| Objective:Since the theory of liver governing and relieving has been put forward,it has always been a key component of the theory of visceral signs,and has played a vital role in the development and improvement of visceral signs,prevention and treatment of diseases.With the innovation of technical means and the deepening of research,the theory of liver governing catharsis is constantly enriched and improved,which promotes the theory from phenomenon description to essence elucidation.Based on the previous work of the research group,focusing on the development of premenstrual dysphoric disorder(PMDD),a modified PMDD rat model of liver qi depression was established.On the basis of our previous work,we focused on the international frontier of premenstrual dysphoria: the ambiguous understanding of "ALLO-mediated GABAAR changes" in the pathogenesis of PMDD.From the perspective of combination of disease and syndrome,we explored the pathogenesis and pathological mechanism of liver qi depression syndrome of PMDD caused by liver dysfunction,and revealed the possible mechanism of "ALLO-mediated GABAAR changes" in brain central lesions of PMDD liver qi depression syndrome,It can promote the theory of liver governing the relieving and abnormal relieving from phenomenon description to essence elucidation,reveal the scientific connotation of liver governing the relieving and fully verify the effectiveness and scientificity of Shuyu capsule,and provide valuable clues for developing targeted therapeutic drugs and enriching the modern scientific connotation of traditional Chinese medicine theory.It can really solve the problem that the drug response rate of international first-line treatment of PMDD is less than 60%,the mental side effects are big,the onset is slow,and some patients will have drug resistance.Methods:Theoretical analysis and experimental research are combined:on the one hand,it systematically combs the related literature of "liver dominating catharsis and catharsis disorder theory" and PMDD disease and syndrome research,and finds the key scientific problems to be solved;on the other hand,according to the above problems and the clinical findings of PMDD,the modified forced swimming test was used to prepare the rat model of liver qi stagnation syndrome of PMDD caused by liver dysfunction;The latency,time and times of suspension immobility in forced swimming test,the times and time percentage of open arm entry in elevated cross maze test,and the latency of light and heat tail flick test were used as the main basis for the evaluation of depression like emotion in PMDD rats with liver qi stagnation syndrome;ELISA,RT-q PCR and Western blot were used to detect the level of ALLO in the peripheral blood and hippocampus of the model rats and the expression of GABAAR? subunit;Injection of adeno-associated virus vector resulted in the low expression or over expression of GABAAR ? subunits,which contradicted the above findings.Shuyu capsule intervention was used to reveal the underlying pathological and neurobiological mechanism of allo mediated GABAAR.(1)The improved forced swimming stress was used to prepare an effective PMDD rat model of liver qi stagnation;fluoxetine and Shuyu capsule were intervened to investigate the improvement of behavior characteristics of the model rats,and to verify the reliability of the model and the effectiveness of the drug.(2)ELISA was used to detect the changes of progesterone,5?-dihydrocorticosterone,tetrahydroprogesterone,5?-reductase,3?-steroid dehydrogenase and other micro indicators in serum and hippocampus of rats in each group,and to investigate the changes of related hormone content and key enzyme content in the process of progesterone metabolism to tetrahydroprogesterone,so as to explore the key micro mechanism of Shuyu capsule in the process of PMDD liver qi depression.(3)Based on the above results of hormone metabolic pathway changes,in order to verify its downstream target,RT-q PCR,Western blot and immunohistochemistry were used to detect the gene and protein expression of GABAAR? subunit.The interference knockdown adenovirus and over expression vector adenovirus of GABAAR? subunit were injected into the hippocampus of PMDD model rats with liver qi stagnation by stereotactic localization,Forced swimming test was used to investigate the improvement of behavioral characteristics of disease and syndrome in model rats,and the gene and protein expression of GABAAR? subunit in hippocampal brain region of rats in each group were detected after injection of GABAAR? subunit interference knockout adenovirus and overexpression vector adenovirus.(4)Shuyu capsule intervened the rats in each group after the injection of GABAAR? interference knock-out adenovirus,and made a series of behavioral evaluation.RT-q PCR,Western blot and immunohistochemistry were used to detect the gene and protein expression of GABAAR? subunit,so as to accurately target the therapeutic target of Shuyu capsule.Results:(1)The results of animal behavior test showed that: forced swimming test showed that the immobility time and immobility times of model group in non acceptance period were significantly increased compared with those in acceptance period(p<0.001,p<0.05),The results of elevated cross maze test showed that the percentage of open arm residence time and the percentage of open arm entry times in the model group in the non acceptance period were significantly lower than those in the acceptance period(p<0.001,p<0.001);the tail flick latency in the non acceptance period was significantly shorter in the model group(p<0.001,p<0.001).After the intervention of fluoxetine and Shuyu capsule,the above abnormal changes were effectively relieved and disappeared(p>0.05),suggesting that the above behavioral parameters of stress group had typical estrous cycle dependent changes,and fluoxetine and Shuyu capsule could effectively improve the symptoms.(2)The results of ELISA biochemical test showed that: there were significant differences in progesterone,5?-dihydrocorticosterone,tetrahydroprogesterone,5?-reductase,3?-steroid dehydrogenase and other indexes between the serum and hippocampus of rats in each group.The indexes of 5?-dihydrocorticosterone,allo,5?-reductase,3?-steroid dehydrogenase in the model group were significantly lower than those in the control group(serum:p<0.05,p<0.01;hippocampus: p<0.05,p<0.05,p<0.01)< The results showed that the content of progesterone in serum and hippocampus of model group was significantly higher than that of normal group(p<0.01,p<0.05),and fluoxetine and Shuyu capsule could effectively improve the above changes.(3)RT-qPCR,Western blot and immunohistochemistry showed that there was no significant difference in the expression of GABAAR? between the control group and the model group(p>0.05);Rats in control group and model group were injected with R?Gabrd expression of GABAAR? subunit in hippocampus was significantly decreased after gabrd interference(p<0.0001).Expression of GABAAR? subunit in hippocampus increased significantly after overexpression of R?Gabrd gene(p<0.0001).After virus intervention,the decrease of GABAAR? expression could induce the behavior phenotype of PMDD with liver qi stagnation syndrome in normal rats.The results showed that the immobility time and times of forced swimming test in non acceptance period were significantly increased compared with those in acceptance period(p<0.01,p<0.01),and the immobility latency was significantly decreased compared with those in acceptance period(p<0.001).The increased expression of GABAAR ? had no effect on the pathogenesis of liver qi depression in PMDD.(4)In the model rats with decreased GABAAR? expression,Shuyu capsule did not significantly improve the time,times and latency of suspension immobility in forced swimming test,the percentage of time and times of entering open arm in elevated cross maze test,and the latency of tail flick in photothermal tail flick test(p>0.05,p>0.05,p>0.05).The expression of GABAAR? subunit was not significantly improved.Conclusion:(1)Changes in the content or activity of 5?-reductase and 3?-steroid dehydrogenase lead to abnormal metabolic pathway from progesterone to tetrahydroprogesterone,which is one of the deep pathogenesis of liver qi depression syndrome of PMDD.Shuyu capsule can improve the abnormal behavior symptoms of PMDD rats with liver qi depression by increasing enzyme activity and regulating hormone content.(2)GABAAR? subunit is one of the key targets for Shuyu capsule to exert its anti Liver qi stagnation syndrome of PMDD caused by abnormal emotion of liver regulating,suggesting that Shuyu capsule may exert its therapeutic effect by regulating the metabolic pathway of tetrahydroprogesterone,thus targeting GABAAR? subunit.(3)The Possible hypothesis of "Shuyu capsule effect" : the extract or combination of effective components of Shuyu capsule,a traditional Chinese medicine compound,contains estrogen and progesterone and its derivatives,which are similar to endogenous estrogen in structure and function.It can regulate the content of estrogen and progesterone,or produce enzyme functional substances,improve the metabolic level of tetrahydroprogesterone,enhance the metabolic pathway from progesterone to tetrahydroprogesterone,and thus have a therapeutic effect on PMDD with liver qi stagnation syndrome.To sum up: hippocampus is the key brain area of PMDD patients with liver qi stagnation syndrome caused by abnormal emotion of liver regulating,and the abnormal changes of pre menstrual allo and its metabolic pathway are the deep pathogenesis of PMDD,GABAAR ? Subunits are the targets of liver regulating dysfunction.Shuyu capsule increased by 5 ? Reductase,3 ?-The content of steroid dehydrogenase can improve the clinical symptoms of PMDD patients with liver qi stagnation syndrome. |
PDF Full Text Request