The effects of NAC, inulin and combined therapy on blood-brain barrier integrity, hippocampal oxidative stress and hippocampal inflammation in rats with Testosterone deprivation. However, treatment with NAC, inulin and combined therapy, but not testosterone, in ORX rats significantly decreased the plasma triglyceride levels, when compared to sham-operated rats and ORX rats treated with vehicle (Table 1). These findings suggest that NAC, inulin, and the combined therapy ameliorated the deleterious effects on the brain in castrated male rats similar to those treated with testosterone. In another in vivo study, a reduction in levels of testosterone and an increase in levels of LH and FSH were seen after NAC supplementation among adult male Wistar rats(Reference Moghadam, Shariati and Naeimi16). In a meta-analysis study, beneficial effects of NAC supplementation on IL-8, malondialdehyde and homocysteine levels have been shown. To investigate blood-brain-barrier permeabilization, the expression of hippocampal tight junction proteins including claudin-5 and occludin were measured by immunoblotting. Similar to insulin sensitivity, plasma triglyceride, plasma cholesterol, plasma HDL and LDL levels were not significant difference between ORX rats and sham-operated rats. ORX rats showed significantly decreased body weight, food intake, and visceral fat weight when compared to those of sham-operated rats (Table 1). NAC, and inulin attenuated gut dysbiosis, but the combined therapy and testosterone replacement had better efficacy to restore the balance of gut microbiota in the testosterone-deprived condition. These findings suggested that testosterone-deprived rats developed gut dysbiosis. Previous study reported that consumption of 10% inulin for 4 weeks markedly shifted the composition of gut microbiota and improved intestinal function . The diet of treated rats was prepared by adding 10% of inulin fiber into the control diet (by weight). Recently, our previous study reported that both microglia and astrocytes also had an amoeboid-like shape in castrated male rats similar to this study . A previous study showed negative microglial morphological changes in high fat diet-fed rats, which were improved with prebiotic, probiotic and synbiotics treatment . Testosterone replacement, NAC, inulin, or the combined therapy in ORX rats equally ameliorated cognitive decline, possibly through anti-oxidative and anti-inflammatory effects of these therapies. These changes led to further brain oxidative stress 28,29, brain inflammation, and microglial activation 11,29,30, resulting in cognitive decline, as shown in the present study. Testosterone replacement, NAC, inulin, and the combined therapy attenuated hippocampal dysplasticity, leading to the improvement of cognition in testosterone-deprived condition. Testosterone replacement, NAC, inulin, and the combined therapy equally improved cognitive function as indicated by the decreased time taken to reach the platform in the acquisition test and the increased time spent in the target quadrant during the probe test in ORX rats (Figure 7C-E). For all multiple comparisons, data were analyzed using a two-way ANOVA followed by post-hoc Tukey’s analysis. Data from each experiment were expressed as mean ± S.E.M. For acquisition tests, the significance was calculated using repeated Two-way ANOVA tests followed by post-hoc Tukey’s analysis. After 30 minutes of habituation, rats were randomly placed in the water at one out of four starting points with their head turned towards the border of the water pool. In this study the protocol of the MWM was modified from that described by Vorhees et al., 2006 . Three fields per brain slice, three brain slices per animal, and six animals per group were measured for the CA1 region of the hippocampus and cortex . The mean fluorescent intensity was also measured and the complexity of the glial cells were measured by Sholl analysis 11,27. Three microglial and astrocyte cells per brain slice from the CA1 region of the hippocampus, three brain slices per animal were measured from all six animals in the group. It must be noted that the GSH levels might be effective in the relationship between NAC and reproductive biomarkers(Reference Atkuri, Mantovani and Herzenberg50). Therefore, NAC, through decreasing lipid peroxidation, could have beneficial effects on reproductive biomarkers. Participants of included studies in seventeen of eighteen studies had PCOS. Regarding TT, studies with shorter duration had smaller sample sizes than studies with longer duration studies. In other investigated variables, extending supplementation duration was ineffective in the overall results. If the study contains a methodological defect that may affect its findings, the study was given a ‘high risk’ score; if there was no defect for that domain, a ‘low risk’ score; if the information was insufficient to determine the effect, an ‘uncertain risk’ score. The Cochrane Collaboration’s risk of bias tool was employed to assess the risk of bias for each study(Reference Higgins, Altman and Gøtzsche19). In addition to searching in databases, we checked the references of related articles for any missing eligible articles (hand-search method).
