Research On The Effect Of Androgen On Immune Function Of Astrocytes

Androgen is a class of carbon-19steroid hormone of the body, which contains Testosterone(T) and Dihydrotestosterone(DHT) and so on. The brain is an important target organ of androgen, besides, it not only has a certain impact on the memory of brain and cognitive function, but also has some protective effects on the nervous system. The current research on estrogen’s protective effects on the nervous system was mostly using neurons. As the most brain glial cells, Astrocytes play an important role on neurons with nutrition, protection, support and regulation, as well as the stability and function of the nervous system. Researching on mechanisms of how androgen regulated astrocytes’ immune function not only could provide new theoretical support for the protection of the nervous system, but also provide an important theoretical value on prevention and treatment of neurological diseases, especially those age-related diseases which related to androgen.To study the immunological mechanism androgen playing on astrocytes in vitro effectively, we nedd to establish a more effective system of cultivating astrocytes in vitro. The primary astrocytes in the study comes from the SD rats aged1-to3-day. After isolating and purificating for serial times, the primary cells were obtained, and the purification rate exceeded99%, The results inferred that the primary cells were obtained could meet the requirement for the research.In our study, we firstly used10μg/ml LPS to stimulate AST for10h, then we used0nM,10nM,100nM and1000nM5a-DHT to deal with astrocytes for24h. The results inferred that as the concentration of5a-DHT increased, the activity of AST significantly decreased. And compared to the group of LPS, when AST was treated with LPS plus100nM5a-DHT, the activity of AST decreased respectively (P<0.05), while there was no significant difference compared with the control group(P>0.05).After stimulating AST using10μg/ml LPS for10h, then we used10nM5a-DHT to deal with astrocytes for24h. After that, we detected astrocyte activity on Oh,2h,4h,8h,12h,24h,36h,48h, The results showed the group with LPS only is14.03%higher than which using LPS+5a-DHT(P<0.05), other groups showed no significant difference.Cell cycle was related to the immune function of cells. In the study, we firstly used10μg/ml LPS to stimulate AST for10h, then we used0nM,10nM5a-DHT to deal with astrocytes for24h and collected cells for cell cycle detection. The results showed that10nM5a-DHT could significantly inhibit the increased ratio of cells in S+G2stages induced by10ug/ml LPS, respectively (P<0.05).The astrocytes occurred inflammation response, which could result the change on ultrastructure of astrocytes. With the purpose of understanding the effect of astrocytes on ultrastructure of AST, we firstly used10μg/ml LPS to stimulate AST for10h, then added10nM5a-DHT astrocytes in the experimental group and added0nM5a-DHT to astrocytes in the control group for another24h. The results showed that10μg/ml LPS made rough endoplasmic reticulum swell, mitochondria expansion and especially been vacuolization. But in the group LPS plus10nM 5α-DHT, the number of exception of the rough endoplasmic reticulum and mitochondria is significantly reduced.we firstly used10μg/ml LPS to stimulate AST for10h, then we used0nM,10nM,100nM and1000nM5a-DHT to deal with astrocytes for24h, and then determination of the level of NO, IL-1β and TNF-α of AST, in order to understand the impact of androgen on the levels of immune cytokine secretion of AST. IT show that, the level of NO, IL-1β and TNF-α which increased by the LPS-induced has decreased,10nM5a-DHT can inhibition the secretion of NO, IL-1β and TNF-α induced by LPS (P<0.05)In order to further determine the AST NO, IL-1β and TNF-a secretion levels decline is the result of androgen effect, the experiment with10u g/ml LPS cultured AST10h, adding a concentration of OnM5a-DHT,10nM5a-DHT,10nM5a-DHT+100nM flutamide medium, and continue to culture24h to determine AST NO, IL-1β and TNF-a level. The results demonstrated that androgen losed inbition to LPS in flutamide medium group. At the same time, detection the level of AST NO、IL-1βand TNF-α between10μg/ml LPS+10nM5a-DHT group and10μg/ml LPS+10nM5a-DHT+100Nm flutamide group at0.5h、1h、2h、8h、12h、24h, the results showed that the level of AST NO、IL-1βand TNF-α in10μg/ml LPS+10nM5a-DHT group gradually decreased but increased in10μg/ml LPS+10nM5a-DHT+100Nm flutamide group.In order to understand the role of androgen signal transduction path, the experiment group with10u g/ml LPS cultured AST10h, adding a concentration of20u M p38inhibitor SB203580and20u M/ERK-1/-2inhibitor U0126before add lOnM5a-DHT30min. At the same time, the control added OμM SB203580and U0126, and continue to culture10min to determine AST NO, IL-1β and TNF-α level. The results found that androgen could adjust AST immune response through ERK-1/2transduction path. Detection the level of AST NO、IL-1βand TNF-α between20μM SB203580and20μM U0126group at5min、10min、20min、30min, the results showed that the level of AST NO、IL-1βhad no significant changes in20μM SB203580, but TNF-α gradually decreased. The level of AST NO、IL-1Band TNF-a in20μM U0126had no obvious changes.To sum up, the experiment can drow the following conclusion:1. The test builds a research model in vitro about the AST function regulated by androgens.2. Androgens can significantly inhibit AST cell activity elevation, AST excessive proliferation induced by10u g/ml LPS, reduced S+G2cell ratios and protect AST normal ultrastructure.3. Androgen via ERK-1/2transduction pathway inhibits AST TNF-a levels caused by10u g/ml LPS.