RA was obtained from Shanxi Hunyuan Wansheng Huangqi Development Co., Ltd. (Shanxi, China) and validated by Prof. Long Dai of Binzhou Medical University (Yantai, Shandong), and the corresponding voucher specimens (voucher number: 20211001) were stored in the herbarium of Experimental Center of Chinese Medicine of Binzhou Medical University. The RA underwent processing using an 800A disintegrator (Yongkang Hardware and Medical Instrument Plant, China). High-performance liquid chromatography (HPLC)-grade solvents were procured from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Potato liquid medium and MRS culture medium were sourced from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, China). Various chemicals such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium carbonate, potassium persulfate, and sodium hydroxide were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Salivary amylase, pepsin, gastric lipase, pancreatic enzyme, and pig bile powder were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Additionally, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-hydrazine-bis (3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS), potassium oxonate, and D-fructose were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Anhydrous ethanol was obtained from Tianjin Yongda Chemical Reagent Co., Ltd. (Tianjin, China). Vanillin was bought from Tianjin Guangfu Fine Chemical Research Institute. Benzbromarone tablets (BT) were procured from Changzhou Kangpu Pharmaceutical Co., Ltd. (Changzhou, China). Biochemical assay kits for UA, triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute, while xanthine oxidase (XOD) assay kits were bought from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Lactobacillus acidophilus CGMCC1.1878 was acquired from Institute of Microbiology Chinese Academy of Sciences. Poria cocos (bio-08656) was acquired from Beijing Bai Ou Bo Wei Biotechnology Co., Ltd. (Beijing, China).

The fermentation of FRA was in accordance with our previous study (21). RA powder and ultrapure water were mixed and sterilized in an YXQ-Sll autoclave (Shanghai Boxun Medical Biological Instrument Co., Ltd., Shanghai, China). Then, the Poria cocos seed fermentation broth was inoculated on the former mixture and incubated in a GSP-9160MBE incubator (Shanghai Boxun Medical Biological Instrument) at 27°C for 10 days.

Supernatants attained from in vitro colonic fermentation with fecal microbiota at 0 h and 48 h were distilled to constant weights in a vacuum environment using a RE-52AA rotary evaporator device (Shanghai Yarong Biochemical Instrument Factory, Shanghai, China). The solids then were weighted.

The antioxidant activities of the samples were carried out through the DPPH free radical scavenging assay. One milligram per milliliter sample solution of 1,600 μL, 800 μL, 400 μL, 200 μL, 100 μL, and 100 μL diluted solutions was mixed with 1 mL DPPH, respectively, and then blended with anhydrous ethanol to 4 mL. After a 30-min incubation in darkness, the absorbance of the mixed solutions was measured at 517 nm utilizing TU-1810 APC UV–Visible spectrophotometer at Beijing Persee General Instrument Co., Ltd. (Beijing, China). The free radical scavenging activities were evaluated according to the following formula:

where A₁ represents the absorbance of the reaction mixture, A₂ represents the absorbance of the blank (without DPPH), and A₃ represents the absorbance of the control (without sample).

The antioxidant activities of the samples were measured through ABTS radical cation (ABTS⁺) scavenging activity. Different concentrations of sample solutions 0.1 mL were mixed with 4 mL ABTS⁺ working solution and incubated in darkness for 6 min. The mixture absorbance was measured at 734 nm utilizing TU-1810 APC UV–Visible spectrophotometer. The free radical scavenging activities were assessed according to the following equation:

where A₀ represents the absorbance of the control (without sample), A represents the absorbance of the reaction mixture, and B represents the absorbance of different concentrations of the sample solution.

All animal experiments were performed as approved by the ARRIVE guidelines and the Guidelines for Care and Use of Laboratory Animals, and the animal experiments were approved by the Animal Ethics Committee of Binzhou Medical University in Shandong, China (ethics approval number: 2024-L030) and complied with the principles of 3R. Male Kunming mice (weight range: 25–30 g) were procured from Jinan Pengyue Laboratory Animal Breeding Limited Company (laboratory animal license number: SCXK (Lu) 20190003). The mice were reared in a specific pathogen-free laboratory characterized by a room temperature of 24 ± 2°C, humidity of 60 ± 5%, and a 12 h light/dark cycle. All mice were acclimated to this environment for 3 days prior to the experiment procedure. The mice were randomly divided into six groups (n = 8), namely, the normal control group (NC), the HUA group (PO), the positive group (PO + BT), the RA group (PO + RA), the FRA group (PO + FRA), and the Poria cocos group (PO + Pc).

Except for the control, all groups were intragastrically injected with 500 mg/kg potassium oxonate once a day to construct the HUA model, which was carried out in accordance with the previous study (37). Mice in the positive group, RA group, FRA group, and Poria cocos group were given benzbromarone (10 mg/kg), RA (2.5 g/kg), FRA (2.5 g/kg), Poria cocos (2.5 g/kg) 1 h after modeling for 24 consecutive days, respectively. The dose of benzbromarone was referred to the previous study (38). The doses of RA, FRA, and Poria cocos were calculated according to the Methodology of Pharmacological Experiment (39). The NC group received distilled water, and the rest received 10% fructose-drinking water. All mice drank and ate freely according to the guidelines set by the China Animal Protection Association.

Mice from six groups were observed and the index of the mice was recorded, which containing the condition of eating, drinking, mental state, and the change in their weights. Orbital blood samples were collected 3 h after administration on 12 d and 24 d and were centrifuged at 3,500 rpm for 10 min and then stored at-20°C refrigerator. The mice fasted after administration on day 24 were anesthetized 3 h after the last administration and then were euthanized by cervical dislocation. The kidneys and livers of the mice were removed and immersed in 4% paraformaldehyde for subsequent use.

Assay kits and ELISA were utilized to measure serum levels of UA, TG, XOD, ALT, and AST.

The tissues of kidneys and livers were fixed in 4% paraformaldehyde, then cut into histopathological slices and stained with hematoxylin and eosin (H & E), and then examine the slices with an optical microscope at 200× magnification.

Different volumes of AG IV standard solutions (0.5 mg/mL) were added to test tubes with stoppers and were replenished with methyl alcohol to 0.5 mL, mixing with 0.5 mL of 8% vanillin ethanol solution and 5 mL of 72% sulfuric acid. After incubating at 62°C for 20 min and cooling to room temperature, the absorbance of solutions was measured at 540 nm using TU-1810 APC UV–Visible spectrophotometer to establish a standard curve. Sample solutions (0.1 mL) prepared in sections 2.3 and 2.4 were determined as the method described above.

The different volumes of the Rutin standard solution (0.2 mg/mL) were added into 10 mL volumetric flasks. Moreover, the Rutin solutions were mixed with 70% ethanol, 5% NaNO₂ solution, 10% Al(NO₃)₃ solution, and 4% NaOH solution, then diluting to volume with 70% ethanol. The absorbance of mixture was measured at 510 nm using TU-1810 APC UV–Visible spectrophotometer. Then, the standard curve was drawn. The sample solutions (1.5 mL) prepared in sections 2.3 and 2.4 were added to 10-mL volumetric flasks and repeated the same method described above.

An ELSD-16 (SHIMADZU, Japan) and a DGU-20A3R HPLC system (SHIMADZU, Japan) with a Phenomenex C18 column (4.60 × 250 mm, 5 μm) were utilized to determine AG IV content. The mobile phases were comprised of acetonitrile (A) and water (B) with a gradient elution as follows: 0-20 min, 96-80% B; 20-40 min, 80-70% B; 40-65 min, 70-40% B; 65-70 min, 40-96% B; 70-80 min, and 96% B, and the flow rate was at 1.0 mL/min with 20 μL of injection volume under 25°C.

The content of calycosin was determined by HPLC, with a Thermo Scientific Dionex Ultimate 3000 series HPLC system (Dionex, USA), a diode array detector (Dionex, USA), and a Kromasil 100-5-C18 column (4.6 by 250 mm). The mobile phases were comprised of 0.05% formic acid (A) and acetonitrile (B) with a gradient elution as follows: 0-5 min, 0-5% B; 5-10 min, 5-20% B; 10-25 min, 20-25% B; 25-35 min, 25-30% B; 35-40 min, 30-35% B; 40-54 min, 35-40% B; 54-55 min, 40-0% B, and the flow rate was at 1.0 mL/min with 10 μL of injection volume under 30°C.

Retrieving compound information of RA and Poria cocos from the TCMSP database,¹ focusing specifically on oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 (40). Subsequently, low-intestinal-activity compounds were eliminated through the SwissADME website. The English names of the active compounds were imported into the PubChem database. Afterward, retrieving SMILES and the Swiss Target Prediction platform² was employed to predict targets with a possibility ≥0 and then entailed screening and collecting targeted compounds targets.

“Hyperuricemia” was utilized as a key word to collect the HUA disease targets from the following databases, such as GeneCards,³ DisGeNET,⁴ and MOIM.⁵ And the targets attained from these databases were merged and the repeating items were eradicated.

The targets of RA, Poria cocos, and HUA were imported into Draw Venn Diagram tool, respectively, extracting common targets among them and painted the Venn diagram.

The information of the components and the data of the targets was imported into Cytoscape 3.9.1 to construct an interaction network diagram of “drug component target.”

A protein–protein interaction (PPI) network was devised utilizing the STRING⁶ online platform database. The function of Centiscape 2.2 in Cytoscape 3.9.1 was used to extract the core targets by calculating degree values, betweenness centrality and closeness centrality.

Gene Ontology (GO) enrichment pathway analysis was executed through the DAVID database to explore molecular function (MF), biological process (BP), cellular component (CC), and other gene target functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis was carried out to discuss the biological pathways of the drug function and analyze the pathway enrichment of the potential targets. The bioinformatics platform was used to show visual analysis.

The 3D structure of the core target protein was sourced from the Protein Database,⁷ serving as protein receptor, and PyMOL was used to remove the ligand and non-protein from the target protein and saved the consequence as a pdb format file. Hydrogen atoms were added to protein receptors with AutoDock Tools 1.5.7, and then, the structure was saved as a pdbqt format file. The 2D structure of AG IV, calycosin, and formononetin was retrieved from PubChem database⁸ and saved as a mol2 format file. We applied AutoDockTools1.5.7 to add hydrogen atoms and examine central node of the molecules and the rotatable keys, saving as a pdbqt format file and served as ligands. AutoDock Vina 1.1.2 was utilized to carry out molecular docking and assessed the binding energy between the ligands and protein receptors, determining the binding activity. PyMOL software was used to perform visual display.

The level of UA served as a key indicator of hyperuricemia and was a significant judge of the success of the model. As shown in Figure 3A, at 24 d, the UA level in the HUA group was 267.61 ± 67.72 μmol/L and 1.40 times as much as the 191.55 ± 0.00 μmol/L observed in the NC group (p < 0.05), indicating the successful construction of the HUA model. After 24 d of treatment, the UA levels in the positive, RA, FRA, and Poria cocos groups were significantly reduced to 174.45 ± 7.69 μmol/L, 183.95 ± 26.68 μmol/L, 143.08 ± 4.81 μmol/L, and 183.09 ± 67.72 μmol/L, representing decreases of 34.81, 31.26, 46.53, and 31.58% compared to the HUA, respectively (p < 0.05). The results show that FRA demonstrates the most striking effect on promoting UA excretion.

As can be observed in Figure 3B, the TG levels in the HUA group were 0.99 ± 0.32 mmol/L and 1.68 times as much as the NC group, which was 0.59 ± 0.05 mmol/L at 24 d (p < 0.05). After 24 d treatment, the TG levels in the positive, RA, FRA, and Poria cocos groups experienced striking decreases to 0.41 ± 0.23 mmol/L, 0.49 ± 0.02 mmol/L, 0.23 ± 0.04 mmol/L, and 0.74 ± 0.05 mmol/L, representing decreases of 58.59, 50.51, 76.77, and 25.25% compared to the HUA, respectively (p < 0.05). TG level in FRA exhibits the most substantial decrease.

As can be observed in Figure 3C, after 24 d of consecutive treatment, the XOD level in the HUA group was 0.96 ± 0.02 U/mL, 1.39 times as much as the NC group’s 0.69 ± 0.03 U/mL (p < 0.05). After 24 d treatment, the XOD levels in the positive, RA, FRA, and Poria cocos groups were 0.43 ± 0.07 U/mL, 0.60 ± 0.03 U/mL, 0.46 ± 0.03 U/mL, and 0.67 ± 0.07 U/mL, reflecting decreases of 55.21, 37.50, 52.08, and 30.21% compared to the HUA, respectively (p < 0.05). The result suggests that the efficacy of FRA in reducing UA was related to restricting the XOD levels and inhibiting UA production. Benzbromarone performed its functions mainly by decreasing reabsorption of UA through inhibiting the URAT1 in kidney tubules (52), while our result indicated that benzbromarone could impact XOD activity, which is in accordance with a previous study (53).

As can be observed in Figure 3D, the ALT level in the HUA group demonstrated a 1.20-fold increase at 32.68 ± 1.89 U/L compared to the NC group’s 27.14 ± 0.54 U/L at 24 d (p < 0.05). After 24 d of treatment, the ALT levels in the positive, RA, FRA, and Poria cocos groups decreased to 14.68 ± 0.27 U/L, 23.29 ± 1.09 U/L, 16.78 ± 1.62 U/L, and 25.79 ± 3.53 U/L, representing decreases of 55.08, 28.73, 48.65, and 21.08% compared to the HUA, respectively (p < 0.05).

As can be seen in Figure 3E, the AST level in the HUA group showed a 1.62-fold increase at 38.21 ± 5.17 U/L compared to the NC group’s 23.62 ± 0.29 U/L at 24 d (p < 0.05). After 24 d consecutive treatment, the AST levels in the positive, RA, FRA, and Poria cocos groups were 20.02 ± 5.37 U/L, 27.27 ± 0.63 U/L, 16.61 ± 2.49 U/L, and 30.78 ± 1.71 U/L, indicating decreases of 47.61, 28.63, 56.53, and 19.45% compared to the HUA, respectively (p < 0.05). These results suggest that FRA was likely to reverse liver damage resulting from HUA.

In Figure 4A, the kidney tissues from the control display a healthy structure with regularly arranged glomeruli, normal renal tubular epithelial cells, and intact medulla kidney. While the HUA and the Poria cocos group exhibit several abnormal changes, including obvious renal tubule dilation, hydropic degeneration of renal tubular epithelial cells, and inflammatory cell infiltration around the local blood vessel. The positive RA and FRA groups attenuated kidney damage and ameliorated hydropic degeneration of renal tubular epithelial cells. These findings demonstrate that RA and FRA could improve kidney histopathological alternations in mice, thus alleviating the adverse effects of HUA.

As shown in Figure 4B, the liver tissues hepatocytes in the control exhibit a normal condition. While the HUA and the Poria cocos group perform severe watery and vacuolar degeneration within the hepatocytes. Treatment with benzbromarone, RA, and FRA show minor watery degeneration of the hepatocytes without inflammatory infiltration. These results indicate that RA and FRA hold the ability to ameliorate the histological changes in hepatocytes induced by HUA, offering beneficial effects on liver tissues.

Collectively, FRA displayed the most striking pharmacodynamic effects on the reduction in UA, TG, and AST levels. Compared with RA and Poria cocos, FRA could remarkably reduce the serum level of XOD and ALT. Meanwhile, RA and FRA could improve the kidney and liver pathological changes caused by HUA. The results are in accordance with other studies on Astragalus membranaceus protection in the kidney (50, 53, 54).

Table 3 demonstrates the selection of 18 active compounds in RA and 8 in Poria cocos based on DL and OB. A total of 544 active compounds and effect targets were obtained through the Swiss Target Prediction website.

The 975 HUA targets were retrieved from the GeneCards, DisGeNET, and MOIM databases. There were 93 common targets were found between HUA and the active compounds of RA and Poria cocos, visualized in a Venn diagram in Figure 5A.

The components and target data were introduced into Cytoscape3.9.1 to obtain the interactive effect network diagram representing “drug component target.” In Figure 5B, the orange rectangles represent drugs, deep green rhombuses represent targets and aqua ovals represent the components.

To construct the PPI network, 875 edges were sourced from the STRING database and the consequence was performed in Figure 5C. The core targets, selected based on degree values, betweenness centrality and closeness centrality, are shown in Figure 5D. The top five degrees values of core targets were TNF, STAT3, CASP3, JUN, and ESR1, showing eminent regulatory potential in HUA treatment. TNF, especially TNF-α, a pro-inflammatory cytokine, plays an important role in the pathological progression of HUA. Previous study revealed that UA could increase the level of TNF-α and stimulate the release of IL-1β (55). STAT3 plays a significant role in many cellular processes, such as cell growth and cell apoptosis (56). It exerts an important influence on the process of renal fibrosis. Inhibiting STAT3 expression could alleviate the kidney disease caused by HUA (57). CASP3 play a key role in the process of cell apoptosis (58). The decrease in CASP3 expression could improve renal tubule dilation and degeneration of epithelial cells (59). JUN is involved in the growth regulation of tumor cells (60). Some studies showed that high level of UA is associated with a high incidence of cancer (61, 62). The abnormal expression of JUN may lead to the increase in UA level. ESR1 can mediate estrogen signals in the body and may exert an influence on PI3K/AKT signaling pathway. Therefore, the results show that RA and Poria cocos play therapeutic role in HUA via multiple target points. The specific mechanism of action needs a further study.

Through GO enrichment pathway analysis, a total of 596 GO terms were obtained, including 457 BP, 50 items of CC, and 98 MF. In terms of BP, the items were involved in the regulation of transcription from RNA polymerase II promoter, the reaction to exogenous stimulus, the positive regulation of genetic expression, and so on. In terms of CC, the items were mainly involved in the plasma membrane, cytoplasm, and nucleus, etc. In terms of MF, the items were mainly related to protein binding, protein binding rate and ATP binding. The top 10 most significant terms of BP, CC and MF are shown, respectively, in Figure 5E. RA and Poria cocos play roles in HUA treatment by regulating various biological processes.

A total of 681 signal pathways attained from KEGG enrichment pathway analysis, consisting of pathways in cancer, the PI3K-Akt signaling pathway, lipids and atherosclerosis, the AGEs-RAGE signaling pathways in diabetic complications, proteoglycans in cancer, and so on. The top 10 important terms are displayed in Figure 5F. A study has proven that AGEs can induce oxidative stress and inflammation in various cells and organs via combining with AGEs receptors (RAGE) (63). HMGB1 is significant mediator of inflammation and a high affinity ligand of RAGE (64). High concentrations of UA strikingly increased extracellular release of HMGB1 (65), promoting the release of IL-6 and TNF-α and other inflammatory cytokines, which lead to renal tubulointerstitial fibrosis and glomerular injury. UA can stimulate the expression of IL-1β, TNF-α, and PTGS2 through PI3K-Akt signaling pathway to induce inflammatory reaction of renal tubular epithelial cells. Inhibiting PI3K-Akt signaling pathway not only can decrease the expression of URAT1 and GLUT9, but also can alleviate kidney inflammatory injuries (66, 67). Therefore, it can be speculated that the treatment of HUA with RA through regulating in multiple signal pathways.

Molecular docking consequence was evaluated based on affinity and was considered as strong interaction when affinity < −5.0 kcal/mol (68–71). The top five degrees values of the core targets performed molecular docking with AG IV, calycosin, and formononetin. Figure 6 shows that calycosin and formononetin bound well to TNF, STAT3, CASP3, JUN, and ESR1, with binding energies below −5.0 kcal/mol. AG IV bound well to CASP3 and ESR1, with binding energies below −3.0 kcal/mol.