| [Research purpose]This project studies and explored the molecular mechanism of the effect of Mongolian medicine Nutmeg-5 flavor pills on nerve regeneration in CUMS rats from the perspective of molecular biology,hoping to reveal the antidepressant mechanism of Nutmeg-5 flavor pills.It will further enrich the theories of Mongolian medicine,provide scientific basis for clinical application of Nutmeg-5 flavor pills and lay a foundation for future research and development of new drugs.[Research methods]1.Grouping and administration:96 healthy SD male rats weighing 200±20g were randomly divided into 6 groups according to their body weight after 1 week of adaptive feeding,with 16 rats in each group.There were blank group,model group,positive control group,high dose,medium dose and low dose groups of cardamom 5 pills.The blank group was fed 8 animals per cage,drinking water and feeding freely without any stimulation.The other groups were raised in single cages and received 28 days of stimulation.All rats were given fluoxetine hydrochloride(3.6mg/kg),cardamo-5 pills(1.6g/kg,0.8g/kg,0.4g/kg)in the positive control group,and normal saline in the blank group and model group.After continuous model administration for 28 days,relevant indicators were detected after the end of stress.2.Modeling method and evaluation:chronic mild unpredictable stress(CUMS)and solitary rearing method were used to replicate the depression model.Body weight and 1%sucrose water consumption were measured on Od,14d and 28d.On day Od and day 28d,the open-field Open box experiment was adopted.After the end of stress stimulation,the methods of tail suspension and Morris water maze learning and memory ability were used for behavioral detection,and the replication model was evaluated.3.Specimen preparation and detection methods:after behavioral observation,the rats were put to death with their heads cut off,the hippocampal tissue was rapidly stripped off on the ice,and the samples were kept under test after rapid freezing with liquid nitrogen.(1)PCNA immunofluorescence method was used to observe the nerve regeneration of DG region in hippocampus of rats in each group.The content of camp in rat hippocampus was determined by ELISA.(3)Immunohistochemical detection:expression of p-crab,CREB and BDNF in rat hippocampal tissue.The expression of Cleburne and BDNFmRNA in rat hippocampal tissue was detected by rt-pcr.The expression of miRNA 132 and miRNA182 in rat hippocampal tissue was detected by qrt-pcr.4.Data statistics:SPSS 22.0 statistical software was used to process data.All data were presented as mean+standard deviation(x+S).Test level p=0.05,p<0.05 was considered to be statistically significant.[Results]1.Effects of Nutmeg-5 flavor pills on behavioral behavior in CUMS rats1.1 Influence on weight change of rats:before the experiment,there was no significant difference in weight between each group,which was comparable.During the experiment,the body weight of the stimulated rats changed slowly.On the 14th day of the experiment,compared with the blank group,the body weight of the rats in the model group changed slowly,showing a statistical difference(P<0.05).Compared with the model group,the body weight of the rats in the positive control group and the medium dose group of Mongolian medicine Nutmeg 5 pills showed a high change(P<0.05).On the 28th day of the experiment,compared with the blank group,the weight change of rats in the model group was significantly slower(P<0.01).Compared with the model group,the body weight of rats in the positive control group and the medium dose group of Nutmeg-5 flavor pills was significantly higher(P<0.01),and the body weight of rats in the high dose group of Nutmeg-5 flavor pills was also significantly higher than that in the model group(P<0.05).The results showed that the weight gain of the model group was slower,and fluoxetine and Nutmeg-5 flavor pill could improve the weight change of CUMS rats.1.2 Effect on 1%sucrose water consumption in rats:there was no statistical difference in 1%sucrose water consumption in rats before the experiment.On the 14th day of the experiment,compared with the blank group,the consumption of sucrose water in the model group was significantly reduced,with a statistical difference(P<0.05).Compared with the model group,the consumption of sucrose water in the medium dose group of fluoxetine and Mongolian medicine Nutmeg-5 flavor pills was significantly higher(P<0.05).On the 28th day,compared with the blank group,the consumption of sugar water in the model group was significantly reduced(P<0.01);compared with the model group,the consumption of sugar water in the fluoxetine group and the high school dose group of Mongolian medicine Nutmeg-5 flavor pills was significantly increased(P<0.01,P<0.05).The results showed that CUMS rats had decreased reward response and anhedonia.Fluoxetine and Mongolian medicine Nutmeg-5 flavor pills have increased sugar water consumption.1.3 Efects on the voluntary activities of rats with open-field behavior:before the experiment,there was no significant difference in the number of horizontal and vertical voluntary activities of rats with open-field behavior(P>0.05).After the experiment,compared with the blank group,the number of horizontal crossing grids and vertical erect times of rats in the model group were significantly reduced(P<0.01).Compared with the model group,the number of horizontal crossing cells was significantly increased in the fluoxetine group and the medium and low dose Mongolian medicine Nutmeg 5 pills group(P<0.01,P<0.05).The number of times of vertical standing in the medium dose group of fluoxetine and Mongolian medicine Nutmeg 5 pills was significantly higher than that in the model group(P<0.05).The results showed that the autonomous activity and exploration ability of CUMS rats in the new environment decreased.Fluoxetine and Mongolian medicine Nutmeg-5 flavor pill can improve the autonomous activity and exploration ability of CUMS rats.1.4 Influence on the immobile time of tail suspension in rats:compared with the blank group on the 28th day of the experiment,the immobile time of the model group was prolonged(P<0.01).Compared with the model group,the immobile time of fluoxetine group and the medium dose group of Nutmeg-5 flavor pills was significantly shorter than that of the model group(P<0.05).The CUMS rats behaved hopelessly.Fluoxetine and Mongolian medicine Nutmeg-5 flavor pills can shorten the immobility time of rats to different degrees.1.5 Effects on the learning and memory ability of water maze:after the stimulation,compared with the blank group,the escape time of the model group was significantly prolonged(P<0.01),and The Times of crossing the platform were reduced.Compared with the model group,the escape incubation period of rats in the fluoxetine group and the medium dose group of Nutmeg-5 flavor pill was significantly shortened(P<0.05,P<0.01).All the drug groups increased The Times of crossing the platform,but there was no statistical difference compared with the model group.The results showed that both fluoxetine and Nutmeg-5 flavor pills had certain effects on enhancing the learning and memory ability.2.Effects of Mongolian medicine Nutmeg-5 flavor pill on hippocampal nerve in CUMS rats:after 28 days of stress,the number of PCNA positive cells and the cell proliferation rate in the SGZ region of hippocampal were significantly lower in the model group than in the blank group(P<0.01).Compared with the model group,the number and proliferation rate of PCNA positive cells in the hippocampus were significantly higher in the fluoxetine group and the high school dose group of Mongolian medicine Nutmeg-5 flavor pill(P<0.01).3.Effect of Nutmeg-5 flavor pills on cAMP-CREB-BDNF signaling pathway in CUMS rats:3.1 Effect of Nutmeg-5 flavor pill on camp content in the hippocampal tissue of CUMS rats:after the end of stress stimulation,camp content in the model group was significantly lower than that in the blank group(P<0.01).Compared with the model group,the content of hippocampal camp was significantly higher in the fluoxetine group and the high-dose Mongolian medicine Nutmeg-5 flavor pill group(P<0.05).Explanation:the Mongolian medicine Nutmeg-5 flavor pill had high content of camp in hippocampus.3.2 Effects of Nutmeg-5 flavor pill on the expression of p-CREB,CREB and BDNF proteins in the hippocampal tissues of CUMS rats:according to the immunohistochemical results,the p-CREB IOD value,the number of CREB positive cells and the number of cells in the model group were significantly lower than those in the blank group(p<0.01,p<0.05).Compared with the model group,the values of p-CREB IOD and the number of CREB positive cells were significantly higher in the fluoxetine group and the high school dose group(p<0.01,p<0.05).The BDNF positive cells in the hippocampus of the blank group were densely arranged and deeply stained.Compared with the blank group,the BDNF positive cells in the model group were sparsely arranged,irregular,mostly in the form of air cannons,with shallow staining of nuclear plasma,and significantly reduced BDNF IOD value(P<0.01).Compared with the model group,fluoxetine group and the high school dose group of Mongolian medicine Nutmeg-5 pills showed a strip-like distribution of positive cells in the granular layer and CA3 area of the hippocampal dentate rus with orderly and deep staining,and the BDNF IOD value was significantly increased(P<0.01,P<0.05).3.3 Effects of Nutmeg-5 flavor pill on the expression of CREBmRNA and BDNFmRNA in the hippocampal tissues of CUMS rats:compared with the blank group,the expressions of CREBmRNA and BDNFmRNA in the hippocampal tissues of the model group all decreased(P<0.05);compared with the model group,the expressions of CREBmRNA and BDNFmRNA in the medium and high-dose groups of fluoxitine and Nutmeg-5 flavor pill were up-regulated(P<0.05).4.Effect of Nutmeg-5 flavor pill on the expression of miRNA132 and miRNA182 in hippocampal tissues of CUMS rats:CUMS stress stimulation can down-regulate the expression of miRNA 132、miRNA182 in hippocampal tissues of CUMS rats.Compared with the blank group,the expressions of miRNA132 and 182 in the hippocampus of rats in the model group were down-regulated,showing a statistical difference(P<0.05).Compared with the model group,miRNA132 expression was up-regulated in the high-dose Mongolian medicine Nutmeg-5 flavor pill group(P<0.05),and miRNA 182 expression was up-regulated in the high-dose Mongolian medicine Nutmeg-5 flavor pill group,but there was no statistical difference(P>0.05).[Conclusion]1.The CUMS and orphan model adopted in this project are widely used animal models of depression at home and abroad.During the experiment,the weight growthrate,sucrose water bias,horizontal and vertical activity times of the model group rats were reduced,the tail suspension time and Morris water maze escape latency were prolonged,and the time of crossing the original platform were reduced,indicating that the depression model was successfully replicated.According to the experimental results,Nutmeg-5 flavor pill can improve the behavioral changes of CUMS rats,indicating that Nutmeg-5 flavor pill has an antidepressant effect.2.Nutmeg-5 flavor pill can promote hippocampal nerve regeneration in CUMS rats.It can up-regulate cAMP content,CREB,BDNF protein and gene expression in hippocampus.It can up-regulate the expression of endogenous regulatory factor miRNA132.3.Nutmeg-5 flavor pill promotes hippocampal nerve regeneration by upregulating cAMP-CREB-BDNF signaling pathway and endogenous miRNA132.4.Mongolian medicine Nutmeg-5 flavor pill promotes hippocampal nerve regeneration in CUMS rats by regulating cAMP-CREB-BDNF signaling pathway and endogenous miRNA 132,and may be one of its mechanisms of antidepressant action. |
PDF Full Text Request