Potassium oxonate (PO, JS250846) and uric acid (UA, IS236490) were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Allopurinol (AP, H31020334) was purchased from Shanghai Xinyi Wanxiang Pharmaceutical Co., Ltd. (Shanghai, China). Kits for the determination of uric acid (UA), creatinine (Cr), and blood urea nitrogen (BUN) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Xanthine oxidase (XOD), dopamine (DA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) detection kits were purchased from Wuhan Elabscience Biotechnology Co., Ltd. (Wuhan, China). H&E staining solution kits and Masson staining solution kits were purchased from Pinofei Biotechnology Co., Ltd. (Wuhan, China). Anti-p53 antibody and anti-MAOA antibody were purchased from Jiangsu Qinke Biological Research Center Co., Ltd. (Liyang, China). Anti-COMT antibody, anti-SERPINE1 antibody, and anti-NLRP3 antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rabbit IgG was purchased from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan, China).

SPF ICR male mice (8 weeks old, 20 ± 2 g) were purchased from Henan Skibbes Biotechnology Co., Ltd. (Laboratory Animal Production License No. SCXK (Yu) 2020-0005). The mice were acclimatized for 1 week under controlled conditions of 25 °C, with ad libitum access to standard rodent chow and water throughout the experiment. All experimental procedures adhered to the guidelines for the care and use of laboratory animals and were approved by the Laboratory Animal Ethics Committee of Wuhan Hualianke Biotechnology Co, Ltd. (approval No. HLK-20250427-001).

After the acclimatization feeding period, the mice were randomly divided into 6 groups (n=12/group). The normal control group (Control) received daily intraperitoneal injections of normal saline as a control. The other groups were injected intraperitoneally with potassium oxonate (250 mg/kg/d) and UA (150 mg/kg/d) to establish the HUA mouse model. After 1 h of modeling, the model group (Model) was administered 0.5% CMC-Na solution via gavage as a control, while the remaining groups received interventions for 14 days: low-dose SLJZ (0.832 g/kg/d), medium-dose SLJZ (1.664 g/kg/d), high-dose SLJZ (3.328 g/kg/d), and allopurinol (AP, 10 mg/kg/d). The SLJZ dosages corresponded to 1x, 2x, and 4x the adult human daily dose. Body weight was recorded every 2 days throughout the experiment. 2 h after the final administration, the mice were euthanized, and blood and kidneys were collected for further analysis.

The collected blood samples were stored overnight in a refrigerator at -4°C. On the following day, the samples were centrifuged at 12,000 rpm for 10 min, and the supernatant was carefully collected. The levels of UA, Cr, BUN, and XOD in the supernatant were measured using commercially available kits according to the manufacturer’s instructions.

Renal tissue sections were stained with H&E to observe the basic structure, cell morphology and inflammatory damage of kidney tissue, while Masson staining was used to analyze renal fibrosis (7). The collected renal tissues were fixed in 10% paraformaldehyde for 24 h, embedded in paraffin, and sectioned (8 μm) for H&E or Masson staining. The sections were visualized and photographed under a microscope (200×).

The intersection of DEGs and targets obtained from network pharmacology was used to identify the action targets of SLJZ for HUA. Additionally, the results of GO and KEGG analyses were integrated to elucidate the potential pathways of SLJZ in treating HUA.

The Autodock Vina was used for molecular docking to explore possible interaction models between active compounds and core target proteins.The crystal structure of the core target protein was retrieved from the RCSB-PDB database (https://www.rcsb.org). Preprocessing of the protein receptor involved the removal of ligands, water and the addition of hydrogen. The 3D structure of the active compound ligand was obtained from the PubChem database. Subsequently, molecular docking of the modified binding site with the ligand was conducted using Autodock Vina, and the resulting docking poses were visualized using PyMOL software.

PBS solution containing protease inhibitors was added to the mouse kidney tissue, followed by homogenization. The mixture was then centrifuged at 5000 rpm for 10 min, and the supernatant was collected. The concentrations of DA, IL-6, IL-1β, and TNF-α in the supernatant were measured using specific kits according to the manufacturer’s instructions.

Kidney tissue sections were analyzed using immunohistochemistry (9). The sections were placed in a citric acid-based repair solution (pH=6.0), within an autoclave, heated until bubbling, and maintained for 2 min. After natural cooling, the sections were incubated in 3% H₂O₂ at room temperature for 20 min to block endogenous peroxidase activity. Subsequently, the sections were sequentially incubated with primary and secondary antibodies for further analysis. Positive staining on each image was quantified by Image J software.

Data analysis was conducted using GraphPad Prism 8.0 software. Results are presented as mean ± standard deviation (SD). Intergroup comparisons were performed using one-way ANOVA, and P < 0.05 were considered to indicate statistical significance.

From the completion of adaptive feeding to the end of drug administration on day 14, the body weights of the mice were continuously recorded ( Figure 1A ). We observed that the body weights of mice in the control group increased significantly and stabilized at a certain level, whereas the mice that had been modeled all began to decrease significantly starting from the fourth day. the model group exhibited the most pronounced weight loss, while the AP group showed the least reduction. The significant reduction in body weight observed in both the low-dose and high-dose SLJZ groups may be explained by the nature of SLJZ as a drug for hyperlipidemia treatment. At the low dose, its insufficient renal protective effect might contribute to this outcome, whereas at the high dose, its pronounced lipid-lowering efficacy likely dominates the observed weight changes. In addition to body weight reduction, the modeled mice exhibited decreased appetite, disheveled fur, depression, and in severe cases, prolonged reflexes. Following drug intervention, all of these conditions were significantly improved.

To evaluate the therapeutic effect of SLJZ on HUA, blood samples from mice in each group were collected at the end of modeling on day 14, and serum centrifugation was performed the following day to detect HUA-related indices. Compared with the control group, the UA levels in the model group were significantly elevated, confirming the successful establishment of the HUA model (10). The UA levels in treated mice decreased significantly ( Figure 1B ). Renal function was assessed using Cr and BUN as markers. We found that Cr levels in the model group were significantly higher than those in the control group, whereas Cr levels in the high-dose SLJZ group were reduced to levels comparable to those in the AP group and significantly lower than those in the model group ( Figure 1C ). Although BUN levels were similar across the low, medium, and high-dose SLJZ groups, they were all significantly lower than those in the model group ( Figure 1D ). XOD is a key enzyme in UA production, converting hypoxanthine to xanthine and subsequently producing UA (11). Figure 1E shows the XOD levels in each group of mice. Compared with the control group, XOD levels in the Mode group were significantly increased. While there was no significant difference between the low-dose SLJZ group and the model group, XOD levels in the medium and high-dose SLJZ groups, as well as the AP group, were significantly lower. Notably, XOD levels in the AP group were even lower than those in the control group. It may be because AP competitively binds to XOD, thereby reducing UA production; however, the strong inhibitory effect of AP on XOD may lead to the accumulation of hypoxanthine and xanthine in the body, potentially causing kidney injury. The results indicate that SLJZ effectively inhibits UA accumulation in HUA mice and provides renal protection.

The progression of HUA is frequently associated with renal damage; therefore, histologic changes in the kidneys were evaluated using H&E staining ( Figure 2 ). The kidneys of mice in the control group exhibited normal structural integrity, with clearly defined glomeruli and well-organized renal tubular structures. In contrast, the renal tissues of mice in the model group displayed pronounced pathological alterations, including degeneration and necrosis of renal tubular epithelial cells, as well as increased inflammatory cell infiltration in the renal interstitium. These pathological changes were significantly ameliorated following interventions with SLJZ and AP.

We also evaluated the role of SLJZ in HUA-induced renal tissue fibrosis using Masson staining ( Figure 2 ). There were fewer blue collagen fibers in the renal tubular interstitium of mice in the control group, and a few collagen fibers were visible in the perivascular area of the blood vessels in the model group whereas there was a significant increase in blue collagen fibers in the renal interstitium. In contrast, both SLJZ and AP treatments significantly reduced collagen deposition in renal tubular interstitium. These findings indicate that SLJZ effectively alleviates renal pathologic changes associated with HUA-induced fibrosis.

Previously, we predicted the potential targets of SLJZ for treating HUA using network pharmacology. Subsequently, transcriptomics was employed to identify the differential genes reversed by SLJZ in the kidney tissues of HUA mice, thereby preliminarily elucidating the mechanisms underlying SLJZ’s uric acid-lowering effects, which involve anti-inflammatory and antioxidant activities. To further explore the core targets of SLJZ in HUA treatment, we overlapped the 44 potential targets predicted by network pharmacology with the 280 DEGs identified through transcriptomics. This analysis revealed two intersecting targets: COMT and SERPINE1. These are key targets of SLJZ in HUA treatment, and their expression levels are presented in Table 2 (compared with the model group).

COMT is primarily involved in dopamine metabolism process in vivo, acting synergistically with MAOA (24). Notably, MAOA has already been identified in previous web-based pharmacological predictions. Dopamine can promote glomerular filtration in vivo, thereby enhancing urate excretion and contributing to UA reduction. Conversely, the up-regulation of COMT and MAOA decreases dopamine levels in vivo, inhibiting renal tubular filtration and impeding UA salt excretion. SERPINE1 serves as the primary inhibitor of urokinase. In conjunction with prior experimental results, urate accumulation in vivo activates NLRP3 inflammasomes, while SERPINE1, as a regulator of inflammatory responses, exacerbates inflammation by suppressing the fibrinolytic system. Additionally, within the p53 signaling pathway, the upregulation of SERPINE1, a downstream protein of p53, further intensifies oxidative damage (25), which subsequently elevates UA levels in vivo. Based on these findings, we selected COMT, MAOA, p53, NLRP3, and SERPINE1 as the core targets for validating SLJZ’s efficacy in treating HUA.

To verify the interaction between the active compounds and core targets, molecular docking was performed to evaluate the binding energy between small molecules and target proteins. Figures 6A–E display the compounds with the highest docking scores for COMT, MAOA, p53, NLRP3, and SERPINE1, respectively. The binding energy heatmap is presented in Figure 6F . The results demonstrate that the seven active compounds exhibit favorable binding affinities with COMT, MAOA, p53, NLRP3, and SERPINE1.

COMT and MAOA are the critical rate-limiting enzymes in the DA metabolic process. Their upregulation promotes the degradation of DA, thereby inhibiting UA excretion. Upon activation, NLRP3 induces the release of IL-6, IL-1β, and TNF-α. Therefore, measuring the levels of DA, IL-6, IL-1β, and TNF-α in the kidneys of mice can indirectly reflect the regulation of COMT, MAOA, p53, NLRP3, and SERPINE1 by SLJZ. Compared with the control group, the DA content in the kidneys of the model group was significantly reduced, indicating that the DA metabolic process was accelerated. This effect was ameliorated after SLJZ intervention ( Figure 7A ). Compared with the control group, the levels of IL-6, IL-1β, and TNF-α in the kidneys of the model group were significantly elevated. In the high-dose group, the levels of IL-6, IL-1β, and TNF-α were decreased to levels comparable to those of the AP group and were significantly lower than those of the model group ( Figures 7B–D ).

The results of the previous experiments suggested that SLJZ might exert its uric acid-lowering effect by regulating the COMT/MAOA signaling pathway and the p53/SERPINE1/NLRP3 signaling pathway. Therefore, the expression levels of the relevant proteins in renal tissues were examined using immunohistochemistry, and the results are presented in Figure 8 . In the immunohistochemical analysis, the target proteins appeared as yellowish-brown staining. As can be clearly observed from the figure, the expression levels of COMT, MAOA, SERPINE1, NLRP3, and p53 in the model group were significantly higher than those in the control group, and these proteins were down-regulated after SLJZ intervention. These findings indicated that SLJZ may reduce UA levels by inhibiting the COMT/MAOA signaling pathway and the p53/SERPINE1/NLRP3 signaling pathway.