Cornus officinalis polysaccharides alleviate diabetic nephropathy via gut microbiota-mediated bile acid metabolism and Slc27a2-PPARα-Angptl4 axis

Cornus officinalis polysaccharides alleviate diabetic nephropathy via gut microbiota-mediated bile acid metabolism and Slc27a2-PPARα-Angptl4 axis | 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 Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed.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. 10 March 2025 V1 Latest version Share on Cornus officinalis polysaccharides alleviate diabetic nephropathy via gut microbiota-mediated bile acid metabolism and Slc27a2-PPARα-Angptl4 axis Authors : Jing Li 0009-0008-6410-0680 , Jiaxin Ye , Rui Ding , Saiya Chen , Feier Yang , Lingjun Ye , Min Hao , … Show All … , Qiyuan Shan , xin Han 0000-0002-1725-7940 , Zhixiang Dong , Lu Wang , Kuilong Wang [email protected] , Gang Cao , and Caihua Sun Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.174157932.28306752/v1 360 views 214 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Diabetic nephropathy (DN), affects 30% diabetic, presents significant global healthcare challenges. Despite advancements in current therapies, their adverse effects underscore demand for safer options. Recent research has indicated that the gut microbiota as a promising therapeutic target for DN. Cornus officinalis, a traditional Chinese medicinal herb widely used for DN management, contains bioactive polysaccharides with gut microbiota-modulating potential. However, the precise mechanisms underlying their therapeutic effects remain poorly understood. Purpose: This study aims to elucidate the mechanisms by which Cornus officinalis polysaccharides (COP) ameliorate DN through microbiota modulation. Methods: DN was induced in Sprague-Dawley rats via streptozotocin injection and high-fat diet. Animals were randomly assigned to receive COP (120, 240, or 480 mg/kg/day), valsartan (positive control), or vehicle. Effects were evaluated through biochemical parameters, renal histopathology, immunofluorescence, and Western blotting. Gut microbiota composition was analyzed via 16S rRNA sequencing. Integrated metabolomic/transcriptomic analyses for critical metabolites and pathways, with in vitro mechanistic validation. Results: COP administration significantly ameliorated renal damage while restoring glomerular filtration function. It enhanced the abundance of Akkermansia muciniphila and restored bile acid (BA) metabolic homeostasis. In vitro studies revealed that Akkermansia muciniphila modulated lithocholic acid (LCA) metabolism. Transcriptomics identified PPAR signaling as the core mechanism, with LCA derivatives mitigating podocyte injury through the Slc27a2-PPARα-Angptl4 axis, defining the nephroprotective pathway. Conclusion: COP ameliorates DN though a multi-mechanistic approach involving gut microbiota modulation, restoration of BA metabolism, and repair of glomerular function via the Slc27a2-PPARα-Angptl4 axis. These findings provide a novel multi-target strategy for DN management. Cornus officinalis polysaccharides alleviate diabetic nephropathy via gut microbiota-mediated bile acid metabolism and Slc27a2 - PPARα - Angptl4 axis Jing Li 1 ,# , Lyu Qiang 1,# , Qiao Yang 1, # , Jiaxin Ye 1 , Rui Ding 1 , Saiya Chen 1 , Feier Yang 1 , Lingjun Ye 1 , Min Hao 1 , Qiyuan Shan 1 , Xin Han 1 , Zhixiang Dong 1 , Lu Wang 1,* , Kuilong Wang 1 ,* , Gang Cao 1,* , Caihua Sun 2,* not-yet-known not-yet-known not-yet-known unknown 1 School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 311402, China 2 The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hosptital of Chinese Medicine), Hangzhou 310006, China * Correspondence, Kuilong Wang, [email protected] ; Gang Cao, [email protected] ; Lu Wang, Lu Wang, [email protected] ; Caihua Sun, [email protected] . # These authors contribute equally to this work Authors e-mail Jing Li, [email protected] Lyu qiang, [email protected] Qiao Yang, [email protected] Jiaxin Ye, [email protected] Rui Ding, [email protected] Saiya Chen, [email protected] Feier Yang, [email protected] Lingjun Ye, [email protected] Min Hao, [email protected] Qiyuan Shan, [email protected] Xin Han, [email protected] Zhixiang Dong, [email protected] Abstract Background : Diabetic nephropathy (DN), affects 30% diabetic, presents significant global healthcare challenges. Despite advancements in current therapies, their adverse effects underscore demand for safer options. Recent research has indicated that the gut microbiota as a promising therapeutic target for DN. Cornus officinalis , a traditional Chinese medicinal herb widely used for DN management, contains bioactive polysaccharides with gut microbiota-modulating potential. However, the precise mechanisms underlying their therapeutic effects remain poorly understood. Purpose : This study aims to elucidate the mechanisms by which Cornus officinalis polysaccharides (COP) ameliorate DN through microbiota modulation. Methods : DN was induced in Sprague-Dawley rats via streptozotocin injection and high-fat diet. Animals were randomly assigned to receive COP (120, 240, or 480 mg/kg/day), valsartan (positive control), or vehicle. Effects were evaluated through biochemical parameters, renal histopathology, immunofluorescence, and Western blotting. Gut microbiota composition was analyzed via 16S rRNA sequencing. Integrated metabolomic/transcriptomic analyses for critical metabolites and pathways, with in vitro mechanistic validation. Results : COP administration significantly ameliorated renal damage while restoring glomerular filtration function. It enhanced the abundance of Akkermansia muciniphila and restored bile acid (BA) metabolic homeostasis. In vitro studies revealed that Akkermansia muciniphila modulated lithocholic acid (LCA) metabolism. Transcriptomics identified PPAR signaling as the core mechanism, with LCA derivatives mitigating podocyte injury through the Slc27a2 - PPARα - Angptl4 axis, defining the nephroprotective pathway. Conclusion : COP ameliorates DN though a multi-mechanistic approach involving gut microbiota modulation, restoration of BA metabolism, and repair of glomerular function via the Slc27a2 - PPARα - Angptl4 axis. These findings provide a novel multi-target strategy for DN management. Keywords Polysaccharides; Diabetic nephropathy; Gut microbiota; Bile acid; Lithocholic acid; Podocytes Highlights 1. COP had ameliorated DN by restoring gut microbiota dysbiosis. 2. The restoration of microbiota balance and regulation of bile acid metabolism were identified as a critical mechanism underlying the improvment in DN. 3. LCA and its derivatives exerted protective effects against DN by modulating the Slca272 - PPARα - Angptl4 axis. Abbreviations : DN: Diabetic nephropathy; COP: Cornus officinalis polysaccharides; LCA: Lithocholic acid; ESRD: end-stage renal disease; UACR: urinary albumin-to-creatinine ratio; A. muciniphila : Akkermansia muciniphila ; 3-oxoLCA: 3-oxolithocholic acid; isoLCA: isolithocholic acid; WT-1: wilms’ tumor 1; CCL-2: C-C Motif Chemokine Ligand 2; PMP: 1-phenyl-3-methyl-5-pyrazolone; FT-IR: Fourier transform infrared spectroscopy; SEM: scanning electron microscopy; HFD: high-fat diet; FBG: fasting blood glucose; TG: triglycerides; H&E: hematoxylin-eosin; PAS: Periodic Acid-Schiff; IF: Immunofluorescence; WB: Western blotting; qRT-PCR: Quantitative real-time polymerase chain reaction; IFN-γ: interferon gamma; BA: bile acid; Slc27a2 : Solute Carrier Family 27 Member 2; PPARα : Peroxisome proliferator-activated receptor α; Angptl4 : Angiopoietin Like 4; MPC5: Mouse renal podocyte cells. not-yet-known not-yet-known not-yet-known unknown Introduction Diabetic nephropathy (DN), the predominant microvascular complication of diabetes, remains a leading cause of end-stage renal disease (Kato and Natarajan, 2019). Its pathogenesis involves podocyte loss, glomerulosclerosis, and tubulointerstitial fibrosis, with podocyte injury initiating glomerular barrier dysfunction and progressive proteinuria (Kato and Natarajan, 2019; Mohandes et al., 2023). Current therapies that primarily target glycemic control (e.g., metformin) or direct renoprotection (e.g., pentoxifylline) often fail to reverse established renal damage (Song et al., 2021). This therapeutic impasse underscores the urgent need for multi-target interventions capable of addressing both metabolic dysregulation and podocyte pathophysiology. Emerging evidence highlights gut microbiota dysbiosis as a key driver of metabolic disorders, including diabetes and cardiovascular diseases (Fan and Pedersen, 2021). Specifically, reduced microbial diversity and altered community structure within the gut microbiota have been closely linked to the pathogenesis of renal failure (Hobby et al., 2019). Under pathological conditions, disturbances in the gut microbiota lead to dysregulation of bile acid (BA) metabolism, resulting in the accumulation of pro-inflammatory and cytotoxic BAs that enter systemic circulation and exacerbate renal disease progression (Lin et al., 2022). Conversely, microbial-derived protective secondary BAs preserve renal homeostasis under physiological conditions (Lin et al., 2023). These findings highlight the critical role of gut microbial homeostasis in renal health and disease, thus emphasizing the need for further investigation into gut microbial interactions to advance DN pathogenesis understanding and develop novel therapeutic strategies targeting the gut-kidney axis. Cornus officinalis, the dried fruit of Cornus officinalis Sieb. et Zucc., is widely used in the clinical management of DN. In traditional Chinese medicine, it is primarily extracted by boiling water, yielding significant quantities of polysaccharides. Pharmacological studies have identified Cornus officinalis polysaccharide (COP) as a bioactive component with hypoglycemic and antioxidant properties (Lan et al., 2024; Wang et al., 2024). Despite low susceptibility to human enzymatic degradation, COP interacts with the gut microbiota to restore microbial homeostasis and exert therapeutic effects (Goto et al., 2022; Yang et al., 2020). Preclinical studies demonstrated COP’s efficacy in ameliorating diabetic hyperlipidemia (Fu et al., 2021), yet key questions remain unresolved: (1) Whether COP is the primary pharmacodynamic component in Cornus officinalis targeting DN; (2) whether its therapeutic effects are mediated through interactions with the gut microbiota, and what the precise underlying mechanisms are. Therefore, we hypothesize that COP may ameliorate renal injury in DN through gut microbiota-mediated metabolites. This study aims to systematically investigate the regulatory network of COP on the gut microbiota-host metabolism axis by establishing DN animal models, integrating metabolomics and transcriptomics technologies, and validating findings through in vitro cellular experiments. The research seeks to elucidate the underlying mechanisms of traditional herbal medicine in multi-targeted DN therapy, provide a theoretical foundation for developing innovative microbiota-modulating therapeutics, and promote interdisciplinary convergence in the modernization of phytomedicine research. Materials and methods Materials and reagents Cornus officinalis was the dried ripe fruits of Cornus officinalis Sieb, et Zucc., collected from Lingi, Chun’an County (Hangzhou, China), and was authenticated by Associate Professor Zhu Bo from the School of Pharmaceutical Sciences in Zhejiang Chinese Medical University. (Hangzhou, China). Streptozotocin and Valsartan were purchased from Sigma-Aldrich (St. Louis, USA). The high-fat diet (HFD) chow was obtained from Wuxi Fanbo Biotechnology Co., Ltd. (Jiangsu, China). Creatinine, albuminuria and triglyceride assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Desmin, podocin, claudin-1, wilms’ tumor 1 (WT-1) and C-C Motif Chemokine Ligand 2(CCL-2) were purchased from proteintech (Wuhan, China). Nephrin was purchased from abcam (Shanghai, China). Preparation of polysaccharides from Cornus officinalis The coarse powder of Cornus officinalis was initially defatted through reflux extraction with 90% ethanol (1:8, w/v) at 90°C for 4 h using a round-bottom flask. Following ethanol evaporation under reduced pressure, the resultant residue was subjected to hot aqueous extraction (1:10, w/v) at 90°C for 4 h. The aqueous extract was concentrated to 25% of its original volume using vacuum evaporation and subsequently precipitated with 95% ethanol (1:4, v/v) at 4°C for 12 h. The precipitated polysaccharides were collected by vacuum filtration and sequentially processed through rotary evaporation at 45°C and lyophilization at -50°C for 24 h. To remove protein contaminants, the lyophilized powder was treated with Sevag reagent (chloroform: n-butanol = 4:1, v/v) through five cycles of vigorous shaking and phase separation. This procedure yielded the crude COP. To ensure reproducibility, the entire extraction protocol was independently replicated three times under standardized conditions, generating three distinct batches designated as COP1-COP3. Monosaccharide composition analysis COP was hydrolyzed with 2 M trifluoroacetic acid (110°C, 6 h), followed by methanol washing and reconstitution. Hydrolysates and monosaccharide standards (mannuronic acid, mannose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose) were separately subjected to 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization with 0.3 M NaOH/10% PMP (70°C, 45 min). After chloroform extraction and filtration (0.22 μm), derivatives were analyzed on Agilent 1260 HPLC equipped with Ultimate LP C18&LP C8 column (150 × 2.1 mm, 3 μm) under isocratic elution (acetonitrile:0.77% ammonium acetate=15:85, 1 mL/min) at 30°C with 254 nm detection. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) FT-IR analysis was performed using the Nicolet IS50 FT-IR spectrometer (Thermo Scientific, Wilmington, USA) with spectral acquisition from 4000 to 500 cm⁻¹. Samples were prepared by homogenizing COP with KBr at a 1:100 (w/w) ratio and compressing into transparent pellets. Morphological characterization was conducted on a Hitachi SU8010 SEM (Hitachi, Japan) operated at 2 kV. Powdered COP was mounted on aluminum stubs, excess particles removed by compressed air, and observed at magnifications ranging from 100× to 5,000×. Animals and treatments Sprague-Dawley rats (3-4 weeks old) were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology (Zhejiang, China) and maintained under a specific-pathogen-free environment in the laboratory animal center of Zhejiang Chinese Medical University (Hangzhou, China, Approval No: IACUC-20220314-16). All procedures were conducted in accordance with the Institutional Animal Care and Use Committee guidelines of Zhejiang Chinese Medical University (Protocol No. 202203-0153). After a 1-week acclimatization period, rats were stratified into two cohorts: normal diet control (NFD, n=6) and HFD groups. HFD-fed rats (body weight 240-280 g) received daily intraperitoneal STZ (40 mg/kg) for 7 days until achieving fasting blood glucose (FBG) ≥11.1 mM. Diabetic rats were then randomized into: Model (Mod, HFD + saline i.g.), Positive control (POS, HFD + valsartan 40 mg/kg/day), COP treatment groups (LCOP/MCOP/HCOP: 120/240/480 mg/kg/day i.g. + HFD). During the 15-week intervention period, body weight and FBG were monitored weekly and biweekly, respectively. 24-hour urine samples were collected using metabolic cages at week 14. Fecal specimens were cryopreserved at -80°C prior to terminal procedures. Following 12 h fasting, animals were anesthetized for cardiac puncture blood collection, euthanized by cervical dislocation, with subsequent collection of renal tissues and colonic contents for analysis. Biochemical parameter and renal index Serum triglycerides (TG), creatinine (Scr), and urea nitrogen levels, along with urinary creatinine, and microalbumin concentrations, were quantified using biochemical parameter test kits. The urinary albumin-to-creatinine ratio (UACR) was calculated as microalbumin/creatinine. Body weight were recorded daily. After the rats were sacrificed, kidneys were excised, rinsed with cold saline, and weighed. Tissue aliquots were either fixed in 4% paraformaldehyde for histology or flash-frozen in liquid nitrogen (-80°C storage). Renal index was calculated as (kidney weight/body weight) ×100%. Renal histopathology Kidney tissues were fixed in 4% paraformaldehyde (4°C, 12 h), paraffin-embedded, and sectioned (5 μm). Consecutive sections underwent: Hematoxylin-eosin (H&E) for histoarchitecture; Masson trichrome for fibrosis (blue area quantified via ImageJ); Periodic Acid-Schiff (PAS) for glycogen deposition and basement membrane integrity. Semiquantitative scoring of glomerular/tubular injury, vascular congestion, and inflammation was performed on sections. Five randomly selected sections per group with 7-10 glomerular fields analyzed ensured statistical validity. not-yet-known not-yet-known not-yet-known unknown Immunofluorescence (IF) and Western blotting (WB) analysis The kidney sections were incubated with anti-podocin (1:200) and Alexa Fluor 488-conjugated secondary antibody (1:500), stained with IF, imaged by fluorescence microscopy, and quantified via ImageJ. Total proteins extracted with RIPA/1% PMSF were quantified by BCA, separated on 10% SDS-PAGE, and transferred to PVDF membranes. After blocking (5% skim milk/PBST, 1 h), membranes were incubated with primary antibodies (4°C, overnight) and HRP-conjugated secondaries (1 h). ECL-detected bands were quantified using ImageJ. 16S rDNA sequencing analysis Fecal DNA was extracted and subjected to 16S rDNA sequencing targeting V3-V4 regions (Illumina NovaSeq platform). Paired-end reads were quality-filtered, merged, and processed through DADA2 pipeline for amplicon sequence variant (ASV) calling. Taxonomic classification was performed using SILVA 138.1 reference database. Alpha-diversity (Shannon/Chao1/ Observed_otus) and beta-diversity (PCA) analyses were computed with QIIME2. Further data analysis and visualization was performed using the OmicStudio tools at https://www.omicstudio.cn/tool. Quantitative real-time polymerase chain reaction(qRT-PCR) Total RNA from kidney tissues/cells was isolated using TRIzol® and reverse-transcribed with Script cDNA Synthesis Kit. qRT-PCR was performed on LightCycler® 96 system with ChamQ SYBR Master Mix under cycling conditions: 95°C/15 min; 40 cycles of 94°C/15 s, 60°C/30 s, 72°C/30 s; melting curve analysis. Relative mRNA levels were calculated by 2−ΔΔCt method with β-actin normalization. The primer sequences used for qRT-PCR analysis are shown in Table 1. UPLC-MS/MS for metabolomics Colon content metabolites were analyzed using Xevo G2-XS Q-TOF/MS system (Waters, Milford, Massachusetts, USA). Lyophilized samples (100 mg/mL) were homogenized in 70% methanol containing deuterated internal standard (2 μg/mL) with ceramic beads, centrifuged (12,000 ×g, 4°C, 10 min), filtered through 0.22 μm membranes, and subjected to LC-MS/MS. Chromatographic separation was achieved on Acquity UPLC HSS T3 column (100×2.1 mm, 1.8 μm) at 0.3 mL/min (40°C). Mobile phases: (A) 0.1% formic acid; (B) 0.1% formic acid/acetonitrile. Gradient program: 0-2 min: 80% A; 2-12 min: 48% A; 12-18 min: 35% A; 18-22 min: 10% A; 22-30 min: 80% A (2 μL injection). RNA sequencing analysis Total RNA from renal tissues was extracted using TRIzol®. RNA integrity (RIN ≥6.5, 28S/18S ≥1.0) and purity (OD260/280=1.8-2.2) were verified via Agilent 5300 Bioanalyzer and NanoDrop ND-2000. Polyadenylated mRNA was enriched using oligo(dT) beads, fragmented, and reverse-transcribed into cDNA (SuperScript™ II kit). Libraries were constructed with Illumina® Stranded mRNA Prep kits, size-selected (300 bp), and sequenced on NovaSeq X Plus (PE150, Majorbio Bio-pharm). Culture and Screening of A. muciniphila growth modulators in vitro A. muciniphila , ATCC BAA-835=CIP 107961, was was cultured anaerobically (37°C, 5% H₂/10% CO₂/85% N₂) using COYB chambers. For A. muciniphila in vitro BA metabolism, when the bacteria grew to OD=600 density for experimental use. LCA (100µM) was added to the medium, aliquots (2 mL) collected at 0/48 h underwent sequential processing: centrifugation (12,000 ×g, 5 min), lyophilization, methanol extraction (10 min sonication), and clarification (12,000 ×g, 10 min). Supernatants were concentrated, reconstituted in 70% methanol containing CA-2,2,4,4-d4 (2 μg/mL), filtered (0.22 μm), and analyzed by LC-MS/MS. Cell culture and experimental design Mouse renal podocyte cells (MPC5, BNCC342021) were purchased from BeNa Culture Collection (Henan, China) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 10 U/mL interferon gamma (IFN-γ) under permissive conditions (33°C, 5% CO₂). Differentiation was induced by temperature shift to 37°C without IFN-γ for 14 days prior to experiments. Differentiation was induced by temperature shift to 37°C (without IFN-γ) for 14 days and confirmed via podocin WB analysis. not-yet-known not-yet-known not-yet-known unknown Cytotoxicity assessment Cell viability was determined via MTT assay. Differentiated MPC5 cells (1×10⁴ cells/well) in 96-well plates were treated with LCA, isolithocholic acid (isoLCA), or 3-oxolithocholic acid (3-oxoLCA) at concentrations ranging from 0 to 25 μM for 48 h. After removing supernatants, cells were incubated with 0.5 mg/mL MTT for 4 h at 37°C. Formazan crystals were dissolved in Dimethyl sulfoxide (150 μL/well, 10 min shaking, protected from light). Absorbance was measured at 490 nm using a microplate reader (BioTek, USA). Viability was calculated as: Cell viability = [A (treatment group) − A (blank group)]/ [A (control group) − A (blank group)] × 100% (n=3 independent experiments). Based on cell viability results, 10 μM BA concentrations (non-cytotoxic) were selected for subsequent studies. High glucose injury model Differentiated mature MPC5 were cultured at 30 mM glucose concentration to stimulate cell injury and incubated in the presence or absence of LCA, isoLCA, 3-oxoLCA (10µM) for 48h. Protein expression of desmin, podocin, and nephrin was analyzed by WB using specific antibodies. Chemical characteristics of COP COP exhibited characteristic polysaccharide parameters (Table 2), with galacturonic acid (39.78%), arabinose (22.90%), glucose (19.36%), and galactose (11.89%) as the dominant components, alongside minor components (≤3.66%). As illustrated in Fig. 1B, COP displayed a rough morphological surface with a dense fibrous structure. The chemical functional groups of COP were analyzed using FT-IR (Fig. 1C). The broad absorption band observed between 3200 cm -1 and 3600 cm -1 corresponds to the characteristic stretching vibrations of -OH groups. The peaks at 2922 cm⁻¹ represent C–H stretching vibrations, including contributions from C–H, C–H₂, and C–H₃ bonds. Additionally, the absorption peak at 1751 cm⁻¹, attributed to the bending vibration of C=O, indicates the presence of aldehyde groups in COP. COP attenuates renal lesions in DN rats To evaluate the renoprotective potential of COP in DN, we administered three titrated doses of COP (low: 120 mg/kg, medium: 240 mg/kg, high: 480 mg/kg) and assessed their therapeutic effects. COP treatment attenuated DN-associated pathological manifestations, including body weight loss, hyperglycemia, and increased 24-hour urine output, in a dose-dependent manner. Moreover, COP significantly ameliorated DN-induced metabolic disturbances, as evidenced by reductions in serum TG and urea nitrogen levels. Notably, COP administration led to a dose-dependent decrease in albuminuria and urinary UACR, as illustrated in Fig. 2D. In addition, DN rats exhibited marked renal hypertrophy (p < 0.001 vs. CON group), which was dose-dependently ameliorated by COP treatment. High-dose COP (HCOP) restored renal morphology and significantly reduced the renal index (p < 0.05 vs. MOD group) (Fig. 2E-F). Histopathological analysis demonstrated COP’s capacity to mitigate glomerular structural damage: H&E staining showed reduced interstitial fibrosis and preserved glomerular architecture, Masson staining indicated decreased collagen deposition, and PAS staining demonstrated alleviated glycogen accumulation (Fig. 2E). Quantitative histopathology scoring confirmed the dose-responsive therapeutic efficacy, with HCOP achieving significant reversal of DN-induced renal injury (p < 0.001 vs. MOD group). These findings underscore the potential of COP in mitigating renal structural damage and fibrosis in DN. COP repairs the glomerular filtration barrier. A hallmark of DN is damage to the glomerular filtration barrier, with podocytes serving as its critical defensive component (Tian and Ishibe, 2016). IF analysis revealed enhanced expression of the slit diaphragm protein podocin in COP-treated groups (Fig. 3A). Double-staining experiments (Claudin-1/WT-1) showed pathological proliferation of WT-1+ cells in Bowman’s capsule of DN rats, which was significantly attenuated by COP treatment (Fig. 3B-C), consistent with the pattern of mural epithelial activation described in the literature (Luna-Antonio et al., 2017). WB analysis further confirmed COP’s regulatory effects on DN-induced cytoskeletal rearrangement (Desmin), tight junction activation (Claudin-1), and inflammatory CCL-2 (Fig. 3D), collectively ameliorating podocyte dysfunction as established in (Shankland et al., 2014). COP reversed the changes in the composition of the gut microbiota of DN rats. Based on the dose-dependent therapeutic efficacy (Fig. 2-3), fecal samples from CON (control), MOD (diabetic nephropathy model), and HCOP (480 mg/kg) groups subjected to 16S rRNA sequencing (V3-V4 region). As illustrated in Fig. 4A-C, the Chao1, observed_otus and Shannon indices, which reflect microbial alpha-diversity, were significantly reduced in the MOD group. While COP treatment did not markedly alter Chao1 and observed_otus indices, it significantly improved the Shannon index compared to the MOD group. Moreover, PCA further revealed distinct clustering patterns in microbial β-diversity among the three groups (Fig. 4D). At the phylum level, COP treatment altered the composition of gut microbiota, downregulating the relative abundance of Firmicutes and Verrucomicrobiota while upregulating Proteobacteria and Desulfobacterota, which were elevated in the MOD group (Fig. 4E-F). At the genus level, the MOD group exhibited increased relative abundance of Lactobacillus, Escherichia-Shigella, Allobaculum, Clostridium, and Dorea, alongside decreased abundance of Akkermansia, Phascolarctobacterium, and NK4A214_group. COP treatment effectively reversed these microbial compositional changes. Differential analysis identified 18 bacteria with significant differences among the three groups, with Akkermansia showing the most pronounced variation (Fig. 4G). Phylogenetic reconstruction further confirmed Verrucomicrobiota/Akkermansia as the primary lineage associated with therapeutic efficacy (Fig. 4H). Spearman correlation network analysis (FDR-corrected) demonstrated Akkermansia abundance strongly inversely correlated with UUN (r=-0.84, q= 0.0001), UACR (r=-0.67, q=0.006), and TG (r=-0.60, q=0.02) (Fig. 4I). Multivariate Corrlink modeling ranked Akkermansia as the top microbiota predictor of DN improvement (p≤0.05) (Fig. 4J). Collectively, these results indicate that Akkermansia may serve as a key bacterium mediating the amelioration of DN by COP. COP altered fecal metabolite profiles in DKD rats Data normalization, retention time correction, and peak picking were conducted using Progenesis QI software. The resulting data matrices were subsequently imported into Metaboanalyst 5.0 for comprehensive network analysis of different metabolites and identification of key metabolic pathways. Metabolomic analysis revealed distinct clustering patterns between experimental groups, as demonstrated by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) (Fig. 5A-B), indicating that COP-mediated restored fecal metabolite profiles in DN rats. Pathway enrichment analysis identified primary BA biosynthesis as the most significantly perturbed metabolic pathway (Fig. 5C). Targeted quantification confirmed COP-induced modulation of key BA metabolites, including LCA, 3-oxoLCA, and isoLCA (Fig. 5D). These metabolites exhibited strong negative correlations with clinical parameters of DN (Fig. 5E), suggesting potential as biomarkers for DN progression and therapeutic response. Transcriptome analysis suggests that COP attenuates renal injury by systematically suppressing the expression of PPAR pathway-related genes To elucidate the molecular mechanisms underlying the therapeutic effects of COP on DN, we conducted comprehensive transcriptomic profiling of renal tissues from three experimental groups. PCA demonstrated distinct clustering among experimental groups (Fig. 6A), with COP treatment shifting global transcriptomic patterns toward those of the CON group states (Fig. 6B). Differential gene expression analysis, performed using stringent criteria (adjusted p-value < 0.05 and fold change ≥ 2), revealed that COP treatment modulated 938 genes compared to the DN model group (654 up-regulated /284 down-regulated), among which 178 were shared targets associated with DN-related dysregulation (Fig. 6C-D). KEGG pathway enrichment analysis identified PPAR signaling as the predominant pathway (p<0.001), involving key target genes such as Slc27a2 , PPARα , and Angptl4 (Fig. 6E). Reduced PPARα expression was reported to contribute to mitochondrial dysfunction and lipid accumulation (Herman-Edelstein et al., 2014). Furthermore, PPARα deficiency exacerbated podocyte apoptosis, accelerating dyslipidemia, proteinuria, and renal failure in animal models of DN (Chen et al., 2023). To validate these findings, a heatmap was generated to visualize the expression patterns of PPAR pathway-related genes from the RNA-seq dataset across the three experimental groups. Subsequent qRT-PCR analysis of rat kidney tissues confirmed that PPARα expression was significantly downregulated in the model group, while the expression of Slc27a2 and Angptl4 was markedly upregulated. These findings suggest that COP may attenuate DN-induced renal injury through the Slc27a2 - PPARα - Angptl4 axis. A. muciniphila in vitro culture regulates LCA and its BA derivatives. A. muciniphila is the sole species within the genus Akkermansia. Therefore, we hypothesized that A. muciniphila modulates the metabolic pathway of LCA metabolism (Fig. 7A). To test this hypothesis, A. muciniphila was co-cultured with LCA (100µM) for 48h, resulting in the generation of 3-oxoLCA and isoLCA (Fig. 7B-C). These findings suggest that A. muciniphila may play a role in the progression of DN by regulating the metabolism of LCA, 3-oxoLCA, and isoLCA. LCA, 3-oxoLCA, and isoLCA have a reparative effect on the damage caused by high glucose stimulation of MPC5, possibly through regulation of the Slc27a2 - PPARα - Angptl4 axis . To evaluate the nephroprotective effects of BA regulated by A. muciniphila , we assessed the impact of LCA, 3-oxoLCA, and isoLCA against HG-induced injury in differentiated MPC5 podocytes. MTT assays revealed that cells viability remained relatively high at BA concentration below 10 µM compared to the solvent control (Fig. 8A), prompting the selection of 10 μM for subsequent experiments. Under sustained HG conditions mimicking DN, BA treatment (48 h) attenuated cytoskeletal disruption and glomerular barrier dysfunction. WB analysis demonstrated that LCA, 3-oxoLCA, and isoLCA reversed HG-induced downregulation of nephrin and podocin while suppressing desmin overexpression (Fig. 8B).Among these BAs, 3-oxoLCA exhibited the most pronounced protective effects. These findings suggest that the three BAs regulated by A. muciniphila may confer renal benefits in DN. qRT-PCR analysis further confirmed that 3-oxoLCA downregulated Slc27a2 and Angptl4 while upregulating PPARα expression under HG stress (Fig. 8C), consistent with renal transcriptomic data. Given theestablished role of Angptl4’s in impairing glomerular charge barriers and promoting proteinuria (Tang et al., 2021), our findings suggest 3-oxoLCA mitigates podocyte injury through the Slc27a2 - PPAR - Angptl4 axis, highlighting its therapeutic potential in DN. Discussion DN remains a global therapeutic challenge, underscoring the urgent need to explore natural bioactive compounds with minimal side effects (Wu et al., 2024). This study demonstrated that COP alleviated DN progression through a sequential “polysaccharide-microbiota-transcriptome-experimental validation” axis. As non-digestible macromolecules, COP bypassed small intestinal degradation to directly enrich A. muciniphila (Verrucomicrobia phylum), a keystone species inversely correlated with hyperglycemia and renal injury markers. This aligns with prebiotic capacity of polysaccharides to selectively promote beneficial taxa (Lee et al., 2022). Critically, A. muciniphila drives biotransformation of LCA into nephroprotective derivatives (3-oxoLCA and isoLCA), as confirmed by in vitro co-culture experiments. These BAs act as key signaling mediators in gut-kidney crosstalk (de Vos et al., 2022). COP restored systemic BA homeostasis, countering DN-associated metabolic inflammation (Chiang and Ferrell, 2019). While A. muciniphila is known to promote the colonization of bile salt hydrolase-producing bacteria (He et al., 2024), our study is the first to elucidate its translational role in LCA derivatization, advancing the understanding of microbiota-BA-host interactions. Transcriptomic profiling identified PPAR signaling pathway as the central mechanism mediating COP’s renoprotection. COP downregulated Slc27a2 (a PPARα upstream regulator) while activating PPARα expression, thereby suppressing Angptl4 -mediated proteinuria-a cascade validated in high glucose-injured podocytes. LCA derivatives directly activated PPARα signaling, mitigating podocyte injury. The convergence of in vivo transcriptomics and in vitro functional assays established a clear link between A. muciniphila -mediated metabolite production (LCA derivatives) and host molecular responses, confirming the “microbiota-metabolite-transcriptome” cascade. The integrated pathway elucidates COP’s renoprotective effects (Fig. 9): (1) COP enriches A. muciniphila , a microbial driver of BA metabolism; (2) A. muciniphila generates LCA derivatives that systemically activate PPARα ; (3) PPAR signaling suppresses Angptl4 , restoring glomerular barrier integrity. This mechanistic cascade aligns with evidence that BA remodeling protects podocytes via PPAR pathways (Yamashita et al., 2020), while addressing a critical gap in understanding how non-absorbable polysaccharides exert systemic effects. This study demonstrates that COP exhibited remarkable potential in ameliorating STZ-induced DN, positioning it as a promising natural therapeutic candidate. To further advance its translational potential, subsequent investigations could focus on the following dimensions: (1) Given the pivotal regulatory role of A. muciniphila in COP intervention, systematic evaluation of COP’s renoprotective effects through microbial colonization experiments in germ free animal models would help clarify specific microbiota-host interactions; and (2) While LCA and its derivatives have demonstrated metabolic regulatory activities in cellular models, their clinical applicability, and validation in animal systems warrant further exploration. These research endeavors will facilitate the transition of COP from mechanistic studies to clinical implementation, establishing both theoretical and practical foundations for developing innovative “gut-kidney axis”-targeted therapies for DN. Conclusions In conclusion, this study elucidates the mechanism of COP in combating DN. COP remodels the gut microbiota, enriching A. muciniphila , which drives the derivatization of LCA to activate PPAR signaling, ultimately restoring glomerular function. By integrating microbial ecology, metabolomics, and transcriptomics with experimental validation, we establish a novel paradigm in which traditional medicine polysaccharides modulate microbiome-host interactions through metabolite-pathway networks. These findings not only advance the therapeutic strategies for DN but also provide a comprehensive framework for developing microbiota-targeting natural products in the treatment of metabolic diseases. Fundings This work has been financially supported by 2024 “Pioneer” and “Leading Goose” R&D Program of Zhejiang (2024C03102) CRediT authorship contribution statement Jing Li : Writing – original draft, Data curation. Lyu Qiang ：Project administration，Writing –review and editing. Qiao Yang : Methodology, Investigation. Jiaxin Ye : Validation, Writing - Original Draft. Rui Ding : Methodology, Investigation. Saiya Chen : Formal analysis, Visualization. Feier Yang : Formal analysis, Visualization. Lingjun Ye : Formal analysis. Min Hao : Investigation, Project administration. Qiyuan Shan : Resources, Supervision. Xin Han : Resources, Supervision. Zhixiang Dong : Resources, Supervision. Lu Wang : Investigation, Project administration. Kuilong Wang : Writing –review and editing, Funding acquisition, Conceptualization. Gang cao : Funding acquisition, Project administration. Data availability Data will be made available on request. not-yet-known not-yet-known not-yet-known unknown Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Keywords metabolomics natural product renal pharmacology Authors Affiliations Jing Li 0009-0008-6410-0680 Zhejiang Chinese Medical University View all articles by this author Jiaxin Ye Zhejiang Chinese Medical University View all articles by this author Rui Ding Zhejiang Chinese Medical University View all articles by this author Saiya Chen Zhejiang Chinese Medical University View all articles by this author Feier Yang Zhejiang Chinese Medical University View all articles by this author Lingjun Ye Zhejiang Chinese Medical University View all articles by this author Min Hao Zhejiang Chinese Medical University View all articles by this author Qiyuan Shan Zhejiang Chinese Medical University View all articles by this author xin Han 0000-0002-1725-7940 Zhejiang Chinese Medical University View all articles by this author Zhixiang Dong Zhejiang Chinese Medical University View all articles by this author Lu Wang Zhejiang Chinese Medical University View all articles by this author Kuilong Wang [email protected] Zhejiang Chinese Medical University View all articles by this author Gang Cao Zhejiang Chinese Medical University View all articles by this author Caihua Sun The First Affiliated Hospital of Zhejiang Chinese Medical University View all articles by this author Metrics & Citations Metrics Article Usage 360 views 214 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jing Li, Jiaxin Ye, Rui Ding, et al. 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