PCOS is the most common endocrine condition affecting approximately 20% of reproductive -aged women. Though some ovulation induction agents, such as letrozole, clomiphene citrate and metformin, have been used to improve reproductive outcomes in clinic, the side effects of these drugs should not be ignored[20, 21]. Thus, numerous studies attempted to guide the development of new and effective therapies for PCOS. Recently, treatment based on traditional Chinese medicine and food ingredients, such as AF extract, soy isoflavones and crocetin, provided a novel therapeutic way for PCOS [22–24]. All these observations suggested rooms for improvement in PCOS therapies. Possession of beneficial effects on animal models of different central nervous system diseases made troxerutin, a rutin derivative, an attractive therapeutic method for us to investigate its possible effect on PCOS. Specifically, troxerutin inhibited cyclin-dependent kinase 1 expression, enhanced type 1 protein phosphatase α dephosphorylation and abolished MEK/ERK1/2/C/EBP β activation, which subsequently reversed the memory impairment in the DA-treated mice[25]. The neuroprotective potential of troxerutin in 6-OHDA rat model of Parkinson's disease was through mitigation of apoptosis ,astrogliosis, oxidative stress and part of its effect was dependent on PI3K/ERβ signaling[26]. Troxerutin and cerebroprotein hydrolysate injection acted as a neuroprotective agent against cerebral ischemia/reperfusion injury via anti-inflammation, anti-apoptosis and blood-brain barrier maintenance[27]. In the present study, we evaluated the possible effect of chronic administration of troxerutin in a DHT-induced PCOS rat model, and two important findings were revealed. First, administering troxerutin 300 mg/kg exerted a beneficial effect of reducing body weight, the elevated levels of LH, testosterone and GnRH in PCOS rats and its effect was superior to troxerutin 150 mg/kg. Second, the potential mechanism behind the observed effect on overweight, polycystic ovaries and endocrine aberration involved altering GABA, glutamate, kisspeptin/kiss1r and NKB/NK3r in the hypothalamic pituitary region. To the best of our knowledge, this is the first study to investigate the possible effect of troxerutin on PCOS using a rat model.
GnRH pulses stimulate the synthesis and secretion of LH and FSH from anterior pituitary. It is well known that although produced in the same cell named gonadotroph, LH and FSH synthesis is regulated by different frequency of GnRH pulses, with LH favored by fast pulse frequencies(> 1 pulse per hour) and FSH favored by slow pulse frequencies(< 1 pulse per 2–3 hour). As for PCOS, a neuroendocrine hallmark is persistent and rapid GnRH pulses, which favor pituitary synthesis of LH and contribute to the increased LH levels[28]. LH is a stimulus for androgen synthesis, so the increased LH drive in turn caused the elevated androgens [29, 30]. Therefore, in terms of biochemical indicators, the most important and characteristic abnormality referring to PCOS is elevated LH, subsequently elevated testosterone and low to normal FSH levels in serum[4, 31]. Consistent with previous studies, PCOS rats displayed similar biochemical abnormality including markedly increased LH, concomitantly increased testosterone levels and non-significant FSH levels as compared to sham rats. Next, we observed the serum LH, testosterone and FSH levels in PCOS and troxerutin groups in order to determine whether troxerutin treatment has any therapeutic effect on PCOS. As the results revealed, troxerutin treatment indeed inhibited the elevated LH and testosterone levels without significant influence of FSH level. In addition, we further estimated the circulating and hypothalamic status of GnRH, which were both significantly increased in PCOS rats. However, there was a significant troxerutin-caused ( 300 mg/kg) decline in serum and hypothalamic GnRH. In regard to the serum hormone levels in rats, many previous studies have reported that LH were generally in the range of 1.5 ~ 3 mIU/ml[32, 33], FSH levels varied from 8mIU/ml to 20mIU/ml [22, 34]and testosterone varied from 0.2 to 10 ng/ml[32, 34–37], which were approximately consistent with the corresponding results in the present study. Conclusively, troxerutin treatment 300 kg/mg to rats for up to 4 weeks inhibited the hyperactive GnRH/LH system in PCOS rats.
Compelling evidence provided the strong support for brain being the culprit in PCOS[38, 39]. The secretion of GnRH from the brain is itself regulated by numerous upstream factors, such regulators include GABA, norepinephrine, dopamine, serotonin and glutamate ,amongst others[40, 41]. While specific impairments in the brain were difficult to assess in humans, rat models greatly facilitated the identification of difference underpinning the hyperactive HPG axis in PCOS. Therefore, the representative GnRH regulators of inhibitory GABA and stimulatory Glu were observed in the present study. In comparison to sham rats, PCOS rats showed a turnover of regulatory neurotransmitters with a significantly decreased GABA and increased Glu, which were both reversed by administering troxerutin 300 mg/kg to rats for up to 4 weeks. As far as the hypothalamic neurotransmitter levels were concerned, previous studies reported that hypothalamic GABA was in range of 12–20 µM/g and hypothalamic Glu was in range of 22–45 µM/g in rats ,each approximately consistent with our findings after unit conversion. To date, one arcuate nucleus population of particular recent interest has been the kisspeptin/neurokinin B/dynorphin expressing 'KNDy' neurons ,which synthesized and released GnRH stimulatory neurotransmitters kisspeptin and NKB. Protrusions of GnRH neurons in the ME express their receptors kiss1r and NK3r. In the present study, troxerutin decreased the expression of kisspeptin1 and NKB in the arcuate nucleus, and also the receptors kiss1r and NK3r in GnRH positive neurons of the ME. These results indicated that the protection of troxerutin against PCOS partially was due to its ability to regulate hypothalamic GABA, Glu, kisspeptin/kiss1r and NKB/NK3r. Although it remains determined whether this troxerutin-caused reversal is permanent, the present study highlighted its therapeutic potential for PCOS. More in depth research will be necessary to determine whether troxerutin targeted at hypothalamic neurotransmitters alone is a promising therapeutic approach for the treatment of PCOS.