| The types of autophagy-related diseases are increasing in recent years,making it a research hotspot in the field of biomedicine.In addition,the scope of research on autophagy-related disease is constantly expanding and the level deepening.This study found that autophagy plays a key role i.n the pathogenesis of certain diseases,and that the change in autophagy levels is closely related to depression and chronic stress injury.Therefore,the focus of this research is mainly on the influence of autophagy on neurons.Since the mechanism of depression is complex,it is of great significance to study the relationship between autophagy and depression.Many antidepressants are involved in the regulation of autophagy and in the interference of the autophagy signaling pathway,making autophagy a potential pharmacological research hotspot for depression.Autophagy dysfunction is closely related to neurological disorders and neuroplastic dysfunction.Multiple signal transduction pathways in vivo can regulate the activity of autophagy,but mTORC1(mammalian target of rapamycin complex 1)signal transduction pathway is one of the most important regulatory pathways in autophagy research.Numerous data suggests that activation of the mTORC1 signaling pathway is essential for antidepressant responses and that many traditional antidepressants have a regulatory effect on the mTORC1 pathway.Therefore,targeting at regulation of the mTORC1 signaling pathway can be a research direction for the treatment of depression.At present,the research on the effect of autophagy on neurons has two sides,with arguments on whether activation of autophagy provides a protective effect on the nervous system or aggravates the damage of neurons instead.ObjectiveThe aim of this study was to investigate the effect of liver regulation recipe Sinisan decoction on autophagy activity and the mTORC1 signal transduction pathway in the hippocampal neurons of rats with depression incduced by chronic unpredictable mild stress,in order to explore the molecular biological mechanism of the anti-depression effect of the liver regulation therapy.The mechanism,demonstrating the relationship between the function of ’liver controlling dispersion5 and the function of autophagy,provides a new perspective for the interpretation and use of the biological connotation of ’liver controlling dispersion’.Methods1.Modeling:A total of 75 Wistar male healthy adult(6 weeks old)rats were selected for the research.According to the initial body weight values at the day before grouping,rats were randomLy assigned to blank group,model group,kidney nourishing recipe group,liver regulation recipe group and spleen tonifying recipe group with 15 rats in each group.Except for the blank group,rats in the other four groups were exposed to chronic unpredictable mild stress for 42 days.The exerted stress mainly included seven activities:stopping food and water feeding(24h),heating(40℃-50℃),ice swimming(6℃-4℃),electric shock(35V-55V),tail clapping(3min-6min),restraint(4h-6h)and stroboscopic stimulating(2h-12h).The stress inducing activities were randomLy arranged on rats every day,with one kind of stress given per day in the first week,1-2 kinds of stress given per day in the second week,and gradually transitioning to two kinds of stress given per day.The same kind of stress was not given for two consecutive days to build a chronic unpredictable mild-stress depression model.2.Drug interventions:rats in the kidney nourishing recipe group,the liver regulation recipe group and spleen tonifying recipe group were given drug intervention respectively before stress inducing activities with the Youguiwan,Sinisan and Sijunzi decoction solutions,and the other two groups were given only water as a control.3.Evaluation indicators:(1)Somatic symptom indicator:body mass.(2)Behavioral indicators:the behavior of rats in each group was evaluated respectively by using the open field experiment(evaluating the independent activities of rats),the sucrose preference experiment(reflecting the rats’sense of pleasure),and the Morris water maze test(evaluating the learning and memorizing ability).(3)Biological indicators:Enzyme-linked immunosorbent assay(ELISA)was used to detect the assay of brain-derived neurotrophic factor(BDNF),serotonin(5-HT)and serum adrenocorticotropic hormone(ACTH)and Cortisol(CORT)in cerebrospinal fluid of rats in each group;HE staining and Nissl staining methods were used to observe the morphology and Nissl bodies of hippocampal CA3 neurons in each group;immunofluorescence technique was used to observe the expression of autophagy-related proteins LC3 in hippocampal CA3 region of rats in each group;real-time fluorescence quantitative PCR(qRT-PCR)detecting technique and Western blot technique were used to detect the expression of autophagy and mTORC1 signaling pathway-related genes and proteins in the hippocampus of individual rats in each group.Results1.Body massBefore the CUMS modeling,there was no statistically significant difference between the body masses of the rats before the model was established.After 42 days of stress modeling,the body mass growth of the model group was far less than that of the normal group(P<0.01).The body mass growth of rats in the kidney nourishing recipe group,liver regulation recipe group and spleen tonifying recipe group was roughly equivalent to that of the model group.2.BehaviorAccording to the results,the sucrose preference rate of the model group was much lower than that of the blank group(P<0.01);in the drug intervention group,only the rats in the liver regulation recipe group present a significantly higher sucrose preference rate than that of the model group(P<0.05),while the sucrose preference rate of the rats in the kidney nourishing recipe group and spleen tonifying recipe group was numerically higher than that in the model group,but showed no statistically significant difference between the three groups.The results of the total distance of horizontal movement in the open field show:the distance of rats in model group were significantly reduced compared with the rats in the blank group(P<0.01);while the total distance of total horizontal movement of rats in the kidney nourishing recipe group,liver regulation recipe group and spleen tonifying recipe group were significantly longer than that in the model group(P<0.01).Morris water maze positioning navigation experiment results showed that there was an interaction between time and grouping factors,the effect of time factors varied from group to group,but there was no statistical difference between groups in each day.Morris water maze space exploration experiment showed that the model group rats significantly reduced their frequency of platform crossing movement,the distance towards target quadrant and duration of staying in target quadrant,compared with the rats in blank group(P<0.01).In the drug intervention group,only the kidney nourishing recipe group shows increase in the target quadrant distance and the target quadrant staying time of the rats compared with the model group(P<0.05).Although the number of times of crossing platform movement,the water maze target quadrant distance,and t.he water maze target quadrant staying time of the Sijunzi decoction group and liver regulation recipe group showed an increasing trend compared with the model group,but there was no statistical difference between the groups.3.Comparison of BDNF and 5-HT levels in rat cerebrospinal fluidThe levels of BDNF and 5-HT in the cerebrospinal fluid of the rats in model group were significantly lower than those in the blank group(P<0.01);the decreasing trend of BDNF in the cerebrospinal fluid of rats in the kidney nourishing recipe group,liver regulation recipe group and spleen tonifying recipe group were effectively reversed after intervention of different prescriptions(P<0.01).Rats in the liver regulation recipe group and spleen tonifying recipe group were able to reverse the decrease of 5-HT content in cerebrospinal fluid of rats observed in the model group after different interventions(P<0.01 or P<0.05).Compaired with the liver regulation recipe group,the BDNF content in the cerebrospinal fluid of rats in spleen tonifying recipe group and the 5-HT content in the cerebrospinal fluid of rats in the kidney nourishing recipe group decreased(P<0.01).4.Comparison of ACTH and CORT levels in serum of ratsThe levels of ACTH and CORT in the serum of the model group were significantly higher than those in the blank group(P<0.01).In the drug intervention group,only the rats in the liver regulation recipe group could reverse the situation of both increased ACTH and CORT level(P<0.01 or P<0.05).The rats in the spleen tonifying recipe group could only reverse the increase of CORT content in serum of rats the model group after intervention with the prescription(P<0.01).Compared with the rats in liver regulation recipe group,the serum levels of CORT in the rats of kidney nourishing recipe group and spleen tonifying recipe group showed no statistical difference.5.Morphological structure and distribution of Nissl bodies in hippocampal CA3 region of ratsIn the blank group,the hippocampal CA3 area had high cell density,thick pyramidal cell layer,normal neuronal structure,and deep staining of Nissl bodies in the cytoplasm in good number.There were obvious pathological changes in the hippocampal CA3 neurons of rats in the model group,which was mainly shown in that the cell density decreased,the number of neurons decreased significantly compared with the blank group,the pyramidal cell gap increased,the neuron structure was fuzzy,the shape was irregular and cytoplasmic Nissl bodies stain lightly or disappear.Compared with the model group,the number of neurons in the CA3 area,the number of Nissl bodies,and the damage of neuronal morphological structure in each prescription-intervention group were improved in different degrees:rats in the liver regulation recipe group and the spleen tonifying recipe group present more obvious improvement,with the number of cells increased,the arrangement was relatively tight and tight,the morphology of neurons mostly returned normal,and the Nissl bodies increased.The morphological structure and the improvement of Nissl bodies in the hippocampal CA3 area of the rats in the kidney nourishing recipe group were relatively weaker,with the number of neurons increased compared with the model group less than that of the liver regulation recipe group and the spleen tonifying recipe group.The arrangement was still tight and orderly.No obvious cell degeneration was observed.The Nissl body was lightly stained and the number was acceptable.6.Comparison of LC3 immunofluorescence results in hippocampal CA3 regionDiffused LC3 green fluorescent spots in the hippocampal CA3 neurons of the blank group were of small amount and appeared darker.The fluorescence intensity of LC3 in neurons in the hippocampal CA3 region of the model group was significantly higher than that of the blank group,and the green fluorescent spots of the model group increased.Compared with the model group,the expression of LC3 green fluorescence of neurons in hippocampal CA3 region of the rats in the prescriptions of kidney nourishing recipe,liver regulation recipe and spleen tonifying recipe was decreased to different extent.Both the number and brightness of green fluorescent spots decreased.While the green fluorescence intensity of LC3 in the hippocampal CA3 region of rats in liver regulation recipe group was relatively lower,the brightness of the kidney nourishing recipe group and the spleen tonifying recipe group was roughly equivalent.7.Comparison of autophagy and expression of key genes in mTORC1 signaling pathway in rat hippocampusCompared with the blank group,the relative expression level of autophagy-related gene LC3 mRNA in the hippocampus of the model group increased,and the relative expression level of p62 mRNA decreased significantly(P<0.01).The relative expression levels of autophagy-related gene LC3 mRNA in rat hippocampus significantly decreased after treatment with different drugs including Sinisan decoction and Si junzi decoction(all P<0.01).The relative expression of p62 mRNA increased in liver regulation recipe group and spleen tonifying recipe group(all P<0.01).Compaired with the liver regulation recipe group,the relative expression level of LC3 mRNA was higher and the relative expression level of p62 mRNA was lower in hippocampus of rats in kidney nourishing recipe group(all P<0.01).The relative expression level of p62 mRNA in hippocampus of rats in the spleen tonifying recipe group was lower than that in liver regulation recipe group(P<0.01).Compared with the blank group,the relative expression levels of mTOR signaling pathway-related genes PI3K mRNA,Akt mRNA,mTOR mRNA and Raptor mRNA in the hippocampus of rats in the model group were significantly lower(P<0.05 or P<0.01).Compared with the model group,and in the drug intervention group,only the relative expression levels of PI3K mRNA,Akt mRNA,mTOR mRNA and Raptor mRNA went up in the hippocampus of rats in the liver regulation recipe group(all P<0.01);while in the kidney nourishing recipe group,the relative expression levels of only mTOR mRNA and Raptor mRNA went upper in the tissues(all P<0.01);in the spleen tonifying recipe group,the expression level of mTOR mRNA was up-regulated(P<0.05).Compared with liver regulation recipe group,the relative expression levels of PI3K mRNA,Akt mRNA and Raptor mRNA in hippocampus of rats in kidney nourishing recipe group and spleen tonifying recipe group were relatively lower(all P<0.01);hippocampus of rats in spleen tonifying recipe group presented a lower relative expression level of mTOR mRNA than that of liver regμLation recipe group(P<0.01).8.Comparison of autophagy and expression of mTORC1 signaling pathway-related proteins in rat hippocampusCompared with the blank group,the relative expression level of p62 protein in the hippocampus of rats in the model group went significantly downward,and the ratio of LC3-Ⅱ/LC3-Ⅰ was significantly higher than that of the blank group(all P<0.01).Compared with the model group,the relative expression level of p62 protein in the hippocampus of kidney nourishing recipe group,liver regulation recipe group and spleen tonifying recipe group was up-regulated,and the ratio of LC3-Ⅱ/LC3-Ⅰ was decreased(all P<0.01).Compared with liver regulation recipe group,the relative expression level of p62 protein in hippocampus of rats in kidney nourishing recipe group and spleen tonifying recipe group decreased,and the ratios of LC3-Ⅱ/LC3-Ⅰ in these two groups ineeased(P<0.05 or P<0.01).Compared with the blank group,the ratio of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR in the hippocampus of rats in the model group and the relative expression of Raptor protein decrease to different degrees,all of which presented significant statistic difference(P<0.05 or P<0.01).Compared with the model group,the ratio of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR in hippocampus of liver regulation recipe group,and the relative expression of Raptor protein were increased(all P<0.01);The ratio of p-PI3K/PI3K in the hippocampus of rats in the prescription group was higher than that in the model group(P<0.01).The ratio of p-PI3K/PI3K and p-mTOR/mTOR in the hippocampus of the Spleen tonifying recipe group was higher than that in the model group(bothP<0.01).Compared with liver regulation recipe group,the ratio of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR in hippocampus of kidney nourishing recipe group and of spleen tonifying recipe group was lower than that of Raptor protein(P<0.01).Compared with the blank group,the ratios of p-p70S6K/p70S6K and p-4EBP1/4EBP1 in the hippocampus of the model group were significantly decreased(all P<0.01).Compared with the model group,the ratios of p-p70S6K/p70S6K and p-4EBP1/4EBP1 in hippocampcis of rats in liver regulation recipe group and spleen tonifying recipe group were increased to different degrees((P<0.05 orP<0.01)).In kidney nourishing recipe group,the ratio of p-4EBP1/4EBP1 in hippocampus of rats was increased compared with the model group(P<0.05),while the ratio of p-p70S6K/p70S6K was approximately the same as that in the model group,and the difference was not statistically significant.Compared with liver regulation recipe group,the ratios of p-p70S6K/p70S6K and p-4EBP1/4EBP1 in hippocampus of rats in kidney nourishing recipe group and spleen tonifying recipe group went significantly down(P<0.05 or P<0.01)).Conclusion1.Chronic unpredictable mild stress modeling methods can cause depression-related behavioral changes in rats,which may result from the abnormal expression of BDNF and 5-HT in rat cerebrospinal fluid,as well as ACTH and CORT in serum of rats,and down-regulation of PI3K/Akt/mTORCl signaling pathway activity in the hippocampus which causes excessive activation of autophagy and in turn damages hippocampal neurons.2.Though the antidepressant effect of Sinisan is better than that of Youguiwan and Si junzi,it fail to improve the body weight and learning-memorizing ability of rats,which indicate that the syndrome of the CUMS depression model rats is mainly characterized by“liver depression"accompanied with "liver depression and spleen deficiency" and "liver depression and kidney deficiency".The TCM syndromes present a dynamic change process of "deficiency caused by depression,deficiency accompanied by depression".3.The antidepressant effect mechanism of Sinisan may be related to modulating the abnormal levels of BDNF and 5-HT in rat cerebrospinal fluid,as well as ACTH and CORT in serum of rats,and the activation of PI3K/Akt/mTORC1 signaling pathway cascades in the hippocampus to inhibit excessive autophagy,thereby alleviating hippocampal neuronal damage.4.The biological connotation of "liver controlling dispersion" may be related to the regulation of autophagy function via mediating mTORC1 signaling pathway. |
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