Dioscin Ameliorates Hyperuricemia-Induced Atherosclerosis by Modulating of Cholesterol Metabolism through FXR-Signaling Pathway

Hyperuricemia is one of the independent risk factors for atherosclerotic cardiovascular disease. Herein, we investigate the association between uric acid and cholesterol metabolism and the effect of dioscin on the prevention of hyperuricemia-induced atherosclerosis. In the potassium oxonate-treated ApoE−/−−/− mice, atherosclerosis was accelerated along with elevated serum cholesterol levels in the hyperuricemic state, which can be ameliorated by dioscin. Together with the in vitro assays, we found that the effect of dioscin was at least partially through the regulation of the farnesoid X receptor (FXR) -small heterodimer partner (SHP) -7α-hydroxylase (CYP7A1) signaling pathway in the liver. Tigogenin (a metabolite of dioscin) suppressed FXR activation and increased CYP7A1, resulting in an increased conversion rate of cholesterols into bile acids. Further clinical study revealed that treatment with a dioscin-enriched preparation decreased serum cholesterol levels in individuals with hyperuricemia. In summary, this study demonstrated a slowdown effect of dioscin on the progression of hyperuricemia-induced atherosclerosis.


Introduction
Hyperuricemia is defined as a serum uric acid level higher than 420 µmol/L (7.0 mg/dL) [1]. Epidemic studies have demonstrated that the prevalence of hyperuricemia in China was 13.3 %, among which, the male and female prevalence was around 19.4% and 7.9%, respectively [2]. The total affected population was up to 180 million in 2017. Hyperuricemia is one of the independent risk factors for arterial atherosclerosis, and has been known to increase the mortality of cardiovascular diseases [3,4]. The 2018 European guidelines for the treatment of high blood pressure highlighted that hyperuricemia is independently associated with the development of atherosclerotic cardiovascular disease [5].
Currently, the most common medications for hyperuricemia include xanthine oxidase inhibitor (XOI) (reducing the production of uric acid) and uricosurics that increase the excretion of uric acid. XOIs such as febuxostat decrease uric acid production by inhibiting xanthine oxidase [6]. However, compared with lifestyle modifications, 24 months of

Animal
All animal experimental designs were approved by the Science and Technological Committee and the Animal Use and Care Committee of TJUTCM (No. TCM-LAEC2021010). Eight-wee-old male C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). ApoE deficient (ApoE −/− ) mice, male, SPF grade, 8 weeks old, were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All animals had free access to standard diet and water and were housed in experimental conditions at 25 ± 2 • C with humidity of 60 ± 5% in a fixed 12 h artificial light period.
The C57BL/6J mice were randomized into the normal control group (n = 8) and hyperuricemia group (n = 8). The mice of the hyperuricemia group received PO (200 mg/kg body weight) and adenine (50 mg/kg body weight) through gastric gavage, and the normal control mice received saline with the same volume. The treatment was applied once a day in the morning for 21 consecutive days. At the end of the administration, mice were euthanized by an overdose of isoflurane; blood and liver samples were collected and stored at −80 • C until analysis.
ApoE −/− mice were randomly allocated into the control ApoE −/− mice group (n = 10), PO-induced hyperuricemic ApoE −/− mice group (n = 10) and dioscin-treated hyperuricemic ApoE −/− mice group (n = 10). All ApoE −/− mice were fed with an HCD. The hyperuricemic ApoE −/− mice group (dose: 250 mg/kg, volume: 20 mL/kg, in saline) was treated with PO intraperitoneal injection once a day and the control ApoE −/− mice received saline injection with the same volume continuously for 3 months. The dioscintreated hyperuricemic ApoE −/− mice received daily treatment of dioscin (100 mg/kg body weight) through gastric gavage for 3 months. At the end of the administration, mice were euthanized by an overdose of isoflurane blood; liver, ileum, and the main artery were quickly and carefully dissected on an ice plate. All specimens were stored at −80 • C for further analysis.

Uric Acid and Lipids Analysis
UPLC analysis was used to determine the serum uric acid level of mice as described previously [18]. The total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels of serum were determined with the commercial kits (BioSino Biotechnology and Science Inc., Beijing, China) according to the manufacturer's instructions. Hepatic TC and triglycerides (TG) were extracted using the previous method [19] and then were determined with the same commercial kits described above.

Histological Oil Red O Staining
Liver sections were fixed in 10% formalin overnight, embedded in paraffin, sectioned, and then stained with Oil Red O, according to the manufacturer's protocol. Images of the sections were obtained with Axio Imager D2 (Zeiss, Oberkochen, Germany).

Atherosclerotic Lesions Analysis
Aortic lesions were determined as previously described [20]. Briefly, after euthanasia, the mouse whole aorta was harvested. The aorta was then fixed in 4% paraformaldehyde. After removal of outside connecting tissue and fat, the aorta was stained with Oil Red O solution, and lesions in en face aorta were quantified by computer-assisted image analysis, and calculated as percent lesion area. To determine the sinus lesions in the aortic root, 5 µm frozen sections of the aortic root were collected and stained with Oil Red O solution. Images of the sections were obtained with Axio Imager D2 (Zeiss, Oberkochen, Germany). The total morphometric lesion area was determined with a computer-assisted image analysis procedure.

Bile acid Profile Analysis
Serum samples of hyperuricemia individuals or mice (100 µL) were mixed with 1 mL ice-cold acetonitrile, and then vortexed vigorously. After keeping on ice for 30 min, the mixture was centrifuged at 14,000× g for 10 min under 4 • C. The supernatant was evaporated under nitrogen flow. The residue was re-dissolved in 50 µL of 100% methanol, and centrifuged at 20,000× g for 10 min under 4 • C. The supernatant was used for liquid chromatography-mass spectrometry (LC-MS) analysis.
For liver and ileum samples of mice, approximately 50 mg liver and 20 mg ileum were homogenized in 5 volumes of ddH 2 O, NDCA (100 ng) was added as an internal standard to 250 µL of tissue homogenate, and vortexed thoroughly. The homogenate was then added with 1.25 mL of ice-cold acetonitrile, and shaken vigorously. Afterward, the processing steps were the same as serum samples.
Detection and quantification were achieved using the Waters UPLC system (Waters Corporation, Milford, MA, USA) coupled to an Exactive™ Plus Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source (ThermoFisher Scientific, Waltham, MA, USA).
The separation was accomplished on a Waters ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm, Waters Co, Milford, MA, USA). The column temperature was 55 • C, the flow rate was 0.4 mL/min and the injection volume was 2 µL. The mobile phase consisted of 0.01% acetic acid in acetonitrile: H 2 O (80:20, mobile phase A) and 0.01% acetic acid in acetonitrile: H 2 O (20:80, mobile phase B). The gradient elution of the mobile phase was shown in Supplementary Table S1.

QRT-PCR
RNA isolation, cDNA synthesis, and qRT-PCR analysis were performed as described previously [18]. The primers used for qRT-PCR were synthesized by Dingguo Bio Co. Ltd., Shanghai, China. Sequences used for qRT-PCR were shown in Supplementary Table S2 (primer for mice) and Supplementary Table S3 (primer for HepG2 cells). Results were presented as levels of expression relative to those of controls after normalization to GAPDH using the 2− CT methods. Analysis was carried out in triplicates.

FXR Mediated Coactivator Recruitment Assay
FXR coactivator recruitment assay was performed according to the manufacturer's instructions (Invitrogen, Waltham, MA, USA). The activity of FXR was determined in Black, 384-well assay plates from PerkinElmer Life Sciences. FXR-LBD (5 nM) was added to the test compounds followed by the addition of a mixture of the fluorescein-coactivator peptide (500 nM) and terbium anti-GST antibody (5 nM); CDCA was included in assays as the positive controls. After an incubation period at room temperature, the fluorescence emission signals were detected at 520 nm and 495 nm. The TR-FRET ratio of 520:495 was used for the calculation of the EC 50 and IC 50 using the curve-fitting software GraphPad Prism version 8.0 (San Diego, CA, USA).

Clinic Trial of Dioscin-Enriched Preparation in Hyperuricemia Individual
A randomized controlled trial was conducted at Metabolic Diseases Hospital, Tianjin Medical University, Tianjin, China, from September 2017 to December 2018, in accordance with the International Council for Harmonization guidelines. Each subject provided written informed consent before participation. The trial was approved by the Tianjin Medical University (DXBYYhMEC2017-23-2) and registered with the Chinese Clinical Trial Registry (number ChiCTR-IPR-17014035).
Sixty adults aged 18-60 with fasting serum uric acid levels higher than 420 µmol/L were enrolled. Participants were excluded if they had serious cardiovascular, respiratory, excretory, digestive, mental illness, or gout. After inclusion, the participants were randomized into two arms, which were the placebo group and the dioscin-enriched preparation (commercial name: Di'ao Xinxuekang Capsule) (DA) group. DA group received 600 mg of DA per day for one month, and the control group received a placebo. DA was a kind gift from Di'ao Group Chengdu Pharmaceutical Co. Ltd. (100 mg/capsule, lot: 1701020.) Serum uric acid and cholesterol levels were determined.

Statistical Analysis
SPSS 20.0 statistical software (version 20, SPSS; IBM, Armonk, NY, USA) was used to perform statistical analysis. Data were expressed as the mean ±S.E.M. Shapiro-Wilk test was used to assess the normal distribution of variables. Pearson's correlation analyses were used to evaluate the correlation between the changes in serum uric acid and TC. Paired t-test was used to compare the differences between day 0 and one month later. For two independent (unrelated) groups, an independent t-test was used to compare the differences. For three or more independent (unrelated) groups, significant differences were evaluated by one-way analysis of variance (ANOVA); LSD and Dunnett's tests were used for post hoc evaluations with the normal distribution of variables. Kruskal-Wallis test was used to assess differences with non-normally distributed data. p < 0.05 was considered to represent a statistically significant difference.

Hepatic CYP7A1 Expression Was Reduced in Hyperuricemia Mice Associated with an Elevation in Serum Cholesterol Levels
As previously reported, hyperuricemia can induce dysfunction of cholesterol metabolism, which was considered a key factor in the occurrence and development of atherosclerosis. However, the mechanism is unclear. Firstly, PO-treated mice were used to clarify the pathophysiological linkage between uric acid and cholesterol.
As shown in Figure 1A,B, compared with the normal group, PO and adenine treatment significantly increased the serum uric acid and cholesterol levels by 61.8% and 15.7%, respectively, in the hyperuricemia group. The high uric acid level did not affect the expression of cholesterol synthesis enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), but reduced expression of CYP7A1 by 70.2% in the liver of hyperuricemia mice ( Figure 1C). These data suggest that the dysfunction of cholesterol metabolism induced by hyperuricemia may be related to the conversion process of cholesterols to bile acid. Figure 1A,B, compared with the normal group, PO and adenine treatment significantly increased the serum uric acid and cholesterol levels by 61.8% and 15.7%, respectively, in the hyperuricemia group. The high uric acid level did not affect the expression of cholesterol synthesis enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), but reduced expression of CYP7A1 by 70.2% in the liver of hyperuricemia mice ( Figure 1C). These data suggest that the dysfunction of cholesterol metabolism induced by hyperuricemia may be related to the conversion process of cholesterols to bile acid.

Elevation of Serum Cholesterol Level was Aggravated in ApoE −/− Mice under Hyperuricemic Condition
Compared with the control ApoE −/− mice group, the serum levels of uric acid and TC were significantly increased by 41.5% and 18.6%, respectively, in the PO-treated hyperuricemic ApoE −/− mice group (Figure 2A,B). In addition, compared with the control ApoE −/− mice group, PO administration also significantly increased the serum LDL-C level (70.9%) and decreased the HDL-C level (24.9%) ( Figure 2C,D). We also determined the hepatic TC and TG levels, and the results showed that PO treatment significantly increased hepatic TC and TG levels by 52.5% and 94.6%, respectively ( Figure 2F,G). Oil Red O staining revealed that the marked hepatic steatosis was aggravated in the hyperuricemic ApoE −/− mice group ( Figure 2E).   We further detected the bile acid profile in different tissues. The contents of conjugated bile acids (TUDCA, TCA, TCDCA, TαMCA, and TβMCA), unconjugated bile acids (CA, αMCA, βMCA, UDCA, and CDCA), and secondary bile acids (DCA and LCA) were measured. Compared with the control ApoE −/− mice, the content of serum βMCA markedly decreased by 62.3% ( Figure 3E); the contents of hepatic CA, βMCA, DCA, and TCA significantly decreased by 31.7%, 36.8%, 67.6%, and 40.5%, respectively ( Figure 3G); and the content of ileal TCA markedly decreased by 72.1% in PO-treated hyperuricemic ApoE −/− mice ( Figure 3I).
These results indicated that the conversion efficiency from cholesterol to bile acid decreased under hyperuricemic conditions, leading to dysfunction of cholesterol metabolism.

Hepatic FXR Was Activated and CYP7A1 Was Down-Regulated in PO-Induced
Hyperuricemic ApoE −/− Mice CYP7A1 is the rate-limiting enzyme of bile acid synthesis in the liver. The expression and activity of CYP7A1 determine the conversion rate of cholesterols to bile acid. The expression of CYP7A1 is mainly regulated through two pathways in the gut-liver axis: FXR-small heterodimer partner (SHP) [21] and fibroblast growth factor (FGF15) -fibroblast growth factor receptor 4 (FGFR4) [22]. In liver, FXR can directly activate the transcription of SHP, and the latter negatively regulates CYP7A1 and other CYPs (oxysterol 7α-hydroxylase (CYP7B1), sterol 12α-hydroxylase (CYP8B1), etc.) expression. In ileum, FXR activation promotes the release of FGF15, which enters the liver via enterohepatic circulation, binds to FGFR4, and then represses hepatic CYP7A1.
As shown in Figure 4, compared with the control ApoE −/− mice, the mRNA levels of hepatic FXR, SHP, and FGFR4 were significantly upregulated by 59.0%, 45.7%, and 105.4%, respectively, in hyperuricemic ApoE −/− mice. The mRNA levels of hepatic CYP7A1, CYP7B1, and CYP8B1 were significantly downregulated by 42.8%, 52.6%, and 28.6%, respectively. The mRNA levels of ileal FXR and FGF15 showed tendencies to increase in hyperuricemic ApoE −/− mice (Figure 4).  Figure 3I). These results indicated that the conversion efficiency from cholesterol to bile acid decreased under hyperuricemic conditions, leading to dysfunction of cholesterol metabolism.

Hepatic FXR Was Activated and CYP7A1 Was Down-Regulated in PO-Induced
Hyperuricemic ApoE −/− Mice CYP7A1 is the rate-limiting enzyme of bile acid synthesis in the liver. The expression and activity of CYP7A1 determine the conversion rate of cholesterols to bile acid. The expression of CYP7A1 is mainly regulated through two pathways in the gut-liver axis: FXRsmall heterodimer partner (SHP) [21] and fibroblast growth factor (FGF15) -fibroblast growth factor receptor 4 (FGFR4) [22]. In liver, FXR can directly activate the transcription of SHP, and the latter negatively regulates CYP7A1 and other CYPs (oxysterol 7α-hydroxylase (CYP7B1), sterol 12α-hydroxylase (CYP8B1), etc.) expression. In ileum, FXR activation promotes the release of FGF15, which enters the liver via enterohepatic circulation, binds to FGFR4, and then represses hepatic CYP7A1.
As shown in Figure 4, compared with the control ApoE −/− mice, the mRNA levels of hepatic FXR, SHP, and FGFR4 were significantly upregulated by 59.0%, 45.7%, and 105.4%, respectively, in hyperuricemic ApoE −/− mice. The mRNA levels of hepatic CYP7A1, CYP7B1, and CYP8B1 were significantly downregulated by 42.8%, 52.6%, and 28.6%, respectively. The mRNA levels of ileal FXR and FGF15 showed tendencies to increase in hyperuricemic ApoE −/− mice ( Figure 4).  These results indicated that the decrease in conversion efficiency under hyperuricemic conditions is at least in part related to the FXR-CYP7A1 axis, thereby disturbing the balance between cholesterols and bile acids.  Figure 2H-K). These results suggested that dioscin prevented the atherosclerosis progress that was exacerbated by hyperuricemia.
Dioscin treatment markedly reduced serum uric acid and cholesterol levels by 18.6% and 23.1%, respectively, compared with the control hyperuricemic ApoE −/− mice group (Figure 2A,B). However, there were no significant changes in the serum LDL-C and HDL-C levels ( Figure 2C,D). In the liver, dioscin treatment significantly decreased the TG level by 34.3% but had no significant effect on the TC level ( Figure 2G). Oil Red O staining revealed marked hepatic steatosis in the liver of the hyperuricemic ApoE −/− mice group, which was attenuated in the dioscin-treated group ( Figure 2E). These results indicated that dioscin improved uric acid and dysfunction of lipid metabolism.  Figure 3G). However, dioscin treatment had no effect on the bile acid profile in the ileum ( Figure 3I). These results demonstrated that dioscin administration modulated the hepatic bile acid profile in hyperuricemic ApoE −/− mice.

Dioscin Treatment Inhibited Hepatic FXR Signaling
Compared with the control hyperuricemic ApoE −/− mice, treatment with dioscin significantly decreased the mRNA levels of hepatic FXR and SHP by 66.3% and 36.8%, respectively, while there was no significant difference in the level of FGFR4. Moreover, the mRNA levels of hepatic CYP7A1, CYP7B1, and CYP8B1 significantly increased by 34.5%, 67.1%, and 56.2%, respectively. However, there were no significant differences in the mRNA levels of intestinal FXR and FGF15 between the dioscin-treated and the control groups (Figure 4). These results indicated that dioscin treatment promoted the conversion efficiency from cholesterols to bile acid by inhibiting FXR, thereby maintaining cholesterol homeostasis.

Dioscin Inhibited FXR through Its Metabolite Tigogenin
The above results demonstrated that the hepatic FXR signaling was suppressed by dioscin. We hypothesized that dioscin or its metabolites may act as FXR antagonists. We performed the TR-FRET FXR coactivator assay to test our hypothesis. Using this system, the EC 50 of CDCA, a natural ligand of FXR, was 10.5 µM. Dioscin and its metabolites (diosgenin and sarsasapogenin) did not show agonistic actions on the activation of FXR by CDCA ( Figure 5A). However, tigogenin, a metabolite of dioscin and an epimer of sarsapogenin, showed a strong competitive inhibition effect on the CDCA (100 µM) -mediated recruitment of SRC2 to FXR, with an IC 50 of 85.1 µM ( Figure 5B).
Nutrients 2022, 14, x FOR PEER REVIEW 12 of 20 CDCA ( Figure 5A). However, tigogenin, a metabolite of dioscin and an epimer of sarsapogenin, showed a strong competitive inhibition effect on the CDCA (100 μM) -mediated recruitment of SRC2 to FXR, with an IC50 of 85.1 μM ( Figure 5B). In addition, the expression levels of FXR target genes in HepG2 cells were determined with or without tigogenin treatment. Consistently, the mRNA levels of FXR and its target genes SHP and bile salt export pump (BSEP) were approximately 0.5-fold, 1.6-fold, and 6.8fold higher, respectively, in the CDCA-treated group than in the control group. Compared to treatment with 50 µM CDCA alone, the mRNA levels of FXR, SHP, and BSEP significantly decreased by 0.6-fold, 0.4-fold, and 1.0-fold, respectively, in the presence of 20 µM tigogenin ( Figure 5C). These results indicated that tigogenin may be the active metabolite of dioscin on suppression of FXR, modulating the conversion of cholesterols to bile acid.

Treatment with Dioscin-Enriched Preparation Ameliorates Hypercholesterolemia in Hyperuricemia Patients
We used a dioscin-enriched preparation, whose commercial name is Di'ao Xinxuekang, a post-marketing herbal capsule in China and the Netherlands, to explore the activity of dioscin on hyperuricemia-induced dysfunction of cholesterol metabolism. The active ingredient (Dioscin) of DA is extracted from the rhizomes of Dioscorea panthaica Prain and Burkill. As literature reported [23], after hydration, ring cyclization, and deglycosylation by intestinal microflora and hepatic drug enzyme, spirostanols in DA were metabolized to dioscin or its aglycon as the main metabolites commonly found in serum.

Description of Patients and Interventions
A total of 60 participants aged 18 to 60 years old whose serum uric acid levels were higher than 420 µmol/L were enrolled. The participants were randomized into two arms, which were the control group (n = 27) and the DA group (n = 33). The DA group received 600 mg of DA per day for one month. A total of 8 out of 33 patients in the DA group and 16 out of 27 patients in the control group did not complete the study. Twenty-five patients (75.8%) in the DA group and eleven patients (40.7%) in the control group were included in the analysis of the primary endpoint ( Figure 6).

Demographic and Baseline Characteristics
The baseline demographics and clinical characteristics of the participants are shown in Table 1. There was no significant difference between the DA group and the control group at baseline. Mean baseline serum uric acid was 497.45 ± 43.73 μmol/L in the control group and 514.22 ± 57.33 μmol/L in the DA group, respectively. The mean baseline cholesterol level was 4.89 ± 0.87 mmol/L in the control group and 5.08 ± 1.01 mmol/L in the DA group, respectively.

Demographic and Baseline Characteristics
The baseline demographics and clinical characteristics of the participants are shown in Table 1. There was no significant difference between the DA group and the control group at baseline. Mean baseline serum uric acid was 497.45 ± 43.73 µmol/L in the control group and 514.22 ± 57.33 µmol/L in the DA group, respectively. The mean baseline cholesterol level was 4.89 ± 0.87 mmol/L in the control group and 5.08 ± 1.01 mmol/L in the DA group, respectively.

Serum Uric Acid and Cholesterol Analysis
DA administration led to decreased levels of serum uric acid from 514.22 ± 57.33 to 488.24 ± 82.61 µmol/L (p = 0.034) during the treatment period. (Figure 7A). However, there was no significant change in the serum uric acid level f in the control group (p = 0.101). There was no significant difference in the serum lipid levels between the DA group and the control ( Figure 7B-E). LDL-C levels had a significant increase from the baseline in the control group, but not in the DA group ( Figure 7C). Furthermore, a subgroup analysis was carried out to evaluate the relationship between UA and cholesterol. The result indicated that patients with a TC > 5.17 mmol/L at baseline showed significant reductions (p = 0.010) in TC and LDL-C levels (p = 0.020) from baseline in the DA group ( Figure 7G,H). However, there was no significant reduction in the serum HDL-C and TG levels ( Figure 7I,J). Of note, Pearson's analysis showed that the change in serum uric acid levels was negatively correlated with cholesterol levels (r = −0.462, p = 0.030) after DA treatment for one month ( Figure 7F).

The BA Profile Changes after Oral Administration of Dioscin-Enriched Preparation
To determine the effect of DA treatment on the bile acid profile, an LC-MS approach was used to analyze the bile acid in the serum of hyperuricemia patients. As shown in Figure 7K-N, in the DA-treated subjects with a serum uric acid TC > 5.17 mmol/L at baseline, there was a tendency for the elevation of serum TBA and CBA (TUDCA and GCDCA, etc.) after the treatment.
The clinical data confirmed the relationship between uric acid and cholesterol, and further clarified that tigogenin was the active metabolite of dioscin on suppression of FXR, modulating the conversion of cholesterols to bile acid.

Discussion
Although hyperuricemia is not a disease, it is known that long-term elevated uric acid levels are associated with gout, hypertension, diabetes, renal diseases, and cardio-cerebral vascular diseases. Clinical studies have shown that every 1 mg/dl increase in serum uric acid level was associated with a 23% higher incidence of atherosclerosis [24]. As a result, several clinical guidelines have listed hyperuricemia as one of the important risk factors for cardiovascular diseases in recent years [5]. However, it has been difficult to implement effective clinical interventions for hyperuricemia-related atherosclerosis because the underlying mechanisms remain unclear.
It is estimated that the prevalence of lipid abnormalities in the overall hyperuricemia individual was approximately 51% [25]. The clinical trials reported that XOI treatment did not play a beneficial role in preventing atherosclerosis [7], which indicates that uric acid is not a direct cause of cardiovascular disease. Hyperuricemia is associated with abnormal lipid metabolism, especially with hypertriglyceridemia and hypercholesterolemia [26]. Given that hypercholesterolemia is a well-documented risk factor for cardiovascular diseases [27], we deemed it is necessary to further investigate the mechanism of hyperuricemia-related dysfunction of cholesterol metabolism, aiming at providing guidance for precise therapy.
We found in this study that hyperuricemia induced the activation of FXR, which lowered the conversion of cholesterols converted to bile acids, causing increased cholesterol levels and subsequent atherosclerosis. Meanwhile, we found that Tigogenin, a metabolite of dioscin, inhibited the activation and upregulation of FXR by hyperuricemia, and improved cholesterol homeostasis. This had been supported by a small-scale clinical trial. This study shed light on the precise treatment of the hyperuricemia-related abnormality of cholesterol metabolism.
Dysfunction of cholesterol metabolism is linked to other primary diseases with various biochemical and pathological mechanisms. Hypothyroidism modifies the content and signaling of lipids by a reduction in the expression of perilipin A and proliferator-activated receptor δ [28]. Patients with chronic kidney disease have downregulation of the expression of hepatic apolipoprotein A-I and lecithin cholesterol acyltransferase, which results in impaired high-density lipoprotein-mediated cholesterol uptake from vascular tissue [29]. Patients with nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis showed significant up-regulation of protease-activated receptor 2, accompanied by increasing de novo lipogenesis, and leading to the dysfunction of cholesterol metabolism [30].
Dysfunction of cholesterol metabolism occurs with different mechanisms [13]. Some clinical studies have shown that the effect of statins on reducing the risk of cardiovascular diseases varied tremendously in treating different types of hypercholesterolemia due to different pathogeneses; although, statins do lower serum cholesterol levels consistently [31].
For example, in a NAFLD patient, statins significantly lowered the cardiovascular risks without unexpected adverse events [32]. By contrast, a clinical trial showed that statins had no significant effect on the primary composite end point of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke in hemodialysis patients [33]. Therefore, the risk of cardiovascular disease can only be effectively reduced by pertinent mechanism-based treatments for hypercholesterolemia.
We demonstrated in this study for the first time that the synthesis and absorption of cholesterols did not change significantly in the hyperuricemic state. However, the hepatic FXR was abnormally activated, which caused the inhibition of CYP7A1 expression and lowered the conversion rate of cholesterols to bile acids. This led to increased cholesterol levels and subsequent atherosclerosis. The abnormal cholesterol metabolism in the hyperuricemic state was mainly related to the dysregulation of the FXR-CYP7A1 axis. Current commonly used medications for hyperlipidemia include, but are not limited to: statins that lower cholesterol synthesis; ezetimibe, which inhibits cholesterol absorption; and proprotein convertase subtilisin/kexin type 9, which lowers LDL receptors. The pharmacological actions of these medications are not through the FXR-CYP7A1 axis. Therefore, it is impossible to provide precise therapy for hyperuricemia-related dysfunction of cholesterol metabolism by using these drugs.
Dioscin is widely distributed in herbal medicine and foodstuff, with anti-fungal, antiviral [34], hepatoprotective [35], and anti-hyperuricemia [16] pharmacological activities. In this study, we found that oral dioscin lowered serum uric acid and cholesterol levels, and delayed the progression of atherosclerotic plaque formation.
As a spirostane glycoside, after gut microbiota and liver metabolism, dioscin would be converted to tigogenin, sarsasapogenin, and diosgenin, which contributed to its bioactivity in vivo [23]. Within the metabolites of dioscin, tigogenin showed inhibition of the effect on FXR activation with an IC 50 of 85.1 µM. In vitro activity screening assay followed by in vivo activity study showed that tigogenin significantly suppressed FXR activation and increased CYP7A1 levels, and enhanced the conversion of cholesterols to bile acids. Interestingly, tigogenin and sarsasapogenin, two main metabolites of dioscin, are epimers that structurally differ only in the spatial arrangement of the 25-methyl group. However, their actions of activating FXR are tremendously different.
Further clinic study revealed dioscin significantly improved cholesterol metabolism in subjects with hyperuricemia after one-month oral administration. Our previous research reported that dioscin could decrease serum uric acid levels through increasing renal and intestinal uric acid excretion [16]. The current study further clarified the therapeutic action of dioscin on hyperuricemia-related dysfunction of cholesterol metabolism. Dioscin is abundant in many natural products. It is apparently a potential drug and/or medicinal food for hyperuricemia-related dysfunction of cholesterol metabolism.
In this paper, the initial design of the clinical research was based on the evaluation of the uric acid-lowering activity of dioscin, and hyperuricemia was defined as the key inclusion criteria of the target population, which led to the small sample size in the subgroup analyses. Our next step is to increase the recruitment of individuals with hyperuricemia as well as hypercholesterolemia to further the prevention effect and mechanisms of dioscin on hyperuricemia-induced dysfunction of cholesterol metabolism.

Informed Consent Statement:
A randomized controlled trial was conducted at Metabolic Diseases Hospital, Tianjin Medical University, Tianjin, China, from September 2017 to December 2018, in accordance with the International Council for Harmonization guidelines. Each subject provided written informed consent before participation. The trial was approved by the Tianjin Medical University (DXBYYhMEC2017-23-2).