Drug abuse, including morphine, is one of the problems of human beings today. Morphine has adverse effects on the physiology of various organs, including the nervous system as well as the reproductive system. Studies have shown that baclofen suppresses morphine self-administration. It reduces the severity of withdrawal symptoms and is used during detoxification and rehabilitation of addicts (Assadi et al., 2003). Thus baclofen may interfere with morphine outcomes. With the hypothesis that the effects of long-term morphine use are significantly different from the effects of acute morphine use, we have recently modeled polycystic ovaries with acute morphine use (Mohammadi, et al., 2020). Because this disorder is associated with oxidative stress (Hashemian, et al., 2020), we aimed to investigate the protective effect of baclofen on oxidative stress in the dorsal hippocampus (dH) in an experimental model of acute morphine-induced polycystic ovary syndrome (PCOS).
Various methods have been used in various studies to induce PCOS: letrozole, testosterone, estradiol valerate, and hydroepiandrosterone, adrenocorticotropin, and long-term use of light (Salvetti, et al., 2004). Morphine has also been introduced as one of the causes of PCOS due to its adverse effect on the reproductive axis (Karami, et al., 2015; Karami and Darban-Fooladi, 2015). In this study, we accordingly found that the ovaries had a polycystic ovary in animals receiving morphine. It has previously been shown that a proinflammatory (nitric oxide( system is involved in ovarian cystogenesis (Karami and Darban Foladi, 2015; Karami et al., 2015; Mohammadi et al., 2020). However, in the present work, except for the marginal lining of the ovary, none of the parts of its main frame showed positive reaction to the biochemical staining of NO.
Studies have shown that about 40–50% of people with PCOS are obese, which causes them to burn more fat. Burning fat produces free radicals that affect the body and bring the ovaries under oxidative stress (Tahmasebi, et al., 2015). Therefore, in this study, we measured the lipid profile in the dH tissue in the morphine-receiving group to evaluate the effect of morphine on the ovarian tissue and dH. Measurements of serum triglyceride, total cholesterol, LDL cholesterol and HDL cholesterol, however, did not show significant change between the groups, identifying a negligible effect.
Studies have shown that hypothalamic-pituitary-adrenal axis activity increases in PCOS (De-Gregori et al., 2012; Hahn et al., 2002). Some researchers believe that HPA and sympathetic activity trigger a stress response, which in turn causes physiological and metabolic changes in the body, and that stress and elevated glucocorticoids in the blood cause oxidative stress (Silva, et al., 2019). Therefore, in order to investigate whether oxidative stress in dH is induced due to increased glucocorticoid receptors, we specifically examined the concentration of glucocorticoid receptors at dH. The results, however, showed no significant change in glucocorticoid receptor levels at dH and therefore their involvement is questionable. The central nervous system in general (De-Gregori et al., 2012) and especially the hippocampus has been introduced as very sensitive to oxidative stress (Hosseini et al., 2010). However, the sensitivity of the hippocampus to oxidative stress in the morphine-induced PCO model has not been demonstrated in previous studies. In the present study, the level of oxidative stress at dH was higher in the morphine-treated groups with polycystic ovaries than in the control group. Thus morphine is involved in the development of PCO and oxidative stress at dH.
In mammals, morphine (i.p.) is mainly metabolized in the liver by the enzyme UGT or UDP (uridine-5-phospho-glucuronosyl transferase) to two glucuronide metabolites (morphine-3-glucuronide and morphine-6-glucuronide). M3G is produced 5 times more than M6G, but only M6G can cross the blood-brain barrier (BBB). M6G has been shown to cross BBB 7.5 times less than morphine. It tends to bind to the opioid µ-receptor, but because of its lower levels than morphine, its analgesic activity is significantly lower than the morphine (De-Gregori et al., 2012). In the present study, to investigate whether morphine or its metabolites were more effective in inducing oxidative stress in the brain, a group received morphine intra-VNH at an effective dose (seen earlier by Karimi, et al., 2018). However, no significant difference in the levels of oxidative stress factors in dH tissue was observed in this group compared to the groups that received morphine (i.p.).
Almost every neuron in the hypothalamus is targeted by GABAergic synapses, including GnRH cells, β-endorphins, and endogenous opioids, and even GABA itself (Lagrange, et al., 1996). The arcuate nucleus (ARN) is the major regulatory center for GnRH neurons. It has been shown that there is a strong anatomical circuit between GABAergic neurons in ARN and GnRH neurons (Kawwass, et al., 2017; Davis, et al., 2002). The cell body of GnRH neurons is located in the ARN. There is no BBB in the ARN (Sapru, 2013). The effects of GABA on GnRH neurons secretion may differ with growth stage, hormonal environment and expression of the GABA receptor subtype (Kawwass, et al., 2017). Baclofen, as a GABAB receptor agonist, modulates LH pulsatile secretion. GABAB receptors regulate the excitability of hypothalamic GnRH neurons (Liu, et al., 2015). GABAB receptors are abundant in the VMH nucleus (Davis, et al., 2002), which contains neurosteroid-secreting neurons. It regulates fertility and sexual acceptance (Correa, et al., 2015; Fanselow and Dong, 2010; Parandin and Rassouli, 2017). This nucleus is also linked to the hippocampus (Canteras, et al., 1994). GABA receptors are found in areas of the brain that are sensitive to sex hormones (Bäckström, et al., 2011). Estrogen is not directly related to GnRH neurons. Therefore, in order for estrogen to affect these neurons, there must be mediating neurons. These mediating neurons are GABAergic. Studies have shown that GABA has protective effects against PCOS-induced metabolic and reproductive disorders (Ullah, et al., 2017). Alavian et al. (2019) have demonstrated that injection of GABAB receptor agonist, baclofen, significantly reduces morphine sensitivity expression in female rats.
In the present study, baclofen as a GABAB receptor agonist interfered with oxidative stress induction, so that oxidative stress at dH was significantly reduced in the baclofen-receiving groups compared to the morphine-receiving groups. In baclofen-treated groups, oxidative stress at dH was slightly reduced compared with the morphine-receiving group, indicating that baclofen may interfere with morphine-induced inflammation.