The Regulatory Mechanism Studys With Aerobic Exercise Effecting Inflammatory Response And Improving The Endogenous H₂s Signaling Pathway In CUMS-depressed Mice

1 PurposeIn this study,we constructed a CUMS depressive mouse model,by chronic stress stimulating factor.The next-generation gene sequencing technology was used to analyze differentially expressed genes to screen differentially expressed genes and miRNAs which were intervened by aerobic treadmill exercise.RT-PCR verified gene expression changes.The purpose consists of four parts.Fist of all,aerobic exercise antagonizes the effects of inflammatory response in depressed mice.Secondly,further analysis explored the relationship between the anti-inflammatory effects of moderate-intensity aerobic exercise on depressed mice and the inflammatory response mediated by H₂S gas signaling molecules.Thirdly,exogenous H₂S has been demonstrated to inhibit the inflammatory response of cardiomyocytes by inhibiting the TLR4 pathway.Therefore,this project studied the mechanism of endogenous H₂S gas signaling involved in mediating aerobic exercise against inflammation in CUMS depressed mice after inhibition of TLR4.Last but not least,it could provide a theoretical basis for H₂S gas signaling pathway to mediate aerobic exercise to improve neuronal damage in patients with depression.2 Method2.1 CUMS depressed mice model and regular system of exercise intervention in modelingFifty SPF-grade male KM mice were randomly divided into a control group?CG,n=12?and a model group?MG,n=30?.MG mice were used by chronic unpredictable stress as an intervention to model mice in depression.Firstly,the 13 factors of CUMS depression modeling were used to generate chronic stress stimulation program by random number method.After 4 weeks of stimulation,neurobehavioral behavior was used to evaluate the depression-like behavior of mice.Modeling group mice were modeled by CUMS depression modeling method.Models of successful mice were evaluated by neurobehavioral evaluation.After the mice with different differences were excluded,the random number method was divided into model group?MG?and model exercise group?ME?,15rats/group.In the ME group,8 weeks of moderate-intensity aerobic treadmill exercise was used as an intervention method.Neurobehavioral evaluation was used to observe the changes of depression-like behavior.Nissl staining was used to notice the pathological changes of hippocampal neurons in mice.High-throughput sequencing screening of cytokines associated with inflammatory and sulfur metabolic gas signaling pathways.The content of inflammatory related factors in blood and hippocampus was detected by ELISA.The protein expression of inflammatory cytokines in hippocampus was detected by immunohistochemistry and western blot.The mRNA transcription level of inflammatory related factors in hippocampus was detected by RT-PCR.2.2 The mechanism of research of CBS/H₂S signaling system mediated continuous system of regular exercise intervention on TLR4inflammatory signaling pathway in CUMS depressed mice.Sixty male SPF male healthy KM mice were randomly separated into control group?CG?,model group?MG?,model exercise group?ME group?,TLR4 inhibitor group?TG?and TLR4 inhibitor+exercise group?TE?.),12/group.Exercise group mice were given moderate-intensity aerobic treadmill exercise for 8 weeks.TLR4 inhibitor group was intraperitoneally injected with 3 mg/kg/day dose of TAK-242,and neurobehavioralevaluationwasperformedafterintervention.Depression-like behavior in rats,blood was taken from the heart after anesthesia with pentobarbital sodium,hippocampus was taken on decapitated ice,frozen at-80°C,used for gene sequencing,H₂S content and RT-PCR.The brain was treated with 4%paraformaldehyde infusion,and the brain tissue of the mice was taken on ice.The formaldehyde was fixed for more than 48 hours.It was used for embedding detection of Nissl staining,HE staining and fluorescence immunity.We used Nissl staining and HE staining to observe the pathological changes of hippocampus in mice,immunohistochemical detection of mouse hippocampus protein expression,ELISA detection of serum protein content in mice,deproteinization method for determination of H₂S content in plasma and hippocampus,Western blotting The expression of hippocampal tissue protein,RT-PCR was used to detect the differential expression of gas signaling molecules and inflammation-related factors,and the expression of miRNAs and the immunological detection of hippocampal neurological function and inflammatory response in mice.3 Results3.1 The results of various indicators in CUMS depressed miceThe body weight of mice with chronic stress stimulation for 4weeks decreased significantly.The number of grids,exercise time and number of modifications across the open field test decreased significantly.The syrup preference index were decreased significantly.The immobility time of forced tail suspension and forced swimming ware significantly extended.The results of Nissl staining showed that the neurons in the hippocampal CA1 area of the model group were sparsely arranged,the gaps were widened,the neurons were lost,and the Nissl body nucleus was severely condensed.The activities of 5-HT and BDNF in serum and hippocampus were significantly decreased,and the protein expression and mRNA expression of BDNF were significantly decreased.It was suggested that CUMS depressed mice could be successfully modeled.3.2 The results of regular system of exercise intervention in CUMS depression mice inflammation3.2.1 The results of Neurobehavioral assessmentThe 8-week aerobic treadmill exercise increased the body weight of mice and the number of erecting times,the number of modifications and the number of crossings in depressed mice,reduced the immobility time of forced swimming and forced tailing,and increased the syrup preference index.There was a significant difference in the model group.3.2.2 The results of pathological changes in hippocampus of mice in each groupNissl staining results also showed that the hippocampal neuron lesions in the exercise group were alleviated,the number of nerve cells increased,and the Nissl staining was clear.3.2.3 The results of high-throughput sequencing for screening about inflammation-related factors in miceThe results of differentially expressed genes in blood and hippocampus of CUMS depressed mice after exercise intervention,showed that the differentially expressed genes involved in blood tissue mainly involved long-term depression pathological changes,oxidatve phosphorylation,Toll-like receptor,NF-kB,inflammatory mediator regulation of TRP channels and TGF-?signaling pathway etc.Hippocampus was mainly enriched in pathogenesis of depression,oxidative phosphorylation,Alzheimer's disease,Toll-like receptor,NF-kB and PPAR signaling pathway,sulfur metabolism,cholinergic synapses,and cAMP inflammatory signaling pathways as estrogen and the differential expression of related genes was significant.The comparison between the GO analysis of blood and hippocampus and the significant enrichment of KEGG showed that it mainly focused on neurodegenerative diseases and inflammation.3.2.4 The results of ELISA of blood and hippocampus in each group of miceThe results of ELISA showed that the pro-inflammatory factors IL-1?and TNF-?were significantly lower than the model group,and the anti-inflammatory factor IL-10 was significantly increased.3.2.5 The results of immunohistochemical for determination of inflammatory cytokines in hippocampus of miceThe results of immunohistochemistry showed that the positive expression of pro-inflammatory factors such as IL-1?,NF-?B and TNF-a in hippocampus of ME group were significantly decreased.The protein-positive expression region of inflammatory factor IL-10 was significantly increased.3.2.6 Detection of gene expression in blood and hippocampus of mice by RT-PCRThe results of RT-PCR detection showed that the expression of factors related to mRNA inflammation such as TLR4,IL-1?,NF-?B and TNF-?was down-regulated,and the expression of 5-HT mRNA was up-regulated.3.2.7 The results of western blot analysis about inflammatory factor protein expression in hippocampusThe results of Western blot showed that the protein expression of coastal inflammatory cytokines decreased in the exercise group.3.3 Mechanism of action of CBS/H₂S-mediated motor intervention on TLR4 signaling molecules in CUMS-depressed mice3.3.1 The results of neurobehavioral assessment and morphological changes in hippocampus neurons in each group miceAfter partial inhibition of TLR4,the neurobehavioral score of depressed mice was improved to some extent,and the morphological examination showed a certain degree of repair,and the effect was similar to that of aerobic exercise intervention.After aerobic exercise with TLR4 were inhibited,the positive rate of 5-HT in the hippocampus of mice was higher than that of the model group,and the positive rates of inflammatory factors such as CD45⁺and PARP were improved,and the intervention effect of aerobic exercise combined with TLR4 inhibitor was the best.3.3.2 The results of detection of H₂S content in mice serum and hippocampusThe H₂S content in the plasma and hippocampus of the depression model group was significantly lower than that of the normal group.The changes of H₂S content in blood and hippocampus of mice in TLR4inhibitor group were not obvious.Even though,the aerobic exercise group could increase the H₂S content in blood and hippocampus,and the combined intervention group had the highest content.3.3.3 The differential expression results of mice blood and hippocampus related factors3.3.3.1 The differential expression results of H₂S signal correlation factorsThe differential expression of H₂S gas signal-related genes in the blood and hippocampus of each group showed that the expression level of CSE mRNA in the model group was down-regulated,and the expression level of CBS mRNA in hippocampus was also down-regulated,but aerobic exercise can increase the activity of blood CSE,hippocampus CBS,etc.The same study also found that after partial inhibition of TLR4,the expression of CSE and hippocampal CBS mRNA was also up-regulated.3.3.3.2 The differential expression of inflammtion-related factorsThe differential expression of inflammatory factors in each group of mice showed that the proinflammatory factors IL-1?,IL-6,TLR4,NF-?B and TNF-?mRNA in the aerobic exercise intervention group and the TLR4 inhibited group.Down-regulation,the expression of IL-10mRNA was up-regulated,and there were significant differences.3.3.4 The expression results of miRNAs regulated by mice blood and hippocampus related factorsThe differential expression of miRNAs associated with gas signaling molecules and miRNAs associated with inflammation were significantly up-regulated or down-regulated after TLR4 was inhibited,such as miR-21,miR-195,and miR-30,miR-455 and miR-665.Significantly down-regulated,analysis of its functions,such as miR-21can be used as a target for H₂S gas signaling molecules,on the one hand by TAK-242,on the other hand it is also involved in the regulation of multiple signal channels.For example,regulation of inflammatory pathways such as IL-6 signal transduction and expression of pathway activation,miR-665 was involved in the regulation of coastal neuron protection and inhibition of apoptosis,and miR-30 may participate in the inflammatory process of target genes.miR-195 can participate in its inflammatory response by inhibiting the nuclear transport process of NF-?B,and miR-455 could not only participate in apoptosis induced by endoplasmic reticulum stress,but also be interfered by gas signal molecules such as H₂S.4 Conclusion4.1 After 4 weeks of chronic unpredictable stress stimulation,it could effectively replicate the depression-like behavior of mice and reduced the content of 5-HT and BDNF in serum and hippocampus.The experiment verified the effectiveness of depression modeling.4.2 Aerobic exercise could significantly improve the depression-like behavior of depressed mice,promoted the repair of hippocampal neurons,reduced the content of pro-inflammatory factors and the expression of protein,mRNA,etc.,and played a good role in anti-depression inflammation.It was confirmed that aerobic exercise played a good role in antidepressive inflammation.4.3 High-throughput sequencing of blood and hippocampus in CUMS-depressed mice with aerobic exercise and its associated enrichment analysis showed that inflammation-related TLR4,IL-1?,TNF-?and CSE and CBS related to H₂S gas signaling pathways.The expression of cytokines was significantly correlated,and miRNAs such as miR-21,miR-30 and miR-455,which were targeted to regulate inflammation and H₂S gas signaling pathway cytokines and significantly enriched.4.4 Both aerobic exercise and TLR4 inhibitors could reduce the inflammatory response of blood and hippocampus in depressed mice to some extent.Although TLR4 inhibitors could not significantly increase the expression of H₂S and its synthetase,it had the expression of inflammatory factors and significantly influence.It suggested that H₂S was involved in mediating aerobic exercise intervention in the regulation of depression inflammatory signaling pathway TLR4/MyD88-NF-?B,but TLR4 had little effect on H₂S content and it's related biosynthetic enzymes.4.5 The regular system of exercise intervened the differential expression of H₂S signaling system related factors such as CBS and CSE by miR-455 and differential expression of miR-21,miR-30 and miR-665,which regulated TLR4/MyD88-NF-?B inflammatory signaling pathway and miR-455 regulates H₂S gas signaling pathways such as CBS and CSE.4.6 The dererential expression of TLR4 and H₂S participated in the molecular mechanism of exercise intervention in inflammatory response in depressed mice.That is,aerobic exercise-induced endogenous H₂S could effectively reduce the inflammatory response of blood and hippocampus in CUMS-depressed mice.The mechanism of action was that H₂S gas signaling molecules participate in the inflammatory signaling pathway of aerobic exercise intervention TLR4/MyD88-NF-?B.