The Studies On Active Peptides To Improve LPS-induced Inflammation-related Depression Model

Objective:The etiology of depression is extremely complex,involving heredity,epigenetics,mood regulation,mitochondrial dysfunction,neuroendocrine and neuroimmune factors.Inflammation leads to the decline of neuronal function,and damaged neurons are the basis of brain diseases.Inflammation is considered to be a risk factor for depression and one of important target for antidepressant therapy.Mitochondrial dysfunction is also an important cause of nerve cells senescence and dysfunction.In this study,the activation of microglial cell line BV2 induced by lipopolysaccharide(LPS)was used to establish a neuroinflammatory model and a mouse model of depression induced by lipopolysaccharide,to explore the effect of an active peptide on depression,which is mediated by microglial activation-induced inflammatory response and to clarify the mechanisms,so as to provide a preliminary experimental basis for the application of active peptide in the prevention and treatment of depressive disorder.Methods:In vitro experiments:(1)In this study,the neuroinflammatory model established by microglial cell line BV2 induced by lipopolysaccharide(100ng/m L): the active peptides that inhibit the increased level of cytokines by microglia were screened by ELISA method,and the cytotoxicity effects of different peptides on BV2 cells were detected by MTT method.(2)The conditioned medium obtained from BV2 cells cultured with/without active peptides to culture neuroblastoma cell line SH-SY5 Y cells for simulate the effect of inflammation on neurons in vitro.The experiment is divided into four groups,namely Ctrl,CM-LPS,CM-pp-3(100μM)and CM-pp-3(200μM).MTT,Hoechst33342,flow cytometry and β-galactosidase staining were used to detect the effects of peptides on SHSY5 Y cells viability,nuclear changes,cell cycle distribution and aging phenotypic changes in the model of inflammation.(3)To study the protective effect of active peptides on lipopolysaccharide-induced injury in BV2 cells: β-galactosidase staining and Hoechst33342 were used to detect the phenotype of premature senility and nuclear changes of BV2 cells;flow cytometry was used to detect cycle arrest,ROS level and mitochondrial membrane potential changes;Griess method was used to detect the level of NO in different groups;immunofluorescence staining was used to detect the activation of NF-κB in BV2 cells.Senescence-related proteins,including Sir T 1,p53,p21,Cyclin D1,were detected by Westernblots.In vivo experiments: This experiment was divided into five groups: control group,model group(LPS,1mg/kg),polypeptide group(25mg/kg,low-dose),polypeptide group(50mg/kg,high-dose)and glutathione group(50mg/kg).The inflammation-related depression model was established by intraperitoneal injection of lipopolysaccharide(1mg/kg)in 8-week-old C57BL/6 male mice,and the protective effect and mechanism of an active peptide were explored for the depression model: depressive behaviors will be carried out,including SPT,OFT,TST and the body weight and the state of the eyes and fur status of back in different groups of mice were compared after 24 h of modeling.ELISA method was used to detect the levels of IL-6,IL-1 β,TNF-α and other cytokines in peripheral and brain tissue,and Western blots was used to detect the expression of inflammation-related proteins in brain tissue,including IL-6,IL-1β,TNF-α,i NOS,NF-κB(in nucleus and cytoplasm);HE staining to detect the morphological changes of neurons in hippocampus.The activation of microglia in hippocampus was detected by immunofluorescence staining and the expression of microglia activation marker Iba-1was detected by Western blots.The expression of COX IV by Western blots and COX IV in hippocampus by immunohistochemical method were detected.The expression of mitochondrial dynamics-related proteins,including DRP 1,OPA 1 and MFN-2,were detected by Western blots.Result:In vitro experiments:(1)In the ELISA test,the series of polypeptides pp-1,pp-2,pp-3,and pp-4 inhibited the levels of IL-6 and IL-1β produced by lipopolysaccharide-induced BV2 cells;among them,pp-1 and pp-3 have not affected the viability of BV2 cells at higher concentrations(MTT method).(2)Compared with the LPS model group,SH-SY5 Y cells in the pp-3(100,200μM)group have enhanced cell viability,less nuclear fragmentation,a relatively lower ratio of cells in the G2/M phase,and the number of SA-β-gal positive cells decreased.(3)Compared with the LPS group,pp-3(100,200μM)reduced the nucleus of BV2 cells from being densely stained in Hochest test;SA-β-gal positive staining decreased;NO and ROS levels decreased significantly,and mitochondrial membrane potential was partially restored.The ratio of G1/S phase arrest was reduced and the expression of cycle-related proteins p53,p21 were increased and Cyclin D1 was upregulated,and senescence-related proteins Sir T 1 was upregulated in pp-3 group.In vivo experiment: Compared with the lipopolysaccharide group,after intraperitoneal injection of polypeptide pp-3,depression-like behavior indicators were reduced,manifested as slowing down the sudden weight loss of mice,improving the condition of the eye and back fur,increasing the sucrose preference rate,and the number of times they enters the central area and the residence time increase in OFT,and the relative immobility time of mice decreased in TST;the levels of IL-1β,IL-6 and TNF-α in peripheral serum and brain tissues decreased;Western blots showed the expression of IL-1β,IL-6,TNF-αand i NOS were down-regulated,and the expression of NF-κB in the nucleus was reduced;HE results showed that the state of nerve cells returned to normal to a certain extent;immunofluorescence showed that the number of Iba-1 positive cells decreased and the expression of Iba-1 protein was down-regulated in western blots.Immunohistochemistry showed that COX Ⅳ in the CA3 region of the hippocampus was up-regulated(Western blots also confirmed);Mitochondrial dynamics related protein COX Ⅳ,MFN-2 and OPA1 were up-regulated,and DRP1 was down-regulated.Conclusion:The cells experimental results showed that the lipopolysaccharide-induced inflammatory response of BV2 cells has neurotoxic effects on SH-SY5 Y cells,such as senescence-related cycle arrest and nuclear fragmentation,and the polypeptide pp-3inhibits the premature senescence phenotype of BV2 cells induced by lipopolysaccharide by p53 pathway.The NF-κB pathway inhibits the secretion of inflammatory mediators,inhibits the level of ROS and protects the mitochondrial membrane potential,which promotes the protection of BV2 cells by pp-3.The results in animal experiments showed that polypeptide pp-3 reduced depression-like behaviors of C57BL/6 male mice,and activation in the hippocampus,and it may involve inhibiting the activation of microglia in the hippocampus,which mediates the NF-κB pathway to prevent excessive inflammation,maintains the balance of mitochondrial division and fusion,and protects the mitochondrial respiratory chain from injury.