK-2 cells were detected using DHE and DCF staining. (J) GSH content (μg/10\u003csup\u003e6\u003c/sup\u003e cell). (K) LDH content (U/10\u003csup\u003e4\u003c/sup\u003ecell). (L) SOD activity (U/10\u003csup\u003e4\u003c/sup\u003ecell). (M) MDA content (nmol/10\u003csup\u003e4\u003c/sup\u003ecell). Data are shown as the mean ± S.E.M. ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/403624a2a8f0cf39fac5744f.jpg"},{"id":58319695,"identity":"19635402-3487-4e87-a649-048080b17226","added_by":"auto","created_at":"2024-06-13 23:02:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16935824,"visible":true,"origin":"","legend":"\u003cp\u003ePUE attenuates COM crystal-induced cell apoptosis.(A-F).Western blot and quantitative analysis of p-PI3K, PI3K, p-AKT, AKT, Cleaved-caspase3, caspase3, Bax and Bcl2. (G) HK-2 cells were stained with TUNEL for detection of apoptosis. The staining shows green fluorescence when the cells are in the process of dying. (H) The effect of PUE on COM-induced apoptosis in HK-2 cells was analyzed by flow cytometry. Data are shown as the mean ± S.E.M. ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/21cabdc181faaa7ad946e9bb.jpg"},{"id":58319694,"identity":"37fe53d6-ba15-4d5e-adc6-c47f1585a69e","added_by":"auto","created_at":"2024-06-13 23:02:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5753529,"visible":true,"origin":"","legend":"\u003cp\u003ePUE protected against CaOx crystal-induced renal injury and inflammation in mice. (A) Change in weight. (B,C) Serum Cr and BUN in the mice with CaOx crystalline nephropathy with or without PUE administration. (D,E) Polarized light microscopy was used to visualize CaOx crystal deposition in kidney sections and quantify it as a percentage. (F,G) PAS staining was used to illustrate and score tubular injury. (H-K) Western blot and quantitative analysis of IL1, IL6 and TNFα. Data are shown as the mean ± S.E.M. ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/af048e6443bdc0fa81cc4c27.jpg"},{"id":58319696,"identity":"87782019-f806-4bf3-9e02-b2b2c0983bc3","added_by":"auto","created_at":"2024-06-13 23:02:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":27392370,"visible":true,"origin":"","legend":"\u003cp\u003ePUE protected against CaOx crystal-induced renal apoptosis in mice. (A-G) Western blot and quantitative analysis of p-PI3K, PI3K, p-AKT, AKT, Cleaved-caspase3, caspase3, Bax and Bcl2. (H) Representative immunohistochemistry images and quantitative analysis of p-PI3K, PI3K, p-AKT, AKT, Cleaved-caspase3, caspase3, Bax and Bcl2. (H) Renal were stained with TUNEL for detection of apoptosis. (I) Molecular interaction between PUE and p-PI3K. Data are shown as the mean ± S.E.M. ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/3763262eba81de3b4a26cddb.jpg"},{"id":58319606,"identity":"f33cbf1c-0475-42cc-8759-2488be12c08d","added_by":"auto","created_at":"2024-06-13 22:54:45","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":8399624,"visible":true,"origin":"","legend":"\u003cp\u003ePUE exerted protective effects by PI3K/AKT pathway. (A-I) Western blot analysis of HK2 cells p-PI3K, PI3K, p-AKT, AKT, Cleaved-caspase3, caspase3, Bax, Bcl2, IL1, IL6 and TNFα in indicated groups. (J) HK-2 cells were stained with TUNEL for detection of apoptosis. (K,L) The levels of superoxide anion and reactive oxygen species in HK-2 cells were detected using DHE and DCF staining. (M) The apoptosis of HK-2 cells was detected by flow cytometry. Data are shown as the mean ± S.E.M. ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/b7be770cc17f57fe19da8a79.jpg"},{"id":58604899,"identity":"189f0f32-b1a6-4681-9bf7-4ba3863c2ee6","added_by":"auto","created_at":"2024-06-18 19:17:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":126215946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4509583/v1/de4803b2-6773-4f61-b705-cf68d715151f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Puerarin ameliorates apoptosis and inflammation induced by kidney stones through the PI3K/AKT pathway: Network pharmacology and experimental validation","fulltext":[{"header":"1. Introduction ","content":"\u003cp\u003eThe prevalence of kidney stones, a frequent urological condition, has been on the rise for the last twenty years[1-3]. Despite notable advancements in less invasive surgical methods, the rate of recurrence within a span of five years following the initial operation remains at 50%[4], resulting in substantial socioeconomic and medical burdens[5]. The main component of kidney stones is calcium oxalate (CaOx) crystals, which account for up to 80% of all kidney stones[6]. CaOx\u0026nbsp;causes kidney damage mainly because of apoptosis, autophagy, inflammation, fibrosis and so on[7-10]. Considering this, the discovery of novel therapeutic targets holds immense theoretical importance and practical value in the treatment and prevention of kidney stones.\u003c/p\u003e\n\u003cp\u003eThe method of treating kidney stones with herbal medicine has a long history and profound cultural heritage. With the deepening of scientific research, more and more studies have confirmed that herbal medicine has significant efficacy in the treatment of kidney stones. Its unique advantage lies in its smaller side effects and high safety, providing a more gentle and effective treatment approach for kidney stone patients[11-13].\u0026nbsp;PUE, derived from Pueraria Mirifica, is\u0026nbsp;a\u0026nbsp;bioactive compound abundant in flavonoids, peptides, and other bioactive substances[14]. An increasing amount of pharmacological research has shown that PUE exhibits a range of biopharmacological properties. PUE has been shown to have anti-inflammatory, anti-injury, and anti-apoptotic effects in vitro in a large number of experiments[15-19]. Not only in vitro experiments, but also in vivo experiments have fully verified its efficacy. PUE attenuates neurological deficits apoptotic pathways in subarachnoid hemorrhage mice[20]. PUE suppresses inflammation and alleviates pain symptom in osteoarthritic mice[21]. PUE protects against myocardial ischemia in rats[22]. The effect of PUE on the kidney has also been demonstrated by a large number of in vivo experiments. The renoprotective effect of PUE has been demonstrated in diabetic nephropathy, renal fibrosis, and cisplatin-induced acute kidney injury[23-26]. Nonetheless, limited research has been conducted on the safeguarding impact of PUE against CaOx-induced renal damage.\u003c/p\u003e\n\u003cp\u003eNetwork pharmacology, as a new research methodology, has been successful in revealing multiple targets and complex mechanisms of drugs in various diseases.Using a network pharmacology method, we examined the possible targets and pathways through which PUE can reduce renal injury caused by CaOx.To validate our hypothesis, we conducted experiments both in vivo and in vitro.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1.Network pharmacology\u003c/p\u003e\n\u003cp\u003eGeneCards (Giftes\u0026ge;5) was used to search through the Swiss target prediction database (www.swisstargetprediction.ch) for potential targets associated with kidney stones. Venny tool draw the Venn diagram, kidney stones and PUE-related targets. Protein-protein interaction(PPI) between potential targets were evaluated using the STRING database (https://cn.string-db.org/). Using Cytoscape 3.7, we built targets and kidney stones of PUE potential targets for shared between PPI, through the CytoHubba plug-in filtered on key targets.\u0026nbsp;The\u0026nbsp;DAVID\u0026nbsp;database (david.abcc.ncifcrf.gov) was used for enrichment analysis of\u0026nbsp;the\u0026nbsp;intersecting\u0026nbsp;genes. Molecular docking was performed using AutoDock.\u003c/p\u003e\n\u003cp\u003e2.2.Chemicals and reagents\u003c/p\u003e\n\u003cp\u003ePUE and LY294002 (PI3K/AKT inhibitor) were purchased from TargetMol (Shanghai, China). Glyoxylate and calcium oxalate monohydrate (COM) were obtained from Sigma (Germany). TNF (26405-1-AP), IL1(16765-1-AP), IL6(21865-1-AP), Akt(10176-2-AP), p-Akt(28731-1-AP), Pi3k(20584-1-AP), Bax(50599-2-Ig), Bcl-2(26593-1-AP) and Caspase3(19677-1-AP) were obtained from Peprotech (Wuhan, China). p-PI3K(#AF3242) was obtained from Affinity Biosciences(Jiangsu, China). Using the Cell Counting Kit-8 (CCK-8) sourced from Beyotime (Nanjing, China) and ELISA kits from Solarbio (China), we assayed various oxidative stress markers\u0026mdash;specifically, MDA, SOD, LDH, and GSH\u0026mdash;in the cell homogenates derived from different treatment cohorts.\u003c/p\u003e\n\u003cp\u003e2.3.Cell culture and treatment\u003c/p\u003e\n\u003cp\u003eThe human kidney cell line HK-2 was obtained from the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences. These cells were cultured in an environment containing 10% fetal bovine serum and a DMEM/F12 mixed medium, maintained at 5% carbon dioxide and 95% humidity for routine cultivation. Before stimulating with COM (1000 mg ml-1), we exposed the HK-2 cells to different concentrations of PUE (1, 2, 4, 8, 16, 32, 64, 128 \u0026mu;M) or LY294002 (10 \u0026mu;M) for 30 minutes. Afterwards, they were transferred to a new environment containing 0.5% fetal bovine serum and a DMEM/F12 mixed medium for overnight cultivation.\u003c/p\u003e\n\u003cp\u003e2.4.Cell viability determination\u003c/p\u003e\n\u003cp\u003eCCK-8 was utilized to assess cell viability.In summary, HK2 cells were seeded in 96-well dishes and prior to stimulation with COM (1,000 mg L-1), they were treated with varying amounts of PUE for 24 hours.Next, 10 microliters of CCK-8 solution was introduced into every well and incubated for a period of 60 to 90 minutes.The manufacturer\u0026apos;s instructions (Multiskan MK3, Thermo, United States) were followed to analyze the optical density at 492 nm.\u003c/p\u003e\n\u003cp\u003e2.5.Animals and experimental design\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe conducted rigorous and cautious animal experiments. In this experiment, we carefully selected male C57BL/6J mice aged between 6 and 8 weeks as the subjects to ensure the accuracy and reliability of the experimental results. It\u0026apos;s worth mentioning that we strictly adhere to animal ethics norms, and this experiment has been officially authorized by the Animal Ethics Committee of Anhui Medical University (numbered LLSC20232250). This committee is committed to ensuring that all scientific research activities involving animals follow the highest ethical standards, fully respecting and protecting the rights and interests of animals. Their authorization provides important guarantees for the compliance and morality of our experiment. To create a mouse model for CaOx renal calcium deposition, a daily intraperitoneal injection of 100 mg/kg glyoxylate was administered, and the mice were euthanized after 7 days of injection.Mice received PUE(10, 50, and 100mg/kg) via oral administration, 12 hours prior to glyoxylate injection, followed by daily administration. We anesthetized the mice with Sodium barbiturate (50 mg/kg) (Sigma, 11715) intraperitoneally, collected blood samples from their orbits for analysis, and killed them after they had been fasted and water deprived for 12 hours. The blood that was acquired underwent testing for Cr and BUN in accordance with the instructions provided by the manufacturer, while the renal tissue was regularly embedded in paraffin and examined for molecular objectives. ImageJ software was used to determine the percentage of CaOx crystal deposition in each kidney section.\u003c/p\u003e\n\u003cp\u003e2.6. Tubular injury determination\u003c/p\u003e\n\u003cp\u003eIn order to assess the damage to tubules, we conducted PAS staining using a PAS staining kit according to the established protocol. Subsequently, we invited three experienced pathologists to collaboratively determine the tubule damage score based on the degree of loss of brush border, tubule dilation and atrophy, as well as the formation of intraluminal casts. Regarding tubular lesions, we have established the following grading criteria[27]: 0 (indicating normal), 1 (\u0026le;10%), 2 (11\u0026ndash;25%), 3 (26\u0026ndash;50%), 4 (51\u0026ndash;75%), and 5 (\u0026ge;76%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.7.Histology and immunohistochemical staining\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eParaffin-embedded sections of mouse kidneys were prepared using standard histopathology techniques, including 4% paraformaldehyde fixation, dehydration, waxing, embedding, and sectioning (4 \u0026mu;m). Kidney sections were then stained with HE and PAS reagent kits using standard protocols (Solarbio, Beijing, China).\u0026nbsp;First,\u0026nbsp;Immunohistochemistry was performed\u0026nbsp;by renal biopsy antigen repair, and then endogenous peroxidase activity was blocked. The antibodies were incubated for 30 minutes at a temperature of 4℃. Tissue samples were stained using the DAB (3,3\u0026apos;-diaminobenzidine) staining method. After staining was completed, all observed images were captured and recorded using an Olympus IX83 microscope (made in Japan).\u003c/p\u003e\n\u003cp\u003e2.8.Western blot analysis\u003c/p\u003e\n\u003cp\u003eUsing 10% SDS-PAGE technology, separate 30 micrograms of tissue lysate and cell samples. The separated samples of cells mixed with tissue lysate are then transferred to a nitrocellulose (NC) membrane using electrophoretic transfer. Block the NC membrane for 2 hours using tris-buffered saline containing 0.05% Tween 20 and 5% non-fat milk. Cells are incubated with a specific primary antibody for 8 hours. Subsequently, the cells are co-incubated with a secondary antibody at 37 degrees Celsius for 90 minutes. Images are captured and analyzed using the Licor/Odyssey infrared imaging system. Finally, the gray values of each band are quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e2.9. TUNEL staining\u003c/p\u003e\n\u003cp\u003eFollowing the guidelines provided by Solarbio (Beijing, China), we conducted the TUNEL assay on 5\u0026micro;m tissue sections and HK2 cells. After completing the assay, we first rinsed the slides twice with phosphate-buffered saline, and then stained them with DAPI (Bioworld). Finally, we carefully examined the slides under a fluorescence microscope (Olympus C-5050, made in Japan).\u003c/p\u003e\n\u003cp\u003e2.10. Cell apoptosis assay\u003c/p\u003e\n\u003cp\u003eThe rate of programmed cell death was measured by utilizing a kit for detecting apoptosis, which involved annexin V/PI double staining (Solarbio, Beijing, China).The cells from every group were gathered, rinsed two times with chilled PBS at 4\u0026deg;C, and suspended again in 1\u0026times;Annexin V solution at a concentration of 1\u0026times;106cells/mL. Next, 5 microliters of both Annexin V-FITC and PI staining solution were introduced into 100 microliters of the cell suspension.The cells were thoroughly blended and dyed for 15 minutes at room temperature, ensuring they were kept away from any source of light.Flow cytometry was conducted within one hour.\u003c/p\u003e\n\u003cp\u003e2.11. Enzyme-linked immunosorbent assay (ELISA)\u003c/p\u003e\n\u003cp\u003eWe used an ELISA kit (provided by Solarbio, Beijing, China) to detect the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and lactate dehydrogenase (LDH). The entire experimental process strictly followed the operating guidelines of the cellular ELISA kit.\u003c/p\u003e\n\u003cp\u003e2.12.Statistical analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using GraphPad Prism 8.3.0 (GraphPad Software, San Diego, CA, USA). The presented data represent the mean with standard deviation. Variations among distinct groups were analyzed via one-way ANOVA.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.Network pharmacology analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter cross-referencing kidney stone targets with PUE targets, we successfully identified 39 common drug-disease targets (Fig.1A). Leveraging the STRING online platform, we constructed an interaction network for these shared targets (Fig.1B). Subsequently, we imported the network diagram and data information obtained from the platform into Cytoscape 3.7.0 software for in-depth analysis, and based on this, we built a PPI network (Fig1C). To determine the key nodes in the network, we used the cytoHubba plug-in to screen for hub genes (Fig1D). Detailed information on these core regulatory genes is listed in Table 1. We performed enrichment analysis using 39 shared drug-disease targets of the GO and KEGG pathways. The top of each part is shown in the bubble graph. Based on the analysis of biological processes (BP), the identified targets are linked to the inflammatory response, promotion of apoptotic process, inhibition of apoptotic process, and apoptosis of epithelial cells (Fig.1E). Cellular component(CC) analysis showed that the target distribution in the nucleus, plasma membrane, cytoplasm, cytosol and nucleoplasm(Fig.1F). Molecular function (MF) show the connections and functions between these targets, such as protein binding, cysteine\u0026minus;type endopeptidase activity involved in apoptotic signaling pathway, cysteine\u0026minus;type endopeptidase activity involved in apoptotic process(Fig.1G). The KEGG enrichment analysis results indicated a strong association between the shared targets and the PI3K/AKT signaling pathway (Fig. 1H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable1\u003c/strong\u003e:Basic topological properties of core regulatory genes.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGeen Symbol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage Shortest PathLength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBetweenness Centrality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCloseness Centrality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeighborhood Connectivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eIL1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.13157895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.02327975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.88372093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e24.24242424\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eSTAT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.07894737\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.03130638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.92682927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e23.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCASP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.10526316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.02567058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.9047619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e24.20588235\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003ePTGS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.13157895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.02462452\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.88372093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e24.12121212\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eMMP9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.10526316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.03072048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.9047619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e23.79411765\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTLR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.23684211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.01227401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.80851064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e25.86206897\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eHIF1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.05263158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.04019006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e23.19444444\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eAKT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.02631579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.04827868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.97435897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e22.7027027\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.05263158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.04119749\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e23.02777778\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.583333333333334%\"\u003e\n \u003cp\u003eBCL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.541666666666666%\"\u003e\n \u003cp\u003e1.15789474\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.958333333333332%\"\u003e\n \u003cp\u003e0.02057588\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.875%\"\u003e\n \u003cp\u003e0.86363636\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.041666666666668%\"\u003e\n \u003cp\u003e24.5625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Molecular docking study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the selected HUB gene, we used molecular docking technology to predict the binding affinity between PUE and its target protein (Fig2). Affinity is shown in Table 2. We found that PUE has strong binding affinity with the target proteins of HUB genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable2\u003c/strong\u003e: Docking score of PUE with each core target.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGene Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDockingscore \u0026nbsp; (kcal mol\u003c/strong\u003e\u0026minus;1\u0026nbsp;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHIF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCAS3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSAT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTLR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-7.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBCL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePTGS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMMP9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-8.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAKT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.PUE attenuated COM crystal-induced HK2 cell injury, inflammation and oxidative stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular formula of PUE is shown in Fig.3A. Initially, the impact of PUE on HK2 cells was assessed for its cytotoxicity, and the outcomes from CCK-8 tests indicated that HK2 cell viability remained largely unaffected by PUE concentrations lower than 64 uM. Furthermore, we conducted additional research on the impact of PUE on the damage caused to HK2 cells by COM crystals(Fig.3B). The CCK-8 experiments indicated that exposure to COM crystals resulted in a notable reduction in the viability of HK2 cells, which was alleviated by PUE(Fig.3C)，PUE concentrations of 8, 16 and 32 \u0026micro;M were more effective in improving HK2 cell viability, so we used these three concentrations in subsequent cell experiments. We then found that COM crystals induced a significant increase in IL1, IL6, and TNF\u0026alpha;, while PUE significantly reduced the inflammatory response(Fig.3D-G). To determine whether PUE causes oxidative stress in HK-2 cells by inhibiting COM, we further examined oxidative stress-related indicators. By measuring COM crystal-induced ROS generation by fluorescence and confocal microscopy, we found that PUE treatment partially attenuated ROS(Fig.3H,I). By assaying MDA, SOD, LDH and GSH we further confirmed that PUE significantly reduced the level of COM-induced oxidative stress as the dose increased(Fig.3J-M).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. In vitro, PUE counteracts the impact of COM on the PI3K/AKT signaling pathway and apoptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePUE has been repeatedly shown to have anti-apoptotic effects[28-30]. In this study, we investigated whether PUE can reduce apoptosis in HK2 cells through the PI3K/AKT signaling pathway in vitro using COM.Initially, it was observed that p-PI3K and p-AKT exhibited a notable decrease in the COM group when compared to the control group. However, this decrease was counteracted by PUE. Additionally, the COM group displayed significant alterations in Cleaved-caspase3, Bax, and Bcl2 levels, which were also reversed by PUE in a dose-dependent manner(Fig.4A-F). This suggests that PUE can reverse the apoptosis produced by HK2 cells caused by COM. We then used TUNEL staining to show that treatment with PUE ameliorated COM-induced apoptosis in HK-2 cells(Fig.4G). We also stained the cells with PI/Annexin V and analyzed them by flow cytometry and found that PUE significantly reduced the number of HK-2 cells that showed COM-induced apoptosis(Fig.4H). We fully demonstrated that PUE alleviated COM-induced apoptosis in HK2 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. PUE treatment for renal calcification is a CaOx disease mouse model has a protective effect on inflammation and damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, we examined the defensive impact of PUE on in vivo renal calcinosis caused by CaOx.Mice were given PUE at doses of 10, 50, and 100 mg/kg/d after being pretreated with gavage 12 hours prior to the injection of glyoxylate.The findings indicated that PUE mitigated the decrease in body weight in a mouse model of CaOx renal calcinosis(Fig.5A) and significantly decreased elevated levels of serum Cr and BUN in a mouse model of CaOx renal calcinosis(Fig.5B,C).Additionally, there was a decline in the formation of CaOx crystals in the renal system, as depicted in Figure.5D, E.PAS staining showed that PUE treatment also partially reversed renal tubular injury(Fig.5F, G). Western blot analysis demonstrated that PUE effectively decreased the protein levels of IL1, IL6, and TNF\u0026alpha; induced by CaOx, as depicted in Figure.5H-K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. In vivo, PUE counteracts the impact of CaOx on the PI3K/AKT signaling pathway and apoptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then observed in vivo the changes in p-Pi3K, p-Akt and apoptosis-related proteins induced by PUE on CaOx.By Western blotting and IHC we found that PUE attenuated the inhibitory effect of CaOx on the renal Pi3K/Akt signaling pathway and alleviated the CaOx-induced changes in cleaved-caspase3, Bax and Bcl2 levels(Fig.6A-F). Immunohistochemistry yielded consistent results(Fig.6G). The results of TUNEL staining also showed that PUE ameliorated CaOx-induced renal apoptosis(Fig.6H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7. PI3K inhibitor reverses the effects of PUE to alleviate COM damage, inflammation and oxidative stress in HK2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo provide additional confirmation of PUE\u0026apos;s ability to mitigate the impact of COM on inflammation, apoptosis, and oxidative stress in HK2 cells through modulation of the PI3K/Akt signaling pathway, we employed PI3K inhibitor(LY294002). For the concentration of PUE we used 32uM, which was the most effective in the in vitro experiments described above. Western blot analysis revealed that LY294002 counteracted the anti-inflammatory and anti-apoptotic impacts of PUE(Fig.7A-I). Subsequently by TUNEL staining we found that LY294002 reversed the anti-apoptotic effect of PUE(Fig.7J). Then for further proof, we further examined oxidative stress-related indicators and found that LY294002 reversed PUE to reduce COM-induced superoxide anion and ROS levels in HK2 cells(Fig.7K,L). Finally, cells were stained with PI/Annexin V and analyzed by flow cytometry to further confirm that LY294002 reversed the apoptotic effect of PUE on COM leading to HK2 cells(Fig.7M).\u003c/p\u003e"},{"header":"4. Discussion ","content":"\u003cp\u003eNetwork pharmacology has been extensively employed to comprehend the intricate mechanisms of medication treatment.\u0026nbsp;Network pharmacology allows rapid screening of drug targets, prediction of pathways of action and systematic analysis of drug-disease interactions[31]. PUE is a major isoflavonoid extracted from the Chinese herb Pueraria Mirifica.Pueraria Mirifica, originating from Southeast Asia, has been utilized for countless centuries as sustenance, remedy, and animal feed. Moreover, it is among the earliest plants employed in ancient China[32]. Through the utilization of network pharmacology, we anticipated the capability of PUE in addressing kidney stones and subsequently validated its efficacy via both in vitro and in vivo experiments. This study revealed a strong correlation between apoptosis and the potential target of PUE in the context of kidney stones. The PI3K/AKT pathway may be associated with the anti-apoptotic mechanism of PUE, as indicated by enrichment analysis of the constructed PPI network. Phosphoinositide 3-kinase (PI3K), a lipid kinase, has a crucial function in both normal and abnormal cellular processes.PI3K controls the regulation of proliferation, differentiation, programmed cell death, and migration by activating protein kinase B (PKB or AKT)\u0026nbsp;[33,34]. The role of Pi3k/Akt pathway in inflammation, oxidative stress and apoptosis in kidney disease has been demonstrated by a large number of studies and plays an important role in kidney disease[35,36]. The PI3K/AKT pathway has been confirmed to be closely related to kidney stones[37,38]. PUE has also been shown to exert its pharmacological effects through the Pi3k/Akt pathway[39,40]. Later we also predicted the affinity of PUE and pathway proteins using molecular docking. Therefore, we believe that PUE can alleviate CaOx-induced renal apoptosis through the PI3K/AKT pathway and \u0026nbsp;proved our hypothesis through in vitro and in vivo experiments.\u003c/p\u003e\n\u003cp\u003eReactive oxygen species (ROS), collectively referred to as free radicals, consist of atoms or molecules possessing unpaired electrons. These highly reactive species are crucial in the modulation of signaling molecules[41]. In addition, they have the ability to chemically alter and breakdown proteins, lipids, carbohydrates, and nucleotides.Prior research has indicated the existence of ROS buildup in renal tissues containing calcium oxalate crystal deposits, indicating that ROS might play a role in the advancement and growth of calcium oxalate kidney stone disorder[42]. In addition, clinical studies additionally validated that the serum of patients with stones exhibited a reduced level of antioxidant enzymes in comparison to the normal group. This indicates that individuals with stones have a diminished antioxidant capacity in their bodies, and their levels of antioxidant enzymes are lower compared to the normal group[43]. The levels of SOD and CAT activity, as well as the levels of MDA and GSH in tissues, are representative of the oxidative status of the tissues, lipid peroxidation, and cellular injury[44-46]. Therefore, we responded to the level of oxidative stress by measuring MDA, GSH,SOD and LDH in cells. Studies have reported differential proteins associated with inflammatory cells and processes in CaOx stone patients and controls[47]. We found that PUE significantly reduced the level of CaOx-induced oxidative stress and inflammatory responses in vivo and in vitro by a series of methods. PUE not only successfully alleviated the renal function of mice with a reduction in serum creatinine and urea but also attenuated the deposition and damage of calcium oxalate crystals.\u003c/p\u003e\n\u003cp\u003eSince the PI3K/AKT pathway is a key hub where PUE acts, we found by western blotting and HIC that PUE activated the PI3K/Akt pathway and restored the reduction in p-PI3K and p-AKT by CaOx. The involvement of apoptosis in kidney disease is substantiated by human studies that reveal the activation of pro-apoptotic pathways within kidney tissue. Additionally, preclinical data suggest that the protective effect is achieved through the disruption of genuine pro-apoptotic proteins[48]. The ability of CaOx to cause apoptosis in the kidney has been well documented[49-51]. In our study, we confirmed our idea by testing the anti-apoptotic effect of PUE by various means in vivo and in vitro.\u003c/p\u003e\n\u003cp\u003eTo further confirm that PUE acts through the PI3K/AKT pathway, we applied an inhibitor of PI3K (LY294002)[52]. We found that the anti-inflammatory, antioxidative stress and antiapoptotic effects of PUE were greatly reduced after inhibiting the PI3K/AKT pathway. This further proves our idea.\u003c/p\u003e"},{"header":"5. Conclusion\t","content":"\u003cp\u003eUsing the method of network pharmacology, we initially examined the mechanism of action of PUE in treating kidney stones.The PI3K/AKT pathway was identified by network pharmacology as the mechanism through which PUE exerts its anti-apoptotic effects.Following that, the in vivo and in vitro experiments confirmed the preventive and therapeutic capabilities of PUE in treating kidney stones.The scientific foundation for the impact of PUE on kidney stones and the practical use of PUE is established.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (82070724) , (82370768).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was carried out in compliance with the ARRIVE guidelines. All animal procedures were approved and conducted according to the Animal Experimentation Ethics Committee guidelines. The animal study was reviewed and approved by The Animal Experimentation Ethics Committee of Anhui Medical University (No:\u0026nbsp;LLSC20232250).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e YX, HL and XM: Data curation, Writing original draft. XS,ZS and DG:Conceptualization, Methodology, Software. ZH, BH and YC: Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors supported the publication of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHesse, A.; Br\u0026auml;ndle, E.; Wilbert, D.; K\u0026ouml;hrmann, K.U.; Alken, P. Study on the prevalence and incidence of urolithiasis in Germany comparing the years 1979 vs. 2000. \u003cem\u003eEur. 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Cinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155. \u003cem\u003eRen Fail\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e44\u003c/em\u003e, 601-614.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Puerarin, Kidney stone, Apoptosis, Network pharmacology","lastPublishedDoi":"10.21203/rs.3.rs-4509583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4509583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Puerarin(PUE), an isoflavonoid extracted from Pueraria root, has anti-apoptotic effects. The objective of this research is to examine the impact of PUE on renal apoptosis and inflammation resulting from renal calculi and to elucidate its mechanism.The approach of network pharmacology and molecular docking was employed to discover potential targets and pathways of PUE. An animal model of calcium oxalate crystal deposition by intraperitoneal injection of glyoxylate and a model of COM-induced human renal tubular epithelial cells (HK2) were used to investigate the pharmacological mechanisms of PUE against apoptosis and inflammation. We used hematoxylin-eosin (H\u0026E) and Periodic Acid-Schiff staining (PAS) to assess the effect of PUE on crystal deposition and damage. The mechanism of PUE was elucidated and validated using Western blotting, histology, and immunohistochemical staining.Network pharmacology findings indicated that the PI3K/AKT pathway plays a crucial role in PUE. We experimentally demonstrate that PUE alleviated COM-induced changes in apoptotic proteins, increased inflammatory indicators and changes in oxidative stress-related indicators in HK2 cells by activating the PI3K/AKT pathway, reduced serum creatinine and urea nitrogen levels in mice caused by CaOx, alleviated crystal deposition and damage, and alleviated apoptosis, oxidative stress and inflammation.Puerarin attenuates renal apoptosis and inflammation caused by kidney stones through the PI3K/AKT pathway.","manuscriptTitle":"Puerarin ameliorates apoptosis and inflammation induced by kidney stones through the PI3K/AKT pathway: Network pharmacology and experimental validation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-13 22:54:40","doi":"10.21203/rs.3.rs-4509583/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a64c0b43-078e-4df6-ba80-6af4b374254a","owner":[],"postedDate":"June 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-18T19:08:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-13 22:54:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4509583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4509583","identity":"rs-4509583","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}