Analysis Of TCM Syndrome Elements In Decreased Ovarian Reserve And The Effect Of Wuyu Decoction On Mitochondrial Energy Metabolism In Mice

With the development of reproductive medicine,the research on decreased ovarian reserve(DOR)is gradually deepening.The number and quality of oocytes in DOR patients are reduced,which clearly affects female reproductive function.Mitochondrial energy metabolism determines the quality of oocytes,and AMPK is the key to regulate energy metabolism in oocytes.This study includes two parts:the experimental part and the clinical spart.Experimental part is to explore the mechanism of Wuyu Decoction of tonifying the kidney and regulating the liver in improving oocyte quality of DOR mice through mitochondrial energy metabolism,oxidative stress and AMPK/PGC-1α pathway.Clinical part of the study is to explore TCM syndrome characteristics and syndrome differentiation of the DOR patients,in order to provide the basis for the disease and treatment.Clinical PartPurpose:To explore the characteristics of TCM syndromes and the law of syndrome differentiation of DOR patients,and to provide the basis for the clinical study of the etiology and pathogenesis of the disease and treatment.Method:In this study,through questionnaires and case review,the "DOR patients information collection form" was developed to collect the morbidity,predisposing factors,course of disease,medical history,symptoms and signs,tongue and pulse,laboratory tests,auxiliary examination results of DOR patients.And according to the DOR syndrome statistical table,DOR syndrome elements statistical table and DOR syndrome elements statistical weight table,the statistical judgm ent and correlation analysis of syndrome and syndrome elements of patients were carried out.Result:1.64.79%of the patients were induced by emotional factors,mainly by high pressure and intense work.Infertility/sterility and irregular menstruation were the main demands of patients,accounting for 50.70%and 35.21%respectively.74.65%of the patients had a course of disease longer than one year,56.34%of the patients were infertile,and 46.48%of them had a history of abortion/operation.2.The main changes of menstruation were menstrual block(43.66%),hypomenorrhea(39.66%),dark red menstruation(29.58%),dysmenorrhea(26.76%),delayed menstruation(15.49%),early menstruation(11.27%),menorrhagia(11.27%),menstrual disorder(9.86%)and amenorrhea(5.63%).Other related symptoms included poor sleep(23.94%),frequent loose stool(22.54%),soreness and weakness of waist and knees(21.13%),lassitude and fatigue(19.72%),insomnia(16.90%),premenstrual breast distension(16.90%),constipation(14.08%),lumbago(12.68%),hands and feet cold(11.27%),and 15.49%of the patients had some symptoms aggravated after activity or fatigue.The main types of tongue pulse were thready pulse(98.59%),slippery pulse(78.87%),dark red tongue(74.65%)and thin white tongue coating(70.42%).3.The main syndrome elements of disease location in DOR patients were uterus(55.43%),liver(17.39%)and kidney(14.13%).The syndrome elements of disease nature were mainly blood stasis(20.81%),qi stagnation(18.50%),blood deficiency(15.03%),yin deficiency(15.03%)and yang deficiency(12.72%).The syndrome elements of the same kind often cause the disease simultaneously,and the syndrome elements of disease location account for 46.55%,mainly including uterus and liver(19.71%),uterus and kidney(14.08%);The syndrome elements of disease nature were 66.18%,mainly including blood stasis with qi stagnation(35.21%),blood stasis with yang deficiency(19.71%),qi stagnation with yin deficiency(19.71%),blood deficiency with qi deficiency(18.31%)and qi deficiency with yang deficiency(18.31%).4.There was no statistical correlation between the syndrome elements and the age of patients,serum b_FSH,AMH and antral follicle count.There was a correlation trend that the distribution integral of syndrome elements of disease location in kidney increased with age,but there was no statistical difference.Conclusion:1.Emotional factors are the main causes of DOR as well as the infertility and irregular menstruation are the main demands of patients.DOR mainly affects the reproductive function of patients and causes menstrual changes.2.The main locations of DOR are uterus,liver,kidney and spleen.The pathological changes of DOR are blood stasis,qi stagnation,blood deficiency,yin deficiency,yang deficiency and qi deficiency.3.There was no statistical correlation between the syndrome elements and the age of patients,serum b-FSH,AMH and antral follicle count.Experimental PartPurpose:The method of tonifying the kidney and regulating the liver was used as the basic therapeutic method for DOR,and the components of Wuyu Decoction drug-containing serum were analyzed.This part is to study the effect of Wuyu Decoction on energy metabolism of mitochondria,oxidative stress and AMPK/PGC-1α pathway in DOR mice,and to analyze the mechanism of Wuyu Decoction.The IVM technology was used to explore the mechanism of Wuyu Decoction in promoting oocyte maturation in vitro,and to confirm the effect of Wuyu Decoction in improving ovarian reserve function,increasing ovarian response,promoting oocyte maturation and improving oocyte quality.Method:1.We analyzed that medicinal components and the pharmacodynamic substance of the medicated serum of the Wuyu Decoction by adopting time-of-flight mass spectrometry;2.In Vivo study,DOR mice were treated with Wuyu Decoction to induce ovulation.Combined with the blocking effect of AMPK inhibitors,the parameters of estrous cycle,ovarian reserve(serum AMH and ovarian histomorphology),ovarian response,reproductive function(oocyte morphology,oocyte retrieval rate and fertilized egg rate),energy metabolism(oocyte ATP,mitochondrial content and morphology,ROS Expressions of α-tubulin and Ki67)and protein expression of AMPK/PGC-1α pathway(AMPKα,PGC-1α,NRF1,NRF2)were detected,to analyze the pathogenesis of DOR and the therapeutic mechanism of Wuyu Decoction;3.In Vitro study,the oocytes of DOR mice were treated with Wuyu Decoction and Cornus officinalis extract by IVM technology.The expulsion rate of the first polar body,energy metabolism(ATP content,mitochondrial content and mitochondrial membrane potential),oxidative stress(ROS content and SOD activity)and protein expression of AMPK/PGC-1α pathway(AMPKα,p-AMPKα,PGC-1α,NRF1 and NRF2)were detected.To investigate the effect and mechanism of Wuyu Decoction on oocyte maturation in IVM.Result:1.Time-of-flight mass spectrometry analysis of Wuyu Decoction drug-containing serum showed that according to the response intensity of mass spectrometry,Wuyu Decoction drug-containing serum was sorted.Linoleic acid,(2S)-2-amino-3-phenylpropionic acid,leucine,bis(6-methylheptyl)phthalate,citric acid,linolenic acid,ethyl palmitate,palmitoleic acid and 3,5-ditertbutyl-4-hydroxybenzaldehyde,1-methionine-35s,glycerol 2-monopalmitate,benzoic acid,erucamide,coumarin,ethyl linoleate,cadinol,myristic acid,stearamide,hexade-canamide,aryl-curcumene.2.In vivo,the estrous cycle of DOR mice was longer than that of normal mice,the serum AMH level was lower(P<0.05),and the maturation rate and fertilization rate of oocytes were lower(P<0.05).The contents of mitochondria and ATP in oocytes were decreased,the contents of ROS were increased,the positive expression rates of α-tubulin and Ki67 were decreased(P<0.05),and the morphology of oocytes and mitochondria were not good.The mRNA and protein levels of NRF1 and NRP2 were decreased(P<0.05);Wuyu Decoction could improve the rate of mature oocytes,increase serum AMH,improve the contents of ATP,mitochondria and ROS in oocytes(P<0.05),and improve the morphology of ovarian tissue and mitochondria.The positive expression rates of α-tubulin and Ki67 and the mRNA and protein contents of AMPK,PGC-1α,NRF1 and NRF2 in ovarian tissue were increased(P<0.05).3.In vitro,the oocytes of DOR mice cultured in vitro had lower rate of first polar body extrusion,fewer mitochondria,weaker membrane potential(P<0.05),less ATP production,more ROS and lower SOD activity(P<0.05).The protein expression of AMPKα,p-AMPKα,PGC-1α and NRF1 decreased(P<0.05),and the oocytes of DOR mice cultured in Wuyu Decoction medium had a higher rate of first polar body extrusion(P<0.05).The number of mitochondria,the content of ATP,the activity of SOD and the expression of AMPKα,PGC-1α,NRF1 were increased(P<0.05),but the membrane potential of mitochondria and the content of ROS were not improved(P>0.05);The mitochondrial content and ATP content of oocytes were also improved in Cornus officinalis extract group(P<0.05).Conclusion:1.Fatty acids accounted for the highest proportion of the effective active ingredients in Wuyu Decoction medicated serum,suggesting that the active ingredients in Wuyu Decoction medicated serum were mainly used to provide energy sources and promote energy metabolism.2.The animal experiment proved that Wuyu Decoction can improve the ovarian reserve function and ovarian response of DOR mice,improve the quality of oocytes and enhance the reproductive capacity of mice.3.Cell experiment proved that Wuyu Decoction could promote oocyte maturation in vitro and improve oocyte quality of DOR mice.4.The mechanism of Wuyu Decoction on DOR is to regulate AMPK/PGC-1α pathway,improve mitochondrial energy metabolism and oxidative stress.