Nourishing Kidney Promoting Ovulation Decoction (NKPOD) Attenuates Polycystic Ovary Syndrome by Downregulating miRNA-224

Background Currently, exploring effective agents is urgently required for polycystic ovary syndrome (PCOS) treatment. Although nourishing kidney promoting ovulation decoction (NKPOD) as a traditional Chinese medicine decoction is widely employed to increase pregnancy rates, whether NKPOD attenuates ovulation disorders in PCOS patients remains unknown. Here, we aim to explore the clinical significance and the underlying mechanisms of NKPOD in ovulation disorders. Methods PCOS patients were recruited to confirm the clinical significance of NKPOD in attenuating ovulation disorder. Subsequently, regulation targets of NKPOD were identified through network pharmacology analysis. Additionally, a series of experiments were performed to observe the impacts of NKPOD on miRNA-224 transcription through transcription factor AR. Results In this study, NKPOD administration improved hormone dysregulation and reproductive outcomes in PCOS patients. Interestingly, 100 potential targets related to NKPOD and PCOS were screened, and transcription regulation was observed to be the most enriched function. Mechanistically, NKPOD inhibited miRNA-224 transcription through reducing AR expression, in which AR as a transcription factor directly regulated miRNA-224 transcription. Conclusions Collectively, these findings highlight the therapeutic effect of NKPOD on PCOS, which could provide promising therapeutic agents for PCOS.


Introduction
Polycystic ovary syndrome (PCOS) is an endocrine disease which is characterized by anovulatory cycles/oligo, polycystic ovaries, hirsutism, and insulin resistance [1]. Accumulating evidence indicates that arrested follicular growth and abnormality of early follicle development are critical factors in the pathogenesis of PCOS [2][3][4]. At present, approximately 5% to 20% of females will develop PCOS during their reproductive age [2,5]. Mounting evidence indicates that PCOS increases the risk of type 2 diabetes, cardiovascular disease, obesity, metabolic syndrome, and endometrial cancer [6][7][8]. Additionally, pregnant women with PCOS are more vulnerable to miscarriages and ovarian hyperstimulation syndrome [9]. Terefore, exploring efective agents is urgently required for the treatment of PCOS.
Traditional Chinese medicine (TCM) has been widely applicable to treatment for PCOS [10], and nourishing kidney promoting ovulation decoction (NKPOD), which is composed of twelve kinds of Chinese herbal medicines, is one of them, including Angelicae sinensis Radix, Paeoniae Radix alba, Rhizoma Dioscoreae, Rehmanniae Radix Preparata, Cortex Moutan, Poria, Semen Cuscutae, Dipsaci Radix, Cuscutae Semen, Cornu Cervi, Faeces Trogopterori, and Carthami fos. Modern pharmacological studies have shown that some active ingredients in NKPOD play a role in regulating oocyte competency and embryo development [10][11][12]. For example, animal experiments showed that Bushen-zhu-yun decoction including Angelicae sinensis Radix signifcantly alleviated pathological changes in the ovary, altered hormone levels of serum, and reduced the apoptotic rate of granulosa cells [13], while an experimental study demonstrated that Cuscuta-Salvia ameliorated the pathological changes in the liver, ovaries, and adipose tissue [14]. Multicomponents and multiple targeting characteristics of NKPOD play a common role in the protective efect of PCOS; thus, expanding the knowledge of NKPOD in PCOS treatment will be interesting.
During the development of the oocyte and embryo, numerous miRNAs are produced, and they function through posttranscriptional gene regulation [15,16]. Several previous studies have demonstrated that miRNA-224 is a marker of PCOS and regulates oocyte competency and embryo development [17,18]. As an example, miRNA-224 is involved in the growth of follicular granulosa cell by transforming growth factor-β1 and estradiol (E2) [19]. Another research of miRNA-224 revealed bisphenol-A exposure changes in serum E2 levels and follicle-stimulating hormone levels through transcriptional regulation on miRNA-224 in preantral ovarian granulosa cells [18]. Tese pieces of evidence suggest that miRNA-224 could be involved in later development of follicles.
In this study, we explore and present the NKPODtreatment-mechanism relationships with clinical research, network pharmacology methods, and in vitro experiments. We use these methods to give a dependable result of the NKPOD on attenuating polycystic ovary syndrome. Furthermore, we investigate whether NKPOD could regulate the expression of miRNA-224 and the underlying mechanism.

Clinical Samples.
From January 2018 to June 2020, the PCOS patients (age <38 years) who underwent in vitro fertilization and embryo transfer and intracytoplasmic sperm injection (IVF-ET/ICSI)-assisted pregnancy in the Reproductive Medicine Department of Jiangsu Traditional Chinese Medicine Hospital were collected. Te basic clinical characteristics, embryo laboratory indexes, and the OHSS incidence of the two groups were analyzed and compared (Table 1). Te diagnostic criteria for PCOS referred to as PCOS formulated by experts of the European Society of Human Reproduction and Embryology and the American Society of Reproductive Medicine (ESHRE/ASRM) in Rotterdam in 2003 were as follows: (a) clinical manifestations of hyperandrogenemia and/or hyperandrogen, (b) anovulation or rare ovulation, and (c) ultrasound showing ovarian polycystic changes (ovarian volume greater than 10 ml in one or both ovaries and/or > 12 follicles with a diameter of 2-9 mm). After excluding Cushing's syndrome, congenital adrenocortical hyperplasia, androgensecreting tumors, and other diseases, if 2 of the above three are consistent, it can be diagnosed as polycystic ovary syndrome. Among them, hyperandrogenemia is based on the fact that the fasting testosterone value in the morning of menstruation D3 is higher than the upper limit of normal. Te standard of rare ovulation was a menstrual cycle >35 days. Te ultrasound image of the polycystic ovary is based on the standard that the number of bilateral ovarian sinus follicles (AFC) shown by transvaginal ultrasound is greater than 12. Te selected patients with normal ovarian function were younger than 38 years and had regular menstrual cycles (28-30 d), normal basic reproductive hormones, and a number of follicles in both ovarian sinuses (unilateral AFC = 6-10). Te two groups had no ovarian cysts, hyperprolactinemia, ovarian tumors, endometriosis, ovarian surgery, chemotherapy and radiotherapy, hypothyroidism/hyperthyroidism, and other endocrine diseases, and the chromosomes of the couple were normal. Te Ethics Committee of the Jiangsu Hospital of Traditional Chinese Medicine (2019NL-KS74) approved this study. Written informed consent was obtained from all participants included in the study.

Sample Collection and Detection.
For collection of serum, blood was collected on the 2nd-5th day of the menstrual cycle and the elbow vein blood of the patient was extracted. After centrifugation (2,000 × g for 5 min; 10,000 × g for 5 min) at 4°C, the serum was taken and stored in the −80°C refrigerator for testing. For collection of follicular fuid, the diameter of the selected experimental follicles was more than 18 mm. Te follicular fuid was obtained when the follicles were punctured, and the eggs were taken under the guidance of vaginal B-ultrasound. About 4 ml of clear follicular fuid without blood pollution was left. After centrifugation, the supernatant was left and stored in the refrigerator at −80°C for testing. For collection of granulosa cells, the eggs were punctured according to the egg taken as the standard, the follicular fuid was retained, human lymphocyte separation solution was added, the granulosa cell layer between the two liquid levels was absorbed after centrifugation, PBS was added, the supernatant was discarded after centrifugation, and the lower layer had granulosa cells. Granulosa cells for experimental research and oocytes for IVF-ET/ICSI were collected. Te granulosa cells Bethesda, MD, USA), we visualized the composition-target network. In addition, protein-protein interactions (confdence score >0.9) were selected using STRING (https:// www.string-db.org/) and Cytoscape software. Te core targets were analyzed using plug-in components (CytoNCA) through analysing betweenness, bloseness, degrees, eigenvectors, LAC, and networks. Te visualization of gene ontology (GO) terms was performed by using the clusterProfler package in R.
2.9. Dual-Luciferase Assay. Te wild-type reporter of miRNA-224 promoters (pmiRNAGLO-WT) or the mutant reporter (pmiRNAGLO-MUT) was cotransfected with pcDNA3.1-NC or pcDNA3.1-AR into HEK293T cells. After transfection for 24 h, HEK293T cells were washed with PBS Evidence-Based Complementary and Alternative Medicine and subjected to the dual-luciferase reporter assay system (Promega) for luciferase level measurement. Luciferase activities were normalized to that of Renilla.

Chromatin Immunoprecipitation (ChIP).
By following the manufacturer's instructions, ChIP assays were conducted using ChIP Assay Kit (Beyotime, Haimen, China). Granulosa cells were crosslinked with formaldehyde and sonicated to an average length of 200-1000 bp. Subsequently, IP was performed with an AR antibody (Abcam) or IgG. Finally, precipitated DNA was detected by qRT-PCR.
2.11. Statistical Analysis. Quantitative data are presented as the mean ± SD and analyzed by using SPSS software (version 22.0, IBM, Chicago, USA). Normality of distribution was assessed using the Kolmogorov-Smirnov test. Te Mann-Whitney U tests or Student's t-test was performed for two-group comparisons. Diferences between the two groups after treatment were measured using covariance analysis. Te threshold for statistical signifcance was set at P < 0.05.

NKPOD Attenuates Ovulation Disorders in PCOS
Patients. In this study, we investigated the function of NKPOD in regulating ovulation disorders in PCOS patients. Interestingly, NKPOD administration resulted in a signifcant decrease of BIM in PCOS patients (P < 0.001; Figure 1(a)). PCOS patients displayed higher levels of AMH, while the increase was partly reversed by NKPOD administration (P < 0.001; Figure 1(b)). Te infuence of NKPOD on T levels in PCOS patients was verifed; the results demonstrated that NKPOD administration signifcantly impaired PCOS-mediated promotion of serum T levels (P < 0.01; Figure 1(c)). Similar results were observed in LH levels (P < 0.05; Figure 1(d)). To further explore the efects of NKPOD on attenuating ovulation disorders, we confrmed the serum of E2. Interestingly, higher E2 levels were observed in PCOS patients receiving NKPOD administration (P < 0.05; Figure 1(e)). Moreover, we found that the increased FSH levels in PCOS patients were partially reversed by NKPOD (P < 0.01; Figure 1(f )). Collectively, these data demonstrate that NKPOD exerts a therapeutic efect on PCOS.

NKPOD Improves Reproductive Outcomes in PCOS
Patients. Subsequently, we further elucidated whether NKPOD infuences reproductive outcomes in PCOS patients. Stimulation duration and total Gn doses were not changed in PCOS patients with NKPOD treatment (P > 0.05; Figures 2(a) and 2(b)). Notably, after exposure to NKPOD, we noted that the changes in the number of oocytes punctured and oocytes retrieved were not signifcant compared with those in the NC group (P > 0.05; Figure 2(c)). However, a higher number of oocytes were found in PCOS patients with NKPOD exposure (P < 0.05; Figure 2(d)). Furthermore, we noted that NKPOD administration signifcantly increased the number of 2PN and 2PN cleavage numbers (P < 0.05; Figures 2(e) and 2(f )). Consistent with 2PN, comparisons analysis demonstrated that NKPOD administration had signifcant efects on increasing the number of embryos transferred and high-quality embryos (P < 0.05; Figures 2(g) and 2(h)). We further evaluated the infuences of NKPOD on reproductive outcomes and found that NKPOD treatment led to a signifcant increase in pregnancy rates in PCOS patients (P < 0.05; Table 2). Furthermore, we assessed the safety of NKPOD administration. Importantly, all PCOS patients were examined for blood, urine, stool routine, ECG, and liver and kidney function after each course of treatment. Te results showed that there were no obvious abnormalities and that there were no serious adverse reactions in all patients. Taken together, our fndings highlight the important role of NKPOD in improving reproductive outcomes in PCOS patients.

NKPOD Component-Target Network Construction.
To elucidate the underlying molecular mechanism, in which NKPOD attenuating ovulation disorders, we investigated the targets of NKPOD through network pharmacology. Te components and potential targets of Angelicae sinensis Radix, Paeoniae Radix alba, Rhizoma Dioscoreae, Rehmanniae Radix Preparata, Cortex Moutan, Poria, Semen Cuscutae, Dipsaci Radix, Cuscutae Semen, Cornu Cervi, Faeces Trogopterori, and Carthami fos, which constitute NKPOD, were identifed, as displayed in Table S1. After screening with OB and DL, a total of 63 components were obtained from the TCMSP. 1857 possible targets of these components were also obtained from the TCMSP (Table S1). Subsequently, a total of 954 genes related to PCOS were obtained from the GeneCards database (1260 genes) and CTD (12894 genes), as shown in Figure 3(a) and Table S2. Interestingly, a total of 954 genes, which were both related to NKPOD and PCOS, were confrmed (Figure 3(b) and Table S3). As demonstrated in Figure 3(c), we structured the compound-target network, including 161 nodes, 61 bioactive compounds and 100 targets. Subsequently, the proposed proteinprotein interactions of 100 targets are constructed and displayed in Figure 3(d). After hiding disconnected nodes, 91 nodes and 686 edges were found in the network. To investigate the crucial nodes, betweenness, closeness, degrees, eigenvectors, LAC, and networks were used to identify 24 nodes and 240 edges. To investigate the potential functions of these targets, we performed functional enrichment analysis and found 1867 GO biological terms (FDR cutof = 0.01, Table S4 and Figure 3(e)). Te results of Figure 3(e) revealed the top 10 enriched GO-BP terms, including nuclear receptor activity, ligandactivated transcription factor activity, DNA-binding transcription factor binding, steroid hormone receptor activity, ubiquitin-like protein ligase binding, RNA polymerase II-specifcDNA-binding transcription factor binding, transcription coactivator-binding serine 4 Evidence-Based Complementary and Alternative Medicine hydrolase activity, serine-type endopeptidase activity, and ubiquitin protein ligase binding.

NKPOD Inhibits AR Expression.
To investigate the possible mechanism of NKPOD on attenuating ovulation disorders, we measured miRNA-224 abundance in PCOS patients. As expected, signifcant increased miRNA-224 abundance was discovered in follicular fuid, granulosa cells, and serum of PCOS patients (Figures 4(a)-4(c)). Particularly, NKPOD showed a stronger ability to lower miRNA-224 abundance in follicular fuid, granulosa cells, and serum of PCOS patients (Figures 4(a)-4(c)). Similar results were obtained for miRNA-224-3p (Figures 4(d)-4(f )) and miRNA-224-5p (Figures 4(g)-4(i)). Taken together, these results imply that NKPOD inhibits miRNA-224 transcription. To understand the mechanism by which NKPOD regulated miRNA-224 transcription, we investigated the transcription factor for miRNA-224. Interestingly, AR was identifed as the only target of miRNA-224 transcription factors and NKPOD regulatory targets (Figure 4(j)). Te predicted sites of AR-binding in miRNA-224 promoters by TransmiRNA v2.0 (https://www.cuilab.cn/ transmiRNA) are shown in Figure 4(k). Additionally, the motif of AR predicated by the JASPAR database (https:// jaspar.genereg.net/) is displayed in Figure 4(l). Next, we explored the efects of NKPOD on the expression of AR in granulosa cells; western blotting was performed and displayed that AR expression was signifcantly upregulated in PCOS patients, whereas the increased AR expression was inhibited by NKPOD treatment (Figures 4(m) and 4(n)). Interestingly, the inhibition efect of NKPOD on phosphorylation of AR was determined, in which the elevated p-AR/AR levels in granulosa cells of PCOS patients were signifcantly attenuated by NKPOD treatment (Figures 4(m) and 4(o)).

miRNA-224 Is Confrmed as the Target of AR.
To further understand the mechanism by which AR regulated miRNA-224 transcription, the ChIP assay was used, and we pointed out that the miRNA-224 promoter was strongly enriched in the AR pellet in granulosa cells (Figures 5(a) and 5(b)). Subsequently, we performed the luciferase reporter assay through constructing the wildtype and mutant-binding sequences ( Figure 5(c)). Te results showed that relative luciferase activity was dramatically enhanced in the miRNA-224promoter-WT + AR overexpression group, but there was no apparent change in the miRNA-224promoter-MUT + AR overexpression group (Figures 5(d) and 5(e)). Next, we further investigated the regulation of AR on miRNA-224 transcription. Te efects of AR overexpression on miRNA-224 transcription were further unveiled. qRT-PCR results showed that miRNA-224 expression was signifcantly upregulated in granulosa cells transfected

Disscussion
Meta-analyses and systematic reviews of observational studies and trials with a small sample size have demonstrated the efectiveness of TCM in improving PCOS's clinical outcomes [12,22,23]. Although some components of NKPOD, as a traditional Chinese medicine decoction, have been widely used in the treatment of infertility and have shown some efcacy [23][24][25], pronounced knowledge gaps remain existed regarding the therapeutic efect of NKPOD on PCOS through systematic research and regulatory mechanisms. In the present study, we discovered that NKPOD administration can alleviate hormone and ovulatory aberrations caused by PCOS, including signifcantly reducing the levels of BMI, AMH, T, LH, and FSH and increasing the abundance of E2. Furthermore, we discovered that NKPOD treatment contributed to gestation in PCOS patients, increasing the number of oocytes, number of 2PN and 2PN cleavage numbers, and number of embryos transferred and high-quality embryos as well as pregnancy rates. Subsequently, we explored the regulatory mechanism and found that NKPOD inhibited AR expression, which indicates that AR expression is positively correlated with miRNA-224 transcription. In particular, AR directly regulated miRNA-224 transcription through binding with the promoter of miRNA-224. Tis study provides novel insights into understanding the efcacy and mechanism of NKPOD in improving ovulation disorders and provides an experimental basis for the clinical application of NKPOD in PCOS ( Figure 5(g)).
Accumulating evidence indicates that an intrinsic abnormality of early follicle development and arrested follicular growth are critical factors in the pathogenesis of PCOS [8,26]. Furthermore, hormonal perturbation will have a profound impact on adult endocrine and reproductive status [27,28]. However, currently, there are still no efective drugs for PCOS, so it is of great signifcance to fnd new drugs for the treatment of PCOS. In this study, we sought to investigate the efect of NKPOD on hormonal levels of PCOS patients. Interestingly, our study provided clear evidence that NKPOD administration can alleviate the hormone dysregulation caused by PCOS, including signifcantly reducing the levels of BMI, AMH, T, LH, and FSH and increasing the abundance of E2. Tese benefcial efects provide a theoretical basis for the application of NKPOD in PCOS treatment. Subsequently, we frst explored the     Cervi, Faeces Trogopterori, and Carthami fos. Although the regulatory targets of some ingredients have been clarifed, the regulatory relationship of NKPOD-target-PCOS has not been clarifed. In recent years, network pharmacology has attracted global attention. Interestingly, network pharmacology provides an efective method for component screening and prediction of drug targets. In our study, we present the NKPOD-target relationships, which give a dependable result of the NKPOD efects on PCOS. A total of 100 potential targets including 24 core targets were identifed to be associated with PCOS. Among the 24 core targets, some of them such as AR [29], epidermal growth factor receptor (EGFR) [30], and RAC-alpha serine/threonine-protein kinase (AKT1) [31] have previously been confrmed through experiments. For example, Jiang et al. [32] reported that miRNA-93 promotes ovarian granulosa cell proliferation through targeting cyclin-dependent kinase inhibitor 1A in PCOS. However, some targets such as peroxisome proliferator-activated receptor alpha and heat shock protein HSP 90-alpha have not yet been confrmed with regard to PCOS through experiments. Terefore, these putative targets that have been discovered provide a foundation for future research. Interestingly, through GO enrichment analysis, we noted that the 100 putative targets were mainly involved in the regulation of gene transcription. Tese potential targets and molecular function provide a basis for further research and the feasibility of TCM theory in guiding clinical NKPOD application. At present, the pathogenesis of PCOS has not been fully elucidated. Changes in miRNAs are obvious pathology in PCOS progression, and miRNAs markers have been used to detect and study this disease [33,34]. miRNA-224 is one of the u-regulated miRNAs located in an intron of a transforming growth factor-β (TGF-β) responsive gene and gamma-aminobutyric acid receptor subunit epsilon [35,36]. Tere are numerous in vitro studies that have shown the potential role of miRNA-224 in the ovaries. miRNA-224 has a regulatory efect on cumulus expansion [17,37]. It has been previously reported that miRNA-224 negatively afected ovulation in the mouse model by decreasing the expression of Ptx3 and Smad4 [32]. Notably, in this study, negative regulation of NKPOD on miRNA-224, miRNA-224-3p, and miRNA-224-5p was noticed. Considering the regulation of NKPOD on putative targets including transcription factors as well as the function of transcription factor activity, we speculated that NKPOD may regulate miRNA-224 abundance by regulating the activity of transcription factors. Using bioinformatic methods, we discovered only one common target (AR), which is related to the transcription factor of miRNA-224, NKPOD targets, and PCOS-related genes. Due to the relationship between AR and PCOS [38], we believe that the regulation of miRNA-224 abundance by NKPOD requires the involvement of AR. As expected, AR was verifed as the transcription factor activating miRNA-224 transcription. Mechanism investigation further validated that NKPOD administration eliminated the stability of AR. Te combined data elucidate a crucial mechanism of NKPOD in improving ovulation disorder in PCOS patients.
However, we are aware of some limitations in this study. First, the sample size of PCOS patients was relatively limited, and larger population is necessary to validate the role of NKPOD. Furthermore, the regulation of NKPOD on other targets lacks experimental data support, so it will be interesting to study them in the future. In summary, to our knowledge, this is the frst report that generally investigates the clinical implication and mechanism of NKPOD in PCOS. Te results present that NKPOD improves ovulation disorder in PCOS patients through AR-mediated transcriptional inhibition of miRNA. Tese fndings provide novel insights into understanding the efcacy and mechanism of NKPOD in improving ovulation disorder and provide an experimental basis for the clinical application of NKPOD in PCOS.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Tis work was approved by the Ethics Committee of the Jiangsu Hospital of Traditional Chinese Medicine (2019NL-KS74).

Consent
Written informed consent was obtained from all participants included in the study.

Conflicts of Interest
Te authors declare no conficts of interest.