Role And Mechanism Of Astrocyte In The Damage Of Dopaminergic Neurons Induced By Lipopolysaccharide

Part I Lipopolysaccharide exerts a dual effect on the growth of astrocytes by upregulating the expression of toll-like receptors and activating MAPK and NF-κB pathwayObjectives: The purposes were to investigate the changes and probable mechanisms of growth of astrocytes administrated with lipopolysaccharide (LPS), the expression of toll-like receptors.Methods: LPS was administrated to rat astrocytes for 60min, 6h, 24h and 48h, and the changes of growth of astrocytes were detected by MTT method. Meanwhile, the long-term changes of growth of astrocytes after administration with LPS for 24h were also studied. Moreover, the effects of inhibitor of NF-κB (SN50) and MAPK pathway (PD98059) on proliferation of astrocytes were observed. The expression of toll-like receptors in astrocytes was detected by immunocytochemistry, western blotting and RT-PCR. NF-κB p65 and phospho-p38 proteins expressed in astrocytes were assayed with western blotting.Results: Changes of astrocytes growth were observed only when LPS had been administrated for 24h. LPS of low concentration could promote proliferation of astrocytes and increase the cell viability(P<0.05 vs control), while LPS of high concentration inhibited the proliferation of astrocytes. Being administrated for 24h, LPS could promote the proliferation of astrocytes in short-term. As for the long-term effects, LPS inhibited the proliferation of astrocytes in a concentration-dependent manner. Pretreatment with SN50 or PD98059 could block the effects of LPS on astrocytes. Astrocytes expressed both cell surface and intracellular TLR3, low-level TLR4 in normal circumstance. LPS increased the expression of TLR4 of astrocytes in a concentration-dependent manner, while the expression of TLR3 kept constant. P-p38 and p65 protein expressed most intensely in 24h group, and decreased subsequently. Pretreatment with PD98059 (inhibitor of MAPK) or SN50 (inhibitor of NF-κB) could block the effect.Conclusion: LPS of low concentration could promote the proliferation of astrocytes in short-term and LPS of high concentration could inhibit it. The expression of toll-like receptors in astrocytes was not homogeneous but rather tailored environmental signal. The molecular mechanism of the changes may be related to the activation of pathway of MAPK and NF-κB.Part II Astrocytes exerted a dual effect on the damage of dopaminergic neurons induced by LipopolysaccharideObjectives: The objectives were to investigate the role of astrocytes in the lipopolysaccharide- induced damage of dopaminergic neurons.Methods: After lipopolysaccharide was applied to the third generation of rat astrocytes for 24h, supernatants of astrocytes were collected. We obtained primary mesencephalic dopaminergic neuron-enriched culture systems by neurobasal and ara-c and established coculture system of both astrocytes and neurons by transwell inserts. We administrated lipopolysaccharide into neuron-enriched systems and coculture systems and detected the change of dopaminergic neurons. At the same time, the supernatants of astrocytes were administrated into the neuron-enriched systems, and the survival of dopaminergic neurons and the expression of tyrosine hydroxylase mRNA were observed.Results: Lipopolysaccharide had a negative effect on the survival of dopaminergic neurons concentration-dependently. Both astrocytes and supernatants of astrocytes promoted the survival of dopaminergic neurons, and the former was better than the latter. In the preoccupation of existence of astrocytes, low-concentration lipopolysaccharide promoted the survival of dopaminergic neurons, while high-concentration decreased. The change of the expression of tyrosine hydroxylase mRNA was similar to the survival of dopaminergic neurons. Conclusions: Astrocytes played a protective role in the damage of dopaminergic neurons induced by lipopolysaccharide, and suitable activation of astrocytes could increase the protective effect while excessive activation of astrocytes could attenuate the effect.Part III IL-6 could medicated the dual effect of astrocytes administrated with lipopolysaccharide on the growth of PC12 by IL-6Objectives: After PC12 cells were treated with astrocyte conditioned medium (ACM) which was dealt with LPS, the changes of growth and the probable mechanisms were investigated.Methods: Firstly, the astrocyte conditioned medium (ACM) was obtained after astrocytes were administrated with lipopolysaccharides (LPS) over 24h. Then, the ACM was stratified by ultrafree MC filter units. Subsequently, the changes of the growth of PC12 cells cultivated with the ACM were detected by MTT method. The cytokines secreted by astrocytes after administration with LPS for 24h were also studied. Finally, the effect of the ACM pretreated with recombinant rat IL-6 or rabbit anti-rat IL-6 monoclone antibody on the growth of PC12 cells was observed. In addition, ACM added IL-6 neutralizing antibody was administrated into the neuron-enriched systems, and the survival of dopaminergic neurons and the expression of TH mRNA were analyzed.Results: ACM treated with LPS of low concentration could promote proliferation of PC12 and increase the cell viability(P<0.05 vs control), while ACM treated with LPS of high concentration attenuated the proliferation of PC12. The ACM of molecular weight from 5 to 30KDa had a biggest effect on the proliferation of PC12. Being administrated for 24h, LPS could promote the astrocytes to secrete IL-6 in a concentration-dependent manner, while the secretions of GDNF, TNF-α, NO had no significant changes. Low concentration in LPS could up-regulated the production of GSH and the activity of GPx, but high concentration down-regulated. Recombinant rat IL-6 could simulate the change, while rabbit anti-rat IL-6 monoclone antibody could block the effect. Anti-rat IL-6 neutralizing antibody itself had no effect on the survival of mesencephalic dopaminergic neurons, but significantly changed the effect of ACM. ACM administrated with IL-6-neutralizing antibody promoted the survival of dopaminergic neurons dose-independently, but significantly lower than that of ACM. Moreover, after ACM was administrated with IL-6-neutralizing antibody, dual effect of ACM disappeared.Conclusion: Astrocytes played a protective role on PC12 cells, and suitable activation of astrocytes increased the proliferation while excessive activation decreased. It is production of IL-6 mainly, not secretion of GDNF, that medicated astrocytic dualism.