Network pharmacology and experimental validation reveal Salvia miltiorrhiza’s renoprotective mechanisms via PI3K-Akt pathway in diabetic nephropathy

Network pharmacology and experimental validation reveal Salvia miltiorrhiza’s renoprotective mechanisms via PI3K-Akt pathway in diabetic nephropathy | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 15 May 2025 V1 Latest version Share on Network pharmacology and experimental validation reveal Salvia miltiorrhiza’s renoprotective mechanisms via PI3K-Akt pathway in diabetic nephropathy Authors : ShengLuo 0009-0003-4767-3210 , DanLuo [email protected] , Tongying Chen , Lin Ma , and FuYun Zhang Authors Info & Affiliations https://doi.org/10.22541/au.174731607.76137462/v1 182 views 109 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract This study aims to systematically investigate the renoprotective mechanisms of Salvia miltiorrhiza (SM;丹参) in diabetic nephropathy (DN) through an integrative approach combining network pharmacology, molecular docking, and experimental validation. Bioactive constituents and pharmacological targets of Salvia miltiorrhiza were identified through systematic bioinformatics mining and protein-protein interaction (PPI) network analysis. Molecular docking simulations validated interactions between key components and targets. Streptozotocin (STZ)-induced DN mice were treated with Salvia miltiorrhiza to assess renal function, oxidative stress, inflammatory markers, and PI3K/AKT/mTOR pathway modulation. Network pharmacology identified 62 overlapping targets between Salvia miltiorrhiza and DN, with TNF, AKT1, and EGFR as core targets. Molecular docking demonstrated strong binding affinities (e.g., luteolin-TNF: −9.2 kcal/mol). In vivo, Salvia miltiorrhiza significantly reduced hyperglycemia, proteinuria, serum creatinine (Scr), and blood urea nitrogen (BUN), histopathology demonstrated reduced glomerulosclerosis and fibrosis, while attenuating oxidative stress and reduced serum levels of caspase-1, interleukin-18 (IL-18), and interleukin-1β (IL-1β). Western blot analysis confirmed significant reductions in phosphorylation levels of PI3K (p-PI3K), AKT (p-AKT), and mTOR (p-mTOR). Salvia miltiorrhiza ameliorates DN progression through multi-target modulation of the PI3K/AKT pathway, suppression of inflammatory responses, and oxidative stress mitigation. These findings mechanistically validate Salvia miltiorrhiza as a pleiotropic therapeutic agent targeting DN pathophysiology, providing translational rationale for clinical development. This study provides a pharmacological foundation for repurposing Salvia miltiorrhiza as a multi-target therapeutic agent in DN management. Network pharmacology and experimental validation reveal Salvia miltiorrhiza’s renoprotective mechanisms via PI3K-Akt pathway in diabetic nephropathy Sheng Luo 1 , Tongying Chen 3 , Lin Ma 4 , FuYun Zhang 1* , Dan Luo 2* 1 Dalian Ocean University, Dalian, 116023, China； 2 Spinal Minimally Invasive Department, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou,510000, China； 3 The Third Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou, 510006, China； 4 College of Physical Education and Health, Guangzhou University of Chinese Medicine, Guangzhou, 510000, China * Corresponding authorsE-mail addresses: [email protected] (D. Luo) and [email protected] (FY.Zhang) Abstract: This study aims to systematically investigate the renoprotective mechanisms of Salvia miltiorrhiza (SM;丹参) in diabetic nephropathy (DN) through an integrative approach combining network pharmacology, molecular docking, and experimental validation. Bioactive constituents and pharmacological targets of Salvia miltiorrhiza were identified through systematic bioinformatics mining and protein-protein interaction (PPI) network analysis. Molecular docking simulations validated interactions between key components and targets. Streptozotocin (STZ)-induced DN mice were treated with Salvia miltiorrhiza to assess renal function, oxidative stress, inflammatory markers, and PI3K/AKT/mTOR pathway modulation. Network pharmacology identified 62 overlapping targets between Salvia miltiorrhiza and DN, with TNF, AKT1, and EGFR as core targets. Molecular docking demonstrated strong binding affinities (e.g., luteolin-TNF: −9.2 kcal/mol). In vivo, Salvia miltiorrhiza significantly reduced hyperglycemia, proteinuria, serum creatinine (Scr), and blood urea nitrogen (BUN), histopathology demonstrated reduced glomerulosclerosis and fibrosis, while attenuating oxidative stress and reduced serum levels of caspase-1, interleukin-18 (IL-18), and interleukin-1β (IL-1β). Western blot analysis confirmed significant reductions in phosphorylation levels of PI3K (p-PI3K), AKT (p-AKT), and mTOR (p-mTOR). Salvia miltiorrhiza ameliorates DN progression through multi-target modulation of the PI3K/AKT pathway, suppression of inflammatory responses, and oxidative stress mitigation. These findings mechanistically validate Salvia miltiorrhiza as a pleiotropic therapeutic agent targeting DN pathophysiology, providing translational rationale for clinical development. This study provides a pharmacological foundation for repurposing Salvia miltiorrhiza as a multi-target therapeutic agent in DN management. Keywords: Diabetic nephropathy; Salvia miltiorrhiza; Network pharmacological analysis；PI3K/AKT/mTOR signaling pathway； Molecular docking 1 | INTRODUCTION Diabetic nephropathy (DN), the primary contributor to end-stage renal disease (ESRD) globally, represents approximately 40% of diabetes-associated ESRD cases 1 . Characterized by glomerular basement membrane thickening, mesangial expansion, and tubulointerstitial fibrosis, DN progresses to irreversible proteinuria and renal function decline 2,3 . While contemporary molecular studies have elucidated its multifactorial pathogenesis—involving metabolic dysregulation (chronic hyperglycemia, insulin resistance), hemodynamic abnormalities (renin-angiotensin system activation), and inflammatory cascades (TGF-β/Smad signaling)—current therapies targeting glycemic control and hypertension remain insufficient to arrest disease progression in 30-40% of patients 1,3 . The burgeoning prevalence of DN, particularly among younger obese populations, necessitates novel therapeutic strategies addressing concurrent pathological mechanisms 4 . Salvia miltiorrhiza Bunge (Lamiaceae), a traditional Chinese herb with centuries of empirical use in microcirculatory disorders, comprises two principal pharmacologically active fractions: (1) water-soluble phenolic acids (e.g., salvianolic acid B) exhibiting antioxidant (ROS IC50: 12.3 μM) and antifibrotic (68% collagen IV synthesis inhibition) properties, and (2) lipophilic diterpenoids (e.g., tanshinone IIA) demonstrating anti-inflammatory (55% NF-κB inhibition at 10 μM) and insulin-sensitizing effects (3.2-fold PPARγ activation) 5-7 . These pleiotropic pharmacological properties establish Salvia miltiorrhiza as a viable therapeutic entity for DN intervention. Network pharmacology, an interdisciplinary paradigm integrating structural drug similarity and biological effector-target interactions, offers a systematic approach to deciphering multi-component herbal mechanisms 8 . This methodology aligns with the systems-oriented therapeutic philosophy of traditional Chinese medicine (TCM), enabling comprehensive analysis of Salvia miltiorrhiza’s bioactive constituents and pharmacological targets in DN 9 . Network pharmacology, an emerging discipline grounded in systems biology, genetic redundancy, and network biology, utilizes in silico methodologies and established databases to perform high-throughput data analysis and precision spatial modeling of genes, target proteins, diseases, and therapeutics 10 . By integrating structural and therapeutic drug similarities with biological effector-target interactions, this approach enables systematic identification and mechanistic investigation of bioactive constituents in traditional Chinese medicine (TCM), as evidenced by Salvia miltiorrhiza’s polypharmacological efficacy against DN. Recent advancements in network pharmacology have transformed TCM mechanistic investigations by elucidating polypharmacological mechanisms underlying multi-component, multi-target therapies 11 . This study integrates multi-omics network analysis, molecular docking, and experimental validation to elucidate Salvia miltiorrhiza’s renoprotective effects in DN. In vivo investigations further confirmed that Salvia miltiorrhiza exerts renoprotective effects through concerted modulation of PI3K/AKT/mTOR-mediated oxidative stress mitigation. These findings not only validate its clinical potential but also advance the development of TCM-derived therapies targeting this pathway, providing a translational bridge for of traditional medicine into modern precision therapeutics. 2 | MATERIALS AND METHODS 2.1 | Materials Six-week-old male C57BL/6 mice (specific pathogen-free-grade) (SPF-grade) were obtained from the Laboratory Animal Center of Guangzhou University of Chinese Medicine (GZUCM). The mice were housed under standard SPF conditions with controlled temperature and humidity, ad libitum access to food and water, and a 7-day acclimation period. All experimental procedures were approved by the Animal Ethics Committee of GZUCM. 2.2 | Screening of the chemical constituent Salvia miltiorrhiza and predictionof related targets Bioactive constituents in Salvia miltiorrhiza were systematically screened using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP)( https://old.tcmsp-e.com/tcmspphp), where pharmacokinetic parameters—notably oral bioavailability (OB) ≥ 30% and drug-likeness (DL) score ≥ 0.18—served as primary selection criteria, as higher values correlate with enhanced bioactive potential 12 . Following a boolean operator-guided search of Salvia miltiorrhiza -associated compounds, structural formulas of these phytochemicals were acquired from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and imported into SwissTargetPrediction(http://swisstargetprediction.ch/) for target identification. Putative drug-component target associations were filtered using a probability threshold of probability score ( p > 0) to ensure biological relevance 13 . 2.3 | Screening targets of DN DN-associated targets targets were retrieved from the GenCards database (https://www.genecards.org/) and the OMIM database (https://www.omim.org/) using the keywords ”diabetic nephropathy” and ”diabetic kidney disease.” Targets were filtered based on relevance scores (median-centric thresholding algorithm) to construct a DN target dataset. All potential targets were standardized in the UniProtKB database (https://www.uniprot.org/) 2.4 | Identification of overlapping targets between Salvia miltiorrhiza and DN The targets of Salvia miltiorrhiza and DN were subjected to intersection analysis using Venny (https://bioinfogp.cnb.csic.es) online tool to identify potential pharmacological targets for DN. 2.5 | Protein-protein interaction (PPI) construction of Salvia miltiorrhiza against DN The common targets were analyzed using the STRING database (https://string-db.org/) to construct a PPI network, which was visualized and optimized in Cytoscape software (https : //cytoscape. org/). Core targets were identified based on degree centrality (top 10 nodes), which might also be the core targets of Salvia miltiorrhiza in the prevention of DN. 2.6 | Gene ontology (GO) function and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of core targets To systematically investigate the biological functions of intersecting target genes and their roles within individual signaling pathways, functional enrichment analysis of overlapping targets was performed using the DAVID database(https://davidbioinformatics.nih.gov). GO terms categorized into biological processes, molecular functions, and cellular components were systematically examined, along with pathways from the KEGG 14 . The analytical results were subsequently visualized and interpreted using the MicrobiomeAnalyst (https://www.bioinformatics.com.cn/) for comprehensive data representation. 2.7 | Network pharmacology modeling The potential pharmacological targets of Salvia miltiorrhiza in DN were systematically investigated through the application of Cytoscape, a network visualization tool, to establish compound-target interactions and visually represent the pharmacological network 15 . 2.8 | Molecular docking The high-degree core targets identified from the protein-protein interaction (PPI) network were selected as candidate receptors. Their three-dimensional crystal structures were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/) and prepared for docking simulations. The primary bioactive constituents of Salvia miltiorrhiza were obtained from the PubChem database in SDF format, followed by molecular mechanics optimization and format conversion to PDB using Open Babel(https://openbabel.org). Molecular preprocessing, including crystallographic water elimination, hydrogen addition, and charge optimization, was performed with AutoDock (https://autodock.scripps.edu). The binding pocket coordinates were defined based on known ligand-binding sites, and parameter files (GPF format) were generated for automated docking. Rigorous molecular docking validation was conducted using AutoDock Vina with exhaustiveness set to 20 to ensure sampling completeness. The optimal docking poses with the highest binding affinities were exported in PDBQT format and structural visualization through PyMOL 2.2.1 for interaction analysis 16 . 2.9 | Animal modeling and grouping After one week of acclimation feeding, the mice were randomly assigned to: Sham-operated (n=8) and Diabetes induction (n=32) groups. After 12-hour fasting with ad libitum water access, type 1 diabetes was induced in modeling group mice via intraperitoneal administration of streptozotocin (STZ; 100 mg/kg dissolved in 0.1 M sodium citrate buffer, pH 4.3), while sham group animals received equivalent volumes of vehicle solution 17 . Fasting glucose was measured after 72h of fasting for 12h, and the success of diabetes modelling was defined by the glucose≥16.7 mmol/L. Post-modeling, animals were randomly allocated into three experimental groups: DN model group(n=8), Salvia miltiorrhiza low-dose group(n=8), and high-dose group(n=8). The administered doses were determined based on preliminary experiments or the commonly used dosage ranges of Salvia miltiorrhiza injection reported in the literature 18 . The low-dose group was injected intraperitoneally with Salvia miltiorrhiza injection (3 mL·kg⁻¹·d⁻¹) once a day, and the high-dose group was administered twice as much as the low-dose group for 8 weeks. 2.10 | 24h Urine collection Following 8 weeks of pharmacological intervention, 24-hour urine collections were performed using metabolic cages with individual housing, with food restriction implemented during the collection period. Ambient temperature was maintained at 22°C to prevent stress-related mortality. Urine samples were transferred to polypropylene centrifuge tubes annotated with total collection volume, then centrifuged at 4°C (3000 ×g, 15 min). The resulting supernatant was aliquoted into 1.5 mL microcentrifuge tubes and cryopreserved at -80°C. Urinary protein concentration was quantified using a commercial urine protein quantification kit, with albumin excretion rates calculated based on 24-hour urine volume and protein concentration. 2.11 | Biochemical analysis Following the experimental protocol, mice underwent 12-hour fasting with water deprivation prior to euthanasia. Serum samples were analyzed using an automated biochemical analyzer(Mindray, China) to quantify serum creatinine (SCr), blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total protein (TP) 17 . 2.12 | Renal histopathology Renal tissues were harvested and fixed in 4% paraformaldehyde (PFA) at 4°C for 24 h, followed by graded ethanol dehydration and paraffin embedding. Serial 4-μm sections were prepared for hematoxylin and eosin (H&E) staining to evaluate glomerular morphology and Masson’s trichrome staining to quantify interstitial collagen deposition. Briefly, the H&E staining protocol was as follows: Tissue sections were deparaffinized in xylene I and II, rehydrated through a graded ethanol series (100% to 70%), stained with hematoxylin for 5 min and eosin for 1 min, dehydrated, and mounted with a coverslip. For Masson’s trichrome staining, sections were sequentially stained with Weigert’s iron hematoxylin for 1 min, differentiated in acid ethanol for 15 s, incubated with Masson blue for 2 min, counterstained with Lichun red for 10 s, treated with phosphomolybdic acid for 1 min, and finally stained with aniline blue for 1 min. Stained slides were imaged under a light microscope. 2.13 | Oxidative stress assays Malondialdehyde (MDA) levels in mouse serum and superoxide dismutase (SOD) enzyme activity were measured using commercial assay kits according to the manufacturer’s instructions, the level of MDA in mouse serum was measured at 532 nm using a microplate reader, and the activity of the antioxidant enzyme SOD was determined at 560 nm. Absorbance values were recorded with the microplate reader, and subsequent analysis was performed through calculations based on standard curves 19,20 . 2.14 | Serum inflammatory factor detection Portions of renal tissue were collected and lysed using RIPA lysis buffer. The lysate was centrifuged at 3000 rpm for 10 minutes at 4°C to collect the supernatant. Protein concentrations were determined using the BCA assay. Following protein transfer, the membrane was blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against PI3K, p-AKT, AKT, P-mTOR, mTOR, and β-actin. After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 hours. Protein bands were visualized using ECL chemiluminescence, and the optical density of each band was analyzed with a gel imaging analysis system. 2.15 | RNA extraction and quantitative real-time polymerase chain reaction(qRT-PCR)assay Total RNA was extracted from kidney tissues (≈100 mg) using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. Briefly, the procedure included the following steps: (1) Tissues were homogenized in 1 mL of TRIzol reagent and incubated at room temperature for 10 min; (2) 200 μL of chloroform was added, and the mixture was vortexed vigorously for 30 s, followed by centrifugation at 12,000 ×g for 15 min at 4°C; (3) The aqueous phase (400 μL) was transferred to an equal volume of isopropanol, incubated for 10 min, and centrifuged at 12,000 ×g for 10 min at 4°C; (4) RNA pellets were washed twice with 75% ethanol (10,000 ×g, 5 min, 4°C); (5) Pellets were air-dried for 10 min and resuspended in 200 μL of DEPC-treated water 21 . List of primers is shown in Table 1 2.16 | Western blot Kidney tissues were lysed in RIPA buffer (with protease inhibitors), centrifuged (3,000 ×g, 10 min, 4°C), and quantified (BCA assay, Pierce). Proteins (30 μg/lane) were separated by SDS-PAGE (10% gel) and transferred to PVDF membranes. After blocking (5% skim milk, 1 h), membranes were incubated overnight (4°C) with primary antibodies, add p-AKT, p-/mTOR, β-actin. following TBST washes, HRP-conjugated secondary antibodies (1:5,000) were applied (2 h, RT). Signals were detected by ECL and quantified with ImageLab 22 . 2.17 | Statistical analyses Data are presented as mean ± standard deviation (SD) derived from at least three independent biological replicates. After confirming normal distribution and homogeneity of variance, one-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for comparisons among three or more groups, and an unpaired Student’s t-test was used for comparisons between two groups. A p -value less than 0.05 was co 23 nsidered statistically significant. 3 | RESULTS 3.1 | Bioactive compound screening in Salvia miltiorrhiza The TCMSP database analysis identified 65 bioactive constituents in Salvia miltiorrhiza . Subsequent target prediction using SwissTargetPrediction revealed 144 potential gene targets associated with these compounds. 3.2 | DN target identification Disease-associated targets were systematically retrieved from three primary databases: GeneCards (yielding 16,892 targets), OMIM (97 targets). To refine the dataset from GeneCards, a tripartite median-based filtering strategy was employed, selecting 993 targets with the highest relevance scores. Following integration of all database entries and removal of duplicates, a total of 2,051 unique disease targets were identified for subsequent analysis. 3.3 | Target intersection analysis Venn diagram analysis (Fig. 1A) identified 62 overlapping targets between bioactive compounds of Salvia miltiorrhiza and DN, suggesting their potential as therapeutic targets for DN intervention. 3.4 | Protein-Protein interaction network The protein-protein interaction (PPI) network (Fig. 1B), constructed using STRING and visualized in Cytoscape, identified TNF, AKT1, IL1B, EGFR, STAT3, and SRC as core targets based on degree centrality ranking. Network topology analysis demonstrated a hierarchical structure, with densely connected inner nodes (dark-colored, larger size) representing high-degree central targets, and sparsely connected outer nodes indicating peripheral targets. 3.5 | Functional enrichment analysis 3.5.1 | Go function analysis GO enrichment analysis systematically characterized the functional attributes of differentially expressed proteins, yielding 887 biological processes (BP), 107 cellular components (CC), and 205 molecular functions (MF), with the ten most significant entries ranked by ascending p-values and graphically represented(Fig. 2A). According to the analysis, many potential targets were mainly enriched in phosphorylation, protein phosphorylation and response to hypoxia among them in the BP category. Plasma membrane, receptor complex, and cell surface were among the CC categories, and nuclear receptor activity, protein tyrosine kinase activity, ATP-binding were among the MF categories. These findings suggest that Salvia miltiorrhiza exerts therapeutic effects on DN by modulating cellular localization, molecular interactions, and pathogenic biological processes. 3.5.2 | KEGG pathway analysis To elucidate the molecular mechanisms underlying DN pathogenesis, core targ 24 ets were mapped to the KEGG pathways. Significant enrichment was observed in lipid metabolism, atherosclerosis, and the PI3K/AKT signaling pathway (Fig. 2B), with bubble size proportional to gene count and color gradient indicating statistical significance. The PI3K/AKT pathway, a key regulator of cell survival and fibrosis, along with lipid metabolism dysregulation, aligns with known hallmarks of DN progression 25 . These findings implicate these pathways as pivotal mediators of Salvia miltiorrhiza’s therapeutic effects, potentially through ameliorating metabolic dysfunction and inflammatory signaling. 3.6 | Compound-Target network Network topological analysis identified hub nodes (e.g.TNF, AKT1) as pivotal therapeutic targets, while edge density analysis revealed synergistic interactions among bioactive constituents through shared target modulation. This architecture reflects the multi-component, multi-target mechanism characteristic of traditional Chinese medicine (TCM) 26 . A compound-target interaction network was constructed using Cytoscape illustrating interactions between Salvia miltiorrhiza’s bioactive constituents and their putative pharmacological targets (Fig. 3). 3.7 | Molecular docking results Molecular docking simulations characterized the interaction patterns between Salvia miltiorrhiza’s bioactive constituents and key targets in DN pathogenesis. Binding energy thresholds were defined empirically: values ≤ -5.0 kcal/mol indicated favorable interactions, while values ≤ -7.0 kcal/mol suggested strong binding affinities 13,27 . Three targets (TNF, AKT1, EGFR) and three constituents (Luteolin, Tanshinone IIA, Cryptotanshinone) were selected for validation based on network centrality. All screened pairs exhibited binding energies ≤ -6.0 kcal/mol (Table 2), with Tanshinone IIA-EGFR (-8.2 kcal/mol) and Luteolin-TNF (-7.9 kcal/mol) showing the strongest interactions(Fig. 4). 3.8 | Effects of Salvia miltiorrhiza on body weight and blood glucose in DN mice Sham group mice exhibited progressive weight gain consistent with normal development. DN model mice exhibited significant weight reduction accompanied by alopecia and lethargy compared to sham group. Salvia miltiorrhiza treatment dose-dependently attenuated weight loss (Fig. 5A). Before treatment, no intergroup differences in baseline blood glucose levels were observed prior to treatment. After intervention with Salvia miltiorrhiza , the blood glucose levels of the model groups were all significantly higher compared to the sham group, and the blood glucose levels of the Salvia miltiorrhiza low and high dosage groups were all significantly lower compared to the model group (Fig. 5B). 3.9 | Renal and hepatic function biomarkers Salvia miltiorrhiza intervention significantly attenuated renal dysfunction in DN mice, with biochemical profiles detailed. Model group displayed elevated serum creatinine (SCr), blood urea nitrogen (BUN), and hepatic transaminases (AST/ALT), indicative of multi-organ injury. Salvia miltiorrhiza dose-dependently reduced SCr and BUN, achieving partial restoration of hepatic function (AST/ALT). Meanwhile, hypoproteinemia in diabetic mice (TP) was ameliorated by treatment.24-h urinary protein excretion, a key DN biomarker, increased in model mice and decreased with Salvia miltiorrhiza treatment. These findings demonstrate Salvia miltiorrhiza’s renoprotective and hepatoprotective efficacy through multi-target modulation of DN pathophysiology (Fig. 6). 3.10 | Renal histopathology Renal histopathological features were evaluated via H&E and Masson’s trichrome staining (Fig. 7). HE staining revealed that sham mice maintained intact renal architecture characterized by uniform glomeruli, patent capillaries, and normal tubular epithelium. In contrast, DN model mice showed marked glomerulosclerosis, tubular proteinaceous casts, and perivascular leukocyte infiltration, Salvia miltiorrhiza treatment dose-dependently attenuated these lesions, with the high-dose group showing near-normal glomerular morphology and minimal interstitial inflammation. Masson’s trichrome staining demonstrated physiological collagen distribution in sham kidneys. DN mice exhibited pronounced tubulointerstitial fibrosis，increase in collagen area compared with sham group, glomerular basement membrane thickening, and peritubular matrix expansion. Salvia miltiorrhiza intervention reduced fibrotic deposition versus the model group. 3.11 | Oxidative stress markers in renal tissue MDA, a lipid peroxidation byproduct, was significantly elevated in STZ-induced DN mice, which were attenuated by Salvia miltiorrhiza pretreatment(Fig. 8A). SOD, a critical antioxidant enzyme catalyzing superoxide anion (O₂⁻)dismutation, showed diminished activity in the model group, Salvia miltiorrhiza treatment restored renal SOD activity dose-dependently(Fig. 8B). These results indicate Salvia miltiorrhiza’s dual efficacy in mitigating oxidative stress via lipid peroxidation suppression and endogenous antioxidant defense potentiation. 3.12 | Anti-inflammatory effects Serum levels of caspase-1, IL-18, and IL-1 showed significant elevation in the DN model group compared with the sham group. Salvia miltiorrhiza treatment dose-dependently significantly attenuated these inflammatory markers, with the high-dose regimen demonstrating superior potency to its low-dose counterpart. Similarly, renal mRNA expression of caspase-1, IL-18, and IL-1β increased in the model group, which was dose-dependently ameliorated following Salvia miltiorrhiza intervention(Fig. 9). These findings suggest that Salvia miltiorrhiza alleviates DN by suppressing inflammatory responses. 3.13 | PI3K/AKT/mTOR pathway modulation Western blot analysis revealed marked hyperphosphorylation of the PI3K/AKT/mTOR signaling cascade in DN mice. Compared with sham group, the protein levels of PI3K, p-AKT/AKT, and p-mTOR/mTOR in the kidneys of mice from the model group were significantly elevated (Fig. 10). Salvia miltiorrhiza treatment significantly suppressed pathway activation, reducing these markers in both high- and low-dose groups compared to the model group. These results provide mechanistic evidence for the nephroprotective efficacy of Salvia miltiorrhiza . 4 | DISCUSSION Diabetic nephropathy, a chronic metabolic disorder stemming from insulin deficiency or impaired pancreatic islet functioninduces systemic hyperglycemia that triggers multi-organ dysfunction, particularly affecting renal, cardiovascular, and neurological systems 22,28 . Converging evidence from preclinical models and omics-based approaches has significantly advanced our understanding of DN pathobiology 1 . Notably, renal fibrosis, a hallmark of chronic kidney disease, drives progression to renal failure and end-stage disease, underscoring the urgency to address DN’s rapid progression, poor prognosis, and socioeconomic burden 17 . Salvia miltiorrhiza , a traditional herb with diverse bioactive constituents, demonstrates therapeutic potential in DN 29,30 . Network pharmacology and experimental validation identified its key mechanisms: attenuating inflammation, restoring metabolic homeostasis, and modulating glucose/lipid metabolism. Enrichment analysis revealed multi-target, multi-pathway characteristics, with TNF emerging as a core target 27,31 . Accumulating evidence indicates that SCR exacerbates renal fibrogenesis by regulating the TGF-β/Smad pathway 32 , which forms a complementary mechanism with the PI3K/AKT pathway inhibition identified in this study.Critical active components mediate Salvia miltiorrhiza’s renoprotective effects by suppressing renal inflammation, improving renal function, and mitigating fibrosis 24 . In STZ-induced diabetic mice, Salvia miltiorrhiza reduced fasting glucose, Scr, BUN, and 24-h urinary protein while enhancing body weight and lowering AST/ALT levels, confirming its systemic and organ-specific benefits. Histopathological analyses (HE/MASSON staining) further validated its capacity to ameliorate renal structural damage and fibrosis. Mechanistically, Salvia miltiorrhiza suppressed inflammatory responses, reducing serum caspase-1, IL-18, and IL-1β levels, thereby attenuating chronic inflammation 17,33,34 . Additionally, it alleviated oxidative stress by lowering renal MDA and boosting SOD activity 17,33 . Crucially, Salvia miltiorrhiza modulated the PI3K/AKT pathway, a pivotal regulator of tissue repair and apoptosis. By downregulating PI3K-Akt signaling, it inhibited inflammatory cytokine release, mitigated tubular epithelial and glomerular basement membrane damage, and enhanced renal recovery. This dual regulation of inflammation and apoptosis highlights Salvia miltiorrhiza’s multifaceted therapeutic strategy 35,36 . 5 | CONCLUSION Integrating network pharmacology, molecular docking and experimental models, this study elucidates Salvia miltiorrhiza’s renoprotective effects via inhibition of the PI3K/AKT pathway, oxidative stress mitigation, and suppression of inflammatory responses. These findings underscore its potential as a multi-target agent for DN treatment, establishing a translational framework that synergizes phytomedicine-derived multi-target strategies with contemporary therapeutic paradigms. FUNDING This study was financially supported by the Research Project of Traditional Chinese Medicine Bureau of Guangdong Province (No. 20221178) DATA AVAILABILITY STATEMENT Data will be made available on request. CONFLICT OF INTEREST STATEMENT The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influencethe work reported in this paper. Table legends Table 1. List of primers Table 2. The binding free energy of components docking with target molecules. Figure legends Fig. 1. (A) Evenn diagram of the intersection between Salvia miltiorrhiza and DN; (B)The protein-protein interactions at intersection targets Fig. 2. (A) DAVID GO enrichment analysis; (B) Bubble diagram of KEGG enrichment analysis Fig. 3. Network diagram of the active ingredient of Salvia miltiorrhiza acting on DN Fig.4. Molecular docking diagram. (A) Luteolin-TNF. (B)Tanshinone IIA-EGFR Fig.5. Body weight and blood glucose measurement results in mice. (A) The body weight at different time points; (B) The blood glucose at different time points; ***p < 0.00l vs Sham group; ##p Fig.6. Primary metabolic parameters in mice.(A) Serum creatinine; (B) blood urea nitrogen; (C) Aspartate aminotransferase; (D) Alanine aminotransferase; (E) Total Protein (F) 24 h urine albumin. **p < 0.01,***p < 0.00l vs Sham group; #p Fig.7. The results of H&E and MASSON staining in all mouse groups Fig.8. Indicators of oxidative stress in mice (A) The malondialdehyde content; (B) The superoxide dismutase content ** p < 0.01,***p < 0.00l vs Sham group; #p Fig.9. Serum inflammatory factor and mRNA expression of renal inflammatory factor in mice (A) Serum caspase-1 content; (B) Serum IL-18 content; (C) Serum IL-1βcontent; (D) caspase-1 mRNA levels; (E) IL-18 mRNA levels; (F) IL-1βmRNA levels **p < 0.01,***p < 0.00l vs Sham group; #p Fig.10. PI3K/AKT/mTOR signaling pathway of renal in mice. (A) Detection of renal PI3K, p-AKT, AKT, P-mTOR and mTOR proteins; (B-D)Quantitative analysis of PI3K/β-action, p-AKT/AKT, P-mTOR/mTOR expression ratios by grayscale densitometry. **p< 0.01 vs Sham group; #p <0.05,##p REFERENCES 1. Sawaf H, Thomas G, Taliercio JJ, Nakhoul G, Vachharajani TJ, Mehdi A. Therapeutic advances in diabetic nephropathy. J Clin Med 2022; 11 . 2. Juin SK, Ouseph R, Gondim DD, Jala VR, Sen U. Diabetic nephropathy and gaseous modulators. Antioxidants (Basel) 2023; 12 . 3. Chen H, Tian X, Yu X. Editorial: association between diabetic nephropathy and diabetic retinopathy or non-diabetic nephropathy. 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Authors Affiliations ShengLuo 0009-0003-4767-3210 Dalian Ocean University View all articles by this author DanLuo [email protected] Guangdong Provincial Hospital of Chinese Medicine Zhuhai View all articles by this author Tongying Chen Guangzhou University of Chinese Medicine Third Clinical Medical College View all articles by this author Lin Ma Guangzhou University of Chinese Medicine View all articles by this author FuYun Zhang Dalian Ocean University View all articles by this author Metrics & Citations Metrics Article Usage 182 views 109 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation ShengLuo, DanLuo, Tongying Chen, et al. Network pharmacology and experimental validation reveal Salvia miltiorrhiza’s renoprotective mechanisms via PI3K-Akt pathway in diabetic nephropathy. Authorea . 15 May 2025. 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