Punicalagin attenuates hyperuricemia 
Hyperuricemia is a metabolic disorder characterized by elevated serum uric acid (SUA) levels and is closely related to a variety of diseases such as gout, chronic renal disease, diabetes mellitus, and other diseases . Hyperuricemia has recently emerged as the second metabolic disorder following diabetes globally, exerting a significant public health concern and threat to global human health , . It was reported that the prevalence was ca . 14.6 % in the United States in 2015–16, 13.4 % in Japan in 2016–17 , and 14 % among adults in China in 2018–19 . The incidence of global hyperuricemia is still steadily increasing, with effective strategies for managing hyperuricemia remain rare. It has been reported that insufficient uric acid (UA) excretion accounts for almost 90 % of primary hyperuricemia cases . The UA excretion is ca . 70 % mediated through the renal pathway, whereas the remaining 30 % through the intestinal tract , both need UA transport proteins . In the kidney, UA is primarily reabsorbed by the urate transporter 1 (URAT1) and glucose transporter (GLUT9), and is mainly excreted by ATP-binding cassette superfamily G member 2 (ABCG2) and organic anion transporters (OATs) . Additionally, ABCG2 and GLUT9 have also been found to mediate UA excretion through the intestine . However, hyperuricemia is always accompanied by severe damage of the kidney and intestine, which is associated with inflammation triggered by UA , . The damage and inflammation of these two organs alter the expression of UA transporters, which induces the under-excretion of UA and finally leads to increased SUA levels , . Therefore, these transporters are considered significant targets for preventing and treating hyperuricemia. However, drugs targeting these transporters in the kidney, such as probenecid and benzbromarone (BenZ), have limited clinical application due to side effect like severe renal toxicity and hepatotoxicity . Additionally, there have been no marketed drugs targeting intestinal UA transporter until now. Therefore, identifying innovative and effective strategies to address hyperuricemia by boosting the extensive excretion of UA through the kidney and intestine is crucial. Recently, it has also been discovered that gut microbiota significantly contribute to hyperuricemia. The structure and composition of the gut microbiota are discovered to be altered by hyperuricemia. For example, short-chain fatty acids (SCFA)-generating bacteria Prevotellaceae_UCG-001 , Muribaculaceae are reduced , , while pro-inflammatory bacteria such as Parabacteroides and Tuzzerella are increased. , , . In turn, the gut microbiota dysbiosis can not only aggravate intestinal inflammation and damage, but also worsen renal inflammation and injury by circulation, which contribute to the dysfunction of these two organs and exacerbating hyperuricemia , . Several studies have demonstrated that hyperuricemia was effectively alleviated through the alteration of the gut microbiota . Thus, adjustment of gut microbiota can represent an efficient and encouraging strategy in treating hyperuricemia. Punicalagin (2,3-(S) hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-d-glucose, PU) is the most abundant ellagitannin in the pomegranate peel and characterized by a high level of hydroxylation ( A). PU has been documented to exert a wide range of biological functions, including anti-inflammation and glycolipid metabolism regulation , , . Previous studies have suggested that PU can improve renal and intestinal functions under various conditions. For example, PU has been proven to have a kidney-protective benefit in kidney injury induced by lipopolysaccharide (LPS) , cisplatin , and diabetes mellitus by anti-inflammation. PU has also been revealed to be effective against intestine inflammation and dysfunction , . Furthermore, PU has been demonstrated to improve gut microbiota homeostasis in diabetic nephropathy , hepatic steatosis , and insulin resistance , contributing to the alleviation of these diseases. These studies suggested that PU has potential for preventing and treating hyperuricemia, albeit its effect and action mechanisms on hyperuricemia remain elusive. In this current study, PU was found to effectively alleviate hyperuricemia and protect against the injury of kidney and intestine in hyperuricemia mice, adding to its several health benefits. Additionally, PU improved UA transporters expression and reduced the activation of the inflammatory signaling pathways in both the kidney and intestine. Moreover, PU was revealed to alleviate renal glycometabolism disequilibrium, and gut microbiota dysbiosis using omics analysis. This is the inaugural report on the UA-lowering effect of PU alongside its action mechanisms, which provides an intriguing strategy for treating and preventing hyperuricemia. 

Materials and reagents PU (98 %) was obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Adenine (AD) and Potassium oxonate (PO) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Mice IL-6 (FF-ED20188), IL-1β (FF-ED20174) and TNF-α (FF-ED20852) ELISA kits were obtained from Jinan Fanfeng Biotech Co., Ltd. (Jinan, China). SUA, serum creatinine (SCr), and blood urea nitrogen (BUN) kits were purchased from Nanjing Jiancheng Biotech Co., Ltd. (Nanjing, China). Antibodies of anti-URAT1 (14937–1-AP), anti-GLUT9 (26486–1-AP), anti-p-JNK (80024–1-RR), anti-JNK (66210–1-Ig), anti-GAPDH (10494–1-AP), and secondary antibody (SA00001-2) were purchased from Proteintech Biotech Co., Ltd. (Wuhan, China); anti-p-p38 (GB153380-100), anti-p38 (GB154685-100), anti-nuclear factor-κB p65 (NFκB p65) (GB11997-100), p-NFκB p65 (GB113882-100) and anti-β-actin (GB111364-100) were purchased from Servicebio Biotech Co., Ltd. (Wuhan, China); anti-p-IκBα (AP1220) and anti-IκBα (A19714) were obtained from Abclonal Biotech Co., Ltd. (Wuhan, China). Western blot analysis The total protein of kidney and intestine was extracted with a RIPA lysis buffer composed of 2 % protease and phosphatase inhibitor. Proteins (30 μg) were isolated in a 10 % SDS-PAGE and moved to 0.2 μm PVDF membranes. The membranes for non-phosphorylated and phosphorylated proteins underwent blocking for 1 h at ambient temperature using 5 % skim milk and 5 % BSA, in that order. The blocked membranes were then incubated overnight at 4℃ with the primary antibody targeting URAT1 (1:1000), GLUT9 (1:600), p-IκBα (1:1000), IκBα (1:1000), p-NFκB p65 (1:1000), NFκB p65 (1:1000), p-JNK (1:1000), JNK (1:1000), p-p38 (1:1000), p38 (1:1000), GAPDH (1:7500) and β-actin (1:1000), respectively. GAPDH or β-actin served as the internal control. Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) for 2 h at room temperature. The protein bands were made visible using an enhanced chemiluminescence (ECL) detection kit and captured via the ChemiDoc™ Touch Imaging System (Bio-Rad, USA). The protein bands were quantified using Image-Pro Plus software. Establishment of the hyperuricemia mouse model and drug administration The male Kunming mice (18–22 g) were purchased from the Jinan Peng Yue Laboratory Animal Breeding Co., Ltd (Jinan, China). Mice were housed under controlled conditions (22-26℃, 50 % relative humidity, 12-h day/dark cycle). Hyperuricemia in mice was established via gavage of 200 mg/kg AD and 300 mg/kg PO once daily for two weeks, which can increase SUA levels by about 2-fold stably with lower death rate . Mice in the normal group were administered 0.5 % carboxymethylcellulose-Na (CMC-Na), according to our previous study , . Prior to PU administration, SUA levels of three mice randomly selected from the normal and model groups were determined and compared to ensure the success of hyperuricemia mice model establishment, as previously reported , . Subsequently, the model group was randomly divided into five sub-groups (n = 10) as detailed in . Previous studies have reported that PU dosages of at 100–200 mg/kg/d effectively reduced blood sugar levels in mice models induced with streptozotocin/high-fat diets . In addition, PU at 100 mg/kg/d exhibited superior anti-mucositis activities in mucositis rat model . Based on the above reports, 100, 200 and 300 mg/kg/d of PU were selected to administrate for hyperuricemia mice in the present study. The mice were administered as detailed in once a day for two consecutive weeks and weighed daily during treatment. Upon concluding the experiment, the mice underwent a 12 h fast, then the blood, kidney and intestine were collected. The blood samples were left at room temperature for 1 h and then centrifuged at 3000 r/min for 10 min to obtain serum. Histopathological examination After dissection, kidney and intestine samples of mice were excised, washed using PBS, preserved in 4 % paraformaldehyde, encased in paraffin, and sliced. Sections were sliced to a 4 μm thickness, kept in an oven at 65℃ for 6 h, and subsequently dyed using hematoxylin and eosin (H&E). The photographs were captured using an inverted microscope (Nikon TS2, Japan) at 200 × magnification under a bright field. The renal and intestinal histopathological scores was calculated using an average scale ranging from 0 (normal) to 4 (serious severity) . Measurement of inflammatory factors Approximately 30 mg of kidney tissues from each mouse were cut and homogenized on ice, and then the mixture underwent centrifugation at 5000 g for 10 min at 4℃. Following extraction of the supernatant, the IL-1β, IL-6, and TNF-α levels in the supernatant were quantified using ELISA kits according to the guidelines provided by the manufacturer. 16S rRNA sequencing analysis For examining the bacterial community's taxonomic composition, 6 fecal samples of each group were chosen for 16S rRNA analysis. Fecal samples were obtained the day before the mice were executed. The MagPure Soil DNA LQ Kit (Magan, China) was employed to extract genomic DNA from fecal samples. Sequencing of DNA samples was performed using the Illumina NovaSeq 6000 system (Illumina Inc., USA). Raw sequencing data were processed using DADA2 adhering to the standard settings of QIIME2, and ASV abundance tables were obtained. The representative reads were annotated using a q2-feature-classifier. The Bray-Curtis distances were analyzed using principal coordinate analysis (PCoA) to represent the beta diversity of the gut microbiota, which were estimated by QIIME2 software. R Package was used to analyze the notable variances among various groups using Kruskal Wallis. Analysis of gut microbiota functions were conducted using KEGG database and Enzyme nomenclature database. The Kruskal-Wallis H test was employed for the differential analysis of gut microbiota and functional relative abundances, as previously reported , , , , . Metabolomics analysis of kidney tissue After dissection, ca. 30 mg kidney tissue of each mice was homogenized with 600 μL 80 % methanol solution containing 4 μg/mL 2-chloro-L-phenylalanine, and 150 μL supernatant was collected. Subsequently, 80 μL pyridine solution containing 15 mg/mL methoxyamine hydrochloride was added to the supernatant, then vacuum-dried and methoximated at 37℃ for 60 min. After adding 50 μL bis(trimethylsilyl)trifluoroacetamide solution and 10 μL internal standards (Excel. S1), the derivatization of samples was fulfilled at 70℃ for 60 min. The derived samples were then analyzed using GC/MS Agilent 7890B gas chromatography system coupled to an Agilent 5977A MSD system (Agilent Technologies Inc., CA, USA). The DB-5MS fused-silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J & W Scientific, Folsom, CA, USA) was utilized to separate the derivatized samples. Helium (>99.999 %) was used as the carrier gas at a constant flow rate of 1 mL/min. A total of 1 μL sample was injected using the splitless mode with the injector temperature maintained at 260℃. The temperature of MS quadrupole and ion source (electron impact) was 150 and 230℃, respectively. The collision energy was 70 eV. The mass spectrometric data was acquired in the full-scan mode ( m / z 50–500), and the solvent delay time was 5 min. The MD-DIAL software was employed for metabolomics data. Post peak alignment, a total of 284 peaks were identified across all samples. Principle component analysis (PCA) and (orthogonal) partial least-squares-discriminant analysis (O)PLS-DA were utilized with the R ropls package to depict the metabolic differences among experimental groups. OPLS-DA and PLS-DA were used for comparisons between two groups and multiple groups, respectively. The metabolites exhibiting variable importance in the projection (VIP) > 1.0 and p < 0.05 were calculated as differential metabolites. Statistical analysis Results were presented as the mean ± standard deviation (SD). If not specified, the significant differences were calculated by one-way ANOVA for multiple groups and student's t -test for two groups, followed by Tukey's post hoc test with the GraphPad Prism software (version 9.5, GraphPad Software, Inc.). Figures were generated with GraphPad Prism software. p < 0.05 was considered a statistically significant difference. 

PU (98 %) was obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Adenine (AD) and Potassium oxonate (PO) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Mice IL-6 (FF-ED20188), IL-1β (FF-ED20174) and TNF-α (FF-ED20852) ELISA kits were obtained from Jinan Fanfeng Biotech Co., Ltd. (Jinan, China). SUA, serum creatinine (SCr), and blood urea nitrogen (BUN) kits were purchased from Nanjing Jiancheng Biotech Co., Ltd. (Nanjing, China). Antibodies of anti-URAT1 (14937–1-AP), anti-GLUT9 (26486–1-AP), anti-p-JNK (80024–1-RR), anti-JNK (66210–1-Ig), anti-GAPDH (10494–1-AP), and secondary antibody (SA00001-2) were purchased from Proteintech Biotech Co., Ltd. (Wuhan, China); anti-p-p38 (GB153380-100), anti-p38 (GB154685-100), anti-nuclear factor-κB p65 (NFκB p65) (GB11997-100), p-NFκB p65 (GB113882-100) and anti-β-actin (GB111364-100) were purchased from Servicebio Biotech Co., Ltd. (Wuhan, China); anti-p-IκBα (AP1220) and anti-IκBα (A19714) were obtained from Abclonal Biotech Co., Ltd. (Wuhan, China). 

The total protein of kidney and intestine was extracted with a RIPA lysis buffer composed of 2 % protease and phosphatase inhibitor. Proteins (30 μg) were isolated in a 10 % SDS-PAGE and moved to 0.2 μm PVDF membranes. The membranes for non-phosphorylated and phosphorylated proteins underwent blocking for 1 h at ambient temperature using 5 % skim milk and 5 % BSA, in that order. The blocked membranes were then incubated overnight at 4℃ with the primary antibody targeting URAT1 (1:1000), GLUT9 (1:600), p-IκBα (1:1000), IκBα (1:1000), p-NFκB p65 (1:1000), NFκB p65 (1:1000), p-JNK (1:1000), JNK (1:1000), p-p38 (1:1000), p38 (1:1000), GAPDH (1:7500) and β-actin (1:1000), respectively. GAPDH or β-actin served as the internal control. Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) for 2 h at room temperature. The protein bands were made visible using an enhanced chemiluminescence (ECL) detection kit and captured via the ChemiDoc™ Touch Imaging System (Bio-Rad, USA). The protein bands were quantified using Image-Pro Plus software. 

The male Kunming mice (18–22 g) were purchased from the Jinan Peng Yue Laboratory Animal Breeding Co., Ltd (Jinan, China). Mice were housed under controlled conditions (22-26℃, 50 % relative humidity, 12-h day/dark cycle). Hyperuricemia in mice was established via gavage of 200 mg/kg AD and 300 mg/kg PO once daily for two weeks, which can increase SUA levels by about 2-fold stably with lower death rate . Mice in the normal group were administered 0.5 % carboxymethylcellulose-Na (CMC-Na), according to our previous study , . Prior to PU administration, SUA levels of three mice randomly selected from the normal and model groups were determined and compared to ensure the success of hyperuricemia mice model establishment, as previously reported , . Subsequently, the model group was randomly divided into five sub-groups (n = 10) as detailed in . Previous studies have reported that PU dosages of at 100–200 mg/kg/d effectively reduced blood sugar levels in mice models induced with streptozotocin/high-fat diets . In addition, PU at 100 mg/kg/d exhibited superior anti-mucositis activities in mucositis rat model . Based on the above reports, 100, 200 and 300 mg/kg/d of PU were selected to administrate for hyperuricemia mice in the present study. The mice were administered as detailed in once a day for two consecutive weeks and weighed daily during treatment. Upon concluding the experiment, the mice underwent a 12 h fast, then the blood, kidney and intestine were collected. The blood samples were left at room temperature for 1 h and then centrifuged at 3000 r/min for 10 min to obtain serum. 

After dissection, kidney and intestine samples of mice were excised, washed using PBS, preserved in 4 % paraformaldehyde, encased in paraffin, and sliced. Sections were sliced to a 4 μm thickness, kept in an oven at 65℃ for 6 h, and subsequently dyed using hematoxylin and eosin (H&E). The photographs were captured using an inverted microscope (Nikon TS2, Japan) at 200 × magnification under a bright field. The renal and intestinal histopathological scores was calculated using an average scale ranging from 0 (normal) to 4 (serious severity) . 

Approximately 30 mg of kidney tissues from each mouse were cut and homogenized on ice, and then the mixture underwent centrifugation at 5000 g for 10 min at 4℃. Following extraction of the supernatant, the IL-1β, IL-6, and TNF-α levels in the supernatant were quantified using ELISA kits according to the guidelines provided by the manufacturer. 

For examining the bacterial community's taxonomic composition, 6 fecal samples of each group were chosen for 16S rRNA analysis. Fecal samples were obtained the day before the mice were executed. The MagPure Soil DNA LQ Kit (Magan, China) was employed to extract genomic DNA from fecal samples. Sequencing of DNA samples was performed using the Illumina NovaSeq 6000 system (Illumina Inc., USA). Raw sequencing data were processed using DADA2 adhering to the standard settings of QIIME2, and ASV abundance tables were obtained. The representative reads were annotated using a q2-feature-classifier. The Bray-Curtis distances were analyzed using principal coordinate analysis (PCoA) to represent the beta diversity of the gut microbiota, which were estimated by QIIME2 software. R Package was used to analyze the notable variances among various groups using Kruskal Wallis. Analysis of gut microbiota functions were conducted using KEGG database and Enzyme nomenclature database. The Kruskal-Wallis H test was employed for the differential analysis of gut microbiota and functional relative abundances, as previously reported , , , , . 

After dissection, ca. 30 mg kidney tissue of each mice was homogenized with 600 μL 80 % methanol solution containing 4 μg/mL 2-chloro-L-phenylalanine, and 150 μL supernatant was collected. Subsequently, 80 μL pyridine solution containing 15 mg/mL methoxyamine hydrochloride was added to the supernatant, then vacuum-dried and methoximated at 37℃ for 60 min. After adding 50 μL bis(trimethylsilyl)trifluoroacetamide solution and 10 μL internal standards (Excel. S1), the derivatization of samples was fulfilled at 70℃ for 60 min. The derived samples were then analyzed using GC/MS Agilent 7890B gas chromatography system coupled to an Agilent 5977A MSD system (Agilent Technologies Inc., CA, USA). The DB-5MS fused-silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J & W Scientific, Folsom, CA, USA) was utilized to separate the derivatized samples. Helium (>99.999 %) was used as the carrier gas at a constant flow rate of 1 mL/min. A total of 1 μL sample was injected using the splitless mode with the injector temperature maintained at 260℃. The temperature of MS quadrupole and ion source (electron impact) was 150 and 230℃, respectively. The collision energy was 70 eV. The mass spectrometric data was acquired in the full-scan mode ( m / z 50–500), and the solvent delay time was 5 min. The MD-DIAL software was employed for metabolomics data. Post peak alignment, a total of 284 peaks were identified across all samples. Principle component analysis (PCA) and (orthogonal) partial least-squares-discriminant analysis (O)PLS-DA were utilized with the R ropls package to depict the metabolic differences among experimental groups. OPLS-DA and PLS-DA were used for comparisons between two groups and multiple groups, respectively. The metabolites exhibiting variable importance in the projection (VIP) > 1.0 and p < 0.05 were calculated as differential metabolites. 

Results were presented as the mean ± standard deviation (SD). If not specified, the significant differences were calculated by one-way ANOVA for multiple groups and student's t -test for two groups, followed by Tukey's post hoc test with the GraphPad Prism software (version 9.5, GraphPad Software, Inc.). Figures were generated with GraphPad Prism software. p < 0.05 was considered a statistically significant difference. 

PU reduced SUA levels in the hyperuricemia mice To evaluate the impact of PU on hyperuricemia, a mice model with hyperuricemia was established as detailed in previously reports , . The establishment of hyperuricemia mice model and administration of PU were demonstrated in B. After model establishment, the body weight of hyperuricemia mice decreased notably but was reversed by BenZ at 10 mg/kg/d, and PU at 200 and 300 mg/kg/d ( C). Compared to the control group (71.21 μmol/L), SUA concentration of hyperuricemia mice significantly raised to 247.12 μmol/L, which was decreased to 69.71 μmol/L by BenZ administration ( D), suggesting that hyperuricemia mice model was established successfully , , . In contrast, SUA level in hyperuricemia mice was significantly and dose-dependently decreased to 158.76 μmol/L, 85.28 μmol/L, and 82.60 μmol/L by PU at 100, 200, and 300 mg/kg/d ( D). The findings suggested that PU is capable of efficiently alleviating hyperuricemia. PU mitigated kidney damage in the hyperuricemia mice Hyperuricemia is typically accompanied by kidney damage . PU has been revealed to alleviate renal damage and dysfunction in several diseases, such as diabetic nephropathy . Thus, the effect of PU on kidney functions in hyperuricemia mice was investigated using serum biochemical parameters alongside histopathological examination. Compared to the control, the kidney coefficients, BUN and SCr levels in hyperuricemia mice increased significantly, which were significantly and dose-dependently decreased in the PU group ( A, B, C). D depicted kidney appearance of the hyperuricemia mice as pale and remarkably increased with white spots, which was returned to ruddy and smooth in both the PU and BenZ groups. Consistent with these results, H&E staining demonstrated that kidneys in hyperuricemia model group indicated glomerular atrophy, and tubular dilatation, which were significantly improved by PU at 200, and 300 mg/kg/d, and BenZ at 10 mg/kg/d ( E). The improvement on renal injury was also verified by histopathological scores ( A). Overall, these findings indicated that PU could effectively alleviate kidney damage in case of hyperuricemia mice. PU restored the altered expression level of UA transporters and suppressed inflammation in the kidney of hyperuricemia mice The UA excretion through the kidney is dependent on specific proteins mediating UA reabsorption and excretion . Therefore, the expression of UA reabsorption and excretion transporters in the kidney was measured using western blotting. As shown in A-B, expression of UA reabsorption proteins URAT1 and GLUT9 were remarkably increased, while UA excretion proteins ABCG2 and OAT1 showed significant decrease in the model group. These results were consistent with previous studies , , suggesting that UA reabsorption was increased, while UA excretion was reduced in hyperuricemia mice kidneys. In contrast to the hyperuricemia model group, URAT1 and GLUT9 were inhibited by PU at 200, and 300 mg/kg/d dose-dependently, whereas the URAT1-targeted inhibitor BenZ significantly inhibited the URAT1 and GLUT9 expression ( A-B). Additionally, PU at medium and high doses, and BenZ at 10 mg/kg/d significantly increased protein expression level of UA excretion transporter ABCG2 and OAT1 in the kidney ( A-B). These results indicated that PU could facilitate renal UA excretion by improving renal UA transporter expression in the hyperuricemia mice. Earlier research has verified a link between hyperuricemia and kidney inflammation, resulting in severe renal damage , . Additionally, inflammation signaling pathway, such as MAPK/NF-κB, could regulate UA transporters expression , . Consequently, the impact of PU on the kidney inflammation in mice with hyperuricemia was assessed. Compared to the control group, the hyperuricemia model mice exhibited a significant increase of phosphorylation levels of IκBα and NF-κB p65 ( C and D). However, treatment with PU at indicated concentrations significantly decreased elevated phosphorylation of IκBα and NF-κB p65 in the kidney ( C and D). Similarly, the increased phosphorylation of JNK and p38 in the kidney of hyperuricemia mice was significantly decreased by PU at 200 and 300 mg/kg/d as well ( C and D). In parallel to alterations in these signaling pathways, PU administration inhibited the elevated levels of IL-1β, IL-6, and TNF-α, in the hyperuricemia mice kidney ( E). Additionally, the elevated phosphorylation levels of IκBα, NFκB p65, p38, and JNK, alongside up-regulated levels of the three inflammatory factors in the hyperuricemia mice kidney were also significantly inhibited by BenZ ( C-E). These results provided evidence that PU could inhibit inflammation as mediated via suppressing MAPK/NF-κB activation in the kidney of hyperuricemia mice, which could contribute to improving renal damage and insufficient UA excretion through the kidney ( F). PU attenuated renal glycometabolism disorder in the hyperuricemia mice To comprehensively assess the protective effect and action mechanisms of PU on the hyperuricemia mice kidney, renal non-targeted metabolomics analysis was fulfilled, and 284 metabolites were identified (Excel. S2). PCA and PLS-DA analysis showed that the PU group was isolated from the hyperuricemia mice model group and close to the control group, which indicated that PU reversed altered renal metabolite composition of the hyperuricemia mice ( A and B). In contrast to the control group, the hyperuricemia model group exhibited 146 differential metabolites ( C and Excel. S2). However, after treatment with PU, levels of 97 differential metabolites were reversed ( D-E and Excel. S2). These data further supported the conclusion that PU improved metabolic disorders in the kidney of hyperuricemia mice. As shown in F-G, the hyperuricemia model group had 5.85- and 3.31-folds abundance of uric acid and creatinine respectively compared to the control group, while were reduced by 79.2 % and 75.6 % post PU treatment, individually, suggesting that the impaired kidney function and kidney-mediated UA excretion were improved by PU treatment. Compared with the control group, decreased D-glucose level was observed, while glucose 6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, D-ribulose 5-phosphate and D-ribofuranose significantly increased in hyperuricemia mice, all of which were reversed by PU, indicating that PU improved glycolysis and pentose phosphate pathways (PPP) in the kidney of hyperuricemia mice ( F and G). Consistently, KEGG analysis demonstrated that the glycolysis was the top one pathway ameliorated by PU, meanwhile pentose and glucuronate interconversion and PPP pathways were also effectively improved ( H). Collectively, renal metabolomics analysis provided evidence that PU could improve kidney function by attenuating the glycometabolism dysequilibrium in hyperuricemia mice. PU relieved intestinal damage and dysfunction in hyperuricemia mice The intestine is a significant UA excretion pathway . However, several studies have shown that hyperuricemia could induce intestinal damage , . Thus, H&E staining of the intestine was performed to assess intestine damage. Compared to the control, the intestinal villus was swollen and arranged disorderly, and the striated borders were blurred in hyperuricemia mice, which was restored by PU administration, indicating that PU ameliorated the intestine damage of hyperuricemia mice ( A), which were further verified by histopathological scores of intestine ( B). To research the impact of PU on intestinal UA excretion, the intestinal UA excretion proteins levels were detected using western blotting. The intestinal ABCG2 and GLUT9 were remarkably decreased in case of hyperuricemia mice, which was reversed by treatment with PU at all three doses ( B and C). However, BenZ as positive drug control did not significantly impact protein expression level of these two UA transporters in the intestine. Likewise to the results obtained from the kidney, the phosphorylation levels of IκBα, NFκB p65, p38, and JNK were significantly up-regulated in the intestine, which was significantly inhibited by PU at tested doses ( D and E). These results demonstrated that PU could promote UA excretion through the intestine by inhibiting MAPK/NF-κB activation and suggested that PU possessed a more extensive promoting UA excretion effect than BenZ in the intestine ( F). PU ameliorated gut microbiota dysbiosis in hyperuricemia mice Gut microbiota are crucial in preventing and treating hyperuricemia through improving inflammation and damage of the intestine and kidney , . Thus, to further investigate whether PU facilitated protecting the intestine and kidney, and mitigating hyperuricemia via gut microbiota, 16S rRNA sequencing was conducted. The PCoA score plot clearly separated the control group and hyperuricemia model group into different clusters, which was reversed by PU, implying that PU restored the composition of gut microbiota ( A). In terms of phylum variances, the hyperuricemia group exhibited a greater presence of Firmicutes and a reduced Bacteroidota than control group ( B and C). Thus, the ratio of Firmicutes to Bacteroidota was higher in the hyperuricemia mice as previously reported ( C) , . However, the increased Firmicutes , decreased Bacteroidota , and elevated Firmicutes / Bacteroidota were significantly reversed by PU, indicating that PU could restore intestinal microbiota homeostasis in the hyperuricemia mice ( B and C). At the genus levels, the pro-inflammatory bacteria Parabacteroides , Oscillibacter , Desulfovibrio , and Tuzzerella were elevated in the hyperuricemia mice and significantly decreased with PU treatment ( B and C). In addition, the short-SCFA-generating bacteria Prevotellaceae_UCG-001 and Muribaculaceae were decreased in the hyperuricemia mice and significantly enhanced after PU treatment ( B and C). Moreover, KEGG was used to examine the possible functional alterations in gut microbiota, and 69 significantly differential pathways were predicted (Excel. S3). Among these, the renin-angiotensin system, arachidonic acid metabolism, and lipopolysaccharide biosynthesis pathway, closely related to inflammation and hyperuricemia, were significantly activated in the hyperuricemia mice and reduced upon PU administration ( D). In summary, the findings suggested a substantial contribution of gut microbiota in the UA-ameliorating and organ-protective effect of PU. 

To evaluate the impact of PU on hyperuricemia, a mice model with hyperuricemia was established as detailed in previously reports , . The establishment of hyperuricemia mice model and administration of PU were demonstrated in B. After model establishment, the body weight of hyperuricemia mice decreased notably but was reversed by BenZ at 10 mg/kg/d, and PU at 200 and 300 mg/kg/d ( C). Compared to the control group (71.21 μmol/L), SUA concentration of hyperuricemia mice significantly raised to 247.12 μmol/L, which was decreased to 69.71 μmol/L by BenZ administration ( D), suggesting that hyperuricemia mice model was established successfully , , . In contrast, SUA level in hyperuricemia mice was significantly and dose-dependently decreased to 158.76 μmol/L, 85.28 μmol/L, and 82.60 μmol/L by PU at 100, 200, and 300 mg/kg/d ( D). The findings suggested that PU is capable of efficiently alleviating hyperuricemia. 

Hyperuricemia is typically accompanied by kidney damage . PU has been revealed to alleviate renal damage and dysfunction in several diseases, such as diabetic nephropathy . Thus, the effect of PU on kidney functions in hyperuricemia mice was investigated using serum biochemical parameters alongside histopathological examination. Compared to the control, the kidney coefficients, BUN and SCr levels in hyperuricemia mice increased significantly, which were significantly and dose-dependently decreased in the PU group ( A, B, C). D depicted kidney appearance of the hyperuricemia mice as pale and remarkably increased with white spots, which was returned to ruddy and smooth in both the PU and BenZ groups. Consistent with these results, H&E staining demonstrated that kidneys in hyperuricemia model group indicated glomerular atrophy, and tubular dilatation, which were significantly improved by PU at 200, and 300 mg/kg/d, and BenZ at 10 mg/kg/d ( E). The improvement on renal injury was also verified by histopathological scores ( A). Overall, these findings indicated that PU could effectively alleviate kidney damage in case of hyperuricemia mice. 

The UA excretion through the kidney is dependent on specific proteins mediating UA reabsorption and excretion . Therefore, the expression of UA reabsorption and excretion transporters in the kidney was measured using western blotting. As shown in A-B, expression of UA reabsorption proteins URAT1 and GLUT9 were remarkably increased, while UA excretion proteins ABCG2 and OAT1 showed significant decrease in the model group. These results were consistent with previous studies , , suggesting that UA reabsorption was increased, while UA excretion was reduced in hyperuricemia mice kidneys. In contrast to the hyperuricemia model group, URAT1 and GLUT9 were inhibited by PU at 200, and 300 mg/kg/d dose-dependently, whereas the URAT1-targeted inhibitor BenZ significantly inhibited the URAT1 and GLUT9 expression ( A-B). Additionally, PU at medium and high doses, and BenZ at 10 mg/kg/d significantly increased protein expression level of UA excretion transporter ABCG2 and OAT1 in the kidney ( A-B). These results indicated that PU could facilitate renal UA excretion by improving renal UA transporter expression in the hyperuricemia mice. Earlier research has verified a link between hyperuricemia and kidney inflammation, resulting in severe renal damage , . Additionally, inflammation signaling pathway, such as MAPK/NF-κB, could regulate UA transporters expression , . Consequently, the impact of PU on the kidney inflammation in mice with hyperuricemia was assessed. Compared to the control group, the hyperuricemia model mice exhibited a significant increase of phosphorylation levels of IκBα and NF-κB p65 ( C and D). However, treatment with PU at indicated concentrations significantly decreased elevated phosphorylation of IκBα and NF-κB p65 in the kidney ( C and D). Similarly, the increased phosphorylation of JNK and p38 in the kidney of hyperuricemia mice was significantly decreased by PU at 200 and 300 mg/kg/d as well ( C and D). In parallel to alterations in these signaling pathways, PU administration inhibited the elevated levels of IL-1β, IL-6, and TNF-α, in the hyperuricemia mice kidney ( E). Additionally, the elevated phosphorylation levels of IκBα, NFκB p65, p38, and JNK, alongside up-regulated levels of the three inflammatory factors in the hyperuricemia mice kidney were also significantly inhibited by BenZ ( C-E). These results provided evidence that PU could inhibit inflammation as mediated via suppressing MAPK/NF-κB activation in the kidney of hyperuricemia mice, which could contribute to improving renal damage and insufficient UA excretion through the kidney ( F). 

To comprehensively assess the protective effect and action mechanisms of PU on the hyperuricemia mice kidney, renal non-targeted metabolomics analysis was fulfilled, and 284 metabolites were identified (Excel. S2). PCA and PLS-DA analysis showed that the PU group was isolated from the hyperuricemia mice model group and close to the control group, which indicated that PU reversed altered renal metabolite composition of the hyperuricemia mice ( A and B). In contrast to the control group, the hyperuricemia model group exhibited 146 differential metabolites ( C and Excel. S2). However, after treatment with PU, levels of 97 differential metabolites were reversed ( D-E and Excel. S2). These data further supported the conclusion that PU improved metabolic disorders in the kidney of hyperuricemia mice. As shown in F-G, the hyperuricemia model group had 5.85- and 3.31-folds abundance of uric acid and creatinine respectively compared to the control group, while were reduced by 79.2 % and 75.6 % post PU treatment, individually, suggesting that the impaired kidney function and kidney-mediated UA excretion were improved by PU treatment. Compared with the control group, decreased D-glucose level was observed, while glucose 6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, D-ribulose 5-phosphate and D-ribofuranose significantly increased in hyperuricemia mice, all of which were reversed by PU, indicating that PU improved glycolysis and pentose phosphate pathways (PPP) in the kidney of hyperuricemia mice ( F and G). Consistently, KEGG analysis demonstrated that the glycolysis was the top one pathway ameliorated by PU, meanwhile pentose and glucuronate interconversion and PPP pathways were also effectively improved ( H). Collectively, renal metabolomics analysis provided evidence that PU could improve kidney function by attenuating the glycometabolism dysequilibrium in hyperuricemia mice. 

The intestine is a significant UA excretion pathway . However, several studies have shown that hyperuricemia could induce intestinal damage , . Thus, H&E staining of the intestine was performed to assess intestine damage. Compared to the control, the intestinal villus was swollen and arranged disorderly, and the striated borders were blurred in hyperuricemia mice, which was restored by PU administration, indicating that PU ameliorated the intestine damage of hyperuricemia mice ( A), which were further verified by histopathological scores of intestine ( B). To research the impact of PU on intestinal UA excretion, the intestinal UA excretion proteins levels were detected using western blotting. The intestinal ABCG2 and GLUT9 were remarkably decreased in case of hyperuricemia mice, which was reversed by treatment with PU at all three doses ( B and C). However, BenZ as positive drug control did not significantly impact protein expression level of these two UA transporters in the intestine. Likewise to the results obtained from the kidney, the phosphorylation levels of IκBα, NFκB p65, p38, and JNK were significantly up-regulated in the intestine, which was significantly inhibited by PU at tested doses ( D and E). These results demonstrated that PU could promote UA excretion through the intestine by inhibiting MAPK/NF-κB activation and suggested that PU possessed a more extensive promoting UA excretion effect than BenZ in the intestine ( F). 

Gut microbiota are crucial in preventing and treating hyperuricemia through improving inflammation and damage of the intestine and kidney , . Thus, to further investigate whether PU facilitated protecting the intestine and kidney, and mitigating hyperuricemia via gut microbiota, 16S rRNA sequencing was conducted. The PCoA score plot clearly separated the control group and hyperuricemia model group into different clusters, which was reversed by PU, implying that PU restored the composition of gut microbiota ( A). In terms of phylum variances, the hyperuricemia group exhibited a greater presence of Firmicutes and a reduced Bacteroidota than control group ( B and C). Thus, the ratio of Firmicutes to Bacteroidota was higher in the hyperuricemia mice as previously reported ( C) , . However, the increased Firmicutes , decreased Bacteroidota , and elevated Firmicutes / Bacteroidota were significantly reversed by PU, indicating that PU could restore intestinal microbiota homeostasis in the hyperuricemia mice ( B and C). At the genus levels, the pro-inflammatory bacteria Parabacteroides , Oscillibacter , Desulfovibrio , and Tuzzerella were elevated in the hyperuricemia mice and significantly decreased with PU treatment ( B and C). In addition, the short-SCFA-generating bacteria Prevotellaceae_UCG-001 and Muribaculaceae were decreased in the hyperuricemia mice and significantly enhanced after PU treatment ( B and C). Moreover, KEGG was used to examine the possible functional alterations in gut microbiota, and 69 significantly differential pathways were predicted (Excel. S3). Among these, the renin-angiotensin system, arachidonic acid metabolism, and lipopolysaccharide biosynthesis pathway, closely related to inflammation and hyperuricemia, were significantly activated in the hyperuricemia mice and reduced upon PU administration ( D). In summary, the findings suggested a substantial contribution of gut microbiota in the UA-ameliorating and organ-protective effect of PU. 

Increasing evidence supports that UA excretion rather than UA production that leads to hyperuricemia. Approximately 90 % of hyperuricemia cases were diagnosed to be caused by insufficient UA excretion . Therefore, promoting UA excretion has been regarded as key target for preventing and treating hyperuricemia. It is estimated that ca. 70 % of UA is excreted through a complex process of reabsorption and excretion mediated by the kidney . The primary transporter involved in UA reabsorption is URAT1, which is expressed at the apical surface of renal tubular cells . GLUT9, localized both to the basolateral membrane and apical membrane, is also identified as crucial in UA reabsorption . ABCG2 and OAT1, which are localized on the apical brush border and basolateral membrane respectively, mainly regulate UA excretion from the blood to the lumen of the proximal tubule . It has been revealed that mice with defective URAT1, GLUT9, ABCG2 or OAT1 in the kidney were susceptible to be hyperuricemia , , supporting the hypothesis that these UA transporters are effective targets for preventing and treating hyperuricemia. The remaining UA excretion, approximately 30 %, is regulated by the intestine, which is also dependent on specific UA transporters . Recent studies suggested that ABCG2 and GLUT9 are crucial in intestinal excretion of UA as confirmed from knocking out of ABCG2 and GLUT9 in the intestine that led to elevated levels of SUA , . Thus, these UA transporters in these two organs have been considered the most promising and effective targets for treating hyperuricemia. However, clinically available medication targeting renal UA excretion, BenZ and probenecid, have been limited for their side effects . Additionally, no medications targeting the excretion of intestinal UA are clinically accessible. Thus, natural bioactive compounds have been examined considering their multi effects to ameliorate hyperuricemia, in addition to their safety. A large number of phytochemical studies targeting renal URAT1 have led to the identification of compounds, such as flavonoids and terpenoids as potential URAT1 inhibitors. At the present, natural products targeting both the kidney and gut have been gaining increasing attention considering their stronger likely effects. Astragali Radix intervention, commonly known for its use in traditional Chinese medicine, was found to ameliorate hyperuricemia by modulating renal and intestinal UA transporters including URAT1, GLUT9, and ABCG2 in hyperuricemia mice . In addition, empagliflozin, an antidiabetic medication, was revealed to attenuate hyperuricemia by promoting protein expression of UA excretion protein ABCG2 in both kidney and intestine . However, there are still few studies reporting on the UA-lowering effect of natural products by simultaneously targeting the kidney and intestine. In this study, PU was found to reduce the URAT1 and GLUT9 levels, while increasing the expression of ABCG2 and OAT1 in hyperuricemia model mice kidney, similar to BenZ treatment. However, PU rather than BenZ was found to significantly induce expression of ABCG2 and GLUT9 in the intestine of hyperuricemia mice, presenting a multi effect in different organs. These results indicated that PU might possess a more effective anti-hyperuricemia effect than BenZ, by promoting more extensive excretion of UA mediated by both kidney and intestine, simultaneously. It is worth noting that in hyperuricemia mice, the expression of the GLUT9 protein was elevated in the kidney but decreased in the intestine, which was reversed by PU. Similarly, Bao et al. reported the different effects of Eurycomanol on the GLUT9 protein expression in these two organs using hyperuricemia mice previously . GLUT9 has been found to have two distinct N-terminal isoforms, GLUT9a (widely expressed) and GLUT9b (primarily in the liver, kidney, and placenta) . The N-linked glycosylation level of these two isoforms has been discovered to be differentially affected by integral membrane protein 2B (ITM2B) , which might cause the protein stability difference between these two GLUT9 isoforms. Thus, the posttranslational modification might lead to the protein expression difference of GLUT9 in the kidney and intestine. Furthermore, the intestine environment is more complex than the kidney, which could be another reason contributing to this phenomenon. To answer this question, further research is needed to investigate how PU improves GLUT9 protein expression in the kidney and intestine respectively. Hyperuricemia is always accompanied by inflammation. It has been revealed that UA and urate crystals could stimulate inflammation and activate inflammatory signaling pathways, which aggravate organ damage and dysfunction . In this study, proinflammatory cytokines were remarkably increased in the kidney of hyperuricemia mice. Correspondingly, the inflammatory MAPK/NF-κB pathways were activated in the kidney and intestine. These results were consistent with previous studies , , . In contrast, PU administration significantly decreased proinflammatory factors levels in the kidney, concurrent with suppressing the activation of NF-κB and MAPK in the kidney and intestine. Additionally, H&E staining analysis demonstrated that PU could effectively alleviate kidney and intestine damage in the hyperuricemia mice. These results suggested that PU could alleviate hyperuricemia mice renal and intestinal inflammatory damage. Moreover, it was discovered that the expression of UA transporters was influenced by the inflammation MAPK/NF-κB signaling pathways. Zhang et al. demonstrated that fucoidan could regulate URAT1 and GLUT9 expression through repressing the activation of NF-κB and JNK MAPK in UA-induced HK-2 cells . Li et al. demonstrated that NF-κB controlled URAT1 in NRK-52E cells . Recently, we reported that nuciferine derivative, belonging to alkaloids, could suppress URAT1 and GLUT9 expression and alleviate kidney damage through regulating NF-κB signaling pathway . Here, PU was demonstrated to significantly inhibit MAPK/NF-κB phosphorylation in the kidney and intestine of hyperuricemia mice, indicating that PU could restore the expression of UA transporters via the MAPK/NF-κB signal pathway. Recently, numerous research efforts indicate a strong link between hyperuricemia and metabolic disorders . Metabolomics is increasingly utilized to explore the underlying mechanisms of diseases through analysis of the comprehensive alteration of endogenous metabolites . Here, renal metabolomics was conducted to assess differences in the metabolism of hyperuricemia mice treated with or without PU. Consistent with serum biomarkers analysis, the concentration of UA and creatinine was found to be significantly increased in hyperuricemia mice kidney and decreased upon PU administration. This indicated that PU effectively attenuated the renal injury and dysfunction induced by UA. Moreover, glycometabolism pathways as well as metabolites associated with these metabolic pathways, such as pentose and glucuronate interconversion and PPP, were significantly activated in the kidney of hyperuricemia mice, which were significantly reversed by PU. The activation of glycolysis was linked to numerous side routes, such as PPP, hexosamine pathway, and lipid biosynthesis , . Moreover, the accumulation of glycolytic intermediates induces the release of pro-inflammatory and pro-fibrotic factors, exacerbating renal inflammation and fibrosis, and thereby worsening kidney damage . Recent studies have found astragaloside IV effectively improved renal glycolysis activation in diabetic mouse models , and isorhamnetin alleviated renal glycolysis activation and fibrosis by inhibiting the TGF-β1-induced expression of PFK . These findings suggested that renal glycolysis might play an important role in the biological effects of natural products. It has been reported that PPP could decrease the levels of glucose 6-phosphatase, while increase the levels of ribose 5-phosphate, the precursor to UA, which could lead to excessive production of UA . Meanwhile, the breakdown of fructose and glucose leads to ATP depletion and renal tubular inflammation, which might also worsen kidney function . Therefore, hyperuricemia could stimulate glucose metabolic disorder in the kidney, which might lead to renal dysfunction and aggravate hyperuricemia. In present study, PU effectively reversed the glucose metabolic disorder in the kidney, which suggested that the hyperuricemia-ameliorating and renal protection effect of PU might partly be attributed to the improved glucose metabolism in the kidney. Additionally, the beneficial effect of PU on hyperuricemia through regulating renal glycometabolism needs to be further verified. There is increasing evidence demonstrated that the hyperuricemia has a bidirectional relationship with gut microbiota. The gut microbiota composition and functions were remarkably altered in hyperuricemia mice , while transplanting fecal bacteria from hyperuricemia model mice into control mice led to the elevation of SUA levels . In terms of action mechanism, gut microbiota dysbiosis could negatively affect intestinal barrier and integrity, thus facilitating pathogenic bacterial invasion and toxins such as LPS from the intestine into the kidney through blood circulation, which contributes to aggravating inflammation, damage, and dysfunction of the kidney, finally resulting in insufficient UA excretion . Herein, PU administration was found to increase the relative abundance of Bacteroidetes while decreasing Firmicutes , reversing the ratio of Firmicutes to Bacteroidetes which was significantly elevated in the hyperuricemia mice. The findings indicated that PU could restore the gut microbiota composition and diversity, which is consistent with previous studies reporting the alleviative effects of PU on gut microbiota dysbiosis , , . Recently, Lacticaseibacillus rhamnosus Fmb14 and Lacticaseibacillus rhamnosus 1155 were demonstrated to restore the ratio of Firmicutes to Bacteroidetes by increasing Bacteroidetes and decreasing Firmicutes . The gut microbiota-derived SCFAs have been found to possess protective effects on hyperuricemia by suppressing inflammation and providing energy for UA excretion through the intestine . Prevotellaceae_UCG-001 , an SCFA producing bacteria, has been demonstrated to alleviate DSS-induced colonic inflammation through increasing the production of acetic acid, propionic acid, and butyric acid . Recently, it was reported that the SCFA-producing bacteria Muribaculaceae might to be associated with the reduction of pro-inflammatory factors IL-1β, IL-6, TNF-α in ulcerative colitis mice . Reduced SCFA-generating bacteria could cause elevated inflammatory factors to enter the kidney through circulation, potentially resulting in renal dysfunction. In the present study, the SCFA-generating bacteria Prevotellaceae_UCG-001 and Muribaculaceae were significantly reduced in the hyperuricemia mice model, which were effectively elevated by PU administration. Furthermore, studies are needed to profile SCFAs to evaluate the improvement of SCFAs alternation in the hyperuricemia mice upon PU administration in future. In contrast, pro-inflammatory bacteria, such as Parabacteroides , Oscillibacter , Desulfovibrio , and Tuzzerella , were significantly increased in hyperuricemia and chronic kidney disease, and have been found to be positively associated with renal and intestinal inflammation and damage , , , . To be more specific, the Parabacteroides might lead to hyperammonemia through enhancing the urea metabolism in the gut microbiota, which could cause renal injury . Oscillibacter has been reported to be correlated with abnormal intestinal permeability . Desulfovibrio was revealed to be enriched in the fecal samples of patients with chronic kidney disease (CKD), and correlated with CKD kidney dysfunction. . In the study of the ameliorative effect of mannuronate oligosaccharides on hyperuricemia, Tyzzerella was found to present a strong positive correlation with pro-inflammatory factors . Therefore, the increase of these pro-inflammatory bacteria might cause kidney dysfunction through various pathways. In present study, these pro-inflammatory bacteria were remarkably enriched in the hyperuricemia mice, which were effectively reversed upon PU administration. Functional analysis of gut microbiota also demonstrated that LPS biosynthesis was up-regulated in hyperuricemia mice, and down-regulated with PU treatment. Collectively, PU might alleviate the hyperuricemia, and mitigate inflammation and injury of the kidney and intestine via remodeling of gut microbiota, and adding to the prebiotic nature of PU . However, the beneficial effect of PU on hyperuricemia and complications through gut microbiota needs to be further verified by gut microbiota transplantation experiments. Research on the UA-lowering effects of ellagitannin is limited. In 2020, Adachi et al. reported the UA-lowering effect of ellagic acid (EA), another major ellagitannin in pomegranates . They showed that EA could significantly reduce UA production in AML12 hepatocytes and effectively cancel the increase in plasma UA level in the model group. However, the UA-lowering mechanism of EA was not explored further. In our study, PU administration was found to effectively ameliorate hyperuricemia in hyperuricemia mice model through restoring the function of kidney and intestine. PU could ameliorate protein expression of UA transporters in the kidney and intestine through suppressing inflammation signaling pathway activation. Additionally, PU was found to ameliorate gut microbiota dysbiosis and kidney glycometabolism dysequilibrium in the hyperuricemia mice model. This study revealed the UA-lowering role of PU on hyperuricemia and investigated the fundamental mechanisms associated with kidney and intestine, posing it as prospective nutraceutical for treating elevated UA in clinical trials especially considering its large safety margin and extensive reports in humans. 

Jin-yue Sun, Ning-yang Li, Chao Liu and Qi-dong Ren supervised the study. Qing-qing Han, Qi-dong Ren and Chao Liu designed the experiments. Qing-qing Han performed experiments and analyzed the data with the help of Xu Guo, Yu-hong Zhang, Meng-qi Zhang, Ying-ying Chen, Shu-tao Sun. Qing-qing Han, Qi-dong Ren and Mohamed A. Farag wrote the manuscript. 

The authors have declared no conflict of interest. 

All animal study procedures were performed in compliance with the guide for the care and use of laboratory animals and were approved by the Ethics Committee of the Shandong Academy of Agricultural Sciences (No. SAAS-2022-SD03). 

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. 

