The Relationship Between Inflammation And Autophagy And Autophagy Related MTOR Signaling Pathway In Schizophrenia And The Anti-neuroinflammatory Effect Of Hippocampus Erectus Ethanol Extracts

Schizophrenia is a complex group of severe mental disorders with unknown etiology.Clinical symptoms include hallucinations,delusions,speech or behavioral disorders,and often accompanied by cognitive dysfunction.Clinical and experimental studies have indicated that proinflammatory cytokines were increased in the blood and the cerebrospinal fluid of schizophrenia patients.By contrast,the expression and function of autophagy related protein are decreased.The mRNA levels of a crucial autophagy-related protein,Beclin 1,was reported to be decreased in the hippocampus of schizophrenia patients.Over-expression of pro-inflammatory cytokines inhibit autophagy,while autophagy regulates inflammation through innate immune signaling pathways,which could remove endogenous inflammasome agonists and negatively regulate the secretion of pro-inflammation.Conversely,downregulated autophagy leads to excessive interleukin(IL-1β)secretion,which in turn,stimulates the autocrine secretion of other pro-inflammatory cytokines and inflammatory response.The kinase mammalian target of rapamycin(mTOR)is a major regulator of the autophagic process,in which phosphatidylinositol 3-kinase(PI3K/Akt)pathway underlies the upstream of mTOR and modulates its activity.Both mTOR and PI3K/Akt expression was up-regulated in schizophrenia.However,the inter-relationship between chronic inflammation and autophagy,and whether inflammation suppresses autophagy via PI3K/Akt/mTOR signaling pathway remains unknown in schizophrenia.The aim of current study is to answer these questions.This dissertation firstly detects neurotransmitters,then tests inflammation,autophagy and autophagy regulatory pathway.Study pro-inflammation induced autophagy,and inhibited autophagy regulated inflammasome.Study the relationship between inflammation and autophagy in schizophrenia.Inflammation and autophagy is a new direction for anti-schizophrenia drugs,and the regulation of immune and brain functions by marine active substances in recent years.This study explored the inhibitory effect of the marine drug Hippocampus erectus on the inflammatory response of lipopolysaccharide(LPS)-induced microglia(BV2 cells)and the possible mechanism.Materials and methods:In clinical study,66 samples of schizophrenia(SZ)and 44 heathy controls(CT)were enrolled in this research and clinical and laboratory data as well as blood samples were collected.High-performance liquid chromatography was used to detect the Concentration of monoamine neurotransmitters,such as dopamine(DA),serotonin(5-HT),noradrenalin(NE)and their metabolism 3,4 dihydroxy-phenyl acetic acid(DOPAC),Homovanic acid(HVA)in plasma of schizophrenia.Proinflammatory cytokines in the plasma of schizophrenia were measured by Milliplex luminex200.The components of autophagy vesicles,marked with MDC,were measured by flow cytometry.The mRNA and protein expression of PI3 K,Akt,mTOR,p70 ribosomal protein S6 kinase(p70S6k),eiF4E-binding protein 1(4EBP1),Beclin 1,microtubule associated protein 1 light chain 3(LC3)-B/LC3 A and lysosome-associated membrane proteins1(LAMP 1),Cathepsin D and p62 were assayed by real-time fluorescence quantitative PCR(q-PCR)and Western Bspartate specific proteinase1(Caspase 1),IL-1? and IL-6 were detected.The pro-inflammatory cytokines IL-1?,IL-6 and anti-inflammatory cytokine IL-10 were also used to stimulate lymphocytes of schizophrenia for 48 h.Autophagy biomarker LC3 B and Beclin 1 were measured.In vitro study,Pre-treated BV2 cells with Hippocampus erectus ethanol extracts for 24 h,then added 100 ng/mL LPS.Pro-inflammations,NO,M1 marker CD68 and Beclin 1 were detected by Elisa kit,Greiss method,and cellular immunofluorescence.Results:The levels of DA and HVA were significantly increased,while the levels of 5-HT,NE,DOPAC and 5-HT/DA,NE/DA were significantly reduced in plasma of schizophrenia.The concentration of proinflammatory cytokines as well as the mRNA expression levels,such as IL-1β,IL-6 and INF-γ,were significantly increased in the plasma of schizophrenia patients.in the SZ,mRNA and protein expressions of Beclin 1,LAMP 1,Cathepsin D and LC3 B/LC3 A in lymphocytes were significantly decreased,while p62 was significantly increased compared with CT.In the lymphocytes of SZ patients,the mRNA expression levels of PI3 K,Akt,mTOR,p70S6 k and 4EBP1 were significantly increased.In parallel,the protein expression of PI3 K,p-Akt,p-mTOR,p-p70S6 k,and p-4E-BP1 were also significantly increased.While,the mRNA and protein expression level of ULK1 were decreased.After the treatment of IL-1?,IL-6 and anti-inflammation(IL-10)to lymphocytes of schizophrenia patients for 48 h,the mRNA levels of LC3 B and protein level of Beclin 1 were decreased.After the treatment of 3-MA in lymphocytes of schizophrenia patients for 48 h,NLRP3,ASC,Caspase 1 and pro-inflammatory cytokines IL-1? and IL-6 were increased.The Hippocampus erectus ethanol extracts can significantly reduce the proliferation of BV2 cells and the levels of NO,IL-1β,IL-6 and TNF-α released in BV2 cell induced by LPS.It also reduces M1(CD68)polarization and increases Beclin 1 expression.Conclusion:1.The results of neurotransmitters indicate that the function of dopamine was active,while the function of 5-HT,NE were down in schizophrenia.5-HT,NE energy system and DA energy system were unbalanced in schizophrenia.2.The results of pro-inflammations suggested that immune system was active.The results of relative autophagy-lysosome biomarkers suggest that the function of autophagy-lysosome was disrupted.And results of relative mTOR pathway suggest that the activation of the PI3K-Akt-mTOR signaling pathway may exert inhibited effects on autophagy in schizophrenia.3.We investigated the activity of autophagy through the treatment of IL-1?,IL-6 and anti-inflammation(IL-10)cytokine to the lymphocytes of schizophrenia patients,then found that inflammation inhibited autophagy,while anti-inflammation promoted autophagy.We also investigated the inflammatory response after the treatment of 3-MA to the lymphocytes of schizophrenia patients,then found that autophagy promoted inflammation via NLRP3 inflammasome.4.The Hippocampus erectus ethanol extracts can reduce the release of proinflammatory cytokines and oxygen free radicals in BV2 cells induced by LPS,which may play an anti-inflammatory role by reducing M1 polarization and increasing autophagy.This study provides a new theory for the pathogenesis of schizophrenia and a new approach to treat schizophrenia with autophagy and inflammation.Also provides a new theory for Hippocampus erectus anti-neuroinflammatory effect.