Shenzhuo formulation ameliorates diabetic nephropathy by regulating cytochrome P450-mediated arachidonic acid metabolism

Abstract

BACKGROUND

Diabetic nephropathy (DN) is a major complication of diabetes, marked by progressive renal damage and an inflammatory response. Although research has investigated the pathological mechanisms underlying DN, effective treatment options remain limited.

AIM

To evaluate the therapeutic impact of Shenzhuo formulation (SZF) on a DN mouse model and to examine its potential molecular mechanisms using transcriptomic and metabolomic approaches.

METHODS

We established a DN mouse model through a high-fat diet combined with streptozotocin (STZ) injection, followed by SZF treatment. We analyzed SZF’s effects on gene expression and metabolite profiles in renal tissues of DN mice using transcriptomics and metabolomics techniques. Additionally, based on transcriptomic and non-targeted metabolomic findings, we further assessed SZF’s influence on the expression of factors related to the cytochrome P450 (CYP450)-mediated arachidonic acid (AA) metabolism pathway, as well as its effects on inflammation and oxidative stress.

RESULTS

SZF intervention significantly decreased hyperglycemia and mitigated renal function impairment in DN mice. Pathological analysis revealed that SZF treatment improved renal tissue damage, reduced fibrosis, and diminished glycogen deposition. Transcriptomic analysis indicated that SZF influenced mRNA expression of CYP450-related genes, including Cyp2j13, Cyp2b9, Pla2 g2e/Cyp4a12a, Cyp4a32, Cyp2e1, and Cyp4a14. Non-targeted metabolomic results demonstrated that SZF altered the levels of metabolites associated with the AA metabolic pathway, including 5,6-EET, 14,15-EET, phosphatidylcholine, and 20-HETE. Further experiments showed that SZF upregulated the expression of CYP4A and CYP2E proteins in renal tissue, as well as CYP2J and CYP2B proteins. Additionally, SZF significantly reduced the expression of inflammatory factors in renal tissue, enhanced antioxidant enzyme activity, and alleviated oxidative stress.

CONCLUSION

SZF exerts anti-inflammatory and antioxidant effects by regulating CYP450-mediated AA metabolism, leading to improved renal function and improved pathological state in DN mice.

Key Words: Diabetic nephropathy; Shenzhuo formulation; Transcriptomic; Metabolomic; Cytochrome P450; Arachidonic acid metabolic; Inflammatory; Oxidative stress

Core Tip: This study aimed to evaluate the therapeutic impact of Shenzhuo formulation (SZF) on a diabetic nephropathy (DN) mouse model and to examine its potential molecular mechanisms using transcriptomic and metabolomic approaches. The findings of this research substantiated the significant potential of SZF in ameliorating renal injury in DN mice. The primary pathway through which SZF mitigates renal injury appears to be the modulation of cytochrome P450-mediated AA metabolism, which contributes to its anti-inflammatory and antioxidant effects.

INTRODUCTION

Diabetic nephropathy (DN) ranks among the most prevalent microvascular complications of diabetes mellitus, characterized by a high incidence and poor prognosis. It has become the leading cause of chronic kidney disease (CKD) and end-stage renal disease[1]. As the global prevalence of diabetes mellitus continues to rise, addressing the prevention and treatment of DN poses a critical challenge for medical research. According to the latest data from the International Diabetes Federation, more than 463 million individuals worldwide had diabetes as of 2019, and this number is projected to reach 700 million by 2045[2]. Consequently, conducting in-depth research on the pathogenesis of DN and identifying effective treatments is increasingly urgent.

In recent years, advances in molecular biology have enhanced our understanding of DN pathogenesis, particularly through transcriptomics and metabolomics, both essential components of systems biology. These methodologies provide powerful tools for investigating the underlying mechanisms of DN[3]. Transcriptomics analyzes changes in gene expression to reveal the functional and state changes of cells and tissues during disease progression[4]. In contrast, metabolomics detects alterations in small molecule metabolites, reflecting dynamic changes and regulatory networks within metabolic pathways throughout the organism[5]. The combined application of transcriptomics and metabolomics offers a comprehensive perspective, elucidating molecular mechanisms and metabolic effects. This approach accurately reveals biochemical changes in disease states and deepens our understanding of the pathomechanisms underlying DN.

In traditional Chinese medicine, herbal compounds have been widely used to treat DN and have demonstrated some efficacy[6]. For instance, Compound Xiancao Granule restores metabolic homeostasis by remodeling intestinal flora and regulating carbohydrate and amino acid metabolism, as well as inflammation-related pathways, thereby aiding in the treatment of DN[7]. Danggui Buxue Decoction may improve DN by influencing lipid metabolism[8]. The Shenzhuo formula (SZF), which includes Salvia miltiorrhiza (S. miltiorrhiza) Bunge, Astragalus mongholicus (A. mongholicus) Bunge, Hirudo, Rheum officinale (R. officinale) Baill. As its primary components, SZF works synergistically to benefit qi, activate blood circulation, resolve blood stasis, and clear collaterals. A retrospective study indicated that SZF could enhance glomerular filtration rate and reduce 24-h urinary total protein (24h-UTP)[9]. Additionally, network pharmacological analyses suggest that SZF may prevent and regulation DN through signaling pathways such as TNF and PI3K-Akt[10].

To further investigate the effects and mechanisms of SZF on DN, this study established a DN mouse model to observe the therapeutic effects of SZF intervention. We employed transcriptomics and metabolomics techniques to explore the potential mechanisms underlying SZF's effects on this model. This study aims to elucidate how SZF regulates relevant signaling pathways to improve the pathological state of DN. These findings provide a scientific foundation for the clinical application of SZF and offer new perspectives and strategies for the pharmacological treatment of DN.

MATERIALS AND METHODS

The experimental reagents, pharmaceuticals, and other materials necessary for this study are described in detail in the Supplementary materials.

Animal model establishment, grouping and treatment

We obtained sixty SPF-grade healthy male C57BL/6 mice, aged 6-8 weeks and weighing 20-22 g, from SPF (Beijing) Biotechnology Co., Ltd. [Animal License No. SCXK (Beijing) 2024-0001]. The mice were housed in groups of five per cage, maintained at 24 ± 2°C with 55% ± 5% relative humidity, and a 12-h light-dark cycle. They had free access to food and water. All experimental procedures followed the Guidelines for Animal Ethics and received approval from Cangzhou Hospital of Integrated Traditional Chinese Medicine and Western Medicine of Hebei Province (Approval Number: CZX2024-KY-129).

After a one-week acclimatization period, we randomly assigned the mice into two groups: 10 mice in the control group (Control), which received a standard diet, and 50 mice underwent 8 wk of high sugar high fat diet feeding before modeling was initiated. We followed previously described methods to induce the model[11]. After fasting for 12 h with free access to water, mice on the high sugar high fat diet received intraperitoneal injections of STZ at 30 mg/kg, whereas the Control received an equal volume of 1% sodium citrate buffer. After 72 h, blood was collected from the tail vein to measure random blood glucose, with glucose levels of ≥ 16.7 mmol/L used as the criterion for type 2 diabetes mellitus (T2DM) modeling. Following T2DM confirmation, we continued feeding and monitored 24h-UTP weekly. We set criteria for DN modeling as random blood glucose levels higher than 16.7 mmol/L and 24h-UTP ≥ 20 mg.

Mice that successfully established the DN model were randomly divided into five groups of ten mice each: The Model group (Model), the Positive Control group (PC), the SZF low dose group (SZFL), the SZF medium dose group (SZFM), and the SZF high dose group (SZFH). The Control and Model received daily gavage of saline at a dose of 0.2 mL/kg, whereas the PC group received Irbesartan at 40 mg/kg daily by gavage[12]. The SZFL, SZFM, and SZFH groups received SZF at doses of 3.7 g/kg, 7.4 g/kg, and 14.8 g/kg by gavage daily, respectively. The SZF doses were calculated using a formula for equivalent dose conversion, with the medium dose corresponding to the human equivalent dose[13]. We administered the treatments continuously for eight weeks, monitoring and recording blood glucose levels and body weights of the mice in each group weekly.

After eight weeks of treatment, we collected 24-h urine samples from each group using metabolic cages. We obtained blood samples through intraocular canthus sampling. Following euthanasia, we opened the abdominal cavity, collected the left renal for fixation in 4% paraformaldehyde, and froze the right renal in liquid nitrogen for storage at -80°C.

Renal function index test

The 24-h urine samples were centrifuged at 4000 r/min for 10 min to separate the supernatant. The 24h-UTP levels were measured in each group of mice according to the kit instructions. Blood samples were centrifuged at 3500 r/min for 15 min to collect serum. Creatinine (Cr) and blood urea nitrogen (BUN) in the serum were measured from each group of mice following the kit protocols.

Renal histopathology and morphology

Renal tissues were removed from 4% paraformaldehyde solution for dehydration, paraffin embedding, and sectioning. Sections were cut to a thickness of 4 μm and stained with hematoxylin and eosin (HE), Masson’s trichrome, and periodic acid-Schiff (PAS) stains. After sealing, the sections were stained under a microscope and images were saved for analysis. HE staining allowed for scoring of pathological morphology of renal tissues[14]. Masson staining was used to assess fibrosis in renal tissues, whereas PAS staining evaluated the degree of glycogen deposition. The average optical density was quantified using Image Pro Plus software.

Transcriptomics assay

Renal tissues were removed from -80 °C and total RNA was isolated. Subsequently, total RNA was extracted from renal tissues using the TRIzol method. The purity of the RNA was assessed using a NanoDrop ND-1000 spectrophotometer, with acceptable samples having an A260/280 ratio between 1.8 and 2.2. The integrity and concentration of the RNA were evaluated using a Bioanalyzer 2100, with requirements of an RNA Integrity Number (RIN) ≥7.0 and a concentration >50 ng·μL^-1. Once the samples met quality standards, library preparation and sequencing was performed using the Illumina platform. Differentially expressed genes (DEGs) were analyzed between the Model and Control groups, as well as between the SZFH and Model, using DESeq2 software. The criteria for identifying DEGs were |Log₂ (Fold Change)| ≥ 1 and P_adj ≤ 0.05. KEGG pathway enrichment analyses were performed on the identified DEGs.

Non-targeted metabolomics assays

Non-targeted metabolomics analysis based on methods described in previous studies[15]. Specifically, mouse renal tissues from each group were ground in liquid nitrogen and the samples were centrifuged. The supernatant was diluted with water and methanol was added to include an internal standard. The resulting supernatant was concentrated to form a dry powder, which was then mixed with a solution of methoxyamine in pyridine and N-methyl-N-trimethylsilane trifluoroacetamide. After thorough mixing, the external standard solution was added, the components were well mixed and the mixture was subjected to machine testing. Quality control samples were prepared by mixing equal volumes from each sample. Additional details regarding non-targeted metabolomics can be found in the Supplementary materials.

ELISA assay

Homogenates were prepared from frozen renal tissues and the expression levels of cytokines Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured by ELISA according to the manufacturers' instructions for the respective kits.

Biochemical indexes

Homogenates were prepared from frozen renal tissues to assess biochemical indices. Biochemical kits were used to detect the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as malondialdehyde (MDA) content in the homogenates from each group of mice, following the protocols provided with the kits.

Western blot

Total protein was extracted from 20 mg of renal tissue and protein concentration was determined using the BCA method. Proteins were then separated by SDS-PAGE and transferred to a PVDF membrane via electro-transfer. The membrane was blocked in 5% skim milk solution for 2 h, followed by incubation with the specific primary antibody overnight at 4 °C. After three washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Following another set of washes, the membranes were developed using ECL chemiluminescence. Quantitative analysis of the protein bands was performed using ImageJ software (version 1.76).

Statistical analysis

Statistical analysis was performed using SPSS 22.0 software. All data are presented as mean ± SD. Inter-group differences were assessed using one-way ANOVA, followed by Tukey's HSD test. A P value less than 0.05 was considered statistically significant.

RESULTS

Therapeutic effects of SZF intervention in DN mice

The blood glucose results indicated that mice in the Model had significantly elevated blood glucose levels compared to the Control. In contrast, the SZFH group exhibited significantly reduced blood glucose levels compared to the Model (Figure 1A). Body weight measurements revealed that the Model had significantly lower body weight than the Control. However, the body weight of mice in the SZFH group was significantly greater than that of the Model (Figure 1B). Renal function tests demonstrated that serum levels of 24h-UTP, Cr, and BUN were significantly higher in the Model compared to the Control. The PC group showed lower levels of 24h-UTP, Cr, and BUN compared to the Model, whereas the SZFH group also exhibited varying degrees of reduction in these parameters (Figure 1C-E).

Figure 1 Shenzhuo formulation intervention improves renal injury in diabetic nephropathy mice. A diabetic nephropathy (DN) mouse model group (Model) was established and Shenzhuo formulation (SZF) intervention was given. A and B: SZF intervention significantly reduced blood glucose (A) and increased body weight (B) in DN mice; C-E: SZF intervention improves renal function in DN mice and reduces levels of 24-hour urinary total protein (C), creatinine (D), and BUN (E); F-G: SZF intervention improves the renal histopathological injury in DN mice; Hematoxylin and eosin staining results showing that SZF improves renal histopathological morphology; H and I: Masson staining results showing that SZF improves renal histopathological morphology and that SZF could reduce renal tissue fibrosis; J and K: PAS staining results showing that SZF could reduce renal tissue glycogen deposition. Data are presented as the mean ± SD. n = 10 per group. ^bP < 0.01 vs control group; ^cP < 0.05; ^dP < 0.01 vs Model; FBG: Fasting blood glucose; UTP: Urinary total protein; Cr: Creatinine; BUN: Blood urea nitrogen; DN: Diabetic nephropathy; HE: Hematoxylin and eosin; PAS: Periodic acid-Schiff; AOD: Average optical density; SZFH: Shenzhuo formulation high dose group; SZFL: Shenzhuo formulation low dose group; Control: Control group; PC: Positive control group; Model: Model group.

HE staining results revealed severe renal pathological damage in the Model, characterized by glomerular hypertrophy and hyperplasia of the thylakoid stroma, when compared to the Control. Conversely, renal damage in the PC group, as well as in the SZFL, SZFM, and SZFH groups, showed varying degrees of improvement (Figure 1F and G). Masson staining indicated that the collagen fiber distribution in the PC group renal tissue, as well as in the SZFL, SZFM, and SZFH groups of mice, showed a relative decrease in blue-stained areas compared to the Model (Figure 1H and I). PAS staining results revealed that the PC group and mice in the SZFL, SZFM, and SZFH groups exhibited reduced thickening of the glomerular basement membrane, less hyperplasia in the thylakoid zone, lighter purple-red staining, and decreased glycogen deposition (Figure 1J and K). These findings demonstrate that the effects of SZF on improving DN are dose-dependent, with the SZFH group showing the most significant improvement, comparable to the effects of positive drug interventions. Therefore, in future experiments, we will utilize a high dose of SZF to investigate the underlying mechanisms.

Transcriptomic analysis of SZF intervention in DN mice

Next, we conducted transcriptomic analysis of renal tissues from the Control, Model, and SZFH groups, screening for DEGs between the Model and Control, as well as between the SZFH and Model. We applied the criteria of |Log₂ (FoldChange)| ≥ 1 and P_adj ≤ 0.05. We performed enrichment analysis on these DEGs (Figure 2A and B). The results indicated that compared to Control, DEGs in the Model were primarily enriched in Metabolism of xenobiotics by cytochrome P450 (CYP450), Drug metabolism-CYP450, Drug metabolism-other enzymes, etc. (Figure 2C), whereas those from the SZFH group were mainly enriched in Metabolism of xenobiotics by CYP450, Drug metabolism-CYP450, Retinol metabolism etc. compared to Control (Figure 2D). Notably, Arachidonic acid (AA) metabolism, Drug metabolism-CYP450, and Metabolism of xenobiotics by CYP450 emerged as intersecting pathways. These findings suggest that SZF may improve DN primarily through these three pathways.

Figure 2 Transcriptomics results of model group vs control and Shenzhuo formulation high dose group vs model group in diabetic nephropathy mice. We performed transcriptomic analysis for control, model group (Model), and Shenzhuo formulation high dose group (SZFH). DEGs of Model vs control and SZFH vs Model were screened by |Log₂ (FoldChange)| ≥ 1, P_adj ≤ 0.05, respectively. A and B: DEGs visualized using volcano plots; C and D: KEGG pathway enrichment analysis revealing that DEGs of Model vs control are mainly enriched in metabolism of xenobiotics by cytochrome P450 (CYP450), drug metabolism-CYP450, drug metabolism-other enzymes, etc. (C), and DEGs of SZFH vs Model are mainly enriched in CYP450, Drug metabolism-CYP450, retinol metabolism etc. (D). AA metabolism, metabolism of xenobiotics by CYP450, drug metabolism-CYP450 are the intersecting pathway. n = 3 per group. DEGs: Differentially expressed genes; Control: Control group; Model: Model group; SZFH: Shenzhuo formulation high dose group.

Analysis of non-targeted metabolomics in DN mice with SZF intervention

The principal component analysis results from the non-targeted metabolomics of renal tissues revealed significant differences in metabolites among the Control, Model, and SZFH groups (Figure 3A). The partial least squares discriminant analysis alignment test yielded an R² value of (0.0, 0.89) and a Q² value of (0.0, -0.70) for the Model vs Control comparison (Figure 3B and C). For the SZFH vs Model comparison, we obtained an R² value of (0.0, 0.92) and a Q² value of (0.0, -0.76), indicating that the statistical model exhibited good fit and predictive capability (Figure 3D and E). Next, we identified differential metabolites among the groups using the following criteria: P value is less than 0.05, VIP is greater than 1, and FC is greater than 1.5 or less than 0.67. We conducted KEGG pathway enrichment analysis of the identified differential metabolites using MetaboAnalyst 6.0, applying the screening criteria for key pathways: P less than 0.05 and pathway impact above 0.1. The analysis revealed AA metabolism identified as the shared pathway for Model vs Control, and SZFH vs Model (Figure 3F and G). It is important to highlight that these pathways exhibit overlap, suggesting that they represent the primary metabolic route through which SZF improves DN.

Figure 3 Non-targeted metabolomic results of renal tissues in diabetic nephropathy mice with Shenzhuo formulation intervention. Non-targeted metabolomic analysis was performed on Control, model group (Model), and Shenzhuo formulation high dose group (SZFH). A: Principal component analysis results showing that the metabolite levels in renal tissues were significantly different among Control, Model, and SZFH groups; B-E: Results of the PLS-DA model and alignment test validation model showing that Model vs Control (B and D) and SZFH vs Model (C and E) exhibits good fit and predictive ability; F and G: KEGG pathway enrichment analysis of Model vs Control (F) and SZFH vs Model (G). Shared pathways between the two are marked in red. Amino sugar and nucleotide sugar metabolism, KEGG ID: 00520; Arachidonic acid metabolism, KEGG ID: 00590; Arginine and proline metabolism, KEGG ID: 00330; Cysteine and methionine metabolism, KEGG ID: 00270; Fructose and mannose metabolism, KEGG ID: 00051; Glycerophospholipid metabolism, KEGG ID: 00564; Riboflavin metabolism, KEGG ID: 00564; Sphingolipid metabolism, KEGG ID: 00600; Purine metabolism, KEGG ID: 00230; Pentose phosphate pathway, KEGG ID: 00030; Biotin metabolism, KEGG ID: 00780; Taurine and hypotaurine metabolism, KEGG ID: 00430. n = 6 per group. SZFH: Shenzhuo formulation high dose group; Control: Control group; Model: Model group.

Effect of SZF intervention in DN mice on CYP450-mediated AA metabolism

In our transcriptomic analysis, we identified three significantly enriched pathways: AA metabolism, drug metabolism-CYP450, and xenobiotics metabolism via CYP450. Metabolomics results also indicated that SZF influences AA metabolism-related pathways. Previous research has demonstrated the critical role of CYP450 in regulating AA metabolism[16]. Therefore, we speculate that SZF may improve DN by modulating CYP450-mediated AA metabolism. We first visualized the differential gene expression related to AA metabolism, Drug metabolism-CYP450, and xenobiotics metabolism via CYP450 following SZF intervention in our transcriptomic data. The results revealed that the SZFH intervention upregulated the mRNA expression of Cyp2j13, Cyp2b9, Pla2 g2e, Cbr3, and Cbr1, while it downregulated the expression of Cyp4a12a, Cyp4a32, Cyp2e1, Cyp4a14, and Gm11771 (Figure 4A). This differential expression suggests that SZF may influence the production of key metabolites in the AA metabolic pathway by modulating the activity of various CYP450 subfamilies. Additionally, we analyzed the expression of metabolites associated with the AA metabolic pathway in the Control, Model, and SZFH groups using heatmaps. These analyses demonstrated that the SZFH intervention upregulated the levels of 5,6-EET, 14,15-EET, and phosphatidylcholine, while downregulating 20-hydroxyeicosatetraenoic acid (20-HETE) (Figure 4B).

Figure 4 Effect of Shenzhuo formulation intervention on cytochrome P450-mediated AA metabolism in diabetic nephropathy mice. A: Heatmap of cytochrome P450 (CYP450) metabolism-related gene expression showing that Shenzhuo formulation high dose group (SZFH) intervention significantly up-regulated Cyp2j13, Cyp2b9, Pla2g2e, Cbr3, and Cbr1 mRNA expression and down-regulated Cyp4a12a, Cyp4a32, Cyp2e1, Cyp4a14, and Gm11771 mRNA expression; B: Heatmap of AA metabolism-related product expression showing that SZFH intervened to up-regulated 5,6-EET, 14,15-EET, and Phosphatidylcholine expression and down-regulated 20-HETE expression; C: Translational relationship diagram of CYP450-mediated AA metabolism; D and E: Western blot results showing that CYP4A and CYP2E expression were significantly down-regulated and CYP2J and CYP2B expression were significantly up-regulated after PMS intervention (Supplementary Figure 1). n = 3 for A; n = 6 for B; n = 3 for D and E. ^bP < 0.01 vs control group; ^cP < 0.05; ^dP < 0.01 vs model group; AA: Arachidonic acid; Control: Control group; Model: Model group; CYP450: Cytochrome P450. The original image of Figure 4D is in the Supplementary materials.

The translational relationship of CYP450-mediated AA metabolism is illustrated in Figure 4C. To further validate the regulatory effect of SZF on CYP450-mediated AA metabolism, we conducted Western blot analysis to assess the protein expression levels of CYP4A, CYP2E, CYP2J, and CYP2B. The results showed that CYP4A and CYP2E protein expression were significantly upregulated in the Model compared to the Control. In contrast, SZFH intervention significantly downregulated CYP4A and CYP2E protein expression and upregulated CYP2J and CYP2B protein expression (Figure 4D and E). These data provide additional support for the notion that SZF affects AA metabolism by regulating CYP450, thereby exerting an ameliorative effect on DN.

Effect of SZF intervention on inflammation and oxidative stress

Extensive research indicates that AA metabolism is closely linked to inflammation and oxidative stress[17]. Both inflammation and oxidative stress play significant roles in the development of DN[18]. Therefore, we evaluated the effects of SZF intervention on inflammation and oxidative stress in renal tissues. Our assay of inflammatory factors revealed that levels of IL-1β, IL-6, and TNF-α significantly increased in the renal tissues of DN mice. In contrast, the SZFH intervention significantly downregulated the levels of IL-1β, IL-6, and TNF-α (Figure 5A-C). Additionally, our analysis of oxidative stress-related indices showed decreased activities of SOD and GSH-Px, along with increased MDA levels in the renal tissues of DN mice. The SZFH intervention reversed these changes in oxidative stress indices (Figure 5D-F). These results suggest that SZF exerts an inhibitory effect on inflammation and oxidative stress.

Figure 5 Shenzhuo formulation intervention in diabetic nephropathy mice suppresses inflammatory response and oxidative stress. A-C: Shenzhuo formulation (SZF) intervention significantly down-regulated the levels of IL-1β (A), IL-6 (B), and TNF-α (C) in renal tissues of diabetic nephropathy (DN) mice, and also attenuated the inflammatory response in DN mice; D-F: SZF intervention significantly up-regulated SOD activity (D) and GSH-Px activity (E), and also down-regulated MDA levels (F). This improved antioxidant capacities and attenuated oxidative stress levels in DN mice. n = 10 per group. ^bP < 0.01 vs control group; ^cP < 0.05; ^dP < 0.01 vs model group; IL-1β: Interleukin-1β; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; SOD: Superoxide dismutase; GSH-Px: Glutathione peroxidase; MDA: Malondialdehyde; SZFM: Shenzhuo formulation medium dose group; SZFH: Shenzhuo formulation high dose group; SZFL: Shenzhuo formulation low dose group; Control: Control group; PC: Positive control group; Model: Model group.

DISCUSSION

DN has emerged as a leading cause of end-stage renal failure, significantly increasing morbidity and mortality among diabetic patients[19]. Currently, the primary clinical strategy for managing DN focuses on tightly controlling blood glucose, lipids, and pressure to slow disease progression[20]. SZF can improve the clinical symptoms of DN[9], A. mongholicus Bunge in SZF has been shown to promote body metabolism, eliminate experimental proteinuria, reduce renal interstitial deposition and microthrombosis, and exert a protective effect on the kidneys[21,22]. R. officinale Baill lower blood glucose and improves renal function[23], and S. miltiorrhiza Bunge and Hirudo can effectively improve microcirculation and reduce oxidative stress[24]. Although the molecular mechanisms of single herbs have been partially elucidated, the mechanism of SZF on DN has not yet been clarified. Therefore, we constructed a DN mouse model and employed transcriptomic and metabolomic analyses to investigate the effects of SZF on gene expression and metabolite profiles in DN mice. Our goal was to uncover potential molecular mechanisms and therapeutic targets associated with SZF. We successfully established a DN mouse model using a high sugar high fat diet combined with STZ injection, a widely accepted method in DN research[25]. Throughout the progression of DN, renal damage results in a gradual decline in renal function, typically characterized by significantly elevated levels of 24h-UTP, Cr, and BUN. Changes in these biochemical indices serve as critical indicators of DN progression and renal impairment[26]. In our study, the model mice exhibited classic DN features, with markedly elevated 24h-UTP, Cr, and BUN levels, indicating compromised renal function. Both SZF and the positive control drug irbesartan effectively mitigated alterations in these indices, suggesting that SZF provides significant protection for renal function in DN mice. Furthermore, pathological analyses corroborated these findings, demonstrating that SZF significantly improved renal histopathological changes, reduced renal fibrosis, and diminished glycogen deposition, thereby supporting its therapeutic potential in treating DN. Besides, our results suggest that SZF treatment at various doses is not nephrotoxic. Furthermore, liver injury could aggravate renal injury. The lower levels of renal injury-related factors may also indicate no hepatic toxicity of high-dose SZF treatment. However, further studies are required to evaluate the safety of long-term SZF treatment in different dosages in order to provide more detailed experimental evidence for the usage of SZF in clinic.

To further investigate the specific mechanisms by which SZF improves DN, we found that SZF significantly regulated the expression of CYP450-related genes in DN mice through transcriptomic and metabolomic analyses. This regulation subsequently influenced the AA metabolic pathway. Notably, SZF significantly upregulated the mRNA expression of Cyp2j13, Cyp2b9, Pla2 g2e, Cbr3, and Cbr1, as well as the metabolites 5,6-EET, 14,15-EET, and phosphatidylcholine. AA undergoes conversion into biologically active metabolites, including prostaglandins, leukotrienes, HETEs, and epoxyeicosatrienoic acids (EETs) through multiple metabolic pathways, such as the cyclooxygenase, lipoxygenase, and CYP450 pathways. These metabolites play crucial roles in inflammatory responses, cardiovascular diseases, diabetes, and other pathological conditions[27]. Specifically, CYP2J and CYP2B enzymes, encoded by Cyp2j13 and Cyp2b9, convert AA into EETs via the CYP pathway[28]. EETs, a class of lipid metabolites with anti-inflammatory properties, inhibit the expression of inflammatory factors and mitigate fibrosis, thereby helping to prevent the progression of DN[29,30]. Additionally, Pla2g2e encodes phospholipase A2 (PLA2), a key enzyme in various biochemical reactions and cellular metabolic processes in vivo[31]. PLA2 is involved in converting phosphatidylcholine to lysophosphatidylcholine[32]. Furthermore, hydrolysis of the sn-2 position of membrane glycerophospholipids is catalyzed by PLA2, releasing AA[33]. The upregulation of PLA2 expression increases the availability of AA substrates, promoting the production of anti-inflammatory metabolites.

Conversely, SZF downregulated the expression of genes associated with pro-inflammatory responses, including Cyp4a12a, Cyp4a32, Cyp2e1, Cyp4a14, and Gm11771. Among these, Cyp4a12a, Cyp4a32, and Cyp4a14 primarily contribute to the production of the pro-inflammatory metabolite 20-HETE[34]. Recognized as a significant mediator of inflammation and fibrosis, 20-HETE plays crucial pro-pathological roles in CKD, including DN[35]. Cyp2e1 metabolizes AA through oxidative reactions to produce various lipid metabolites, including 19(S)-HETE[36]. This enzyme is one of the most active cytochromes involved in generating reactive oxygen species (ROS)[37,38] and mediating inflammatory responses[39]. Previous studies indicate that knockdown of Cyp2e1 reduces high glucose-induced apoptosis and oxidative stress[40]. Therefore, SZF may significantly attenuate renal inflammation and fibrosis via downregulation of pro-inflammatory gene expression. To confirm the changes in gene expression at the protein level, we performed Western blotting. Compared to the Model, SZF intervention significantly decreased the protein expression of CYP4A and CYP2E and markedly increased the protein expression of CYP2J and CYP2B. These results further elucidate how SZF influences AA metabolism by regulating the CYP450 enzyme system. This finding offers a new perspective for understanding the molecular mechanisms underlying traditional Chinese medicine's efficacy in treating DN.

Given that AA metabolism plays a crucial role in inflammatory responses and oxidative stress[17], both of which significantly contribute to the development of DN[18], the effects of SZF were evaluated on inflammation and oxidative stress in the DN mice renal tissue. IL-1β, IL-6, and TNF-α drive the progression of DN. These factors promote renal inflammatory cell infiltration and inflammatory mediator production through the activation of various signaling pathways, ultimately leading to glomerulosclerosis and tubulointerstitial injury[41]. In DN, the hyperglycemic state induces the expression of inflammatory factors and amplifies the inflammatory response, creating a vicious cycle [42]. Additionally, under hyperglycemic conditions, processes such as glucose auto-oxidation, oxidative phosphorylation, and protein glycosylation generate significant amounts of ROS[43]. The accumulation of ROS results in lipid peroxidation of cell membranes and increases the production of MDA, a key biomarker of lipid peroxidation[44]. The activities of SOD and GSH-Px indicate the body's capacity to scavenge ROS[45]. Furthermore, hyperglycemia mediated the shift in the AA metabolic pathway. Studies showed that hyperglycemia could promote the conversion of AA into pro-inflammatory mediators such as 20-HETE catalyzed by CYP4A and CYP2E enzymes, while inhibiting its conversion into anti-inflammatory mediators like EETs, thereby exacerbating inflammatory responses and oxidative stress[30,46,47]. Our results indicated that SZF altered the AA metabolic pathway, shifting its metabolism from pro-inflammatory mediators towards anti-inflammatory mediators. Meanwhile, SZF intervention significantly reduced the levels of inflammation and oxidative stress in the renal tissues of DN mice. This suggested that SZF inhibited both inflammation and oxidative stress by modulating CYP450-mediated AA metabolism, thereby alleviating renal tissue injury in DN mice.

Our study provided new insights into the metabolic mechanism of SZF in the treatment of DN and identified candidates for the development of DN therapeutic drugs from the perspective of inflammation and oxidative stress-related metabolism. Considering that SZF has been widely used in clinic, future research should focus on two aspects. On the one hand, novel techniques such as targeted metabolomics and single-cell sequencing are required to elucidate the in-depth mechanism of SZF on DN. On the other hand, novel dosage forms can be developed based on modern people's physique and lifestyle habits. With the bidirectional development of molecular mechanisms and clinical applications, SZF has the potential to become a candidate therapy for DN worldwide.

CONCLUSION

In summary, our study confirmed the significant potential of SZF in ameliorating renal injury in DN mice. The primary pathway through which SZF mitigates renal injury appears to be the modulation of CYP450-mediated AA metabolism, which contributes to its anti-inflammatory and antioxidant effects (Figure 6). However, this investigation serves as a preliminary study on the effects and mechanisms of SZF. To comprehensively explore the mechanisms underlying SZF's treatment of DN, we must combine our findings with multiple validation methods, including in vitro experiments and single-cell RNA sequencing. This approach will provide reliable support for the clinical application of SZF.

Figure 6 Shenzhuo formulation attenuates inflammatory responses and oxidative stress levels via cytochrome P450-mediated arachidonic acid metabolism, which ameliorates renal injury in diabetic nephropathy mice. DN: Diabetic nephropathy; IL-1β: Interleukin-1β; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; SOD: Superoxide dismutase; GSH-Px: Glutathione peroxidase; MDA: Malondialdehyde (Created by FigDraw).