Sensitivity Change Of PAG Region In PMDD Liver-Qi Depression Rat Model And Intervention Effect Of ShuYu Capsule

Purpose:In this study,we took periaqueductal gray(PAG)sensitivity changes as the entry point,and based on the confirmation of the efficacy of Shuyu capsule in improving PMDD liver qi depression.Firstly,we investigated whether PMDD is accompanied by altered sensitivity of the PAG region through the detection of relevant behavioural indicators.The mechanism of the altered sensitivity of the PAG region was further investigated by detecting c-fos expression in the PAG region and interfering with its GABA_AR activation,and whether Shuyu capsule could treat and improve PMDD liver qi depression by affecting the activation of neurons in the PAG region and the GABA_AR distributed on the neuronal membrane.Methods:(1)The phenotype of PMDD liver Qi depression model rats was identified and screened by Forced Swimming Test(FST),and the effect of Shuyu capsule on improving the symptoms of PMDD liver qi depression was evaluated:female Wistar rats were induced to have synchronized and regular erogenous cycles by exogenous estrogen after ovariectomy,and two consecutive regular cycles were selected.FST was performed in late diestrus(LD)and estrus(E),and the rats were divided into control group and model group according to the difference between the two detection indexes.The PMDD liver qi depression syndrome model group was the rats sensitive to stress only in LD,and rats in the model group were given the Shuyu capsule and fluoxetine intervention respectively.The rats were administered two consecutive cycles and the antidepressant efficacy of the drug was evaluated by FST again in LD and E.(2)Behavioral tests of altered sensitivity of the PAG region were performed in each group of rats and evaluation of the intervention effect of Shuyu capsule:the PAG region sensitivity test was performed by tail-flick test and the detection of specific behaviors triggered by electrical stimulation,respectively.The light-heat tail flick test was conducted after the efficacy evaluation of FST,and the test was conducted for one consecutive cycle,all1h after the rats were given the drug.The changes in the latency of tail-flick in each group of rats after different estrous cycles of stress were observed to evaluate the changes in PAG region sensitivity and the intervention effect of Shuyu capsule in the model rats.Rats tested for electrical stimulation in the PAG region,bipolar stimulation electrodes were implanted after the evaluation of the efficacy of FST,4 days after recovery,the rats were electrically stimulated 1 h after the administration of the drug in different estrous cycles,and the changes in the current threshold for triggering specific behaviors were observed to evaluate the changes in the sensitivity of the PAG region in the model rats and the efficacy of Shuyu capsule on the improvement of the sensitivity of the PAG region.(3)To observe the changes in functional activation of c-fos positive neurons of PAG in each group of rats after stress and the effects of GABA_AR agonist and antagonist injection on PAG sensitivity during the LD:For the rats with c-fos positive neuron activation detection,after the efficacy evaluation of FST,the rats that did not respond to tail-flick stimulation were excluded,and the rats in each group were subjected to mild vibration stress in different estrous cycles and sampled with brain tissue perfusion and fixation.The c-fos positive neurons in the PAG region were immunohistochemically stained.For the rats that underwent microinjections,the efficacy evaluation of FST,stereotaxic injection was performed in LD,and GABA_AR agonists,antagonists and saline were injected into the PAG region respectively.The effects of GABA_AR agonists,antagonists and saline were observed in different groups of rats after stress,and the neural mechanism of the change of sensitivity in the PAG region was further investigated.Results:(1)The depression-like behavior of PMDD rats with liver Qi depression syndrome showed estrous cycle-linked in forced swimming test,and Shuyu capsule intervention could improve the estrous cycle dependent depression-like phenotype of PMDD rats with liver Qi depression syndrome.Specifically,in LD,compared with control group,the immobility duration and number of rats in model group were significantly increased;In E,there was no significant difference between the two groups in the immobility duration and number.After Shuyu capsule and fluoxetine intervention in two estrous cycles,compared with model group,the immobility duration and number in Shuyu group and FLX group were significantly reduced.(2)In PMDD rats with liver Qi depression syndrome,the sensitivity of PAG region increased in LD,which could be improved by Shuyu capsule intervention.The specific performance is:In the tail flick test before and after mild vibration stress in different estrous cycles,the tail flick latency of model group rats in LD decreased after stress.After Shuyu capsule and fluoxetine intervention in two estrous cycles,compared with model group,the tail flick latency of Shuyu group and FLX group increased significantly.In the tests of the PAG region of different estrous cycles,the current threshold of the model group induced the freeze and escape behavior during LD was significantly reduced.After the intervention of Shuyu Capsule and fluoxetine during two estrous cycles,compared with the model group,The current threshold of Shuyu group and FLX group induced freeze and escape behavior increased significantly.(3)PMDD rats exhibit estrous cycle-linked changes in PAG region sensitivity after stress may be related to reduced functional activation of PAG region neurons and increased activation of GABA_AR,and the ameliorative effect of Shuyu capsule on model rats is also related to the above mechanism.Specifically,in LD,the number of c-fos positive neurons in the PAG region was decreased in the model group rats after stress compared with the control group rats.The number of c-fos positive neurons in the PAG region was increased after the intervention of Shuyu capsule and FLX.In the experiment of microinjection of GABA_AR agonist and antagonist in the PAG region of rats during the LD,the tail-flick latency after stress was significantly decreased in the control group after GABA_AR agonist injection in the PAG region,while the tail-flick latency after stress was significantly increased in the model group after GABA_AR antagonist injection,and the intervention of Shuyu capsule and FLX reversed the decreased tail-flick latency after GABA_AR.The reduced tail-flick latency after GABA_AR agonist injection was reversed by the intervention of Shuyu capsule and FLX.Conclusions:(1)Shuyu capsule can improve the estrous cycle-linked depression-like behavior in PMDD rats with liver Qi depression syndrome.(2)Shuyu Capsule can correct the increased sensitivity of PAG region in LD of model rats.(3)The mechanism of Shuyu capsule to improve the sensitivity of PAG region in model rats may be related to the effect of GABA_AR receptor on Gabaergic neurons.