Study On The Neuroprotection And Mechanism Of Progesterone Against Aβ Induced Neuroinflammation In Astrcocytes

Alzheimer’s disease(AD) is a progressive neurodegenerative disorder that results in a gradual decline in memory and cognitive processes. The deposition of amyloid-β(Aβ) has been considered as an extremely critical factor for AD development, however, the mechanism underlying Aβ-induced neurotoxicity is not fully understood.It was recently recognized that neuroinflammation is a prominent feature in the modulation of AD processing. In the early stages of AD, AD patients begin to increase the expression of inflammatory genes and synthesis of inflammatory cytokines in the brain.Nevertheless,epidemiological and prospective population-based studies show an association betweensuppression of inflammation and reduced risk for AD. Neuroinflammation is considered to be another important factor regulating the development of AD.Astrocytes are the most abundant neuroal cells in the central nervous system. Under normal physiological conditions, astrocytes dynamically endow neurons with trophic support and modulate information processing, in the meanwhile, implicate in mediating homeostasis in the brain. Once activated, astrocytes strongly produce proinflammatory mediators, including cytokines, growth factors, complement molecules, and chemokines. The released cytokines, particularly interleukin(IL)-1β,IL-6,andtumor necrosis factor(TNF)-α, are the major effectors of neuroinflammatory mediators and affect neurophysiologic mechanism regarding cognition and memory. Remarkably, a large number of excessive reactive astrocytes appear in the cerebral cortex and subcortical structures of AD patients. Indeed, these reactive astrocytes revolve around senile plaques in the AD brain, and release a large number of pro-inflammatory mediators.This constitutes a significant pathological change appearingin the early stages of AD, suggesting that astrocyte is a potent targets for the treatment of brain inflammation. However, the mechanistic connection between Aβ deposition and astrocytic inflammation is not fully understood.Therefore, to discuss the pathogenesis mechanism of AD comprehensively and search effective therapeuticstrategies have become one of the major problems.Neurosteroids, steroid substances with neuroprotective activity, are mainly generated and secreted from various precursors in glial cells and neurons by the action of steroidal enzymes or cholesterol from peripheral circulation into the nervous system. Numerous studies show that neurosteroids not only involve in regulating the development of nervous system, and have an impact on the function of the nervous system, but also supply the nervous system with nutrients, and promote neurogenesis and neuronal survival.Neurosteroids progesterone(PG) as an endogenous neuralactivated steroids, has an obvious effect on regulation of neural cells function. Bashour found that progesterone quickly suppressed the activity of GnRH neurons, axons growth regulation, and regulated the synthesis and release of neurotrophic factor BDNF. Sha showed that subcutaneous injection of PG significantly improved cognitive abilities of AD rats. With many evidence of the function of neurosteroidsrecovered,one particular focus of previous studies is the neurosteroid PG and its neuroprotective capacity in neurodegenerative disorderslike AD.However,its exact mechanism in mediating AD remains poorly understood. Previous studies in our laboratory found that PG inhibited Aβ-induced mitochondrial apoptotic and represented a neuronal protective mechanism, but the neuroprotective mechanism involving the regulation of astrocytic function warrant further investigation.As an important regulator of neural cellsin the central nervous system, astrocytesplay an important role in the regulation of inflammatory responses in the brain.In this study,neuroinflammation AD cell model was established based on Aβ-induced excessive activation of primary cultured rat cortical astrocytes, and discussed the molecular mechanisms of Aβ-induced astrocyte dysfunction and further demonstrated the role of PG and theneuroprotective mechanism involving the regulation of Aβ-induced astrocytic dysfunction and possible regulatory mechanism.Part I β induces astrocytes excessive activationObjective:To research the effect of Aβ on primary cultured astrocytes function, andfurther evaluate Aβ-induced excessive activation of astrocytes.Methods:The cerebral cortex tissue was separated from the brain of newborn SD rats.The primary cultured astrocytespurified and cultured in vitro.Astrocytes marker protein GFAP wasdetected to identify its purity by Immofluorescence method. Immofluorescence, Western blot, ELISA method was used to detect the effect of Aβ on astrocytic morphological changes, GFAP protein expression, proinflammatory cytokines IL-1β and TNF-α secretion levels.Results:1 Aβ exacerbates IL-1β and TNF- ɑ production in astrocytesAstrocytes were treated with Aβ for 0, 3, 6, 12 or 24 h. The levels of IL-1βand TNF-ɑin the medium of treated astrocytes were assayed by ELISA. The results showed that Aβ upregulated proinflammatory cytokines IL-1β and TNF-α levels, compared with non-treated group, Aβ significantlyincreased IL-1β and TNF-α production following 6h treatment.2 Aβ intracellular abnormal accumulates in astrocytesCultured astrocytes were exposed to Aβ for the indicated times(0 h, 3 h, 6 h, 12 h), and the cells were immunostained with antibodies against Aβ. The results showed that Aβ accumulation began to formulate inastrocytes after 3h Aβ treatment.Aβ protein aggregation were found to increase in the vicinity of nucleus or cytoplasm of astrocytes following longer period of timeAβ treatment. These results suggest large number of formation of Aβ accumulation may severely interrupt the astrocytes degradation capacity.3 Aβ induces morphological changes in astrocytesTo assess possible alterations to the astrocytic morphology inresponse to Aβ treatment.Astrocytes were exposed to Aβ for 6h treatment, compared with non-treated group. Immofluorescence results showed that astrocytes obvious appeared morphological changes after 6h Aβ treatment, characterized by an enlarged body and shorter filopodium-like processes.4 Aβ upregulates GFAP expression in astrocytesTo further access the effect of Aβ onastrocytes function, we detectedthe level of GFAP expression,which related tothe activation of astrocytes. The results showed that Aβ increased GFAP expression, compared with non-treated group, Aβ significantlyincreased GFAP expressionfollowing 6h treatment.Conclusions:Aβinducesexcessive activationof astrocytes andcauses a large number of secretion of pro-inflammatory cytokines IL-1β and TNF-α. Part II tudy on the mechanism of Aβ induced neuroinflammation inastrocytesObjective:To discuss whether inflammatory responses are concomitantly involved in Aβ-induced astrocytes activation and explore potential molecular mechanism. To evaluate whether exogenous inflammation signal inflammasome NLRP3 is activated in Aβ treated astrocytes and investigate the relationship between inflammasome NLRP3 and Aβ-induced neuroinflammatory responses.Methods:The cerebral cortex tissue was separated from the brain of newborn SD rats.The primary cultured astrocytespurified from the cortex tissue and cultured in vitro. Immofluorescence, Western blot method was used to detect NLRP3 expressionand Caspase-1 activation in Aβ treated astrocytes. To determine whether NLRP3/Caspase-1 exogenous inflammatory signaling pathways activationis concomitantly involved in Aβ-induced the release of neuroinflammatory cytokines from astrocytes by ELISA method.Results:1 Aβ increases NLRP3 expression in astrocytesAstrocytes were treated with Aβ for different period of time(0h, 3h, 6h, 12h). Immofluorescence and Western blot was used to detect inflammasome NLRP3 expression in astrocytes. Immofluorescence results showed that Aβ up-regulated inflammasome NLRP3 expressionfollowing different period of time Aβ treatment. The expression of inflammasome NLRP3 was obviously increased after 6h Aβ treatment. Western Blot results showed that Aβsignificantlyupregulated the level of inflammasome NLRP3 expressionin astrocytes following 6h treatment, compared with non-treated group.2 Aβ inducesCaspase-1 activation in astrocytesTo further access the effect of Aβ oninflammatory responses regulatory protein Caspase-1 activation, we detectedthe expression of Caspase-1in astrocytes.Results showed that Aβ induced Caspase-1 activation by time-dependent manner. Compared withnon-treated group, Aβ significantly upregulated Caspase-1 p20 expression following 6h treatment. After 12 h Aβ treatment, Aβ furtherinduced Caspase-1 activation(Caspase-1 p20 expression increase), but there was no significant difference between 6h Aβ treatment and 12 h Aβ treatment.3 Aβ-induced NLRP3/Caspase-1 activation triggers IL-1β and TNF- ɑproduction in astrocytesTo determine whether NLRP3/Caspase-1 exogenous inflammatory signaling pathways activationis concomitantly involved in Aβ-induced the release of neuroinflammatory cytokines from astrocytes. The results showed that Aβ significantly upregulated proinflammatory cytokines IL-1β and TNF-α levelfollowing 6h treatment. Caspase-1 inhibitor Z-YVAD-FMK significantly reduced Aβ-induced IL-1β and TNF-α production.Conclusions:Exogenous inflammatory signaling pathways NLRP3/Caspase-1 activationis involved in Aβ-induced neuroinflammation in astrocytes. Part III tudy on the neuroprotective mechanism of PG against Aβ induced neuroinflammation in astrocytesObjective:To research the neuroprotective effects of neurosteroids PG on Aβ-inducedneuroinflammation in astrocytes, further evaluate its neuroprotective mechanism involving the regulation of neuroinflammation and possible inflammatory regulatory mechanism.Methods:The cerebral cortex tissue was separated from the brain of newborn SD rats and cultured in vitro. To determine whether PG exerts neuroprotection against Aβ-induced neutoinflammation in astrocytes by ELISA method. Western blot was used to detect the effect of PG on the expression of NLRP3 in Aβ-treated astrocyte, as well as evaluated inflammatory signaling regulated protein Caspase-1 activation.Results:1 PG suppresses the Aβ-induced release of IL-1β and TNF-α in astrocytesAstrocytes were treated with Aβ in the absence or presence of different doses of PG(0.25, 0.5, 1, and 2 μM) for 6h. The levels of IL-1β and TNF- in ɑthe medium of the treated astrocytes were assayed by ELISA.The results showed that 1μM PG significantly reduced Aβ-induced IL-1β and TNF-α production in astrocytes. In addition, we treated Astrocyte with Aβ for 6h and further treated with or without PG for different periods of time, and detected the levels of IL-1β and TNF-ɑin astrocytes. The results show, PG decreased Aβ-induced release of IL-1β and TNF-α, compared with the Aβ-treated group,1 μMPG 6h treatment significantly inhibited Aβ-induced IL-1β and TNF-α production.2 PG suppresses Aβ-induced activation of NLRP3 inflammasome complex in astrocytesWestern Blot was used to detect the effect of PG on the expression of NLRP3 in Aβ-treated astrocyte. The results showed that PG significantly decreased NLRP3 expression in Aβ-treated astrocytes. Compared with Aβ-treated group, the expression of inflammasome NLRP3 showed remarkable differences in astrocytes after 6h PG treatement.3 PG suppresses Aβ-induced activation of Caspase-1 in astrocytesWestern Blot was used to detect the effect of PG on the activation of Caspase-1(Caspase-1 p20 expression) in Aβ-treated astrocyte. The results showed that PG significantly reduced Caspase-1 p20 expression following 6h treatment in Aβ-treated astrocytes,compared with Aβ-treated group.Conclusions:Neurosteroids PG significantly inhibits the expression of NLRP3 inflammasome in astrocytes and suppresses Aβ-induced neuroinflammation by inhibiting Caspase-1 activation. Part Ⅳ tudy on the role of autophagy in mediating PG neuroprotection against Aβ-induced neuroinflammation and potentialmolecular mechanism in astrocytesObjective:To research the effect of neurosteroids PG on autophagy in astrocytes and further discuss whether autophagy play a vital role in mediating the neuroprotective effects of neurosteroids PG against Aβ-induced neuroinflammation in astrocytes.Methods:The cerebral cortex tissue was separated from the brain of newborn SD rats and cultured in vitro. To determine the effect of autophagy on PG neuroprotection against Aβ-induced neutoinflammation in astrocytes by ELISA method. Western Blot was used to detect the effect of PG on the activation of autophagy in Aβ-treated astrocyte, andevaluated the effect of autophagy on NLRP3/Caspase-1 inflammatory signaling pathways in astrocytes following Aβ treatment.Results:1 Autophagy activation regulated PG protection against Aβ-induced release of IL-1βand TNF-ɑin astrocytesTo evaluate whether autophagy mediated the protective effects of PG against Aβ-induced inflammatory response and potentialmolecular mechanism. ELISA method was used to detect the effect of autophagy inducer Rapamycin on the neuroinflammation induced by Aβ treatment in astrocytes. The results showed that Rapamycin significantly reduced Aβ induced IL-1β and TNF-α release. However, Autophagy inhibitor 3-MA significantly inhibited the protective effects of PG against Aβ induced IL-1β and TNF-α production, compared with PG protection group, IL-1β and TNF-α showeda significantly increase in astrocytes.2 PG improved autophagy activation in Aβ-treated astrocytesTo further explore the effects of PG on autophagy in astrocytes following Aβ treatment. Western blot results showed that PG activated autophagy in Aβtreated astrocytes.Compared with Aβ treated group, PG upregualted LC3-II expression and inducedthe conversion of LC3-I to LC3-II. In accordance with increased LC3-II expression, another key autophagy regulating proteins Beclin-1 was also significant increased after PG treatment.3 PG induced autophagy activation in Aβ-treated astrocytesWe detected autophagic degradation substrates regulatory protein P62 expression by Western blot method. The results showed PG induced the functional activity of autophagy in astrocytes under Aβ treatment.Compared with Aβ treated group, PGsignificantly decreased P62 protein expression. Additionally, autophagy inhibitor 3-MA blocked the regulatory functions of PG on autophagy activity in astrocytes,demonstrated by low conversion of LC3-I to LC3-II, LC3-IIexpression decrease,and the P62 protein expression increase reversely.4 PG activated AKT/mTOR pathway in Aβ-treated astrocytesTo gain further insight into the molecular mechanism of PG involving in autophagy activation, we investigated expression of AKT/mTOR signaling.The results showed PG significantly inhibited AKT/mTOR signaling pathway in Aβ-induced astrocytes.5 Autophagy activation regulated PG protection against Aβ-induced NLRP3 inflammasome complex in astrocytesTo evaluate whether autophagy activation mediated the expression of inflammasome NLRP3 in Aβ-induced astrocytes. The results showed that autophagyinducer Rapamycineffectively induced autophagy activation and accelerated the degradation of NLRP3 in Aβ-treatedastrocytes. Autophagy inhibitor 3-MA significantly blocked the regulatory functions of PG on NLRP3 expression.Compared with the PG treated group, NLRP3 protein showed a significantly increased in Autophagy inhibited group astrocytes.6 Autophagy activation regulated PG protection against Aβ-induced Caspase-1 activation in astrocytesTo further evaluate the effects of autophagy on regulating the inflammatory signaling pathways, we examined the expression of inflammatory regulatoryprotein Caspase-1. The results showed that autophagy inducerRapamycin significantly inhibited Aβ-induced Caspase-1 activation, demonstrated by low conversionof Caspase-1 p20/pro-Caspase-1. In PG treated group, PG significantly induced Caspase-1 activation in Aβ treated astrocytes. However, autophagy inhibitor 3-MA blocked PG regulatory function on Caspase-1 activation.Conclusions:PG significantly improves autophagy activation in Aβ-induced astrocytes, and acceleratesthe degradation processing ofthe autophagy substrates, especially inflammasome complex NLRP3 expression.However, PG attenuated Aβ-induced inflammatory signaling NLRP3/Caspase-1 dependent neuroinflammation by mediating autophagy activationin astrocytes.In summary, in this study,neuroinflammation AD cell model was established based on Aβ-induced excessive activation of primary cultured rat cortical astrocytes.To research the neuroprotective effects of neurosteroids PG on Aβ-inducedneuroinflammation in astrocytesand possible molecular mechanism.The study demonstrates PG attenuatesAβ-induced neuroinflammation in astrocytes. 1 μMPG 6h treatment significantly inhibits Aβ-induced IL-1β and TNF-α production.Our study further demonstrates PG downregulates Aβ-induced inflammasome NLRP3 expression and inflammatory signals regulatoryprotein Caspase-1 activation,and further attenuates Aβ-induced neuroinflammation in astrocytes.That suggests that PG exerts protection against Aβ-induced neutoinflammation in astrocytes by inhibiting exogenous inflammatory signaling pathways NLRP3/Caspase-1 activation.Furthermore, we also illuminates that PG upregulates autophagy activation in astrocytes following Aβ treatment, which may cause astrocytes autophagy dysfunction.These resultsindicate that PG exerts a neuroprotective effect against Aβ-induced NLRP3/Caspase-1inflammatory signaling pathwaysactivation,and significantly upregulatesautophagy activation, which is an important mediator of the neurotoxicevents occurring in Aβ-induced neuroinflammatory responses in astrocytes. Taken together, neurosteroids PG plays a vital role in regulating Aβ-induced neuroinflammation, and its neuropretective effects may facilitate the development of therapies to ameliorateAD.