| Part 1 Clinical research ObjectiveTo observe the clinical effect of JiajianYijingdecoction(JYD)on the treatment of patients of diminished ovarian reserve(DOR)with kidney deficiency and heptic depression type(KDAHDT)and to discuss the mechanism of effectiveness on this prescription by insulin-like growth factor-1(IGF-1)and stem cell factor(SCF).Methods1.63 patients in the outpatient department who met the diagnostic criteria,have been randomly divided into Chinese medicine group(CG)32 cases and Western medicine control group(WG)31 cases.The patients of CG were treated with JD;the patients of WG were treated with estrogen and progestogen sequentialtherapy(EPST).2.CG:Start taking the JDon the 5th day of menstruation,daily dose,3 menstruation cycles for 1 course of treatment.WG:Start taking theProgynova(2mg/d X 21d)+ Dydrogesterone(20mg/d X 10 d,plus Dydrogesterone taking,in the twelfth day)on the 5th day of menstruation,3 menstruation cycles for 1 course of treatment.3.The serum TCM(traditionalChinesemedicine)syndromescore,bFSH,bLH,bE2,IGF-1,SCF,AFC,and the pregnancy rate of the two groups were observed and compared between the two groups before and after treatment.Results1.Comparison of TCM syndrome score: Before treatment,the TCM syndrome integral difference were not statistically significant between the two groups(P>0.05).The scores of menstrual cycle,menstrual period,menstrual quantity,low libido,chest tightnessof two groups after treatment were both lower than those beforetreatment,and the difference wasstatistically significant(P < 0.05).The effect of western medicine regulating menstrual cycle and menstrual period is better thantraditional Chinese medicine(P<0.05).Chinese medicine is superior to western medicine in increasing menstruation and improving chest tightness(P < 0.05);butthere was no difference between the two groups in improving libido(P > 0.05).Chinese medicine treatment can also improve the patient’s lumbar acid distension,breast tenderness,irritability and dizziness(P<0.05),while the western medicine treatment improved but the difference was not statistically significant.After treatment,the comparison between the two groups showed that TCM treatment was better than western medicine in improving the patients’ lumbar acid distention,breast tenderness,irritability and dizziness(P<0.05).2.Comparison of the serum bFSH,bLH,bE2:Comparing of the level of bLH、bFSH and bE2 between the two groups before treatment,there were not significantly differences(P>0.05).After treatment,bFSH and bLH were both lower than before,and bE2 was higher than before,and the difference was statistically significant before and after treatment(P<0.05).There was no statistically significant difference between bFSH and bLH group after treatment(P>0.05).Although the treatment of western medicine increased bE2,the difference was not statistically significant compared with TCM(P>0.05.3.Comparison of the serum IGF-1:Comparing of the level of IGF-1 between the two groups before treatment,there were not significantly differences(P> 0.05),Igf-1 was higher after treatment(P < 0.05);but there was no statistically significant onIgf-1 difference between the group after treatment(P>0.05).4.Comparison of the serumSCF:Comparing of the level of SCF between the two groups before treatment,there were not significantly differences(P >0.05),SCF was lower after treatment(P<0.05);but there was nostatistically significant difference on SCF between the group after treatment(P>0.05).5.Comparison ofAFC:Comparing of AFC between the two groups before treatment,there were not significantly differences(P > 0.05),AFC was moreafter treatment(P < 0.05);but there was nostatistically significant difference on AFC between the group after treatment(P>0.05).6.Comparison of the two groups after treatment of pregnancy rate: Pregnancy rate was no statistically significant difference between the two groups(P>0.05).7.Comparison of the comprehensive effects of two groups:The total effective ratewas no statistically significant between the two groups(P>0.05);the difference between the efficacy index distribution was also not statistically significant(P>0.05).Conclusion1.Both JYD and PST can treat KDAHDT DOR patients,and the comprehensive effect is comparable;with improving the clinical symptoms of DOR patients,reducing bFSH and bLH,elevating bE2,increasing AFC,and elevating IGF-1 and reducing SCF.2.JYDhas an advantage in improving breast pain,chest distress,irritability,swelling of the waist,dizziness and increasing menstrual volume.3.JYD may regulate the secretion of the hypothalamic-pituitary-ovary(H-P-O)axis and the secretion of local factors such as igf-1 and SCFthrough multi-link,multi-system and multi-target,improving ovarian reserve function,improving fertility to some extent.Part 2 Experimental study Objective1.To verify the effectiveness of JYD and Progynova on DOR model rats for 2 weeks of treatment.2.Thegenome-wide expression chip technology was used to detect the rat ovarian tissues of between model group and JYD group toobtain differential gene expression profiles;toscreen out the genes related to the JYD mechanism.3.The obtained differential gene expression profile data was imported into the correlation map(CMAP)databasetoscreen out the compound mostlyrelatedto the curative effect of JYD on DOR.The possible therapeutic effect of this side is fully revealed from the molecular level.Methods1.The 40 femalerats of 4~5 days of estrus cycle.and 12 weeks of rat age were selected for experiment.According to the principle of weight balance,it was randomly divided into blank group,model group,JYD group,and Progynova group.A single intraperitoneal injection was performed with 1ml of 0.9% normal saline,and the remaining 3 groups were injected with a single intraperitoneal injection with cyclophosphamide(CTX)75mg/kg.The rats were fed respectively with physiological saline volume、physiological saline volume、23.58 g/kg JYD,0.09 mg/kg Progynova,we put to death between groups in estrogen interphase ratsafter 2 weeks.2.Observe the general situation and the change of estrus cycle in rats.3.Observe the morphological structure of ovary in rats with returninga intact ovaries of rats from modle group and JYD group to bedetectde by the genome-wide expression chip technology.4.Determine the serum FSH,LH and E2 of rats by enzyme-linked immuneosorbent assay(ELISA)method.5.Agilent Sure Print G3 Rat GE 8x60 K Microarray was used to detect for gene expression on the rat ovarian tissues of model group and JYD group.After expressed genes waselected,we analyzed the expressed geneswith the use of SAM software,andthen the bioinformatics analysis were performed.The differentially expressed genes of these two groups were screened out,then searched and analyzed.6.The obtained differential gene expression profile was made into a file in query signature format,and the relevant compound of the improved DOR condition was obtained through the online tool CMAP database.Retrieve literatureon several related compounds with large correlation degree,thenanalyze them.Results1.Effects on the general condition of rats.After modeling,the rats in the model group were depressed,undulated,decreased activity,weak limbs,and slow response.After 2 weeks of treatment,the rats’ mental state,appearance signs,behavior activities,drinking water and other conditions were obviously restored,similar to the blank group rats.2.Effects on the estrus cycle of rats.After modeling,the estrus cycle of the rat rule disappeared from 4~5 days to 6~12 days,and the period of estrus was extended from 1.5~2.5 days to 4~6 days.Compared with the model group,the estruscycle and interphase were shortened after 2 weeks of treatment.3.Effect of morphological structure of rat ovarian tissue.After modeling,rat ovarian tissue medulla atrophy,cortexhyperplasia,and interstitial fibrosis,granular cell layers,loosely arranged,cavitation,even primary follicles,secondary follicles in the ovaries,atresia follicles increased,primitive follicles,mature follicle.The structure of the rats’ ovarian tissue of JYD and Progynovawas clear,the degree of medullary atrophy and interstitial fibrosis was reduced,the number of granulocytes increased,the follicles decreased,and the mature follicle increased.4.Changes of serum FSH,LH and E2 in rats.Compared with the blank group,the level of FSH and LH in the model group was significantly increased,which was statistically significant.(P<0.01);Compared with the model group,JYD and Progynova can reduce the FSH and LH of rats.(P < 0.05 or P < 0.01);There was no statistical significance onthe difference of the FSH or LH in JYD and Progynova group after treatment(P>0.05).Compared with the blank group,the E2 level in the model group decreased significantly,which was statistically significant.(P<0.01);Compared with the model group,JYD and Progynova can elevate the E2 levelof rats(P<0.05 or P<0.01).There was no statistical significance onthe difference of the E2 in JYD and Progynova group after treatment(P>0.05)。5.Results.ofgene chip detectionAccording to the standards above,we have selected expressed genes of 10126.Among them,280 differential expression of genes were obtained,of which 65(23.21%)were up-regulated and 215(76.79%)were down-regulated in rat ovarian tissues of JYD group,with the use of SAM software according to the differentially significantstandard of fold changes > 1.5 times,or <0.66 times,and q-value < 5%.Generally,totalgene expression in rat ovarian tissues of JYD group is down regulated than inmodel group.Genes inrat ovarian tissues of JYD group show degenerative expression based on differential gene expression6.Literature search and summary results of differentially expressed genes.It was found that up-regulated gene Dab2 and down-regulated gene Calr,Cxcr4,Igfbp4 and Stat3 were correlated with follicular development and ovarian function,and all five genes were involved in the immune regulation,of the body.7.Result of the difference expression gene in the CMAP databaseand literature searches for related compounds.The top fifteen small molecular compounds that negatively related with the treatment effect.of JYD according to the enrichment ofnegative scores from high to low were mepacrine、mitoxantrone、H-7、Ethinylestradiol、Kanamycin、Digitoxigenin、Alfadolone、benzbromarone、ampyrone、Metrizamide、metformin、acacetin、Ethisterone、Ellipticine、fluticasone。The more relevant molecular compounds on the treatment effect.of JYD through literature retrieval were Ethinylestradiol、Ethisterone、Fluticasone、Metformin、Mitoxantrone、Acacetin、Ellipticine.Conclusion1.Both JYD and Progynova could reduce the FSH and LH of the DOR model,increase the number of E2 and mature follicle,and improve the ovarian function of DOR rats,and the effect was similar after 2 weeks of treatment.2.There are obviously difference in the level of m RNA of the rat ovarian tissuesbetween model group and JYD by groupgenome-wide expression chip technology which generally showeddegenerative expression.JYD possiblyimprove the function of ovarian function by up-regulating the Dab2 gene and down-regulating Calr,Cxcr4,Igfbp4 and Stat3 genes.3.In the CMAP database,the difference expression gene was queried.It was found that Estradiol and Ethisterone is more related to JYD from the related compounds obtained.JYD may have the effect of Ethinylestradiol and progesterone,which may be the molecular mechanism for the treatment of DOR and the improvement of fertility.4.Fluticasone has a certain correlation with JYD,and it may also have certain glucocorticoid effects.5.Metformin also has a significant correlation with JYD,and it is speculated that JYD may have similar effects on Metformin.This may be the mechanism by which the prescription reduces the discomfort of breastenlargement,suggesting that the prescription may be beneficial to metabolism.6.JYD may also have the effect of Mitoxantrone,Acacetin and Ellipticine,which are protective and anti-tumor compounds,andmetforminalso has an anti-tumor effect,suggesting that the prescription may have preventive and anti-tumor effects. |
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