Research On The Inflammatory Mechanism Of ASMT In The Antidepressant Effect Of Exercise

Background: Depression is a widespread and life-threatening mental illness that affects more than 300 million people worldwide.In 2017,the World Health Organization(WHO)reported that 300 million people worldwide currently suffer from depression.Depression may be the main cause of disability and the global burden of disease.Melatonin(MT)plays a key role in synchronizing circadian rhythm,and the main feature of depression is sleep disturbance and MT secretion disorder caused by circadian rhythm disorder.N-acetyl-5-hydroxytryptamine-methyltransferase(Nacetyl-5-hydroxytryptamine-methyltransferase,ASMT)is a key rate-limiting enzyme for MT synthesis.The decrease in MT levels in patients with depression may be related to the decrease in ASMT expression and activity related.Long-term use of MT can reduce neuroinflammation in the brain of mice and exert a neuroprotective effect.The pathophysiology of depression and the onset of antidepressants are related to the improvement of neuroinflammation.The neuroinflammatory manifestation of depression is that the level of pro-inflammatory cytokines(such as IL-1? and IL-6)is increased,and the level of anti-inflammatory cytokines(such as IL-4 and IL-10)is reduced.The microecological imbalance of intestinal flora will destroy the host's immune response and induce various inflammatory diseases.Intestinal flora may participate in the neuroinflammatory mechanism of depression.The study found that MT played an important role in regulating biological rhythm and improving depressive behavior.In addition,exercise can inhibit the occurrence of brain diseases by regulating nerve inflammation and improving intestinal flora.However,whether the antidepressant effect of exercise is related to ASMT and its regulatory role in neuroinflammation and intestinal flora remains to be further explored.Objective: This study explored the effects of ASMT gene knockout and exercise on depressive behavior,neuroinflammation and intestinal flora in mice,and on this basis,further explore whether exercise can improve neuroinflammation,regulate intestinal flora,and then alleviate ASMT knockout Induced depressive behavior.Methods: This experiment selected 12 female homozygous ASMT knockout mice,14 female wildtype C57BL/6 mice in the same litter,6-7 weeks old,18-20 g.The ASMT knockout mice were randomly divided into a knockout quiet group(KO group,n=6)and a knockout exercise group(KE group,n=6);wild-type mice were randomly divided into wild quiet group(WT group,n=7)and wild sports group(WE group,n=7).The mice in the WE group and the KE group received 12-week treadmill training from the age of 2 months,and collected feces during the training.After the treadmill training,all groups of mice were subjected to behavioral tests,including forced swimming test(FST),tail-hanging test(TST),and sugar water preference test(SPT).After that,the mice were anesthetized and their necks were killed.The serum,prefrontal cortex and hypothalamus were taken.Elisa was used to detect the content of serum CRP,IL-6,5-HT;RT-PCR was used to detect the genes related to TLR4/NF-?B signaling pathway such as TLR4,NF-?B,IL-18,IL-1?,TLR2 and IL-6,TNF?,IFN-?,CASP1 and other pro-inflammatory factors and IL-10,IL-13 and other anti-inflammatory factors.Western blotting was used to detect the protein levels of hypothalamic TLR4,p-IKB?,p65,p-p65 and IL-I?;16SrRNA gene sequencing was used to detect the composition and abundance of bacterial groups in mouse stool samples.Results:(1)The results of the behavioral experiment of depression showed that compared with the WT group,the KO group's immobility time in the forced swimming experiment was significantly increased(P<0.01),the struggling time was significantly reduced(P<0.05),and the immobility time in the tail suspension experiment was significantly increased(P<0.05),struggling time was significantly reduced(P<0.05);compared with the KO group,the KE group 's immobility time in the forced swimming test was significantly reduced(P<0.05),in the tail suspension test The immobility time was significantly reduced(P<0.01),the struggling time was significantly increased(P<0.05),and the sugar water preference was significantly increased(P<0.01).(2)The results of Elisa showed that compared with mice in WT group,IL-6 concentration in KO group was significantly increased(P<0.01),5-HT concentration was significantly reduced(P<0.01),and 5-HT concentration in WE group was significantly increased(P<0.05);Compared with mice in KO group,the concentrations of CRP(P<0.05)and IL-6(P<0.01)in KE group were significantly reduced,and the concentration of 5-HT was significantly increased(P<0.05).(3)The Real-time PCR results of prefrontal cortical inflammation factor showed that compared with WT group mice,CASP1 mRNA levels in WE group were significantly reduced(P<0.01);compared with WT group mice,KO group mice mRNA levels of TNF? and IFN-? were significantly increased(P<0.05),mRNA levels of IL-10(P<0.05)and IL-13(P<0.01)were significantly reduced;compared with mice in the KO group,In the KE group,the mRNA levels of IL-6(P<0.05),TNF?(P<0.05),IFN-?(P<0.01),and CASP1(P<0.05)were significantly reduced,and IL-13 mRNA levels were significantly increased(P<0.05).(4)The Real-time PCR results of TLR4/NF-?B signaling pathway-related genes in the prefrontal cortex showed that the mRNA levels of TLR4(P<0.05),NF-?B(P<0.01),IL-18(P<0.05),IL-1?(P<0.05),TLR2(P<0.01)in KO group were significantly higher than those in WT group,and the mRNA levels of TLR4(P<0.05),NF-?B(P<0.01),IL-18(P<0.01),IL-1?(P<0.05)and TLR2(P<0.05)in KE group were significantly lower than those in KO group.The western blotting results of hypothalamic TLR4/NF-?B signaling pathway-related proteins showed that compared with WT group,the protein level of TLR4 and p-p65 in KO group was significantly higher(P<0.05),and the phosphorylation level of p65 was significantly higher(P<0.01);compared with Ko group,the phosphorylation level of p65 in KE group was significantly lower(P<0.05).(5)The results of 16 SrRNA gene sequencing showed that in the fourth week of exercise intervention,the chao1 diversity index of the KO group was significantly lower than that of the WT group(P<0.05),and the chao1 diversity index of the KE group was significantly higher than that of the WE group(P<0.01);in the eighth week of exercise intervention,the chao1 diversity index of the KE group was significantly higher than that of the KO group(P <0.05),compared with the WE group of mice,the KE group The chao1 diversity index of mice increased significantly(P <0.01).The relative abundance results of the bacterial community showed that at the phylum level,Bacteroides phylum(66.7%),Thickwall phylum(23.4%),Proteobacteria phylum(5%),and epsilonbacteraeota(2%)in the stools of the four groups of mice Relatively high abundance;at the family level,Muribaculaceae(52.1%),Trichosporonaceae(9.8%),Verrucobacteriaceae(8.7%),and Prevotellaceae(6.9%)have relatively high abundance.The evolutionary branch of the differential species shows that during the fourth week of exercise intervention,the Lactobacillus family plays a major role in the WT group of mice,the Helicobacteraceae,Flavobacteriaceae,Atopobiaceae,and Christensenellaceae play the major role in the WE group of mice,and the KO group of mice Bifidobacteriaceae,Vibriobacteriaceae,Sphingomonasceae,Paecilomycetes,Eubacteriaceae,Clostridiaceae,Azospirillaceae,Anaeroplasmataceae play a major role,Mycoplasmaceae,nitrosation in the KE group of mice Monasceae,Desulfurinaceae,Magnetospiraceae,Muribaculaceae play a major role;in the eighth week of exercise intervention,Bifidobacteriaceae and Peptostreptococcaceae play a major role in the WT group of mice.Mycobacteria,desulfurized Vibrioceae,and Veillonellaceae play a major role,and the KO group mice in the family of Aspergillus,Burkholderiaceae,Bacillus,Christensenellaceae,Marinilabiliaceae,Micropepsaceae,Erysipelotrichaceae play a major role Corynebacteriaceae,Mycoplasmaceae,Pasteurellaceae,Klebsiellaceae,Clostridia and Methyloligellaceae play a major role in the KE group of mice.Conclusion:(1)ASMT knockout can cause depression in mice,and exercise can alleviate depression caused by ASMT knockout.(2)Depressive behavior and central inflammation caused by ASMT knockout are related to the activation of the TLR4/NF-?B signaling pathway,and exercise can reverse the inflammatory response by inhibiting the TLR4/NF-?B signaling pathway,thereby alleviating depressive behavior.(3)The knockout of ASMT gene affects the diversity of intestinal flora,and its mechanism of mediating depression pathology may be related to the disturbance of intestinal flora induced by the activation of TLR4/NF-?B signaling pathway;The inflammation-brain axis improves intestinal microflora disorder and alleviates depressive behavior caused by ASMT gene knockout.