Study On The Efficacy And Mechanism Of Bushen Shugan Decoction Combined With Five Elements Music On Ovarian Reserve Dysfunction

Objective1.Explore the psychological status of DOR patients,analyze the relationship between their clinical symptoms and psychological status,and observe the clinical efficacy of Bushen Shugan Decoction combined with Five Elements Music in the treatment of patients with kidney deficiency and liver depression type DOR,and explore its mechanism of action,with a view to prevention and treatment of traditional Chinese medicine DOR provides a theoretical basis.2.Select Tripterygium wilfordii polyglycoside tablets combined with CUMS method to establish a DOR rat model of kidney deficiency and liver depression,observe the general condition of the rat during the modeling process and the function of the ovaries after modeling,and summarize the preparation experience of the model(drug dosage,stimulation time Etc.)to provide evidence for the follow-up pharmacodynamic experiment of Bushen Shugan Decoction.3.Observe the effect of Bushen Shugan Decoction combined with Five Elements Music on the improvement of ovarian function in DOR model rats with kidney deficiency and liver depression,and use the Bcl-2,Bax,Caspase signaling pathway as an entry point to study Bushen Shugan Decoction combined with Five Elements Music from a molecular perspective The mechanism of action on ovarian function.Methods1.Clinical study:96 patients with kidney deficiency DOR who meet the criteria of intake and discharge were divided into Chinese medicine group(Bushen Shugan Decoction group)48 cases,Chinese medicine combined with Five Elements Music group(Bushen Shugan Decoction combined with Five Elements Music group)according to the random number table method 48 cases.The Chinese medicine group was given Bushen Shugan Recipe for 3 consecutive menstrual cycles;the Chinese medicine combined with the five elements music group was given Chinese medicine(the method is the same as the Chinese medicine group),and the five elements music was appreciated every day,and the middle angle tone and feather tone of the five sounds were randomly assigned,Once a day,45 minutes each time,3 consecutive menstrual cycles.Observation indicators are TCM symptom score,psychological evaluation scale,serum bFSH,bLH,bFSH/bLH,bE2,AMH levels and changes in ovarian AFC,analyze the patient's psychological status,and observe the effects of Bushen Shugan Decoction combined with Five Elements Music on kidney deficiency and liver.The curative effect of depression type DOR patients,and explore its mechanism of action.2.Animal model preparation:24 SD rats with regular estrus cycle were fed adaptively for one week,and they were divided into experimental group and control group according to the random number table method,with 12 rats in each group.The experimental group used Tripterygium Glycoside tablets combined with chronic mild unpredictable stress(CUMS)and a slightly improved method for modeling.The stimulating factors included 10 types:solitary cage feeding;moist litter,tail-clamping stimulation for 1 min,Swimming in ice water at 0?for 5 minutes,fasting for 24 hours;water-free for 24 hours,black and white upside down(dark 12h,day 12h),fasting water for 24h;tilted squirrel cage at 45°,foreign body stimulation for 24h(such as hardwood sticks,rags).Ten kinds of stimuli were randomly arranged so that the rats could not predict the occurrence of stimuli.Once a day,they were given tripterygium wilfordii poly glycoside tablets 50mg/kg/d by gavage for 21 consecutive days.The control group was given an equal volume of normal saline by gavage for 21 consecutive days with adequate water and light,and fed quietly.After the experimental operation,anesthetize and obtain materials.During the experiment,the general condition of the rat was observed daily,and the estrus cycle of the rat was detected.Before and after the experiment,the syrup partiality test and the open field experiment were carried out,and the rat serum E2,FSH,LH,5-HT were measured by radioimmunoassay.HE staining to observe the morphology of the ovary,calculate the ovarian index,systematically analyze the experimental results to verify the rat model,and observe the ovarian function of the rat after modeling.3.Experimental study:A DOR rat model of kidney deficiency and liver depression was established by gavage of Tripterygium Glycoside tablets combined with CUMS and slightly improved methods.According to the random number table method,50 rats successfully modeled were divided into 5 groups,each group 10 rats,respectively:model group,western medicine(bu jia le)group,Chinese medicine(bu shen shu gan decoction)low-dose combined music group,Chinese medicine(bu shen shu gan decoction)medium-dose combined music group,Chinese medicine(bu shen shu gan decoction)High-dose combined music group;10 normal-bred rats served as the blank group.Observe and study the effects of Bushen Shugan Decoction in low,medium and high doses combined with Five Elements Music on rats,and detect ovarian tissue by observing the general conditions of rats,behavioral testing,estrus cycle,ovarian HE staining,serum sex hormones,and Western Blot.The protein expression of Bcl-2,Bax,Caspase-9,and Caspase-3 in the medium,and the possible mechanism of action of the drug was analyzed.Results1.Clinical study:After treatment,compared with the traditional Chinese medicine group,the traditional Chinese medicine combined with the five elements music group scored on"breast pain,lower abdomen and chest and hypochondriac pain,sighs,irritability,insomnia and more dreams" points,total depression scores and factor scores,and depression self-evaluation standards The points were significantly reduced(P<0.05),and there was no significant difference in the points of "menstrual cycle,menstrual volume,dark menstruation,blood clots,sore waist and knees,and decreased libido"(P>0.05).Comparing the traditional Chinese medicine group before and after treatment,there was no significant difference in the points of "dark menstruation and blood clots"(P>0.05).In terms of "menstrual cycle,menstrual volume,waist and knee pain,loss of libido,breast tenderness,chest pain,In terms of "happy sigh,irritability and irritability",depression total score and factor score,and depression self-evaluation standard score,the points were significantly reduced after treatment(P<0.05).There was no significant difference in the scores of "menstrual cycle,dark menstruation,and blood clots" in the traditional Chinese medicine combined music group before and after treatment(P>0.05).In terms of "menstrual flow,waist and knee pain,loss of libido,breast tenderness,and chest and thigh swelling" In terms of pain,sigh,irritability,irritability,insomnia and more dreams,the scores of depression total scores and factor scores,and depression self-evaluation standard scores were significantly reduced after treatment(P<0.05).Compared with before treatment,bFSH and bLH in the traditional Chinese medicine group were significantly reduced after treatment(P<0.05),bE2,AMH,and AFC were both significantly increased(P<0.05);after treatment,bFSH and bFSH/bLH in the traditional Chinese medicine combined music group were both significantly Decrease(P<0.05),bE2,AMH,AFC were all significantly increased(P<0.05).After treatment,compared with the traditional Chinese medicine group,the traditional Chinese medicine combined with Wuxing music group had no significant difference in bFSH,bLH,bFSH/bLH,bE2,AMH,and AFC(P>0.05).2.Animal model preparation:The rats in the control group were in good condition all the time,with smooth coat,normal diet and drinking;compared with the control group,the rats in the experimental group had dull,sparse coat color,easy to fall off,lethargic,timid and easily startled,hunched back,curled up or irritable,decreased activity,loose stools,reduced diet and drinking water.Before modeling,there was no significant difference in weight,estrus cycle,open field test and sugar water test between the two groups(P>0.05),after modeling,compared with the control group,the weight of the experimental group increased slowly(P<0.05),the estrus cycle is disordered,which is manifested as shortening,prolonging or no obvious cycle of the estrus cycle.The disorder rate is 83%(P<0.05),the total distance to the center is significantly reduced(P<0.05),the degree of syrup partiality is significantly reduced(P<0.05),FSH,LH,5-TH increased significantly,E2 decreased(P<0.05),ovarian index decreased significantly(P<0.05),ovarian tissue cortical hyperplasia,medulla atrophy,interstitial fibrosis-like changes,and granular cell arrangement loose and disordered,primary follicles,secondary follicles and atretic follicles increase,and mature follicles decrease.3.Experimental research:(1)In general,rats in the blank group are in good condition,with smooth coat,and normal diet and drinking water.After the treatment,compared with the blank group,the weight of the rats in the model group increased slowly,compared with the model group,the weight of the Chinese medicine combined with the music group increased faster,and there was no significant difference between the other groups(P>0.05).(2)Behavioral comparison,before the experiment,there was no significant difference in the total distance to the center of the rats and the preference for sugar and water in each group(P>0.05),the total distance to the center of the model group was shortened and the preference for sugar and water decreased,After the medication,compared with the model group,there was no significant difference in the total distance to the center and the syrup partiality of the other groups(P>0.05).(3)Estrus cycle:before the experiment,the estrus cycle of each group was normal;After the model was completed,the estrus cycle of the rats in the model group was disordered,which showed prolonged,stagnant and no cycle;On the 42nd day,the estrus cycle of the model group was disordered(P<0.05);Compared with the model group,the disturbance rate of estrus cycle in the middle and high dose music group decreased(P<0.05),and there was no significant difference in the other groups(P>0.05)(4)Ovarian index:the ovarian index of model group was significantly decreased;Compared with the model group,the ovarian index of the medium and high dose music group and the western medicine group was higher(P<0.05);There was no significant difference in ovarian index between the three groups(P>0.05)(5)Serum sex hormone:FSH and LH increased significantly in model group,E2 and 5-HT decreased significantly.After treatment,compared with the model group,FSH and LH in the medium and high dose music group and Western medicine group were significantly decreased(P<0.05),while E2 in western medicine group was significantly increased(P<0.05);The 5-HT of the low,medium and high dose music group was significantly increased(P<0.05)(6)He staining of ovary:the structure of ovary in the low,medium and high dose music group and Western medicine group was better than that in the model group,the fibrosis and medulla atrophy of interstitial decreased,the number of granular cells increased,the distribution of blood vessels increased,the number of original follicles and mature follicles increased,and the number of locked follicles decreased.The western medicine group and the medium and high-dose music group were more obvious(7)Western blot analysis showed that the expression of Bcl-2 protein was increased by lowering Bcl-2 protein expression in the combination music group and Western medicine group,and the ovarian function was improved in the western medicine group and the high dose Music Group(P<0.05).Conclusions1.Clinical study:Bushen Shugan Decoction and Bushen Shugan Decoction combined with Five Elements Music can all improve the symptoms of menstrual flow,waist and knee weakness,and loss of libido,and can reduce bFSH,increase bE2,AMH,AFC;Bushen Shugan Decoction combined with Five Elements Music It is better than Bushen Shugan Decoction alone in improving breast pain,lower abdomen and chest pain,sighing,irritability,insomnia and dreaminess and depression scale score.2.Animal model preparation:it is successful to replicate the model of kidney deficiency and liver depression dor rats by 50mg/kg/d Tripterygium glycoside tablets suspension and cums.3.Experimental study:Bushen Shugan recipe combined with five elements music can reduce FSH and LH in dor model rats and improve the ovarian function of the model rats;The level of 5-HT could be increased and the depression state of the model rats could be improved.The expression of Bcl-2 protein decreased and Bax,caspase-3 and caspase-9 increased after model making.It was suggested that the reason for the ovarian function decline might be that it promoted the apoptosis of granulocyte mediated by mitochondrial apoptosis pathway.It is one of the mechanisms of Bushen Shugan recipe combined with five elements music to improve the Dor of kidney deficiency and liver depression by improving the mitochondrial apoptosis pathway,increasing Bcl-2 protein expression,reducing the expression of Bax,caspase-3 and caspase-9 protein.