Study On The Effect And Mechanism Of Erxian Decoction On Rat Model Of Perimenopausal Syndrome Based On CAMP/PKA/CREB Pathway

Objective: Perimenopausal syndrome seriously affects women's life and work,reduces their quality of life,and endangers their psychosomatic health.This article through the perimenopausal syndrome theory discussion,summarizes the perimenopausal syndrome etiology and pathogenesis characteristic,induces the effective treatment prescription,analyzes Erxian decoction medicine composition and the characteristic.The network pharmacology method was used to investigate the common target of Erxian decoction and perimenopausal syndrome and to screen the possible signaling pathway.After screening for possible signaling pathways,animal experiments were conducted to verify the results.After validation in animal experiments,Meta analysis was performed to evaluate the efficacy and safety of Erxian decoction in the treatment of perimenopausal syndrome,and further provide clinical evidence support.From the basic to the clinical verification,this paper provides the research foundation for the research on the mechanism of perimenopausal syndrome and the research and development of the traditional Chinese medicine treatment for perimenopenopausal syndrome,and attempts to make the modern interpretation of the basic theory of traditional Chinese medicine.Method:1.Theoretical discussion: To review and comb the literature of Chinese medicine related to perimenopausal syndrome,and clarify the etiology and pathogenesis of perimenopenopausal syndrome from the perspective of traditional Chinese medicine,so as to clarify the theoretical basis for the prevention and treatment of perimenopenopausal syndrome by traditional Chinese medicine.The solution of Erxian decoction and its composition were analyzed to clarify its indications and its correlation with the pathogenesis of perimenopausal syndrome.2.Meta-analysis:The literatures related to perimenophenic syndrome were selected through retrieval of databases such as CBM,VIP,wanfang data,CNKI,Embase,Pub Med and Cochrane Library,and meta-analysis of the scores of efficiency,E2,FSH,LH and Kupperman were performed to systematically evaluate the efficacy and safety of Erxian decoction in the treatment of perimenophenic syndrome in women.3.Network pharmacology research:TCMSP,Dis Ge NET,OMIM and Gene Cards databases were used to screen the active components of Erxian decoction and the related target genes of perimenopausal syndrome,and the network diagram of "drug-component-target-disease" was constructed with the software of Cytoscape 3.7.2.The protein interaction network was constructed by string database,and the key target of Erxian decoction in the treatment of perimenopausal syndrome was analyzed by David database for Gene Ontology analysis and KEGG analysis to obtain the mechanism of Erxian decoction in the treatment of perimenopausal syndrome.4.Experimental study: Sixty 15 months old female Sprague Dawley(SD)rats of SPF grade were randomly divided into model group,pentanoic acid estradiol group and Erxian decoction low dose group,medium dose group and high dose group.10 2-month-old female SD rats were set as the blank group.They were examined with smear of vaginal exfoliated cells for 15 days,as to ensure accord with perimenopausal syndrome model evaluation standard.After the group was divided into two groups,the blank group and the model group were given pure water of equal volume,the estradiol valerate group was given estradiol valerate tablet solution,the low,medium and high dose group were given Erxian decoction for 4 weeks.The general situation,water consumption,food intake and voluntary activities of rats in each group were observed during the administration period.The autonomic activity of rats was detected by the autonomic activity recorder.The wet weight and wet weight index of uterus were recorded.The expression levels of 5-HT,?-EP,E2,FSH and LH in serum of rats were detected by enzyme linked immunosorbent assay(ELISA).HE staining was used to observe the pathological changes of ovary,uterus,hypothalamus and hypophysis.The levels of NO,SOD,MDA,CAT and GSH-Px in serum were detected by biochemical kit.Terminal Deoxynucleotidyl Transferase-mediated d UTP Nick-End La Beclin1g(TUNEL)staining was used to observe the apoptosis of rat ovarian granulosa cells.The expression of Bcl-2,Bax and Caspase-3in rat ovary was detected by immunohistochemistry and Western Blot.Real-time PCR and Western Blot were used to detect the m RNA and protein expression of c AMP,PKA and CREB in the ovaries of rats.Western Blot was used to detect the protein expression of LC3-II and Beclin1 in rat ovary.Result:1.Theoretical research results The name of perimenopausal syndrome is not uniform.The disease is due to deficiency of kidney essence.The basic pathogenesis is deficiency of kidney essence,while interfered with hyperactivity of ministerial fire.Erxian decoction is a formula for warming kidney yang,tonifying kidney essence,purging pathogenic hyperactivity of ministerial fire,regulating chong and ren meridians,which is compatible with the pathogenesis of perimenopausal syndrome and is an effective prescription for the treatment of perimenopausal syndrome.2.Results of meta-analysis18 articles were retrieved from literature search,including2003 patients.The effective rate showed that the curative effect of Erxian decoction group was better than that of western medicine treatment group,and the difference was statistically significant[OR=3.52,95%CI(2.52,4.93),P<0.00001];The improvement degree of E2,FSH and LH is better than that of western medicine control group.Kupperman score results indicate that the therapeutic effect of the treatment group and the western medicine control group in improving Kupperman score is equivalent,and the difference is not statistically significant [MD=-3.95,95%CI(-8.51,0.60),P=0.74].In terms of adverse reactions,no adverse reactions were found in Erxian decoction treatment group.3.Results of network pharmacology experiment Through screening 7,23,20,2,37 and 15 active components of Rhizoma Curculiginis,Herba Epimedii,Radix Morindae Officinalis,Radix Angelicae Sinensis,Cortex Phellodendri and Rhizoma Anemarrhenae in Erxian decoction respectively,?-sitosterol,kaempferol,stigmasterol,quercetin,icaritin,rutaecarpine,diosgenin and dehydrotanshinone II A are the key active components of Erxian decoction for intervening perimenopausal syndrome.Moreover,protein kinase AKT1(AKT1),interleukin 6(IL6),vascular endothelial growth factor(VEGF),amino-terminal kinase(JUN),cysteine-containing aspartic protease 3(CASP3),Myc protooncogene protein(MYC),epidermal growth factor receptor(EGFR),prostaglandin endoperoxidase receptor(PTGS2),estrogen receptor1(ESR1),mitogen activated protein kinase 1(MAPK1)are the key targets.Through biological analysis of key targets,it is found that Erxian decoction intervention on perimenopausal syndrome may be related to cancer pathway,PI3K-Akt signal pathway,c AMP signal pathway,MAPK signal pathway,Rap1 signal pathway and other pathways.4 In vivo experimental results(1)Experiment 1 results Compared with the model group,the Erxian tang group and the estradiol group improved the hair luster,shedding,tail and paw color,spirit,expression and reaction.Increased water intake.As the time and distance of spontaneous activity increased,the uterine wet weight and uterine index of the rats increased,the neurotransmitters 5-HT and ?-EP in the serum increased,the hormone E2 in the serum increased,and the FSH and LH in the serum decreased.The level of granulosa cells in the ovary increased,the blood vessels increased,and the number of follicles and corpus luteum also increased.The endometrium thickened,the number of glands increased,and the gland atrophy improved compared with the model group.The number of hypothalamic nerve cells increased slightly,and the nerve cells were arranged in a slightly orderly manner.Adenohypophysis cells increased and were arranged in a slightly orderly manner,cell cytoplasm decreased and the vacuol-like changes around neurohypophysis cells decreased.(2)Results of experiment 2Compared with the model group,NO and MDA were decreased in the serum of Erxian decoction group and the estradiol group,while SOD,CAT and GSH-Px were increased.TUNEL staining showed a decrease in the number and density of green fluorescence in the ovary,a decrease in the apoptosis index,an increase in the number of positive ovarian Bcl-2 cells,and a decrease in the number of positive Bax and Caspase-3 cells.The results of transmission electron microscopy indicated that the mitochondrial swelling and deformation of ovarian granulosa cells were improved and the number of autophagosomes increased.(3)Results of experiment 3Compared with the model group,the expression levels of c AMP,PKA,CREB protein and m RNA in ovaries of rats in Erxian tang group and estradiol group increased to different degrees.The protein expression levels of CREB were increased to varying degrees.The protein expression levels of Bcl-2 in the ovaries of rats were increased,while the protein expression levels of Bax and Caspase-3were decreased.Protein expression levels of LC3-II and Beclin1 increased.Conclusion:1.The perimenopausal syndrome is due to deficiency of kidney essence.The basic pathogenesis is deficiency of kidney essence,while interfered with hyperactivity of ministerial fire.Erxian decoction is a formula for warming kidney yang,tonifying kidney essence,purging pathogenic hyperactivity of ministerial fire,regulating chong and ren meridians,which is compatible with the pathogenesis of perimenopausal syndrome and is an effective prescription for the treatment of perimenopausal syndrome.2.Erxian decoction has the characteristics of multi-level,multi-system and overall intervention in improving perimenopausal syndrome,in which cancer pathway,PI3 K Akt signaling pathway,c AMP signaling pathway and MAPK signaling pathway are the main signal regulatory pathways.3.Erxian decoction can improve the behavior and pathological damage of perimenopausal syndrome model rats.4.Erxian decoction can inhibit the apoptosis of ovarian granulosa cells in perimenopausal syndrome model rats,and its mechanism may be related to improving mitochondrial autophagy of ovarian granulosa cells and up regulating c AMP/PKA/CREB signal pathway.5.Erxian decoction has obvious effect in the treatment of perimenopausal syndrome.It can reduce the level of FSH and LH,improve Kupperman score and raise the level of E2.The incidence of adverse reactions is also lower than that of western medicine control group.