Red Clover Isoflavones Influence Estradiol Concentration, Exercise Performance, and Gut Microbiota in Female Mice

In red clover (Trifolium pratense L.; RC) the main compound is isoflavones, which are selective estrogen receptor modulators for maintaining female health. Isoflavones exert antifatigue effects during exercise in high-temperature environments. This study aimed to investigate the effect of RC supplementation on gut microbiota composition to determine whether it improves intestinal barrier function and exercise performance. Female ICR mice were divided into four groups (n = 8 per group) and orally administered RC once daily for 6 weeks at 0 (vehicle), 308 (RC-1X), 615 (RC-2X), and 1,538 (RC-5X) mg/kg. RC supplementation decreased the fat mass and increased exhaustive swimming time, grip strength, and muscle glycogen in female mice. In the RC supplementation group, serum levels of lactate, ammonia, and creatine kinase decreased after swimming. The estradiol and progesterone levels were higher in the RC group than in the vehicle group. Regarding gut microbiota composition, the RC-2X group may increase intestinal health related to the microorganisms Pseudobutyrivibrio and Parabacteroide. Thus, the use of RC supplements as nutraceuticals could have positive effects on athletes' gut and overall health.


INTRODUCTION
Red clover (Trifolium pratense L.; RC) is one of the most important leguminous plants of temperate zones in Europe and Asia, and it is also a short-term perennial herb (1,2). RC contains high levels of isoflavones (genistein, daidzein, biochanin A, and formononetin) (3). With the discovery of the medicinal value of RC, many studies have shown that the chemical properties of isoflavones extracted from RC are similar to those of estrogen and can thus regulate estrogen levels in the human body. RC is one of the major sources of isoflavones (4,5). In recent years, RC has been used in ameliorating menopausal symptoms, such as hot flush, night sweats, urogenital atrophy, depression, and anxiety, in postmenopausal women (6). In addition, RC can contribute to postprandial blood glucose level reduction, improves hypoglycemia, is an antioxidant, and has effects against obesity in a rat model (7) and osteoporosis in postmenopausal women (3,(7)(8)(9)(10).
Estrogen plays physiological roles in reproductive organs and nonreproductive organs/tissues (11). Nagai et al. (11) showed that estrogen is a sex steroid hormone beneficial for exercise endurance and energy expenditure in female mice. Studies have shown that estrogen can promote the expression of myogenic differentiation 1 mRNA and muscle protein synthesis in postmenopausal women (12). It has a positive effect on the growth and regeneration of skeletal muscles and can promote recovery after sports injury (13,14). Furthermore, estrogen is beneficial in maintaining muscle strength (15). Estrogen therapy or phytoestrogen supplementation may enhance the effects of exercise on bones and muscles mass (16).
Some studies have shown that strenuous exercise can reduce blood flow to the intestinal wall (17)(18)(19). Endurance athletes in particular are more likely to develop gastrointestinal diseases (20). Supplementation of isoflavones through a fermented red clover extract reduces markers of protein degradation (21), increases lean body mass and muscle mass in postmenopausal women (22).
Thus, we used RC, which has detectable estrogen activity and contains a high amount relative effect on estradiol (E2), as a supplement to determine the relationship between exercise performance and the gut microbiota. This study demonstrated that RC improved exercise performance and did not harm the intestinal health of female mice.

Animals and Experiment Design
The RC extract (T. pratense) was purchased from NOW FOOD (IL, USA). Female ICR mice (6 weeks old) grown under specific pathogen-free conditions were purchased from Liaoning Changsheng Biotechnology (Shenyang City, China). The animal protocol (IACUC-19003) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin Sport University, Changchun City, China. All mice were provided a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) and distilled water ad libitum. Housing conditions included a 12-h light-dark cycle and air maintained at room temperature (22 • C ± 1 • C) with 30-40% humidity. The 1X dose of RC used for humans is typically 1,500 mg per day. The 1X mouse dose (308 mg/kg) we used was converted from a human-equivalent dose (HED) based on body surface area according to the US Food and Drug Administration formula: Assuming a human weight of 60 kg, the HED is 1,500 (mg)/60 (kg) = 25 × 12.3 = 308 mg/kg; the conversion coefficient 12.3 is used to account for differences in body surface area between mice and humans, as previously described. In total, 32 mice were randomly assigned to four groups (eight mice/group) for daily vehicle/RC oral gavage for 6 weeks. The four groups were vehicle, 308 (RC-1X), 615 (RC-2X), and 1,538 (RC-5X) mg/kg. The vehicle group received an equivalent volume of solution base.

Exercise Performance Test
Exercise performance protocol was identical to our previous manuscript (23). A low-force testing system (PicoScope 2000, Pico Technology Limited, Cambridgeshire, UK) was used to measure the forelimb grip strength of mice after 5 weeks of vehicle or RC treatment and 1 h after the last treatment dose was administered. The maximal force (in grams) recorded by this low-force system was used as the grip strength. To evaluate endurance times, the swim-to-exhaustion test was conducted, with loads corresponding to 5% of mouse body weight (BW) attached to the tails.

Fatigue-Associated Biochemical Indices, Serum Biochemical Index, and Hormone Index
Fatigue-associated biochemical indices in the serum were determined using an autoanalyzer (SYSMEX XT-2000iv, Sysmex corp., Kobe, Japan). The effects of RC on serum lactate, ammonia, creatine kinase (CK), glucose, lactate dehydrogenase (LDH), and FFA activity were evaluated postexercise. One hour after the last dose of the 41-day supplementation, a 15-min swimming test was performed without weight loading. After 1 h of the last intragastric administration of RC, in all mice serum samples were collected by cardiac puncture and analyzed using an autoanalyzer (SYSMEX XT-2000iv, Sysmex corp., Kobe, Japan). The hormones E2 and progesterone (PGN) were determined using the Roche Cobas e411 analyzer (Roche Diagnostics, Mannheim, Germany).

Tissue Glycogen Determination and Visceral Organ Weight
Glucose is stored as glycogen, mostly in liver and muscle tissues. Liver and muscle tissues were excised after the mice were euthanized and weighed for glycogen content analysis as described previously (23). The liver, heart, lung, kidney, Ovarian fat pad (OPF), gastrocnemius muscle, and brown adipose tissue (BAT) were collected after the last intragastric administration of RC and euthanized.

Magnetic Resonance Imaging and Histological Staining of Tissues
Magnetic resonance imaging (MRI) experiments were conducted on a 0.5T mice MRI scanner (MesoMR, Niumag analytical, Suzhou, China) after 1 h of intragastric administration of RC before anesthetizing. The mice were laid flat on a wooden board, their limbs and trunk were fixed with tape, they were placed in a nuclear magnetic resonance testing room; we selected the third cross-section for MRI imaging. Hematoxylin and eosin (H&E) staining was used for histological and pathological evaluations. After anesthetizing the mice, their tissues were fixed with 10% formalin for histopathological analysis. A clinical pathologist then stained the tissue sections with H&E and examined them under a light microscope with a CCD camera (BX-51, Olympus, Tokyo, Japan).

Bacterial DNA Extraction and 16S rRNA Sequencing
The procedures for sample preparation and extraction were previously described (23). Microbiome bioinformatics were mainly performed with QIIME 2 (24), whereas the OTU clustering procedure was performed with the Vsearch (v2.13.4) (25) pipeline described here (https://github.com/torognes/ vsearch/wiki/VSEARCH-pipeline). Briefly, raw sequence data were demultiplexed using the demux plugin followed by primer cutting with a cutadapt plugin (26). The differences in microbiota composition were analyzed using the orthogonal partial least square-discriminant analysis (OPLS-DA). After the groups were defined, a supervised the OPLS-DA model was applied to all microbiota. The Raw sequence reads project databases are available at https://www.ncbi.nlm.nih.gov/sra/ PRJNA673999.

Statistical Analysis
All data are expressed as mean ± SEM. Homogeneity of variance was examined by Levene's test, and the normality of the data was examined by the Shapiro-Wilk test. Statistical differences between groups were analyzed using one-way analysis of variance (ANOVA) was employed to calculate the significance differences between multiple groups with Duncan's test, and the Cochran-Armitage test was used for the dose-effect trend analysis. All statistical analyses were performed using SPSS version 18.0 (SPSS, Chicago, IL, USA), and p < 0.05 was considered statistically significant.

Effect of 6-Week RC Supplementation on
Tissue Weight, BW, Body Composition, and Diet Intake Table 1 shows the BW, food and water intake, and tissue weight of the mice receiving RC supplementation for 6 weeks. The initial BW, final BW, and water intake were not significantly different between the groups. However, food intake exhibited a significant increase in RC-5X mice (p = 0.0033) compared with the vehicle group. Tissues and organs were obtained from the sacrificed mice after the study period and weighed. No significant difference was observed in the liver, lung, kidney, heart, kidney, muscle, and brown adipose tissues of the mouse groups. Furthermore, the relative weights of all tissues were the same except brown adipose tissue (BAT). Both BAT and relative BAT weight had a dose-dependent decrease (all p < 0.03) by RC supplementation. MRI images, presented in Figure 1A, clearly show that the free fat mass (FFM) was predominantly located in lower bodies of mice, particularly around the genitals. Furthermore, no adverse events were observed in RC-1X, RC-2X, and RC-5X mice that received 6-week supplementation. The details of body composition data were obtained using a mice body composition analyzer procedure (mesoQMR, Niumag analytical, Suzhou, China; Figure 1B). The FFM was significantly higher in the RC-1X (1.06-fold, p = 0.0195) compared with the vehicle group. The fat mass (FM) was significantly lower in the RC-1X (20.38%, p = 0.005), RC-2X (32.53%, p = 0.0020), and RC-5X (23.75%, p = 0.0188) groups than in the vehicle group. The mice exhibited increased BW stability during the study period, and no significant difference was observed between the groups in each week ( Figure 1C).   group. The exhaustive swimming times of the vehicle, RC-1X, RC-2X, and RC-5X groups were 9.9 ± 1.5, 16.7 ± 2.3, 15.7 ± 3.4, and 13.1 ± 2.5 min, respectively, with the value of the RC-1X group being 1.67-fold higher than that of the vehicle group (p = 0.0412). The grip strength had a significant dose-dependent increase (p < 0.0001) in RC supplement whereas the swimming time had no significant difference in dose-dependent effect (p = 0.1131).

Effect of 6-Week RC Supplementation on Tissue Histopathology and Biochemical Variables
At the end of the study period, mice were sacrificed and major organs and tissues removed for histopathological analyses. Representative histopathological sections of the liver, lung, muscle, heart, kidney, and ovarian fat pad (OFP) tissues are shown in Figure 5. The results showed that no morphologic changes of sinusoid and hepatic cords in the liver of RC treatment groups compared with vehicle group. There were no hypertrophy or hyperplasia response in heart cardiomyocytes and myofibers of gastrocnemius muscles. No markedly difference in kidney of renal tubules and glomerulus with RC treatment and no alteration in the alveolar, bronchial and interstitial space in lung. The adipose tissue morphology and size of white adipocytes were not shown to be different between each group. The levels of CK and LDL in the RC group were significantly lower than those in the vehicle group. In addition, RC supplementation exhibited a dose-dependent decrease in triacyl glycerol (TG), CK, and LDL levels ( Table 2).

Effect of 6-Week RC Supplementation on Gut Microbiota
We analyzed gut microbiota composition in vehicle and RCsupplemented mice using 16S rRNA gene sequencing. Figure 6A shows the OPLS-DA. The data provide insights on differences between the vehicle and RC-2X groups. Specifically, the microbial population of the RC group was different from that of the vehicle group, suggesting that RC supplementation significantly influences the gut microbial population. Figure 6B shows that individual bacterial groups were analyzed based on LDA effect size (LEfSe). Several bacterial taxa were identified as being significantly different among the groups. The RC group exhibited an increase in the genera Pseudobutyrivibrio, Enterobacter, Nautella, Parabacteroides, Virgibacillus, and Sulfuricurvum; order Rhodobacterales; and families Porphyromonadaceae, Erysipelotrichaceae, and Prevotellaceae.

DISCUSSION
The result shows that a 6-week RC supplementation increased the FFM in mice lower bodies and decreased their FM. This is consistent with a previous study that demonstrated how isoflavone intake (75 mg/day for 12 months) significantly reduced FM in the trunk in postmenopausal (28). In addition, brown adipose tissue was significantly lowered by RC treatment. The BAT has beneficial effects on control of body composition and metabolism in 12-week-old male C57BL/6 mice (29), gut microbiota could modulate the lipid metabolism in BAT. BAT stimulates lypolysis and plays an important role in inhibiting lipogenesis in mice (30). Thus, RC supplementation could affect on lipid metabolism and have change gut microbiota. In our study, RC supplementation increased the FFM in mice lower bodies and decreased the FM. Therefore, RC increases the muscle glycogen level and thus reduces exercise fatigue and improves exercise performance. Lactate, ammonia, glucose, CK, and LDH levels are the most commonly used biomarkers for studying muscle fatigue (31,32) in humans. A possible explanation for the observed effects is that estrogen can repress mitochondrial uncoupling and reduce muscle fatigue through increased generation of ATP molecules through mitochondrial respiration (11). The gut microbiome may play a role in reducing the accumulation of damage-induced metabolites during exercise. Previous observations indicate that the isoflavone molecular form and the intestinal microbiome active in isoflavone uptake, metabolism, and ultimately therapeutic efficacy have crucial roles in postmenopausal osteopenic women treated with 60 mg of isoflavone aglycones (33). Moreover, studies have demonstrated the potential role of RC in inhibiting chronic inflammation (34). Thus, RC supplementation may possibly relieve muscle soreness and prevent delayed-onset muscle soreness. Our results showed that 6-week RC supplementation increased estrogen and PGN levels in mice. This result may be attributed to isoflavones. Isoflavones are phytoestrogens with potent estrogenic activity (35). This may be due to estrogen receptor-α and estrogen receptor-β. Furthermore, several studies have confirmed that estrogen and PGN contribute to improving endurance exercise performance (11), muscle strength in women (15), and muscle mass (36) and reducing bone loss (3). Moreover, estrogens influence fiber atrophy, specifically fast-twitch muscle rather than slow-type muscle (37). Thus, RC contributes toward improving muscle mass and exercise performance.
In the present study, RC supplementation decreased the levels of CK, LDL, and TG in mice. Moreover, Luís et al. (8) concluded that supplementation with phytoestrogens, through their inhibition of 7a-hydroxylase and the estrogenic effects on the LDL-receptor, modulates enzyme PCSK9 to decrease LDL. This may help explain our results RC decreased the LDL. Furthermore, CK is a crucial clinical biomarker for muscle damage. TG and LDL are the main clinical biomarkers for blood lipids. The decreases in the levels of CK, LDL, and TG indicate that RC supplementation is beneficial for reducing exercise fatigue and blood lipid level. Recent studies have found RC to be effective in treating several diseases; for instance, RC is safe clinically and in vitro for premenopausal patients with hormonesensitive breast cancer (38). Furthermore, a study showed that RC extract (250 mg/Kg) has a significant effect in a rat model of type 2 diabetes (39).
Flavonoids can strengthen intestinal tight junction functions and enhance barrier integrity (40). Furthermore, they can significantly upregulate the relative abundance of Pseudobutyrivibrio and a species of butyrate-producing bacteria present in the rumen (41). Parabacteroides was enhanced in the RC-2X group in a previous study; Parabacteroide is a gram-negative bacterium and acts as a commensal symbiont, promoting mucosal barrier health and preventing increases in several proinflammatory cytokines (42). Virgibacillus is Frontiers in Nutrition | www.frontiersin.org gram-positive bacteria that can be isolated from human intestinal microbiota (43), and they have been found in fish sauce fermentation (44). Virgibacillus is a proteinase applied in functional food ingredients that has immunomodulatory activity (45). Sulfuricurvum is the name for sulfur-oxidizing bacteria present in the normal microflora of the skin surface of humans and mammals in general (46). The family of Erysipelotrichaceae was found to increase in the host with the intake of low-fat diets and hydroxypropyl methylcellulose (47). In our data, FM and TG level decreases may be correlated with Erysipelotrichaceae increase due to a change in lipid metabolism. Overall, RC supplementation improved intestinal barrier integrity through inducing a shift in intestinal microorganism composition. The intestinal barrier function is affected by microbiota's metabolism, whereas a decrease in barrier function during exercise might lead to changed blood flow in the gut, heart, and skeletal muscles, thus affecting exercise performance (19). In general, strenuous exercise promotes alterations in gastrointestinal permeability, thus enhancing intestinal barrier function, which reduces endotoxin release from the gut to reduce heatstroke (48). By contrast, with an increase in gastrointestinal permeability, the temperature of the core increases and blood flows away from the splanchnic area, which can directly open tight junctions and damage the enterocyte membrane, causing tissue hypoxia and oxidative stress (49). RC is an appropriate supplementation with the potential to counteract exercise-induced intestinal dysfunction by shift the positive gut microbiota.
The limitation of this study was we did not control for all female mice estrous cycle to reduce this effect, but the estrous cycle was not the main factor to alter on the exercise performance. A previous study found that estrous cycle seems to influence running economy and estrus impaired exercise heat loss, without no concomitant effect on the endurance exercise performance and grip strength in mice (50). In addition, the estrous cycle did not influence female mice muscle strength, but the estrous cycle undermined the running economy, which change the VO 2 max in low-intensity exercise, and alter the exercise-induced thermoregulation (51). Another limitation was the fact that our intervention lasted only 6 weeks; this time frame was chosen to detect the short-term effects of RC.
Overall, RC supplementation is still an appropriate supplementation with the potential to counteract exercise-induced intestinal barrier dysfunction.

CONCLUSIONS
In conclusion, RC supplementation improved the intestinal health by shifting microbiota composition and exerting antifatigue effects by reducing fatigue-related serum markers. According our data, RC increases forelimb grip strength and swimming time and decreases FM in female mice. RC might be useful in reducing physical fatigue, with the intestinal health contributing a critical role in physical performance, possibly enhancing the genera of Pseudobutyrivibrio, Parabacteroide, and Virgibacillus and the family Erysipelotrichaceae. Thus, our results show that RC is advantageous in shifting the gut microbiome and providing positive effects on female athletes' gut and overall health.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih. gov/bioproject/PRJNA673999/.

ETHICS STATEMENT
The animal study was reviewed and approved by the animal protocol (IACUC-19003) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin Sport University, Changchun City, China.