The Molecular Mechanism Of Major Depression Disorder With Suicidal Behavior

Backgroud: Suicidal behavior was defined that the pantient take their own lives by kinds of methods of their own intention,when the psychological crisis is aggravation.The suicide not only harming the patients' family,but also bringing heavy financial burden to the society and country,it has been one of the most serious public health issues.There is mounting evidence showing that the Major depression disorser(MDD)patients are the high-risk group of suicide,accounting for more than half of all suicides,and suicide is the most serious outcome of MDD.To explore the biomarkers which can predict the suicide behavior of MDD is a hotspot.Assessing suicide attempt/behavior of MDD is based on screening scale so far,which is highly depends on subjective factors,and the people always hide their suicidal attempt,An improved understanding of the factors contributing to suicidal risk in MDD should provide direction for suicide predictor development.Recently,numerous researches have reported the pathophysiology underlying the suicide behavior of MDD,for example: the deregulation of 5-HT of suicides' cerebrospinal fluid,the abnormal of dexamethasone suppression test,and the dysfunction of cholesterol??-3 fatty acid and brain-derived neurotrophic factor of plasma,but are not yet any biomarkers available.Objective: A complementary plasma proteomic approach(2-DE analysis and MALDI-TOF/TOF MS identification combining i TRAQ-LC-MS/MS analysis)was applied to identify differential proteins from drug-na?ve majorly depressed suicide attempters(MDD-SA),majorly depressed suicide non-attempters(MDD-NA),and healthy control(HC)subjects,and using WB blotting and ELISA to validatet the identified proteins,using the animal models to explore the underlying molecular mechanism.In order to improve our understanding of the biological factors that contributs to suicidal risk in MDDMethods:1.According to the inclusion and exclusion criteria,HC,MDD-SA and MDD-NA subjects were recruited in the psychiatric department of the First Affiliated Hospital,and HC subjects were recruited from the medical examination center at Chongqing Medical University(Chongqing,China).Standardizing the collection of samples,and completing the clinical record of patient.2.2-DE analysis and MALDI-TOF/TOF MS identification combining i TRAQ-LC-MS/MS analysis was applied to identify differential plasma proteins from MDD-SA relative to MDD-NA and HC subjects.MetaCore analysis was conducted to build biological networks and illustrate statistically plausible biological linkages between the identified proteins.3.The differential proteins were validated by WB and ELISA in MDD-SA?MDD-NA and HC subjects,4.The animal model was used to investigate the molecular mechanisms of the disease.Result 1: 61 MDD-NA subjects(including 40 drug-na?ve subjects),61 MDD-SA(drug-na?ve)and 61 HC subjects were finally recruited.Result 2: Pooled plasma samples were generated by combining equal volumes of the 12 individual plasma samples from each of the three groups(i.e.,drug-na?ve MDD-SA,drug-na?ve MDD-NA,and HC candidates).According to the manufacturer's instructions,a 420 ?l volume from every pool was depleted of the most abundant proteins with a MARS-human 7 HPLC column for 2-DE.Using 3-10 NL and 4-7 L pH gels to found the 2-DE maps,nearly 1100 protein spots were distinguished on the 3-10 NL pH gels,while 1200 protein spots were detected on the 3-10 NL pH gels,Through PDQuest analysis,33 differential spots were identified(6 spots upregulated and 27 spots downregulated)as MDD-SA compared with MDD-NA.From the foregoing differential spots,MALDI-TOF/TOF MS yielded only 25 unique differential proteinsResult 3: Using the same samples as 2-DE analysis,pooling plasma samples in groups,and a 420 ?l volume from every pool was depleted of the most abundant proteins with a MARS-human 14 HPLC column for i TRAQ,Differentially regulated proteins(±1.2 fold-change with p<0.05)were selected for further analysis.These cut-offs were selected based on literature investigating the reproducibility of iTRAQ? quantification.Finally,20 were differentially expressed in both MDD groups relative to the HC group.No proteins were simultaneously identified by 2-DE and i TRAQ-LC-MS/MS.Result 4: MetaCore analysis was conducted to build biological networks and illustrate statistically plausible biological linkages between the 45 previously identified proteins(i.e.,25 proteins from 2-DE and 20 proteins from iTRAQ-LC-MS/MS).The 'blood coagulation' and 'immune response' pathway maps showed the highest statistical significance(p<0.001).Moreover,the 'blood coagulation' and 'inflammation' process networks showed the highest statistical significance(p<0.001).By manual count,28 of the 45 identified proteins possess established coagulatory and/or inflammatory functions.Result 5: From the 45 identified proteins,25 candidate proteins were selected for Western blotting validation.only four proteins – SAA1,CRP,F?,and PCI – demonstrated significantly altered levels in MDD-SA subjects relative to both MDD-NA and HC subjects.The differential expression of the other proteins found by 2-DE or iTRAQ-LC-MS/MS did not reach statistical significance.Result 6: Expression of seven extrinsic pathway proteins – soluble TF,TFPI,F?,APC,F?,prothrombin,and F1+2 – were assessed using individual samples from the MDD-SA,MDD-NA,and HC subjects containing both drug-na?ve and antidepressant-treated MDD subjects(n=147).F?,APC,F?,TF,and TFPI were all significantly increased in MDD-SA subjects relative to both MDD-NA and HC subjects with no significant differences observed between MDD-NA and HC subjects.F1+2 was significantly increased in MDD-SA subjects relative to MDD-NA but decreased relative to HC subjects.There were no significant differences in prothrombin among the three groups.Result 7: The Gama Function was applied to assess the relative mass-action ratio of prothrombinase,the result show the special profiles: HC(2.13)>MDD-SA(1.34)>MDD-NA(1.00).Result 8: The aPTT levels were measured in plasma samples obtained from all rats immediately after drug administration.Then aPTT levels were significantly elevated in the heparin-treated subjects as compared to control subjects(p=0.0237),the FST immobility time was significantly elevated in heparin-treated subjects as compared to control subjects(p=0.0295).Conclusions:1.Strict inclusion and exclusion criteria is a sinificant foundation for further research of screening the molecular biomarkers and pathogenesis.2.Complementary plasma proteomics was conducted to identify differential proteins,it can maximaize the advantages of proteomics of detecting the biomarkers,and avoid missing the significant proteins at beginning.3.The result of MetaCore analysis show that 45 differential proteins mapped to coagulation and inflammation,it means the pathway of coagulation and inflammation were play significant roles underlying the suicide of MDD,and 28 of the 45 identified proteins which possess established coagulatory and/or inflammatory functions can be considered as a biomarlers for further research.4.The result of WB analysis show that,suicidal behavior in MDD is associated with a more pronounced proinflammatory state,and accompanied by a phenotypic “middle ground” between hypothrombotic depressed non-attempters and euthrombotic healthy individuals.And the result of ELISA analysis reveals that,an extrinsic pathway biomakers that can be applied in predicting and monitoring suicidal risk in MDD patients.5.The FST immobility time was significantly elevated in heparin-treated rats compared to control subjects,this means that the heparin-treated with higher hopelessness,and the level of hopelessness was considered as one of the main risk factors of suicidal behavior of human beings.Therefore,we have further demonstrate the coagulation system is closely related to the risk factors of suicidal behavior of MDD.