Integrated Data Mining and Animal Experiments to Investigate the Efficacy and Potential Pharmacological Mechanism of a Traditional Tibetan Functional Food Terminalia chebula Retz. in Hyperuricemia

Introduction

Hyperuricemia (HUA), a prevalent metabolic disorder, is a growing health concern in China owing to its association with a range of serious conditions. HUA can be caused by excessive uric acid production or insufficient renal and intestinal excretion. It is not only a risk factor for gout but is also closely associated with the onset and progression of conditions such as hypertension, diabetes, and kidney disease. Epidemiological studies have revealed a steady increase (by as much as 17.4%) in the prevalence of HUA in China.¹ It is currently the second most common metabolic disorder, occurring only in patients with diabetes. Moreover, the affected population is becoming progressively younger, which is expected to place a significant burden on China’s healthcare system in the coming years. The general strategy for HUA primarily focuses on the use of xanthine oxidase (XOD) inhibitors, such as allopurinol and febuxostat, to lower uric acid levels, and the use of uricosuric agents, such as probenecid, benzbromarone, and sulfinpyrazone, to promote uric acid excretion. However, adverse reactions associated with XOD inhibitors and uricosuric agents, including diarrhea, headache, rash, severe allergies, and nephrotoxicity, often limit their clinical use. Furthermore, these treatments focus narrowly on serum uric acid reduction, failing to address other critical aspects of HUA pathology, such as renal protection and gut microbiota modulation. This underscores the pressing need for safer and more holistic therapeutic approaches.²

Recent studies highlight the crucial role of the gut-kidney axis in uric acid metabolism and HUA pathology. The gut influences systemic uric acid levels by degrading purines, producing short-chain fatty acids, and maintaining intestinal barrier integrity. Meanwhile, the kidneys, responsible for the majority of uric acid excretion, share closely linked inflammatory and metabolic pathways with the gut. Dysregulation of this axis can exacerbate HUA and associated complications. Recognizing its importance, researchers are increasingly exploring treatments for HUA by targeting the gut-kidney axis. For instance, the Cichorium intybus L. formula has been shown to regulate gut microbiota dysbiosis and provide renal protection in HUA nephropathy rats.³ Similarly, the traditional Chinese medicine (TCM) formula CoTOL demonstrated uric acid reduction, potentially through its regulation of intestinal flora.⁴ These findings highlight the therapeutic potential of targeting gut-kidney interactions.

Tibet is a high-altitude region characterized by elevated atmospheric pressure and low air density. The Tibetan population predominantly leads a nomadic lifestyle with dietary preferences for beef, barley, and butter tea. Nearly 50% of Tibetan adults consume alcohol. The incidence of HUA in the Tibetan region is significantly higher than in other parts of China, with a crude prevalence rate as high as 37.2%. Traditional Tibetan medicine, with its unique theoretical framework, is an essential component of traditional Chinese medicine. Over the years a wealth of traditional prescriptions for the treatment of HUA have been utilized.⁵ For example, RuPeng 15 powder,⁶ Shi Wei Ru Xiang powder,⁷ and TongFeng Tangsan^8,9 have been widely used in Tibetan hospitals to treat HUA and their efficacy confirmed by clinical studies.

This study initially screened Tibetan medical prescriptions for HUA treatment described in classical Tibetan medical books. Through data mining, T. chebula was identified as a potential candidate anti-HUA medication. It is worth noting that no existing literature has reported on the use of T. chebula extract for the treatment of HUA. Subsequently, the therapeutic effects of T. chebula were validated by establishing a HUA rat model, and the potential mechanisms of action were explored. This study serves as a theoretical reference for understanding the biological functions of T. chebula and its application in HUA treatment.

Materials and Methods

Data Mining of Tibetan Medicine Prescriptions for the Treatment of HUA

To systematically analyze and quantify traditional Tibetan medicine knowledge, extensive data mining of prescriptions for hyperuricemia (HUA) treatment was conducted. Key sources included the National Standard for Tibetan Medicine, rGyud-bzhi, Crystal Beads Materia Medica, Chinese Tibetan Medicine, and Chinese Materia Medica-Tibetan Medicine. Using the keyword “huangshui”, historically associated with HUA, relevant prescriptions were identified and extracted. These prescriptions were digitized and structured into a comprehensive dataset using Microsoft Excel. The data were standardized based on the 2020 edition of the Chinese Pharmacopoeia to ensure consistency and comparability. Unlike traditional qualitative interpretations, we used modern data-driven methods to uncover patterns and relationships within the dataset. The frequency of each Tibetan medicine was calculated using Excel pivot tables, transforming the dataset into a transactional format suitable for advanced analysis. Subsequently, we imported this data into IBM SPSS Modeler 18.0 for co-occurrence analysis, enabling us to explore patterns of simultaneous use among Tibetan medicines. Finally, to gain deeper insights, we performed hierarchical cluster analysis using IBM SPSS Statistics 20.¹⁰

T. chebula Extract Preparation

T. chebula fruits were sourced from Lincang, Yunnan, China, and identified by Professor Ma Hongyan (School of Traditional Chinese Medicine, Guangdong Pharmaceutical University). A voucher specimen (GDPUHTC01) is stored in the Herbarium of the Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances. The fruits were dried at 60 °C, ground into powder, and extracted three times by reflux with distilled water (ddH₂O) for 0.5 hours per cycle. The extracts were concentrated under vacuum at 60 °C to remove the solvent.

Qualitative Analysis of T. chebula by UPLC-QE-Orbitrap-MS

Qualitative analysis of T. chebula was performed using ultra-high-performance liquid chromatography (UPLC) coupled with a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Chromatographic separation was achieved on a Thermo Gold C18 column (1.7 μm, 2.1×100 mm) at 40 °C, with a mobile phase comprising 0.1% formic acid in water (A) and methanol (B). The gradient elution program was as follows: 0–5.0 min, 20% B; 5.0–45.0 min, 20–100% B; 45.0–56.1 min, 20–100% B; and 56.1–60.0 min, 20% B.

The mass spectrometric analysis employed an electrospray ionization (ESI) source in both positive and negative modes with a mass range of m/z 200–1000. The resolution was set to 35,000 (primary) and 17,500 (secondary). Key operational parameters included a spray voltage of 3500 V, capillary temperature of 320 °C, probe heater temperature of 350 °C, sheath gas flow rate of 35 units, auxiliary gas flow rate of 10 units, and collision energies of 20, 25, and 30 eV.

Animal Experiments

36 SPF-grade male Sprague-Dawley rats (180 and 220.0 g) were obtained from Guangdong Medical Laboratory Animal Center (Certificate number: 44007200101564). After a one-week acclimatization period, rats were randomly divided into six groups (n = 6/group): Control, Model, allopurinol (ALLO, positive control, GKH Pharmaceutical Ltd., lot: H44021695), and high-dose (HZHI), middle-dose (HZME), and low-dose (HZLO) T. chebula groups. Following the method described by Yang et al,¹¹ all groups except the Control group were administered 10% fructose solution via oral gavage from weeks 1 to 6. Through preliminary experiments, we determined the dosing regimen for T. chebula. To induce the HUA rat model, potassium oxonate solution (600 mg·kg⁻¹) was also orally administered from weeks 2 to 6. During this period, the Control and Model groups received an equivalent volume of 0.5% CMC-Na solution, the ALLO group received allopurinol solution (50 mg·kg⁻¹), and the HZLO, HZME, and HZHI groups were given T. chebula solutions at doses of 45 mg·kg⁻¹, 90 mg·kg⁻¹, and 180 mg·kg⁻¹, respectively, for 4 weeks.

Histopathological Examination and Immunohistochemistry (IHC)

Kidney and intestine tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 4 μm. For histological analysis, sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome stain. Immunohistochemistry (IHC) was used to assess the expression of Claudin-1, Occludin, and ZO-1. Stained sections were examined under a light microscope (Olympus, Tokyo, Japan), and the percentage of positively stained areas was quantified using ImageJ software.

Biochemical Detection/ Plasma Biochemical Assay

Serum was obtained by centrifuging blood at 3800 rpm for 10 min at 4 °C. Serum levels of uric acid (UA), blood urea nitrogen (BUN), creatinine (Cre), and xanthine oxidase (XOD) activity were measured using commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu Meimian Industrial Co., Ltd., Yancheng, China) were used to quantify lipopolysaccharide (LPS), TNF-α, IL-6, diamine oxidase (DAO), and D-lactate (D-Lac).

RNA Extraction, Reverse-Transcription, and Quantitative PCR

Total RNA from intestinal tissues was extracted using an RNA extraction kit (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s protocol. RNA concentration was measured with a micro-spectrophotometer (Allsheng Biotech Co., Ltd., Hangzhou, China), and cDNA was synthesized using a two-step RT-qPCR kit (Seven Biotech Co., Ltd., Beijing, China). Gene expression levels of Claudin-1, Occludin, and ZO-1 were analyzed on a real-time PCR system, with β-Actin as the internal control. The RT–PCR protocol included 95 °C for 30s, followed by 40 cycles of 95 °C for 30s and 60 °C for 30s. Gene expression was calculated using the 2−^ΔΔCt method. Primer sequences are listed in Table 1.

Table 1 Primer Sequences of RT-qPCR

Intestinal Microbiota Analysis

On the final day of the experiment, fecal samples were collected from each rat group and stored at −80 °C. The samples were sent to Shanghai Meiji Biological Technology Co., Ltd. (Shanghai, China) for intestinal microbiota analysis. The V3–V4 region of 16S rDNA was amplified using universal primers (338F: ACTCCTACGGGAGGCAGCA; 806R: GGACTACHVGGGTWTCTAAT). Amplified DNA fragments were purified with an Axygen DNA Gel Recovery Kit (Axygen Inc., Massachusetts, USA) and used to construct an Illumina library with the TruSeq™ DNA Sample Prep Kit (Illumina Inc., San Diego, CA, USA). Sequencing was performed on the Illumina MiSeq™ System to obtain microbiota sequencing data.

Statistical Analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism (version 8.0.1). One-way ANOVA was employed to analyze differences among multiple groups, followed by Tukey’s HSD (Honestly Significant Difference) test as the post hoc method to identify significant pairwise group differences. A p-value < 0.05 was considered statistically significant.

Results

Data Mining of Tibetan Medicine Prescriptions for the Treatment of HUA

From various sources, we retrieved 122 prescriptions aimed at treating HUA, including the National Standard for Tibetan Medicine, rGyud-bzhi, Crystal Materia Medica, Chinese Tibetan Medicine, and Chinese Materia Medica-Tibetan Medicine (Tables S1 and S2). These prescriptions included 248 herbal drugs. Unlike traditional qualitative descriptions, we applied modern data mining techniques to analyze and quantify the use of these herbs systematically. Using frequency analysis, we identified the most commonly used Chinese medicines for HUA treatment, including T. chebula, Juemingzi, Ruxiang, Shexiang, Muxiang, Doukou, Huangkuizi, Maohezi, Shuichangpu, and Yuganzi (Figure 1A). Notably, T. chebula emerged as the most frequently cited species, occurring 54 times and accounting for 44.3% of those identified. Furthermore, co-occurrence analysis revealed that T. chebula exhibited the highest co-occurrence rates with Muxiang (n=26), Maohezi (n=20), Shuichangpu (n=19), Juemingzi (n=22), Yuganzi (n=19), Ruxiang (n=21), Huangkuizi (n=18), and Shexiang (n=21) (Figure 1B). These analyses not only validated T. chebula’s central role in traditional prescriptions but also uncovered potential synergistic relationships with co-occurring herbs, which are not explicitly documented in historical texts. The configuration of different Tibetan medicine combinations was determined using the hierarchical clustering method, which classifies herbs based on their relationships with other Tibetan medicines. In our study, this hierarchical clustering process resulted in the stratification of Tibetan medicines into three distinct and effective prescriptions (Figure 1C). To validate the potential therapeutic value of the most frequently prescribed medicine for HUA, T. chebula, we conducted in vitro assays to assess its inhibitory activity against XOD. The results demonstrated that T. chebula exhibited a dose-dependent reduction in XOD activity, further supporting its use as a promising candidate for HUA treatment (Figure 1D).

Figure 1 Data mining of Tibetan medicine prescriptions in the treatment of UA. (A) High-frequency Chinese medicines in prescriptions. (B) Herbs co-occurrence diagram. (C) Hierarchical cluster analysis of herbs. (D) The inhibitory activity of T. chebula on XOD in vitro.

Qualitative Analysis of Phytochemical Constituents in T. chebula

UPLC-QE-Orbitrap-MS was used to identify the major phytochemical constituents of T. chebula. The total ion chromatograms (TICs) of T. chebula are shown in Figure 2A and B, and their chemical structures are shown in Figure 2C. Through high-resolution UPLC-MS/MS analysis, 47 constituents were rapidly identified from T. chebula based on their accurate MS and MS/MS data, together with comparisons with references (Table S3). These constituents included phenols, terpenoids, flavonoids, sulfonamides, fatty acids, glucosides, and steroids (Figure S1).

Figure 2 Chemical composition analysis and identification of T. chebula. (A) Total ion chromatograms of T. chebula extract in positive mode. (B) Total ion chromatograms of T. chebula extract in negative mode. (C) The structure of the chemical composition identified in T. chebula..

T. chebula Decreased UA Level in Hyperuricemic Rats

To further validate the potential therapeutic value of T. chebula in HUA, we induced a rat HUA model using fructose-water and potassium oxonate (Figure 3A). As shown in Figure 3B, the body weight of all rats increased steadily throughout the experimental period, with no significant differences observed among the six groups. In the third week, the serum UA level of the model group was approximately three times higher than that of the normal group, a statistically significant difference as determined by Tukey’s HSD post hoc test (P < 0.01). This result confirms the successful establishment of the HUA rat model. After 4 weeks of treatment, both T. chebula and allopurinol significantly reduced serum UA levels and XOD activity compared with those in the model group (UA: P = 0.000, P = 0.000, P = 0.000, all P < 0.01, Tukey’s HSD; XOD: P =0.003, P =0.005, P =0.004, all P < 0.01, Tukey’s HSD) (Figure 3C and D). In clinical practice, HUA is often accompanied by an inflammatory response. Elevated serum UA levels can trigger the release of pro-inflammatory cytokines, thereby increasing the risk of renal inflammation and injury. In our study, serum proinflammatory cytokines IL-6 and TNF-α were significantly increased in HUA rats compared to those in normal rats (IL-6: P =0.000, P < 0.01, Tukey’s HSD; TNF-α: P =0.006, P < 0.01, Tukey’s HSD). Notably, T. chebula significantly restored IL-6 and TNF-α levels (IL-6: P =0.000, P =0.004, P =0.001, all P < 0.01, Tukey’s HSD; TNF-α: P =0.006, P =0.012, P =0.012, P < 0.01 or P < 0.05, Tukey’s HSD) (Figure 3E and F).

Figure 3 The therapeutic effects of T. chebula on HUA rats. (A) Schematic experimental design for T. chebula treatment. (B) Body weight. (C) Serum UA. (D) Serum XOD activity. (E) Serum IL-6. (F) Serum TNF-α. Values are presented as mean ± SD (n = 6 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test. **P< 0.05 vs Control group; #P < 0.05, ##P < 0.05 vs Model group.

T. chebula Protected Renal Function of Hyperuricemic Rats

Numerous studies have documented the potential of HUA to induce renal dysfunction and pathological alterations in the kidney, thereby posing a considerable threat to patient well-being. The kidney plays an indispensable role in the regulation of uric acid balance. Figure 4A presents the results of the H&E and Masson staining of kidney sections derived from the six groups of experimental rats. In the control group, the kidney morphology exhibited a normal appearance without any histopathological deviations. Conversely, HUA rats displayed noticeable dilation of the tubular lumen and observable infiltration of inflammatory cells within the kidney tissue. Masson’s staining revealed banded interstitial fibrosis along with a proliferation of collagen fibers. Remarkably, T. chebula administration at varying doses resulted in kidney morphology that closely resembled that of the normal control group. Moreover, there was a significant reduction in banded interstitial fibrosis, which implies the effective mitigation of histopathological kidney damage in HUA rats. Figure 4B–4D illustrate the renal index, serum BUN, and Cre levels across all experimental groups. After 42 days of HUA induction, a substantial increase in the renal index, serum BUN, and Cre levels was observed in the model group compared to that in the control group (Renal index: P =0.001, P < 0.01; BUN: P =0.000, P < 0.01; Cre: P =0.000, P < 0.01, Tukey’s HSD). However, subsequent treatment with T. chebula extract at various concentrations led to a significant reduction in the renal index, serum BUN, and Cre levels compared to those in the model group (Renal index: P =0.000, P =0.000, P =0.000, all P < 0.01, Tukey’s HSD; BUN: P =0.000, P =0.000, P =0.000, all P < 0.01, Tukey’s HSD; Cre: P =0.000, P =0.000, P =0.001, all P < 0.01, Tukey’s HSD). These findings provide compelling evidence of the effectiveness of T. chebula extract in mitigating renal damage induced by HUA.

Figure 4 T. chebula protected renal function of hyperuricemic rats. (A) H&E staining and Masson staining of kidney tissue (blue denotes collagen fibers, bar = 50 µm). (B) Renal index. (C) Serum Cre. (D) Serum BUN. Values are presented as mean ± SD (n = 6 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test. **P< 0.05 vs Control group; ##P < 0.05 vs Model group.

T. chebula Improved Intestinal Barrier Integrity in Hyperuricemic Rats

The intestine plays a vital role in uric acid excretion. Histological examination using hematoxylin and eosin (HE) staining revealed that high-purine diets and excessive serum uric acid (UA) levels disrupted gut morphology, resulting in shortened villi, villus shedding, and reduced crypt depth. Conversely, treatment with T. chebula effectively reversed these morphological changes, as demonstrated by improved villus length and crypt depth ratio (Figure 5A). Serum levels of D-Lac, DAO, and LPS were used as markers to assess gut permeability. As depicted in Figure 5B-5D, in comparison to the control group, the model group showed a significant increase in the serum levels of D-Lac, DAO, and LPS (D-Lac: P =0.000, P < 0.01; DAO: P =0.000, P < 0.01; LPS: P =0.000, P < 0.01, Tukey’s HSD). Notably, in the HZHI, HEME, and HZLO groups, the levels of these markers were significantly reduced compared with those in the model group (D-Lac: P =0.000, 0.000, 0.000, all P < 0.01, Tukey’s HSD; DAO: P =0.000, 0.000, 0.001, all P < 0.01, Tukey’s HSD; LPS: P =0.000, 0.000, 0.000, all P < 0.01, Tukey’s HSD). These findings suggest that T. chebula effectively repairs gut mucosal damage and enhances intestinal barrier permeability.

Figure 5 T. chebula improved intestinal barrier integrity in hyperuricemic rats (A) H&E staining of gut (bar = 100 µm). (B) Serum DAO. (C) Serum D-Lac. (D) Serum LPS. Values are presented as mean ± SD (n = 6 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test. **P< 0.05 vs Control group; ##P < 0.05 vs Model group.

Intestinal tight junctions play a crucial role in maintaining the gut barrier function and intestinal homeostasis. The results of the immunohistochemistry experiments showed that the expression levels of ZO-1, Occludin, and Claudin-1, which are proteins associated with intestinal tight junctions, were significantly reduced in the model group (ZO-1: P =0.037, P < 0.05; Occludin: P =0.000, P < 0.01; Claudin-1: P =0.009, P < 0.01, Tukey’s HSD). However, treatment with T. chebula significantly upregulated the expression of these proteins (ZO-1: P =0.006, 0.003, 0.005, all P < 0.01, Tukey’s HSD; Occludin: P =0.001, 0.017, P < 0.01 or P < 0.05, Tukey’s HSD; Claudin-1: P =0.004, 0.019, 0.018, P < 0.01 or P < 0.05, Tukey’s HSD) (Figure 6A), indicating that T. chebula can effectively maintain gut barrier function. Notably, allopurinol treatment did not affect the expression of these proteins. Consistent with the immunohistochemistry results, T. chebula treatment significantly upregulated the mRNA expression of ZO-1, Occludin, and Claudin-1 (ZO-1: P =0.000, 0.000, P < 0.01, Tukey’s HSD; Occludin: P =0.000 0.000, P < 0.01, Tukey’s HSD; Claudin-1: P =0.003, 0.002, P < 0.01, Tukey’s HSD) (Figure 6B and C). Collectively, these findings suggest that T. chebula treatment preserves gut barrier function by enhancing tight junction protein expression.

Figure 6 T. chebula improved tight junction proteins expression in intestinal. (A) ZO-1, Occludin, and Claudin-1 immunohistochemical staining in intestinal (bar = 50 µm). (B) Statistical analysis of the percentage of the ZO-1, Occludin, and Claudin-1 positively stained area. (C) ZO-1, Occludin, and Claudin-1 mRNA expression by RT-PCR. Values are presented as mean ± SD (n = 6 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test. *P< 0.05, **P< 0.05 vs Control group; #P < 0.05, ##P < 0.05 vs Model group.

T. chebula Regulated UA-Induced Gut Microbiota Dysbiosis in Hyperuricemic Rats

To assess the impact of T. chebula on the intestinal flora of HUA mice, we conducted a comprehensive analysis of gut microbial communities by sequencing the V3-V4 regions of the 16S rRNA gene. Our α-diversity analysis revealed no significant differences in the Shannon and Simpson indices (measures of diversity) among the control, model, and HZHI groups (Figure 7C and D). However, the Chao index and Sobs index (markers of richness) showed a reduction in the model group compared with the control group, whereas the HZHI group exhibited a notable increase in both the Chao index and Sobs index (P =0.034, P =0.008, P < 0.05, Tukey’s HSD) (Figure 7A and B). At the phylum level, the dominant microbiota primarily consisted of Firmicutes, Bacteroidetes, Actinobacteria, Patescibacteria, Desulfobacteria, and Cyanobacteria (Figure 8A). At the genus level, the microbiota was predominantly composed of Romboutsia, Clostridium_sensu_stricto_1, norank_f_Ruminococcaceae, Candidatus_Stoquefichus, Lactobacillus, and norank_f_Muribaculaceae (Figure 8B). Importantly, substantial differences were observed in the species composition at both the genus and phylum levels. Compared to the model group, the HZHI group showed significantly reduced Actinobacteriota and Bacteroidota levels and elevated Firmicutes levels (Figure 7E–H). In summary, these findings demonstrate that T. chebula effectively alters the composition and structure of microbiota in HUA rats.

Figure 7 The influence of T. chebula on the structure of intestinal flora in HUA rats. (A) Sob index. (B) Chao index. (C) Shannon index. (D) Simpson index. (E) The relative abundance of Firmicutes. (F) The relative abundance of Actinobacteriota. (G) The relative abundance of Desulfobacterota. (H) The relative abundance of Bacteroidota. Values are presented as mean ± SD (n = 4 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test. *P< 0.05 Control group; #P < 0.05, ##P < 0.05 vs Model group.

Figure 8 The influence of T. chebula on the species composition of intestinal flora in HUA rats at both the genus and phylum levels. (A) Percent of community abundance on Phylum level. (B) Percent of community abundance on Genus level. Values are presented as mean ± SD (n = 4 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test.

To explore the relationship between alterations in gut microbiota and HUA remission, Spearman correlation analysis was employed to investigate the associations between bacteria and HUA-related metabolic variables at the genus level. The results revealed that Clostridium_innocuum_group, Clostridium_sensu_stricto_1, Erysipelatoclostridium, Subdoligranulum, Megamonas, Faecalitalea, and UBA1819 were positively associated with UA, XOD, IL-6, TNF-α, BUN, Cre, DAO, and D-LAC but negatively correlated with ZO-1, Claudin-1, and Occludin protein levels. In contrast, Monoglobus, DNF00809, and Roseburia were inversely correlated (Figure 9A). Furthermore, T. chebula treatment led to significant reductions in the levels of Clostridium_innocuum_group, Erysipelatoclostridium, UBA1819, Subdoligranulum, and Clostridium_sensu_stricto_1 (P =0.009, P =0.047, P =0.042, P =0.010, P =0.012, P < 0.01 or P < 0.05, Tukey’s HSD) (Figure 9B–F) and significant increases in the levels of Monoglobus, DNF00809, and Roseburia in the model group (P =0.049, P =0.000, P =0.042, P < 0.01 or P < 0.05, Tukey’s HSD) (Figure 9G–I). These findings provide valuable insights into the potential of T. chebula treatment to induce HUA remission by affecting the composition of gut microbiota.

Figure 9 Changes of key gut microbiota among groups. (A) Spearman correlation analysis between intestinal flora and HUA index. (B) The relative abundance of Clostridium_innocuum_group. (C) The relative abundance of Erysipelatoclostridium. (D) The relative abundance of UBA1819. (E) The relative abundance of Subdoligranulum. (F) The relative abundance of Clostridium_sensu_stricto_1. (G) The relative abundance of DNF00809. (H) The relative abundance of Monoglobus. (I) The relative abundance of Roseburia. Values are presented as mean ± SD (n = 4 per group). Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test. *P< 0.05, **P< 0.05 vs Control group; #P < 0.05, ##P < 0.05 vs Model group.

Discussion

Owing to changes in modern lifestyles, the prevalence of HUA has been steadily increasing every year, making it the second most common metabolic disorder after diabetes.¹² Hence, there is an urgent need to develop new anti-HUA drugs that not only reduce uric acid levels but also address other pathological aspects of HUA. The gut-kidney axis has emerged as a critical regulatory mechanism in uric acid metabolism, linking gut microbiota, intestinal barrier integrity, and renal function in a bidirectional relationship. Approximately two-thirds of uric acid is excreted by the kidneys, while the remaining one-third is cleared via the intestines. Impairments in either organ can exacerbate HUA pathology, with disruptions in intestinal homeostasis contributing to systemic inflammation and increased renal burden. This interconnection highlights the importance of therapies that target both gut and kidney health.

In this context, we identified T. chebula as a potential anti-HUA medicine through data mining and experimental validation. Compared to conventional therapies, T. chebula offers a broader therapeutic scope. While medications such as allopurinol primarily target uric acid reduction through xanthine oxidase (XOD) inhibition, T. chebula demonstrated additional benefits, including renal protection, improved intestinal barrier integrity, and modulation of gut microbiota. These multifaceted effects align with the gut-kidney axis framework, addressing key aspects of HUA pathology that are not effectively targeted by existing drugs. By modulating the gut microbiota, enhancing tight junction protein expression (eg, ZO-1, Occludin, and Claudin-1), and alleviating renal inflammation, T. chebula restores balance in the gut-kidney axis, highlighting its potential as a safer and more holistic treatment option.

Huang Shui is considered synonymous with HUA in Tibetan medicine.¹³ Therefore, Huang Shui was chosen as the primary keyword for searching classical Tibetan medical texts and gathering relevant prescriptions. In the frequency analysis, the top 20 high-frequency herbs included T. chebula, Juemingzi, Ruxiang, Shexiang, Muxiang, Doukou, Huangkuizi, Maohezi, Shuichangpu, and Yuganzi, most of which were known to lower uric acid levels.^14–17 For example, Yuganzi extract demonstrated protective effects against acute gouty arthritis by reducing uric acid levels, inhibiting inflammation, and decreasing expression of MMP13.¹⁸ Shuichangpu ethanolic extract reduced serum uric acid, calcium, phosphate, blood urea nitrogen, and creatinine levels in a urolithiatic rat model. Our analysis of Tibetan prescriptions used to treat HUA identified T. chebula as the Tibetan medicine with the highest frequency of occurrence. Surprisingly, there is currently no literature available on the uric acid-lowering effects of T. chebula.

The fruits of Terminalia chebula Retz., commonly referred to as “Hezi” in Chinese, belong to the “medicine food homology” plant and occupy a significant place in traditional Tibetan medicine.¹⁹ Its usage is not limited to Tibet but extends to Southeast Asia and South Asia. T. chebula has various attributes in traditional medicine, including alterative, astringent, carminative, purgative, and stomachic properties.²⁰ T. chebula is well documented for its richness in phenolic compounds such as gallic acid, ellagic acid, hydroxycinnamic acids, and their derivatives; tannins, including terflavin A, terchebulin, punicalagin, chebulagic acid, chebulinic acid, corilagin, and others; and flavonoids such as rutin, quercetin, and methylated derivatives of quercetin.²¹ Existing literature has outlined the various pharmacological actions of T. chebula, including its neuroprotective effect,²² anti-atopic dermatitis activity,¹⁹ antimicrobial properties, anti-joint inflammatory potential,²³ and anti-tumor effects.²⁴ Importantly, this plant does not appear to induce debilitating or toxic side-effects.¹⁵ In the context of Chinese medicine, T. chebula has been found in several prescriptions aimed at addressing hyperuricosis, including formulations such as Ershi WuWei LvXue, Wuwei SheXiang,²⁵ ShiWei RuXiang, ShiWuWei RuPengSan,⁸ and TongFeng TangSan. However, it is noteworthy that there are no reported instances of T. chebula being used for the treatment of HUA.

In this study, we initially assessed the in vitro inhibitory activity of T. chebula on XOD, and the results demonstrated dose-dependent inhibition of XOD by T. chebula. Subsequently, we established a HUA rat model using fructose and potassium oxonate to investigate the uric acid-lowering effects of T. chebula in vivo. Our findings not only confirmed a significant reduction in uric acid levels following T. chebula supplementation but also highlighted its role in restoring gut-kidney axis balance. T. chebula ameliorated renal inflammation by reducing systemic levels of pro-inflammatory cytokines such as IL-6 and TNF-α, likely through the modulation of gut microbiota and the enhancement of intestinal barrier function. These improvements reduce endotoxin translocation, alleviating systemic inflammation and mitigating renal burden.

The kidneys are the primary organs responsible for uric acid excretion, and approximately two-thirds of uric acid is excreted through them.²⁶ HUA progression is often accompanied by renal injury. Elevated UA concentrations can increase renal burden, cause serious renal damage, and even cause chronic kidney disease. Cre and BUN levels are common clinical indicators of renal function, and their elevation often reflects severe renal insufficiency and various other kidney diseases.²⁷ Our research showed that HUA rats experienced serious renal damage, as reflected by significant increases in Cre and BUN levels. The decrease in serum BUN and Cre levels after T. chebula administration in the HUA group prompted T. chebula to effectively alleviate the kidney lesions. These results were confirmed by renal histopathological examination. Collectively, these results suggest that T. chebula supplementation can effectively alleviate renal dysfunction in HUA rats.

The intestine is an important organ for UA excretion, and approximately one-third of the circulating UA is cleared by intestinal enterocytes. UA excretion depends on intestinal homeostasis that is maintained by the intestinal mucosal barrier.²⁸ Serum DAO, D-Lac, and LPS levels are markers of intestinal permeability. DAO is an intracellular enzyme in the intestinal epithelium, whereas D-LAC and LPS are bacterial metabolites produced by intestinal flora.²⁹ Elevated serum levels of DAO, D-Lac, and LPS indicate increased intestinal permeability. After T. chebula intervention, the levels were significantly reduced. The apical junctional complex, a structure that holds intestinal cells together, is essential for maintaining the barrier that prevents harmful substances from entering the bloodstream. Claudin-1, Occludin, and ZO-1 are primary tight junction proteins. In the present study, immunohistochemistry and Q-PCR results showed that T. chebula supplementation significantly increased the reduced protein expression of claudin-1, occludin, and ZO-1 induced by HUA. These results indicate that administration of T. chebula contributes to the restoration of the intestinal barrier in HUA rats.

Furthermore, disruption of intestinal barrier integrity can lead to the translocation of bacteria or bacterial products, such as lipopolysaccharide (LPS), into the bloodstream, triggering the innate immune system and causing chronic systemic inflammation and metabolic disorders.³⁰ In the model group, serum levels of inflammatory cytokines (IL-6, IL-1β, and TNF-α) were significantly higher than those in the control group, confirming the presence of systemic low-grade inflammation. Furthermore, studies have shown that chronic inflammation induced by elevated serum LPS levels is often accompanied by increased XOD activity.³¹ Administration of T. chebula effectively alleviated inflammatory reactions and XOD activity in rats with HUA.

Numerous studies have shown that intestinal flora is an important factor related to HUA.^32–34 According to current research, the effect of intestinal flora on HUA is mainly achieved in three ways: participation in purine metabolism, decomposition of uric acid, and reduction of uric acid levels.³⁵ Metabolites produced by intestinal flora promote the excretion of uric acid, repair the intestinal barrier, and reducing inflammation associated with HUA.³⁴ In our study, T. chebula treatment significantly changed the composition and structure of intestinal flora in HUA rats. Consistent with previous studies,^36–38 at the phylum level, rats in the model group showed a lower abundance of Firmicutes and an increased abundance of Bacteroidetes than rats in the control group. Intervention with T. chebula has the potential to reverse such variations in Firmicutes and Bacteroidota.

At the genus level, our correlation analysis revealed that several bacteria, including Clostridium innocuum, Clostridium_sensu_stricto_1, Erysipelatoclostridium, Subdoligranulum, Megamonas, Faecalitalea, and UBA1819, were risk factors for HUA, while Monoglobus, DNF00809, and Roseburia were protective factors. These findings are consistent with those of previous studies reporting similar results. For example, previous research has demonstrated a positive correlation between Clostridium spp. and uric acid levels.^39,40 Erysipelatoclostridium, a potential opportunistic pathogen, was enriched in patients with gout,⁴¹ and its relative abundance increased after exposure to uric acid, whereas JL-3 treatment reduced its abundance.²⁹ Additionally, Liu et al found that Subdoligranulum was more abundant in the intestinal tract of rats with high purine-induced HUA than in normal rats.⁴² In contrast, our study also observed variations in UBA1819 levels, as end-stage diabetic kidney patients exhibited a higher abundance of this bacterium, which was inversely correlated with UA levels.⁴³ Our results demonstrate that T. chebula can effectively reduce the levels of these harmful bacteria, including Clostridium_innocuum_group, Erysipelatoclostridium, UBA1819, Subdoligranulum, and Clostridium_sensu_stricto_1. Additionally, an in vitro study showed that T. chebula extract displayed strong inhibitory activity against Clostridium perfringens with no adverse effects on the growth of several probiotics.⁴⁴

One major limitation of this study is the reduced sample size (n = 4) for microbiota analysis, which limits the statistical power and generalizability of the findings. This small sample size was due to sample contamination and logistical constraints during the experimental process. While the results are consistent with previous literature on hyperuricemia-related gut microbiota changes, they should be interpreted as exploratory. Future studies with larger cohorts are essential to confirm the observed microbial shifts and establish stronger statistical significance.

In addition, T. chebula significantly increased the levels of certain beneficial bacteria including Monoglobus, DNF00809, and Roseburia. It has been reported that both Monoglobus and Roseburia possess the ability to ferment dietary fiber, facilitating butyrate production.^45,46 Butyrate can enhance intestinal barrier function by inducing the expression of tight junction proteins such as ZO-1 and Occludin.⁴⁷ In our study, the increased expression of ZO-1 and Occludin was attributed to the increased production of butyrate. Roseburia-derived butyrate has been shown to suppress gout flares by reducing the production of IL-1β, IL-6, and IL-8 induced by monosodium urate (MSU) crystals.⁴⁸ Furthermore, Roseburia is associated with improved renal function.⁴⁹ These findings provide valuable insights into the potential of T. chebula treatment to induce HUA remission by affecting the composition of gut microbiota. However, further research is needed to fully understand the mechanism by which T. chebula regulates the gut microbiota.

While our study highlights potential gut microbiota changes linked to T. chebula treatment, the limited sample size weakens the statistical robustness of these findings. As such, these results provide a foundation for future research rather than definitive conclusions about the relationship between microbiota modulation and HUA treatment. To address the limitations of this study, future research should involve larger cohorts to increase statistical power and confirm the observed microbial shifts. Additionally, integrating functional metagenomics or metabolomics could provide deeper mechanistic insights into how specific microbial changes mediate the therapeutic effects of T. chebula.

Conclusion

In conclusion, our study revealed that T. chebula is a promising therapeutic agent for HUA treatment based on the rich heritage of traditional Tibetan medicine. Using data mining and experimental validation, we found that T. chebula extract demonstrated efficacy in reducing serum uric acid levels, mitigating renal injury, enhancing intestinal barrier function, and modulating gut microbiota composition in a HUA rat model. These findings highlight T. chebula as a safe and effective herb for the management of HUA. Furthermore, our research underscores the value of traditional Tibetan medicine in addressing metabolic disorders, and offers a promising avenue for developing alternative treatments for HUA and related conditions.