Potential neuroprotective effect of Androst-5-ene-3β, 17β- diol (ADIOL) against experimentally-induced Parkinson’s disease in rats

Abstract

Several mechanisms are implicated in PD that finally lead to a common final pathway which is degeneration of DA neurons, decline in striatal DA level and, accordingly, to PD. Recent studies focus on neuroinflammation as a major component implicated in the progression of neurodegeneration. Though it is not fully understood whether neuroinflammation is a primary cause or secondary effect, it was illustrated through previous studies that inflammatory mediators are elevated in the activated microglia and reactive astrocytes in the CNS in PD. Recently, it was shown that the ER agonist, ADIOL, is capable of reducing neuroinflammation through an ERβ-CtBP repression pathway. Hence, this prospective study was designed aiming to reveal the potential neuroprotective effect of 3 different doses of ADIOL through detecting the changes in motor behavior, various biochemical parameters including inflammatory genes expression, histopathological examination and the final impact on the neurodegeneration progression in a rotenone-induced PD in rats. The rat groups were divided as follows; • Group 1: Control rats, injected the vehicle of ADIOL (1 ml/kg/day; s.c.), followed -one hour later- by sunflower oil (1 ml/kg/day; i.p.) for 30 days. • Group 2: Positive control rats, receiving ADIOL (3.5 mg/kg/day for 30 days; s.c.). • Group 3: Rotenone-treated rats (1.5 mg/kg/day for 30 days; i.p.). • Group 4: Rotenone-treated rats (1.5 mg/kg/day for 30 days; i.p.) receiving daily dose of ADIOL (0.35 mg/kg/day for 30 days; s.c.) one hour earlier. • Group 5: Rotenone-treated rats (1.5 mg/kg/day for 30 days; i.p.) receiving daily dose of ADIOL (3.5 mg/kg/day for 30 days; s.c.) one hour earlier. • Group 6: Rotenone-treated rats (1.5 mg/kg/day for 30 days; i.p.) receiving daily dose of ADIOL (35 mg/kg/day for 30 days; s.c.) one hour earlier. The results of the present study showed that the middle ADIOL dose (3.5 mg/kg/day) improved mitochondrial function more than the low (0.35 mg/kg/day) and high (35 mg/kg/day) doses, where intact mitochondria with apparent cristae were observed upon EM examination; a finding that was further reflected in the elevated ATP level in both of the striatum and SN upon treatment with the same dose. The link between enhanced mitochondrial function and reduced accumulation of α-synuclein aggregates due to improvement in the UPS was illustrated in the ability of ADIOL to reduce α-synuclein density even at the low dose, where the middle ADIOL dose showed less α-synuclein immunoreactivity than the high dose, in agreement with the previous results. The end result of declined α-synuclein aggregation was further illustrated through the changes in the apoptotic pathway. It was shown that the pro-apoptotic markers; caspase-3, Bax as well as Bax/Bcl-2 ratio were declined with elevation in the anti-apoptotic marker; Bcl-2 in both of the striatum and SN of rotenone-treated rats upon pre-treatment with the 3 doses of ADIOL. The middle ADIOL dose reduced the striatal and nigral levels of pro-apoptotic markers more than the low dose, with no superior effect for the high dose. Contrarily, the nigral caspase-3 level was even reduced upon administration of the middle dose more than the high one. Degeneration of DA neurons in the SN and the decline in DA release in the striatum is the normal end-stage for ATP decline, formation of α-synuclein aggregates and activation of apoptosis. The 3 administered doses of ADIOL effectively elevated the nigral TH, which emphasizes the protective effect of ADIOL upon this DA precursor and, in turn, the progression of PD, where the middle dose showed the best effect. Although the high dose of ADIOL managed to ameliorate TH density, yet its effect was the least among the other 2 doses. Subsequent to the previous finding, the middle ADIOL dose effect upon striatal DA level and DA turnover was better than the low dose, which was not shown regarding the level of DOPAC. Yet, the middle dose of ADIOL produced no change regarding HVA metabolite concentration, unlike the low dose. The high ADIOL dose could not show any better effect, except for the HVA level that was better than the middle dose but similar to the low one. Histopathological examination of both of the striatum and SNpc further illustrated the protective effect of ADIOL against neurodegeneration, even when administered at the low dose. However, the middle dose of ADIOL was the best one to reduce neurodegeneration which was observed in the increased number of viable neurons in both of the striatum and SNpc of rats. This could be related to the findings obtained following EM examination of the SNpc; where the middle dose showed more intact structures like the nuclei and mitochondria, relative to the findings obtained upon administration of the low and high doses of ADIOL. The positive impact of pre-treatment with ADIOL on DA levels was reflected in the improvement of motor behavior. The low dose of ADIOL administered was only capable of decreasing the time spent inactively sitting upon performing open field test. However, the middle ADIOL dose managed to increase the number of squares crossed, number of stops, activity index, and rearing frequency in addition to reducing the inactive sitting time. The activity index and rearing frequency were also enhanced upon administration of the high dose of ADIOL together with a decline in the time spent inactively sitting, without showing any further improvement than the middle dose. On performing bar and grid tests to test for catalepsy, pre-treatment with the low and high doses of ADIOL could not alleviate rigidity. However, daily administration of the middle dose of ADIOL managed to lessen the descent latency upon performing bar test, but not in the grid test, in rotenone-treated rats.