3 alpha-diol, a testosterone metabolite, has anti-inflammatory properties in the experimental autoimmune encephalomyelitis model

Multiple sclerosis (MS) is an autoimmune pathology characterized by inflammatory and demyelinating phases. Clinical observations indicate an involvement for sex hormones in thepathology, so that, recently, a therapy for MS based on hormones has been proposed. A clinicaltrial, involving male MS patients, revealed improvement in cognitive performances after one yeartreatment with testosterone (Sicotte et al., 2007). In the experimental autoimmune encephalomyelitis (EAE) mice, a MS animal model, neuroprotective effects of testosterone, and of its metabolite dihydrotestosterone (DHT), were observed (Dalal et al., 1997; Palaszynski et al.,2004). In the same model, castration has deleterious effects on clinical course, suggesting a protective role for androgens (Bebo et al., 1998). Moreover, in vitro evidences showed that testosterone is able to reduce inflammatory cytokines produced by human macrophages (D’Agostino et al., 1999) and monocytes (Li et al., 1993; Liva and Voskuhl, 2001). These antiinflammatory properties may be mediated by the conversion of testosterone into estradiol, or, alternatively, by some direct effects on androgen receptor (AR), mediated by DHT. This steroid can be subsequently converted into 3 alpha-diol, a GABAA receptor agonist, acting as antiinflammatory agent. Based on these evidences, we were interested in explore the protective effects of androgens treatment in a rat EAE model characterized by acute inflammatory events. We induced male Lewis rats with myelin basic protein in order to develop EAE; animals were treated with 3 alpha-diol or vehicle (sesame oil) every other day for two weeks, whereas control rats (i.e., rats not induced with EAE) received in same days only vehicle. Neurological deficits were assessed throughout the experiment (i.e., until 15 days post induction; dpi), and, at 15dpi, spinal cords were dissected. The clinical course and the weight loss of EAE rats treated with 3 alpha-diol were not different when compared to vehicle treated animals. Significant differences between these two groups were instead observed in the spinal cord after immunostaining analysis for the glial fibrillary acidic protein and the major histocompatibility complex class II, where 3 alpha-diol treatment was able to reduce the inflammatory parameters. In agreement with this observation, gene expression for tumor necrosis factor alpha, a pro-inflammatory cytokine, was decreased after 3 alpha-diol administration compared to oil-treated EAE animals. This effect seems specific for proinflammatory cytokine, because the expression of transforming growth factor beta 1, an antiinflammatory cytokine, was not different between vehicle and 3 alpha-diol treated EAE rats. To asses the metabolic fate of the injected 3 alpha-diol, liquid chromatography tandem mass spectrometry analysis were performed in spinal cord from all groups. 3 alpha-diol levels were significantly increased in steroid treated animals; moreover, a significant raise in DHT levels was observed in the same animals, indicating a retroconversion of 3 alpha-diol into DHT. The data we obtained may suggest that the reduction of inflammatory parameters could be due by both the action of DHT on AR and/or of 3 alpha-diol on GABAA receptor. These observations pointed out, for the first time, the effects of 3 alpha-diol on inflammatory parameters in EAE Lewis rats. Future studies will be aimed to elucidate the involvement of androgen and/or GABAA receptors on the observed effects.