Naringenin, a Flavonoid, Modulates Gut Microbiome and Ameliorates Hormone Levels to Improve Polycystic Ovary Syndrome in Letrozole-induced Rats

Background: Polycystic ovary syndrome (PCOS) is a common female endocrinopathy, which severely affect the menstruation and fertility of patients. Naringenin, a natural avanone, has emerged as a potential therapeutic agent for the management a variety of diseases. However, the underlying mechanism of naringenin in anti ‐ PCOS is unclear. This study was focused on investigating the effects of naringenin on body weight, ovarian tissue, serum hormone level, glucose metabolism level and gut microbiome in letrozole-induced PCOS model rats. Methods: First, we administered letrozole gavage to 10-week-old SD female rats for 4 weeks to induce PCOS rats model, the estrus cycle was observed through the vaginal smear of rats to determine the establishment successful of a PCOS rat model. Then, the successfully modeled PCOS rats were treated with naringenin for 2 months. Finally, observed the changes of rat body weight, ovarian tissue, serum hormone level, glucose metabolism level and gut microbiome after naringenin treatment. Results: The naringenin treatment ameliorate the hormone levels, such testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), improve insulin resistance and the ovarian tissue pathological changes, reduced body weight in the PCOS model rat. Meanwhile, through the detection of rDNA in the faeces of the PCOS model rat, we found some benecial microbes such [Ruminococcus], Faecalibacterium, Butyricimonas, Lachnospira, Parabacteroides, Butyricicoccus and Roseburia were enrichment in naringenin group when compared with the PCOS rats. Conclusions: In summary, our results indicated that naringenin could play an anti-PCOS role, and its mechanism may be closely related to regulating the benecial microbes of gut microbiome. Our research could provide a new perspective for the treatment of PCOS and its related disease. species annotation (genus level), taxonomic_tree, alpha diversity analysis, OTU abundance Venn diagram and heat map of species composition. Species annotation: the top 20 species of taxonomic level of each group were plotted with accumulative histograms to visualize the species of relative abundance and their proportion at different taxonomic levels. The taxonomic_tree can display the composition of all taxa at the same time. Alpha analysis was the of single sample. In this study, Chao1 the levels of hormone, glucose metabolism on letrozole-induce PCOS


Background
Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases in women of childbearing age, with the prevalence of 6-21% [1]. Its main features are anovulation, hyperandrogenism, polycystic ovarian morphology, and the main clinical manifestations include menstrual disorders, infertility, hirsutism, metabolic syndrome comprising obesity, dyslipidemia, insulin resistance (IR) [2], which places women at an increased risk for development into cardiovascular disease and diabetes mellitus [3]. Although several studies have con rmed that genetic, gestational environment and lifestyle factors are connected with the development of PCOS [4][5][6], the exact underlying mechanism triggers for these key biochemical and metabolic disorders in PCOS is still largely unclear. Moreover, due to the side effects of anti-androgens or ovarian function inhibitors, the clinical use of such drugs is generally not accepted. Because development the effective drugs or operations are still a challenge in the treatment of PCOS, other therapies need to be used. So, it is necessary to formulate alternative treatment strategies for women with PCOS, more and more studies using natural plant-based products to treat PCOS.
In recent years, the gut microbiome has become a key factor affecting health and disease, and has made major contributions to metabolic diseases. Studies showed that the gut microbiota can induce or regulate insulin resistance and chronic in ammation, and can also regulate sex hormone levels [7][8][9]. Meanwhile, with the deepening of research, accumulating evidence showed that intestinal ora is closely related to the occurrence and development of PCOS [1,8,10]. Such as, transplanting feces of PCOS patients into mice can cause PCOS phenotype in mice [10], both PCOS patients and animal models, PCOS-induced changes in the gut microbiota alpha and beta diversity can occur to a certain extent [3]. In addition, the clinical manifestations of PCOS were signi cantly reduced in obese women treated with low-fat diet [11], and emerging evidence showed that the composition of the intestinal ora may affect women's metabolic homeostasis and reproductive tness [12]. Based on the above research, we calculate that the gut microbiome may be play an important role to PCOS.
Naringenin, a citrus avonoid that possesses various biological activities, which can be extracted from grapefruit and widely exists in the plant kingdom, and has been isolated from several plants [13,14], and the natural plant extract of naringenin has emerged as a potential therapeutic agent for the management a variety of diseases [15]. Meanwhile, it has been shown that naringenin regulates lipoprotein metabolism, it may be used in the management of diabetes, atherosclerosis and insulin resistance, which was extensively discussed in a previous review [16]. Similarly, naringenin has been shown to have hypoglycemic activity in type 2 diabetic rats [17]. In addition, naringenin has good prospects as an ideal therapeutic agent, a study showed that naringenin treatment in a rat model of PCOS signi cantly increased the levels of the reactive oxygen species (ROS) scavenging enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), prevented weight increase and reduced serum glucose levels, normalized estradiol and testosterone levels and maintained the normal anatomy of the ovaries [18,19]. However, whether and how naringenin regulates the diversity of gut microbiome to anti-PCOS is still unknown. Therefore, we aimed to study the role of naringenin in regulating gut microbiome, glucose metabolism, sex hormone levels, ovarian morphology and body weight of letrozoleinduced PCOS rat, to initially reveal the potential mechanism of its anti-PCOS effect.

Animal
Female Sprague-Dawley (SD) rats, ten-week-old (body weight, 270-300 g), were speci c pathogen-free (SPF) level inbred and obtained from the professional Laboratory Animal Company (Beijing Vital River Laboratory Animal Technology Co., Ltd.). They were randomly divided into two groups, including normal group (n = 6) and PCOS group (n = 24). All rats need 2 weeks to adapt the new environment before the experiment was started, all animals were fed in standard animal room (room temperature: 22 ± 2 •C; relative humidity: 30-40%), free dry pellet diet and water were supply. All protocols in this study were approved by the Animal Care Committee of Beijing University of Chinese Medicine (BUCM), China, and these animals were treated according to the Animal Care Committee.

Induction of PCOS and Diane-35 and Naringenin drugs administration
All animals were under a periodic cycle, the letrozole (Jiangsu Hengrui Medicine co.,ltd, gyzz H199991001), with the concentration of 1 mg/kg ,were gavage for rats to induce PCOS per day, with 28 consecutive days. The estrus cycle was monitored by using vaginal smear, and observation changes in the estrous cycle under the microscope. After, the 18 successful letrozole-induced PCOS rats were randomly divided into the PCOS group (MX), Diane-35 group (YY), naringenin group (YP) with 6 rats in each group. The Diane-35(Bayer Weimar GmbH und Co.KG, gyzz j20140114) as a positive control group (0.2 mg/kg/day) and the naringenin (BioRuler, USA,100 Mill Plain Road Danbury, CT06811) as a treatment group (20 mg/kg/day) were respectively intragastric administration for YY and YP group.
Meanwhile, the normal group (ZC) and PCOS group (PCOS) were given same volume of saline. During the whole experiment period, the body weight (BW) was checked every week, the fasting blood glucose (FBG) was tested after the animals were ambrosia for eight hours. With the experiment termination, all animals were sacri ced in anesthetized deeply, the serum, bilateral ovaries, kidney, and liver were collected for further detection.

Hematoxylin and Eosin(HE) staining
With 4% paraformaldehyde were used to xed ovaries tissue and then embedded in para n, sliced for 5microns, staining with hematoxylin and eosin (H&E). Finally, these tissue samples were removed under a light microscope (Olympus, Japan) with blindly observe and photograph. All histological experimentation were manipulated at the specialties laboratory of BUCM.

Serum hormone analysis
The blood samples of trunk circulation were successfully collected and allowed to stand at room temperature for 2 hours for incubation. All samples were centrifuged (3,000 rpm, 15 min, 4 • C), then the serum was collected into 1.5 ml EP tube, kept at − 80 • C for subsequent experiments.
The basic serum hormone testing including testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin resistance index(FINS). All serum levels were measured by using the radio Immunosorbent Assay (Tianjin Jiuding Medical Bioengineering Co., Ltd.). According to the speci cations to use all kits, repeatability analysis for each sample. The homeostasis model of assessment for insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) are an effective way to determine the insulin-resistance and insulin sensitivity index in the rat model. So, the HOMA-IR was We collected fresh fecal samples from four groups rats before all animals were sacri ced, fresh fecal were placed in a 1.5 ml sterile EP tube with quickly freeze in liquid nitrogen, then store at − 80 °C waiting for further experiments.

Fecal DNA extraction and PCR ampli cation
Total microbial genomic DNA samples were extracted using the DNeasy PowerSoil Kit (QIAGEN, Inc., Netherlands), following the manufacturer's instructions, stored at − 20° C before further analysis. The quantity and quality of extracted DNAs were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA) and 1.2% agarose gel electrophoresis, respectively. Microbial DNA was extracted from fecal samples using the Illumina company TruSeq Nano DNA LT Library Prep Kit (Qiagen, Valencia, CA, United States), according to manufacturer's protocols. The V3-V4 region of the bacteria 16S ribosomal RNA genes was ampli ed by PCR (98 •C for 2 min, followed by 25

Sequencing and data process
Sequence data analyses were mainly performed using QIIME and R packages (v3.2.0). In this study, the main selected sequencing analysis includes usable raw reads, operational taxonomic units (OTUs), species annotation (genus level), taxonomic_tree, alpha diversity analysis, OTU abundance Venn diagram and heat map of species composition. Species annotation: the top 20 species of taxonomic level of each group were plotted with accumulative histograms to visualize the species of relative abundance and their proportion at different taxonomic levels. The taxonomic_tree can display the composition of all taxa at the same time. Alpha analysis was analysis the species diversity of a single sample. In this study, Chao1 and Observed species indexes were used to indicate abundance, Shannon and Simpson indexes were used to indicate diversity, Faith's PD indexes characterizes the diversity based on evolution, Pielou's evenness indexes characterizes uniformity, Good's coverage indexes represents coverage. The different species analysis including OTU abundance Venn diagram and the heat map of species composition, the Venn diagram was to study which species were shared among different sample groups and which were unique, the heat map further compares the differences between species composition and samples, realized the display of the species abundance distribution trend of each sample.

Statistical analysis
The experimental data was processed by SPSS (version20.0, IBM SPSS Statistics), the results were expressed as mean ± standard error of the man (SEM). Statistical differences were determined by Student's independent t-test or One-way ANOVA, the signi cance was accepted at P values < 0.05. Signi cance was shown as *p < 0.05, **p < 0.01vs normal group; #p < 0.05, ##p < 0.01vs PCOS group.

Characteristic of Letrozole-induced PCOS rat model
In this study, we used letrozole for 4 weeks to induce the PCOS rat model and monitored the estrous cycle through vaginal smears. The results showed that we successfully established a rat model of PCOS induced by letrozole, which was con rmed by the irregular estrous cycle (Fig. 1.A). Generally speaking, the estrous cycle was longer than 5 days, or there was no obvious cycle, or it continued to be in the per-estrus, or the interests' period, these are usually considered to be disordered. As shown in Fig. 1.A, the vaginal smear of the normal group showed an obvious and regular estrous cycle. But compared with the normal group, the estrus cycle showed irregular changes and the most common cell type was leukocytes under microscope, suggesting vaginal smears of letrozole-induced model group rats exhibited a disorder of the estrus cycle ( Fig. 1.B).

Effects of Naringenin on body weight in PCOS rats
In order to observe the in uence of naringenin on the body weight of PCOS rats, we conducted a statistical analysis of body weight in this experiment, founding the body weights of the PCOS group was signi cantly increased when compared to the normal group, (*p < 0.05, **p < 0.01vs control; #p < 0.05, ##p < 0.01vs PCOS). Interestingly, the naringenin groups exhibited obviously decreased than Diane-35 group when compare with the PCOS group, but the difference was not statistically signi cant. This suggests that naringenin had a good effect for lose weight on letrozole-induced PCOS rats by eight-week intervention.

Effects of Naringenin on histological changes in PCOS rats
With 4% paraformaldehyde-xed and para n-embedded of rat ovaries, sectioned at 5-µm slices for H&Estained (Fig. 3A). In PCOS group, the ovaries showed pathological changes, no or very few corpus luteum structures, a large number of expanded cystic follicles, no radioactive crowns and oocytes were seen, the ovaries showed typical polycystic changes. But the naringenin group had large number of corpus luteum and had different levels of follicle at different developmental stages, the granulosa cell layer was multilayered with neatly arranged and normal in structure. Based on the above description, we found the pathological changes of the ovarian tissue have been greatly improved after 8 weeks of treatment by naringenin. Meanwhile, the ovaries resembled with the naringenin rats in all aspects after Diane-35treatment.

Effect of Naringenin on serum hormonal level in PCOS rats
To study whether naringenin could ameliorate letrozole-induced hormonal levels in PCOS rats, we tested levels of T, E2, FSH and LH. The result showed that the levels of serum T, LH and FSH were signi cantly increased and the level of serum E2 were signi cantly reduced in the PCOS group when compared with the normal group. After 8 weeks treatment, we found that the levels of serum T, LH and FSH were signi cantly reduced and the level of serum E2 was signi cantly increased. (*p < 0.05, **p < 0.01vs control; #p < 0.05, ##p < 0.01vs PCOS; Fig. 4.). However, it was noticed that compared to the PCOS group, only the level of serum E2 difference was not statistically signi cant by Diane-35 treatment.

Effect of Naringenin on glucose metabolism in PCOS rats
To explore whether naringenin could improve letrozole-induced metabolic abnormalities in PCOS rats, we tested levels of FPG and fasting insulin (FINS) and further evaluated the effect of naringenin treatment on IR and insulin sensitivity in PCOS rats. The result showed that the FBG and FINS levels were signi cantly increased when compared with the normal group, but after the naringenin treatment, the serum levels of FBG and FINS were signi cantly decreased when compared to the PCOS group (*p < 0.05, **p < 0.01vs control; #p < 0.05, ##p < 0.01vs PCOS; Fig. 5.A/B). The FBG level had a decline in the Diane-35 group, but the difference was not statistically signi cant. In addition, compared with the normal group, the HOMA-IR was signi cantly increased and ISI was signi cantly reduced in letrozole-induced PCOS group. Interestingly, the levels of HOMA-IR and ISI were changed signi cantly by naringenin treatment when compared with the PCOS group, including signi cantly decreased in HOMA-IR and signi cantly increased in ISI. (*p < 0.05,**p < 0.01vs control; #p < 0.05, ##p < 0.01vs PCOS; Figure.5.C,D).

The statistics of sequencing
We ful lled gene sequencing on the V3-V4 region of 16S rRNA to assess the impact of naringenin on the dysbiosis of gut microbiome in letrozole-induced PCOS rats. In total, 233,2605 usable raw reads (200,6653 valid sequences), 113,7408 high-quality sequence reads, and 8,2283 OTUs with a similarity of 97% were received from all 24 samples.

Overall structural changes of gut microbiome
In our study, we analyzed the taxonomic compositions of bacterial communities in genus level among the normal, PCOS, naringenin and Diane-35 groups. The proportion of the microbial genus in the naringenin group and Diane-35 were very different from the PCOS model rats. In naringenin group, the microbial genus of Oscillospira, Blautia, Helicobacter, [Ruminococcus], Coprococcus, Parabacteroides, Faecalibacterium, Paraprevotella were higher when compared with PCOS group. In Diane-35 group, the microbial genus of Oscillospira, Coprococcus, Allobaculum, Desulfovibrio, Streptococcus and [Prevotella] were higher than PCOS group (Fig. 6C). Meanwhile, we also analyzed the species classi cation hierarchy tree with abundance information, the naringenin group showed that the abundance of Faecalibacterium prausnitzi, Helicobacter rodentium, Ruminococcus albus, Ruminococcus avefaciens, Blautia product, [Ruminococcus] gnavus, Prevotella copri and Helicobacter rodentium were signi cantly increase(species level) these were consistent with the genus level results. In Diane-35 group, we found the abundance of Ruminococcus bromii (species level), Anaerostipes, Turicibacter, Allobaculum, Desulfovibrio (genus level), Peptostreptococcaceae and Coriobacteriaceae (family level) were remarkably increase (Fig. 6D).

The alpha diversity analysis
According alpha diversity analysis to observe the Chao1 index, Simpson index, Shannon index, Pielou_e index, Observed_species index, Faith_pd index and Goods_coverage index, we found that the alpha diversities were signi cantly changed all groups. When compared with the control group, the Chao1 index, Observed_species index and Faith_pd index were signi cantly reduced, but the Goods_coverage index was signi cantly increased in other three groups. (Fig. 6E, p < 0.05). While no difference was found for the Simpson's diversity index, Shannon index and Pielou_e index.

Key phylotypes of gut microbiome corresponding to Naringenin treatment
There was a clear difference in species difference analysis and marker species screen. First of all, the Venn diagram showed some common and unique species among different groups, as shown in Fig. 1.A, the unique species OTUS in the control, PCOS, Diane-35 and naringenin groups were respectively 9449(25.04%), 6534(17.32%), 7089(18.79%), 7212(19.11%), it was no di cult found that the unique species OTUS was increase after treatment by naringenin when compared with the PCOS group (Fig. 6F). Secondly, we analyzed the heat map that represent the gut microbiome changes on the genus level of different groups, it showed that some bene cial species were signi cantly abundant by naringenin treatment 8 weeks, such as the genus of Parabacteroides, Lactobacillus, Faecalibacterium, Bacteroides, Roseburia, Butyricicoccus, Streptococcus were enrichment. In the Diane-35 group, the Lactobacillus, Anaerostipe,[Prevotella] Bi dobacterium, Streptococcus were abundance. Interestingly, these bene cial species were not emerged in PCOS group (Fig. 6G).

Discussion
PCOS is a metabolic syndrome, obesity, chronic in ammatory, dyslipidemia and IR are usually accompanied [2,21]. In recent ten years, relevant population-scale studies have illustrated the potential role of the intestinal microbiome in changing the health of the host, and the human gut microbiome is necessary for maintaining the human health and immune system [22]. Recently, several studies have revealed intestinal ora is closely linked to the occurrence of PCOS, the diversity and composition of the gut microbiota will undergo certain changes when the case of metabolic disorders [23][24][25]. Previous studies have found that gut microbiome can induce or regulate insulin resistance, obesity and chronic in ammation, and can also regulate sex hormone levels [26,27]. In our study, similar to the results of these studies, we found the naringenin treatment changed the intestinal ora, body weight, ovarian tissue morphology, the levels of hormone, glucose metabolism on letrozole-induce PCOS rats.
Consistent with existing literature, in the current study, we observed the irregular estrous cycles, the ovary pathological change, levels of sex hormone were abnormal such as LH, estradiol, testosterone and FSH in PCOS group of letrozole-induced when compared with the normal group [19,28,29]. All those features illustrated the model of PCOS were established. Interestingly, a avanone, naringenin extracted from citrus fruits and exist in many traditional Chinese herbal medicines, has been displayed to have multiple effective effects on cells both in vivo and in vitro [30]. The result of naringenin treatment was the estradiol level and ISI increased, the T, LH, FSH, FBG, FINS and HOMA-IR levels decreased when compared to the levels in PCOS group. It was noticed that naringenin treatment not only had a signi cant amelioration in serum hormone levels, but also improved insulin resistance, prevented weight increase and maintained the normal anatomy of the ovaries in rat model of letrozole-induced PCOS. Those changes showed that naringenin maybe play an important role to adjustment the hormone levels in treatment the pathological change of ovulation disorder in PCOS.
There was a connection between the PCOS and gut microbiome, studies showed that the intestinal microbiota can regulate sex hormone level in the host, such as a decrease in alpha diversity, the change key phylotypes of gut microbiome corresponding, an increase of bene cial bacteria [36,37], In order to research the role of the intestinal microbiota between naringenin with PCOS, we used 16S rDNA analysis technology to evaluate changes in the intestinal ora with naringenin in the PCOS rats of letrozole-induce. In our study, observing the disorder in the gut microbiota and revealing changes in the relative abundances of speci c species in the gut microbiome in naringenin group. We found 7 differentially abundant bacterial taxa in the naringenin group, the genus of [Ruminococcus], Faecalibacterium, Butyricimonas, Lachnospira, Parabacteroides, Butyricicoccus and Roseburia were enrichment, these also explain why the unique species OTUS was increasing after treatment by naringenin when compared with the PCOS group. The result showed the naringenin treatment can increase the proportion of bene cial bacteria, and normalized the intestinal ora. Interestingly, in the species level of gut microbiome, we noticed that the PCOS rats lack bene cial microorganisms such Faecalibacterium prausnitzii and the disease-related microbes Prevotella copri was appeared. But the Faecalibacterium prausnitzii of bene cial microbes was signi cantly abundance in naringenin group. Gut microbes such as Faecalibacterium prausnitzii is generally regard as "good bacteria" that has function for health-promoting [32]. As well as, in naringenin group, in the genus level of gut microbiome, we found some new bene cial bacteria were enrichment, such as the microbial genus of Faecalibacterium, Roseburia and Parabacteroides which can reduce the in ammation and obesity to promote intestinal microbiota homeostasis [33,34]. Meanwhile, The SCFA-producing bacteria Roseburia, Lachnospira, Butyricicoccus, Lactobacillus and the dominant genus associated with SCFAs including Streptococcus [35][36][37] were abundant after naringenin treatment. In addition, the bacteria Lachnospira and Roseburia of butyrate-producing were also more abundant in the naringenin group, and the content of butyric acid, a bene cial substance in the intestine, can prevent and treat obesity-related diseases [38].
Several studies have demonstrated that various bene ts of probiotics to the host have been shown in numerous human clinical trials, the role of these organisms is to improve the balance of the intestinal ora and increase the production of short-chain fatty acids(SCFAs), which can directly or indirectly play the role of anti-in ammatory, protect the intestinal barrier function, regulate human metabolism and immunity [39][40][41]. Especially, (SCFAs) play an important role in the treatment of obesity, insulin resistance, diabetes and other metabolic diseases [42].As well as study showed that SCFAs can improve intestinal health by promoting the increase of intestinal barrier function and reducing the translocation of bacterial endotoxins on the intestinal wall, this is one of the reasons it reduces in ammation and improves insulin resistance [43]. So, we conjecture the levels of FBG, FINS and HOMA-IR declined, the level of ISI increase may be regulate by gut microbiome, nally contributes to improve insulin resistance.
From what has been discussed above, we speculate that the regulation of gut gut microbiome may be a potential mechanism for naringenin to prevented weight increase, reduction in serum glucose levels, improve insulin resistance, and promoting ameliorate the hormone levels in the PCOS model rat.

Conclusions
In conclusion, our research showed naringenin can prevent letrozole-induced PCOS-related symptoms such as obesity, insulin resistance, and sex hormone disorders, which may be related to the alteration of gut microbiome. Although an ant-PCOS activity of naringenin has been reported, no study to focus on exploring the relationship between naringenin and gut microbiome of PCOS through 16S rRNA sequencing analysis. Our research lls this gap, it suggest naringenin may be a therapeutic effect on PCOS-related metabolic disorders by adjusting the composition of gut microbiome. This provides a new train of thought for the pathogenesis and treatment of PCOS. But its speci c mechanism still needs further experiments, which is also our future research direction.  Line chart of the effect of naringenin on letrozole-induced PCOS in rats, the abscissa represents the intervention time (weeks)( the rst 2 weeks is the adaptation period, 2-6 weeks is the letrozole-induced PCOS period, and 6-14 weeks is the naringenin treatment period), and the ordinate represents the weight, which are distinguished by different colors, as shown in the gure. ZC: normal group, MX: PCOS group, YY: Diane-35 group, YP: naringenin group.