Role Of Macroautophagy In Regulating Microglia-mediated Neuroinflammation And Dopaminergic Neuron Injury

AbstractObjective:To investigate the potential role of macroautophagy in the regulation of microglia- mediated neuroinflammation and dopamine neuron injury.Methods:Tumor necrosis factor(TNF-α) and bacterial lipopolysaccharide(LPS) were used to stimulate BV2 cells and primary microglial cells to establish in vitro inflammation model.Part I: Western blotting analysis were applied to examine the protein expressions of autophagy-related markers including LC3(microtubule-associated protein light chain 3), p62 and Beclin1; Q-PCR were used to measure the M1 and M2 m RNA level changes;Part II: Q uantitative and reverse transcription PC R were used to detect M1 phenotypic marker(i NOS, IL-1β) and M2 phenotypic marker(Arginase, Ym1/2) changes in m RNA levels; Making use of autophagy tool drugs(rapamycin, resveratrol, 3-methyladenine), serum deprivation and autophagy-related gene Atg5 knockdown, Q-PCR were used to detect changes in M1 and M2 phenotype marker m RNA levels;Part III: The primary microglial cell culture supernatant(microglia conditioned medium) and neurons culture medium were mixed at the ratio of 1: 2, and after that the the mixed medium were added in primary neurons co-cultured for 24 h, CCK8 was used to detect the different groups viability of primary midbrain neurons. By immunofluorescence with anti-MAP-2 labeled neurons were observed changes in the projections of neurons.Results:Part I: TNF-α(1ng/ml and 5ng/ml) treatment caused significant increases in autophagy-related proteins such as LC3, Beclin1 and p62(P <0.05). The m RN A levels of microglia M1 marker(i NOS, IL-1β) increased(P <0.05) while that of M2 marker(Arginase, Ym1/2) decreased in TNF-α(5ng/ml)-treated microglia(P <0.05).Part II: In TNF-α(5ng/ml)- and LPS(100ng/ml)- treated microglia, with the study autophagy drugs increases autophagy can be found that the m RNA levels of microglia M1 marker(i NOS, IL-1β)(P <0.05) decreased and the M2 marker(Arginase, Ym1/2) m RNA levels(P <0.05) increased. And reduce autophagy observed the opposite changes.Part III: Though the CCK8 assay detecting neuron viability found that co-cultured with TNF-α-treated microglia medium, the neuro ns viability decreased. Inhibition of autophagy in microglia culture supernatants and co-cultured with neurons also found that the viability of neurons decreased. At the same time, inhibition of autophagy group detected by immunofluorescence with MAP-2 labeled neurons were significantly shorter in the projections; and enhancement of microglia autophagy’s co-culture supernatant were observed the opposite changes.Conclusion:Macorautophagy can regulate microglia- mediated neuroinflammation, and the enhancement of autophagy can promote the microglia polarization toward to M2 phenotype.