A Study On The Antidepressant Mechanism Of Oviductus Ranae Based On MAPK Signaling Pathway

Objective: The antidepressant effect of Oviductus Ranae and the regulation of MAPK signaling pathway were investigated by preparing a mouse depression model by chronic unpredictable mild stress method and culturing mouse hippocampal neuronal cells HT-22 and mouse microglia BV2 in vitro,and the effects of Oviductus Ranae on metabolic pathways and microbial composition in vivo were investigated by tissue metabolomics and intestinal flora analysis,and investigated the antidepressant effect and mechanism of Oviductus Ranae in vivo and in vitro,further elaborated the modern scientific connotation of the antidepressant effect of Oviductus Ranae,and provided theoretical basis for the development and application of Oviductus Ranae antidepressant products.Methods : A mouse depression model was prepared by the chronic unpredictable mild stress method,and the behavioral changes of each group of mice were investigated by the sugar water consumption test,forced swimming test,tail suspension test,open field test and MORRIS water maze test;the levels of CORT,IL-6,IL-1β and TNF-α were measured by ELISA;the structural morphology and apoptosis of hippocampal tissues were detected by HE staining and Tunel staining;the distribution and expression of Iba-1 in hippocampal tissues were detected by immunofluorescence;the expression of MAPK signaling pathway-related proteins in hippocampal tissues was detected by protein immunoblotting.HT-22 cells were cultured in vitro,induction of HT-22 cell damage model by CORT,the cell viability of HT-22 cells was detected by MTT,the apoptosis of HT-22 cells was detected by Hochest33258 staining and flow cytometry,and MAPK signaling pathway-related protein expression of HT-22 cells was detected by Western blot,in order to explore the protective effect of Oviductus Ranae on HT-22 cell injury.BV2 cells were cultured in vitro,induction of BV2 cell inflammation model by LPS,the levels of IL-1β,IL-6 and TNF-α in cell supernatant were detected by ELSIA,the expression of Iba-1 in BV2 cells was detected by immunofluorescence,and the expression of MAPK signaling pathway-related proteins in BV2 cells was detected by Western blot,in order to explore the anti-inflammatory effects of Oviductus Ranae on the model of inflammation in BV2 cells.The LC-QTOF was used to analyze the differences in metabolites in brain and kidney tissues of mice in each group,and to explore the effect of Oviductus Ranae on metabolic pathways and potential biomarkers in depression model mice.The Paired-End sequencing method was used to investigate the differences in the intestinal flora of each group of mice and to investigate the intervention effect of Oviductus Ranae on the intestinal flora of mice in the depression model.Results : Compared with the control group,the body weight of the mice in the model group was reduced(P<0.01),the proportion of sugar water drinking was decreased(P<0.01),the immobility time of the mice in the forced swimming experiment and the tail suspension experiment was prolonged(P<0.01),the movement distance and central activity time in the open field experiment were shortened and the static time and corner activity time were prolonged(P<0.01),the escape latency in the water maze experiment was prolonged,the number of times of crossing the platform,the number of times of entering the platform quadrant and the retention time of the platform quadrant were shortened(P<0.01),fluoxetine and Oviductus Ranae could increase the body weight of mice(P<0.05 or P<0.01),increase the proportion of sugar water drinking(P<0.05 or P<0.01),decrease the immobility time in forced swimming test and tail suspension test(P<0.01),increase the movement distance and central activity time of mice in open field test were,and decrease the resting time and corner activity time(P<0.05 or P<0.01),reduce the escape latency in water maze test,and increase the number of platform crossing,platform quadrant entry and platform quadrant retention time;compared with the control group,there was no significant difference in cerebral blood flow among the model group,fluoxetine group,and Oviductus Ranae 0.2 g/kg group(P>0.05),Oviductus Ranae 0.8 g/kg group showed a significant increase in cerebral blood flow(P<0.01),Oviductus Ranae 0.4 g/kg group showed an increasing trend in cerebral blood flow,but there was no statistical difference(P>0.05),compared with the model group,there was no significant difference in cerebral blood flow among the fluoxetine group,Oviductus Ranae0.4 g/kg,and 0.2 g/kg groups(P>0.05),there was a significant increase in cerebral blood flow in the 0.8 g/kg group of Oviductus Ranae(P<0.05),compared with the fluoxetine group,there was no significant difference in cerebral blood flow in the 0.4 g/kg and 0.2 g/kg groups of Oviductus Ranae(P>0.05),the 0.8g/kg group of Oviductus Ranae showed a significant increase in cerebral blood flow(P<0.05);compared with the control group,the levels of CORT,IL-6,IL-1β and TNF-α in the serum of the model group were increased(P<0.01),compared with the model group,fluoxetine and Oviductus Ranae could reduce the levels of CORT,IL-6,IL-1β and TNF-α in the serum of mice(P<0.05 or P<0.01);HE staining results showed that Oviductus Ranae could improve the structural damage of hippocampal CA1,CA3 and DG regions in mice caused by modeling,tunel staining results showed that this effect was related to the inhibition of apoptosis in hippocampal CA1,CA3 and DG regions of mice;compared with the control group,the number of Iba-1 positive cells in the hippocampal CA1,CA3 and DG regions of the model group increased,compared with the model group,fluoxetine and Oviductus Ranae could reduce the number of Iba-1 positive cells in the hippocampus of mice;compared with the control group,the expression of BAX,Caspase3,Caspase9,NF-κB,p-p38 and p-JNK protein in the hippocampus of the model group increased,and the expression of Bcl-2,IκB-α and p-ERK1/2 protein decreased(P<0.01),compared with the model group,the expression of BAX,Caspase3,Caspase9,NF-κB,p-p38 and p-JNK proteins in the hippocampus of mice in the fluoxetine and Oviductus Ranae groups decreased,and the expression of Bcl-2,IκB-α and p-ERK1/2 proteins increased(P<0.01).Compared with the control group,the viability of HT-22 cells in the model group decreased(P<0.01),the proportion of apoptosis increased(P<0.01),the expression of BAX,Caspase 3,Caspase9,p-p38 and p-JNK protein increased(P<0.01),and the expression of Bcl-2 and p-ERK1/2protein decreased,compared with the model group,Oviductus Ranae could increase the viability of HT-22 cells induced by CORT(P<0.05 or P<0.01),reduce the proportion of apoptosis of HT-22 cells(P<0.01),decrease the expression of BAX,Caspase 3,Caspase9,p-p38 and p-JNK protein(P<0.01),and increase the expression of Bcl-2 and p-ERK1/2protein(P<0.01).Compared with the control group,the Iba-1 positive cells of BV2 cells in the model group increased,the levels of IL-1β,IL-6 and TNF-α increased(P<0.01),the expression of NF-κB,p-p38,p-JNK and p-ERK1/2 protein increased(P<0.01),and the expression of IκB-α protein decreased(P<0.01),compared with the model group,Oviductus Ranae could decrease the Iba-1 positive cells of BV2 cells,decrease the levels of IL-1β,IL-6 and TNF-α(P<0.05 or P<0.01),decrease the expression of NF-κB,p-p38,p-JNK and pERK1/2 protein(P<0.01),and increase the expression of IκB-α protein(P<0.01).In brain tissue,a total of 15 metabolites were found to be potential biomarkers,mainly involved in the regulation of unsaturated fatty acid biosynthesis,glycine,serine and threonine metabolism,sphingomyelin metabolism,glycolysis/glucose production,linoleic acid metabolism,valine,leucine and isoleucine biosynthesis and ABC transport;In renal tissue,a total of 14 metabolites were found to be potential biomarkers,mainly involved in the regulation of sphingomyelin metabolism,glycine,serine and threonine metabolism,unsaturated fatty acid biosynthesis,linoleic acid metabolism,α-linolenic acid metabolism and glyoxylic acid and dicarboxylic acid metabolism.The results of the study on the composition of intestinal microorganisms show that Oviductus Ranae has a certain regulatory effect on intestinal microorganisms in mice,and it mainly has a significant increase in the abundance of Lactobacillus,Lactobacillus,Lactobacillus in intestinal microorganisms.Conclusion : Oviductus Ranae can improve the depression-like behavior of depression model mice,which may play a role by promoting cerebral blood flow,inhibiting the excessive activation of HPA axis,inhibiting the inflammatory response,improving the structural damage of hippocampus,and regulating the structural composition of intestinal flora,the mechanism may be related to the regulation of MAPK signaling pathway,biosynthesis of unsaturated fatty acids,glycine,serine and threonine metabolism,sphingomyelin metabolism,glycolysis/glucose production,linoleic acid metabolism,valine,leucine and isoleucine biosynthesis,ABC transport,α-linolenic acid metabolism,and glyoxylic acid and dicarboxylic acid metabolism.