Research On The Antidepressant Mechanism Of Cang-Ai Volatile Oil By Regulating The Polarization Of Microglia

Objective: Cang-ai Volatile Oil(CAVO)is a compound preparation of volatile oil made by clinically famous Chinese medicine doctors.It has been clinically found to have a good antidepressant effect.This study intends to use lipopolysaccharide(LPS)to establish a mouse depression-like model and a BV-2 cell activation model to explore the antidepressant mechanism of CAVO by evaluating animal behaviors,microglia polarization,brain-derived neurotrophic factor(BDNF),inflammatory factors and key proteins of BDNF/c AMP-response element binding protein(CREB)signaling pathway.Our research results will provide further experimental basis for CAVO treatment of depression.Methods: 1.The effect of CAVO on LPS-induced depression-like behavior and hippocampal and cortical neurons in mice A mouse depression-like model was established by intraperitoneal injection of LPS(0.5 mg/kg).CAVO was nebulized inhalation,the positive drug ketamine(10 mg/kg)were injected intraperitoneally and celecoxib(52 mg/kg)was administered intraperitoneally.The body weights were meassured before and after 24 h and 48 h of LPS injection.the experiment was performed Forced Swimming Test(FTS),Tail suspension test(TST),Sucrose preference Test(SPT),Nissl staining were tested to observe the antidepressant effects of CAVO.2.The effect of CAVO on the phenotypic polarization of microglia in depression-like mice induced by LPS Immunofluorescence staining was used to detect the co-expression of Iba1 and CD16/32 in hippocampal and cortical microglia.ELISA was used to detect the changes of hippocampal inflammatory factors interleukin-6(IL-6)and interleukin-1β(IL-1β),Tumor Necrosis Factor-α(TNF-α),Interleukin-4(IL-4),Interleukin-10(IL-10)and BDNF;Pearson analysis was used to study the correlation between the contents of proinflammatory cytokines,BDNF and depression-like behavior in mice.3.The effect of CAVO on the BDNF/CREB signaling pathway of BV-2 cells activated by LPS CCK-8 method was used to evaluate the effect of CAVO on viability of BV-2 cells and the LPS activated BV-2 cells.The changes in cell morphology after CAVO treatment were observed under the microscope.The Griess method was used to detect the level of nitric oxide(NO).BDNF,p-CREB/CREB,p-AKT/protein kinase B(AKT),(p-ERK1/2)/extracellular regulated protein kinases 1/2,ERK1/2),NF-κB p65 and pNF-κB p65 protein expression were detected Western Bloting.Results: 1.The effect of CAVO on LPS-induced depression-like behavior and hippocampal and cortical neurons in mice After 24 h of LPS injection,the body weight of mice was lower than control group(P<0.01).The body weight of mice in CAVO groups increased significantly than model group(P<0.01).Compared with the control group,the resting time of FTS and TST in model group raised(P<0.01),while the sucrose preference rate decreased(P<0.01).Compared with model group,the resting time of FTS in the CAVO small,medium and large dose groups was shortened(P<0.05,P<0.01).The TST immobility time of CAVO middle and large dose groups were significantly reduced(P<0.01).The sucrose preference rate of mice in CAVO middle and large dose groups increased(P<0.01).2.The effect of CAVO on the phenotypic polarization of microglia in depression-like mice induced by LPS Compared with the control group,the number of Nissl bodies in hippocampus and cortex of the model group decreased,the staining was lighter and the particles were not clear,which indicated the neuron damage.Neuronal damage was reduced in a dosedependent manner after CAVO administration.The number of positive cells colocalized with Iba1 and CD16/32 in the hippocampus and cortex of the model group was significantly increased than that of the control group(P<0.01),while the number of positive cells in the CAVO large and medium dose groups were significantly decreased than that of the model group(P<0.01).The levels of hippocampal pro-inflammatory cytokines IL-6,IL-1β,and TNF-α in the model group were significantly higher than those in control group(P<0.01).Compared with model group,the content of IL-6 of CAVO large,medium,and small dose groups were decreased(P<0.01,P<0.05).The contents of IL-1β and TNF-α in the CAVO large and medium dose groups decreased(P<0.01).At the same time,the levels of IL-4 and IL-10 in the hippocampus of mice in the CAVO large-dose group increased significantly(P<0.01).The BDNF content in the hippocampus of the model group was significantly lower than that of the control group(P<0.01).Compared with the model group,the BDNF content in the CAVO large-dose group was significantly increased(P<0.01).FST immobility time was positively correlated with IL-6,TNF-α content(P<0.05)and IL-1β content(P<0.01).TST resting time was significantly positively correlated with IL-6,IL-1β,and TNF-α content(P<0.01).SPT sucrose preference rate was negatively correlated with the content of IL-6,IL-1β and TNF-α(P<0.01)and positively correlated with the content of IL-10 and BDNF(P<0.01).3.The effect of CAVO on the BDNF/CREB signaling pathway of BV-2 cells activated by LPS Compared with the control group,CAVO had no effect on the viability of normal and LPS-activated BV-2 cells.After the effect of LPS,the number of activated BV-2 cells increased compared with the control group,and CAVO can inhibit the transition of BV-2 cells to activated state.LPS can increase the release of NO from BV-2 cells(P<0.01)and CAVO can reduce the release of NO(P<0.05).Compared with the control group of the control group,the expression of BDNF,phosphorylated CREB,and phosphorylated AKT protein in the model group decreased(P<0.05),the level of phosphorylated ERK1/2 increased(P<0.01),and the expression of phosphorylated NF-κB p65 increased(P<0.01).Compared with the model group,CAVO can significantly up-regulate the expression of BDNF protein(P<0.01)and increase the expression of phosphorylated CREB and phosphorylated AKT protein,while down-regulating the expression level of phosphorylated ERK1/2,and also downregulating the expression level of phosphorylated NF-κB p65(P<0.01).Conclusion: CAVO has the effect of improving depression-like behavior in mice induced by LPS.At the same time,CAVO can inhibit the M1 phenotype polarization of microglia in the brain of model mice,increase the release of BNDF,and reduce the levels of proinflammatory cytokines IL-6,IL-1β,TNF-α and increase the release of antiinflammatory cytokines IL-4 and IL-10,thereby improving neuronal damage.The antidepressant effects of CAVO is the result of synergistic regulation of the PI3K/AKT and MAPK/ERK pathways of the BDNF/CREB signaling pathway.