| Depression,with high morbidity,disability and mortality,is currently one of the major disability burden worldwide.The pathogenesis mainly includes monoamine neurotransmitter hypothesis,neural plasticity hypothesis,and inflammatory reaction.Studies have confirmed that microglia and astrocytes are important components for controlling nerve growth and conduction.However,under stress stimulation,when both astrocytes and microglia convert to pro-inflammatory state,they usually activate neuroinflammatory response and release a large number of pro-inflammatory cytokines.This process can damage nerve conduction and synaptic connections,resulting in decreased neural plasticity.Chronic stress model induces damage to the length and branches of neuronal dendrites,as well as decreased number and function of spinous synapses in medial PFC pyramidal neurons.Currently,many clinically commonly used drugs for treating depression have the defects of slow onset,low remission rate,easiness in relapse,more adverse reactions,and the like.Shenwei Ningyu Tablets,as the first pure Chinese medicinal small compound preparation approved into clinical trials for the treatment of depression in China,can regulate the immune inflammatory response of the chronic restraint stress rat model.Clinical trials confirmed that the drug had a significant effect on the treatment of depression due to deficiency of both qi and yin.Therefore,through the establishment of rat depression model based on solitary confinement and chronic restraint stress,and on the basis of evaluating the efficacy of the antidepressant,we conducted an in-depth study on the antidepressant mechanism of the drug.This study mainly included two parts:Verifying the antidepressant effect of shenwei ningyu tablets based on the depression model of solitary confinement combined with chronic restraint stress:To explore the antidepressant mechanism of shenwei ningyu tablets based on the depression model of solitary confinement combined with chronic restraint stress.Part Ⅰ:Pharmacodynamic evaluation of Shenwei Ningyu Tablets on depression model induced by solitary confinement and chronic restraint stressObjective:to establish a classical depression model of solitary confinement combined with chronic restraint stress,and evaluate the antidepressant effect of shenwei ningyu tablets overall through body weight test,sugar water preference and open field test.And lays a foundation for further mechanism research.Methods:Open field test was performed before the start of the experiment,and the rats with acceptable behavior were selected and divided into normal control group,model group,fluoxetine hydrochloride group and Shenwei Ningyu high,medium and low dose groups.In addition to the normal group,the depression models of solitary confinement combined with chronic restraint stress were established in all the other groups and given drugs continuously during the modeling.The body weight,sucrose preference and open field tests were performed on the 0 th,7 th,14 th and 21 th days of the experiment,respectively.To evaluate the efficacy of Shenwei Ningyu Tablets on depressive behaviors induced by this model.Result:1.In terms of body weight detection,there was no significant difference among groups before modeling.On the 21st day of modeling,compared with the normal group,the body weight of animals in the model group showed significant differences(P<0.01).Compared with the model group,the fluoxetine hydrochloride group and the high,medium and low dose groups of Shenweiningyu(CVNingyu)(P<0.05,P<0.01,P<0.01).2.In the sugar water preference experiment,the sugar water preference rates of animals in each group before the start of the experiment were basically the same.On the 21st day of modeling,the sugar water preference rate of the model group was lower than that of the normal group,but there was no significant difference.Compared with the model group,the sugar water preference rates in other groups were increased,and the fluoxetine hydrochloride group and Shenwei Ningyu low dose group showed significant differences(P<0.01,P<0.05).3.In the open field test,the indicators of animals in each group in the open field test were basically the same before the start of the experiment.On the 21st day of modeling,compared with the normal control group,the indicators such as total distance,total number of spans,standing times and central region residence time in the model group were decreased to different degrees,and there were significant differences(P<0.01).Compared with the model group,the total distance traveled,central region residence time and standing times were increased in different degrees in the fluoxetine hydrochloride group and the Shenwei Ningyu high dose group,with significant differences(P<0.01,P<0.05).Conclusion:Shenwei Ningyu Tablets can significantly improve the depression symptoms such as weight loss,loss of pleasant sensation and reduced exercise caused by the depression model of solitary confinement combined with chronic restraint stress,and has good antidepressant effect.Part Ⅱ:Study on the mechanism of Shenwei Ningyu Tablets on the depression model of solitary confinement combined with chronic restraint stressObjective:From the perspective of glial cell activation-neuron injury,to study the inhibitory effect of Shenwei Ningyu Tablet on overactivated glial cells and neuroinflammation induced by them,as well as the protective effect on neuron structure,so as to explore its mechanism of action in the treatment of depression.Method:1.The immunohistochemical experiment of microglia marker Iba-1 in rat hippocampus,Western blot experiment of type A1 astrocyte marker C3/GFAP immunofluorescence double labeling,and neuritis indicators NF-κB p65,TNF-α,and IL-1β were conducted to explore the inhibitory effect of Shenwei Ningyu Tablet on glial cell activation and neuroinflammatory response.2.The protective effects of Shenwei Ningyu Tablets on neuronal structure were jointly explored through histopathological observation of the hippocampus and immunofluorescence single-label experiments with the neuronal nuclear marker Neun,the structural stability index MAP2 of synapses,and the density index PSD95.Result:1.In the immunohistochemical experiment of microglia marker Iba-1,the number of positive cells was increased in the model group when compared with that of the normal group.Compared with the model group,the number of positive cells in each administration group showed a decreasing trend.The decrease in the high dose group of Shenwei Ningyu was more significant,but it did not show a significant difference.2.In the double immunofluorescence labeling experiment with C3/GFAP,a type A1 astrocyte marker,the co-expression of C3/GFAP in the normal group was relatively weak,and the co-expression in the model group was relatively strong as compared with that in the normal group(red-green fluorescence overlaid with yellow on the image,P<0.05).Compared with the model group,the positive fluorescence co-expression of each administration group was decreased,and the high and medium dose groups of Shenwei Ningyu had significant differences(P<0.01).3.In Western blot experiment of neuritis indicators,compared with the normal group,the NF-κB p65,TNF-α and IL-1β neuroinflammation indicators in the model group were significantly increased and had significant differences(P<0.01 or P<0.05).Compared with the model group,fluoxetine hydrochloride and Shenwei Ningyu high and medium dose groups were significantly reduced and had significant differences(P<0.01 or P<0.05).4.In the observation experiment of hippocampal histopathology of rats,HE staining was used to observe the pathological changes of hippocampal tissue.The results showed that in the normal group,the neurons had clear layers,compact structure,clear nucleoli,and reddish cytoplasm of cell clusters.Compared with the normal group,there were significant changes in the model group,such as the cracking and pyknosis of neuronal nuclei as well as the ambiguity of the boundary between cytoplasm and nucleus,the loosening of hippocampal tissue structure,and the swelling and disorganization of nerve and glial cells.Compared with the model group,the above pathological pHenomena in each administration group were significantly improved after 21-day administration of intervention.5.In the immunofluorescence single-labeling experiment of neuron nucleus Neun,synaptic structure stability index MAP2 and synaptic density index PSD95,the results showed that there was no significant difference in the number of neuron nuclei among different groups.Compared with the normal group,the fluorescence intensity expressions of MAP2 and PSD95 in the model group were weak.Compared with the model group,the positive fluorescence expressions of two indicators in each administration group were increased.Among them,fluoxetine hydrochloride and Shenweining Tablet depression high dose group had significant differences(P<0.01 or P<0.05).Conclusion:Shenwei Ningyu Tablet has obvious antidepressant effect in the depression model of solitary confinement combined with chronic restraint stress.Through inhibiting the activation of glial cells and neuroinflammatory reaction,it further plays the role of protecting the neuron structure,and provides a reference for the research on the treatment of degenerative diseases with traditional Chinese medicine compound preparation. |
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