Expression Alternations Of Secretogranins Are Involved In Paraquat-induced Reactive Astrocyte In Vitro

Objective:Paraquat（PQ）is a rapidly eradicating herbicide.Due to the high toxicity and no special antidote,it has been severely restricted by many countries.Toxicology studies have proved that PQ causes neuroinflammation and oxidative stress,producing non-selective inhibition of all respiratory chain complexes in the mitochondria.Moreover,chronic PQ exposure leads to massive loss of dopaminergic neurons in the substantia nigra,resulting in silmiliar pathological change of PD.Astrocytes participate in the regulation of central nervous system（CNS）homeostasis by secreting a large number of signaling molecules.Astrocyte is activated under the conditions of injury and stress,which is characterized by the alteration of intermediate filament proteins（IFs）.In addition,different categories of functional genes and molecules have been modulated in reactive astrocytes that exert both protective and detrimental roles to neurons.In vivo studys have found that environmental toxin PQ exposure can cause astrocyte activation.However,whether PQ exposure impacts the astrocytic secretory pathway remained undecided.Secretogranins anchor in large dense core vesicles（LDCVs）of the nervous and neuroendocrine system.They are often used as markers to study the mechanism of LDCV secretion in the subsequent experiments.By binding with functional moleculars through specific domains,secretogranins act as a molecular bridge between the core aggregate and the cholesterol-rich secretory granule membrane,playing an important role in their trafficking and subsequent release through the regulated secretory pathway（RSP）.Studies have shown that secretogranins are involved in the synthesis and sorting of peptide hormones in the nervous system and the neuroendocrine system.An imbalanced availability of secretogranins and their derived peptides have been identified in a wide array of neurological and psychiatric disorders,which means the impaired secretogranins-associated RSP may possibly be responsible for the progression of disease.Further study has evidenced that secretogranin II（SCG2）positive secretory granules are involved in gliotransmitters sorting and release through a Ca²⁺-dependent regulated secretory pathway.And the expression of SCG3 in reactive astrocytes were significantly increased in the model of perforated brain injury and Alzheimer’s disease（AD）.It is speculated that the rapid increase of SCG3 represented the preparation for timely storage and the sorting of the peptides.Moreover,the astrocytes that overexpressed SCG3 accumulated around amyloid deposits and disability neurons,suggesting that the dysregulation of SCG3-mediated of the regulated secretion may be one of the mechanisms of brain injury and AD.Based on above results,we use PQ as an inducer to insult U118 astrocytoma cell line and investigate the reaction of astrocytes under environmental toxins PQ.In the model of PQ-induced reactive astrocyte,we examined the changes of SCG2 and SCG3 expressions,confirming their involvement in the sorting and trafficking of inflammatory,neurotrophic factors and neurotransmitters in U118 cells.These data provide new insights into the PQ cytotoxicology and make good preparation of the further study of PD.Methods:1.In curent study,we selected U118 astrocytoma cell line as the object and investigated the reative change of astrocytes under the treatment of PQ.The viability of U118 cells were estimated by MTS assay and morphological changes were observed by inverted phase contrast microscope.2.Expression levels of pro-Casp-3 and PARP were detected by Western blot after PQ exposure.3.The apoptosis of U118 cells treated with PQ was confirmed via Annexin V/PI double staining by flow cytometry.4.The expressions of GFAP and S100βin U118 cells were observed by immunofluorescence analysis after PQ exposure and the average cell area and morphology of U118 astroglia were also analyzed to ensure the successful estamblishment of PQ-induced reactive astrocyte model.5.The cell cycle of U118 cells was evaluated by flow cytometry and the expression levels of multiple astrocytic factors were analyzed by Realtime quantitative PCR in this model.6.The expression levels of SCG2 and SCG3 were analyzed by Realtime quantitative PCR.7.The intracellular expression levels of SCG2 and SCG3protein were determined by Western blot in PQ-activated U118 astroglia.8.The subcellular locations of SCG2 and SCG3 positive vesicles were exhibited by immunofluorescence analysis in PQ-induced U118 astroglia.9.The expression levels of multiple astrocytic factors were analyzed by Realtime quantitative PCR after SCG2 and SCG3 were knocked down by siRNA in U118 astroglia.10.The expression levels of IL-6and GS protein were determined by Western blot after SCG2 was knocked down in U118astroglia.11.The colocalisations between SCG2 positive vesicles,IL-6 and GS were observed by double staining immunofluorescence analysis.12.The colocalisations between SCG3 positive vesicles,IL-6 and BDNF were also observed by double staining immunofluorescence analysis.Results:1.PQ caused a concentration and time-dependent decrease of cell viability.Under the median lethal dose（IC50）,PQ increased cell number and led U118 astroglia to a bushy and swelling phenotype with twisted cellular processes.However,it induced remarkably cell body shrinkage and cell loss as time extends.2.PQ-induced U118 cells showed no significant activation of Casp-3 system under IC50.The content of pro-Casp-3 was significantly reduced with its substrate PARP cleaved after 72 h of treatment.The result indicated that PQ induced delayed apoptosis in U118 cells and suggested that PQ does not exert the toxic effect on astrocytes by directly induction of apoptosis.3.The result of flow cytometry showed that the percentage of cells stained Annexin V（+）/PI（+）began to increase significantly after 72 h of PQ treatment while the ratio of Annexin V（+）/PI（-）did not change.It confirmed that PQ induced delayed apoptosis in U118 cells,which is mainly late apoptosis.4.After 48 h of PQ treatment,U118 cells gradually transformed from bipolar to multipolar phenotype and the average area of the cell body increased.The expression of activation markers GFAP and S100βwere also increased indicating that U118 astroglia was activated.5.After PQ treatment for 48 h,cell ratio of S phase increased.The percentage of cells in S phase increased significantly with G2/M phase decreased as time extended,indicating that PQ lead U118 cells to a S phase cell cycle arrest.6.mRNA expression levels of astrocytic proteins including functional protein GFAP,S100βand GS,inflammatory cytokines TNF-α,IL-1βand IL-6 and neurotrophic factor BDNF and glial derived neurotrophic factor（GDNF）were increased in PQ-activated U118 astroglia.In the late phase,the expression of astrocytes function proteins and nutrition decreased while inflammatory factors continued to rise.The results showed that various astrocytes markers were up-regulated in U118 cells under PQ-induced activation.7.The mRNA and protein expression levels of SCG2 in U118 cells increased first and then decreased,reaching the peak at 48 h of PQ treatment,while the expression levels of SCG3 increased gradually compared with the control group.8.SCG2 and SCG3 proteins were abundantly expressed in the cytoplasm and processes of U118 cells.After PQ induction,the number of SCG2 and SCG3-positive vesicles were significantly increased compared with the control group,mainly in the perinuclear region.It suggested that a large number of secretogranin-positive vesicles were synthesized in the endoplasmic reticulum under PQ exposure.9.SCG2 secretion was low in U118 cells,while a great amount of SCG3proteins in the cytoplasm could be secreted to the extracellular space and SCG3 secretion per unit time was significantly increased after activated by environmental toxin PQ.10.No stable down-regulation of the characteristic astrocyte expression was found after SCG3 silencing,while the levels of IL-6 and GS mRNA decreased when SCG2 was knocked down,indicating that SCG2 may have potential links with IL-6 and GS in U118cells.The level of IL-6 protein expression decreased while of GS remained unchanged after SCG2 silencing.11.Immunofluorescence co-localization analysis showed that SCG2-positive vesicles were co-localized with GS at a lower degree but with IL-6 to a moderate extent.The overlaps were uniformly distributed in the cytoplasm and synapse terminals and increase after PQ treatment,especially in perinuclear space.Combined with Western blot results,the result suggested that the newly synthesized SCG2 vesicles were involved in IL-6 sorting and secretion in U118 cells and SCG2 contributed in GS regulation on the transcription level.12.Immunofluorescence co-localization analysis showed that SCG3-positive vesicles were co-localized with IL-6 at a lower degree but with BDNF to a moderate extent.The overlaps were uniformly distributed in the cytoplasm and synapse terminals and increased after PQ treatment,especially in perinuclear space.We supposed that the newly synthesized SCG3 vesicles were partically involved in BDNF trafficking and secretion in U118 cells.Conclusion:1.PQ induced a dose-and time-dependent decrease of the viability of U118cells,which was result from the activation and delayed apoptosis.2.PQ induced up-regulation of a variety of astrocytic factors expression,and led to S phase cell cycle arrest in activated U118 astroglia.3.The expressions of SCG2 and SCG3 increased significantly in PQ-activated U118 astroglia.4.SCG3 protein was secreted by U118astroglia.The number of SCG2 and SCG3 positive vesicles increased obviously in PQ-activated cells and a large number of newborn secretory granules appeared in perinuclear region of PQ-activated U118 astroglia.5.SCG2 regulated GS expression at transcriptional level and SCG2-positive vesicles were partially involved in the trafficking and secretion of IL-6 in U118 astroglia.SCG3 was not involved in the regulation of the expression of astrocyte-specific factors in the current study.SCG3-positive vesicles may play a role in BDNF trafficking and secretion.