| Part I Clinical trialObjectiveTo observe the clinical efficacy of Jiajian Yijing Decoction in the treatment of patients with diminished ovarian reserve(DOR),and to explore the possible mechanism of improving ovarian function by regulating Th17/Treg immune balance.MethodsSixty-three DOR patients who met the diagnostic criteria and visited the outpatient department of Guangzhou Women and Children's Medical Center from March 2020 to May 2021 were randomly divided into traditional Chinese medicine group(32 cases)and western medicine group(31 cases).Among them,2 cases were removed from the Chinese medicine group and 1 case was removed from the western medicine group.In the Traditional Chinese medicine group,jiajie Yijing Decoction was taken on the first day after the first menstruation,one dose per day,and stopped during menstruation(didrogesterone 20mg/d×10d was taken in case of menstruation more than 45 days),and the next menstrual cycle was taken on the first day after the next menstruation,for a total of 6 menstrual cycles.western medicine group:starting from the 5th day of menstruation,bugarela 2mg/day×21 days+dydrogesterone 20mg/day×10 days(dydrogesterone tablet was added from the 12th day of bugarela),a total of 6 menstrual cycles.TCM symptom scores of the two groups before and after treatment were compared,Blood-based follicle-stimulating hormone(bFSH),basal luteinizing hormone(bLH),basal estradiol(bE2),anti-Muller's hormone(AMH),regulatory T cell(Treg),helper T cell 17(Thl7)and interleukin-10(IL-10),interleukin-17A(IL-17A),tumor necrosis factor-a(TNF-?),interferon-?(IFN-?),the number of sinus follicles(AFC)and the difference of pregnancy rate between the two groups after treatment.Results1.Comparison of TCM symptom scores between the two groups:there was no statistical difference in TCM symptom scores between the TCM group and the western medicine group before treatment(P>0.05),indicating comparability.The scores of TCM symptoms in TCM group and western medicine group were significantly lower after treatment than before treatment,the difference was statistically significant(P<0.05).With them,the scores of "menstrual volume,menstrual blood quality,lumbosacral soreness,breast tenderness and breast pain" in the Chinese medicine group were significantly lower than those in the western medicine group,with statistical significance(P<0.05).The "menstrual cycle" score in the western medicine group was significantly lower than that in the Chinese medicine group,with statistical significance(P<0.05).In terms of total score,there was no significant difference between the TCM group and the western medicine group before treatment(P>0.05),indicating comparability.The total score of TCM syndrome in TCM group and western medicine group after treatment was significantly lower than before treatment,the difference was statistically significant(P<0.05).The total score of TCM syndrome in the TCM group was significantly lower than that in the western medicine group after treatment,with statistical significance(P<0.05).2.Comparison of serum bFSH,bLH,bE2 and AMH between the two groups:there were no significant difference in bFSH,bLH,bE2 and AMH between the Chinese medicine group and the western medicine group before treatment(P>0.05).After treatment,bFSH and bLH in TCM group and western medicine group were significantly lower than before treatment,while bE2 and AMH were significantly higher than before treatment,with statistical significance(P<0.05).After treatment,there was no significant difference in bFSH,bLH and AMH between the TCM group and the western medicine group(P>0.05),while bE2 in the western medicine group was significantly higher than that in the Chinese medicine group(P<0.05).The difference between before and after bE2 treatment in bujiale group was significantly higher than that in the plus or minus Yijing Decoction group,and the difference was statistically significant(P<0.05),while the difference in serum bFSH,bLH and AMH between the TCM group and the western medicine group was not statistically significant(P>0.05).3.Comparison of serum Treg and Th17 between the two groups:there was no statistical significance in Treg and Th17 before treatment between the Chinese medicine group and the western medicine group(P>0.05).Treg in the TCM group and the western medicine group was significantly increased after treatment compared with before treatment(P<0.05),and Th17 was significantly decreased compared with before treatment,with statistical significance(P<0.05).There was no statistical significance in Treg after treatment between the TCM group and the western medicine group(P>0.05).Th17 in the Chinese medicine group was significantly lower than that in the western medicine group,with statistical significance(P<0.05).There was no significant difference in Treg before and after treatment between the Chinese medicine group and the western medicine group(P>0.05).The difference of Th17 before and after treatment in Chinese medicine group was significantly higher than that in western medicine group,with statistical significance(P<0.05).4.Comparison of serum IL-10,IL-17A,TNF-? and IFN-? between the two groups:there was no statistical significance in il-10,IL-17A,TNF-? and IFN-? before treatment between the Chinese medicine group and the western medicine group(P>0.05).After treatment,IL-17A,TNF-? and IFN-? were significantly decreased in the TRADITIONAL Chinese medicine group and the western medicine group compared with before treatment(P<0.05),while IL-10 was significantly increased in the traditional Chinese medicine group and the western medicine group,with statistical significance(P<0.05).After treatment,il-17A,TNF-? and IFN-? in Chinese medicine group were significantly lower than those in western medicine group,with the differences were statistically significant(P<0.05).The difference of IL-17A,IL-10,TNF-? and IFN-? in TCM group before and after treatment was significantly higher than that in western medicine group,with the difference was statistically significant(P<0.05).5.There was no statistical difference in AFC between the Chinese medicine group and the western medicine group before treatment(P>0.05),indicating comparability.After treatment,AFC in the TCM group and the western medicine group was significantly higher than before treatment,with statistical significance(P<0.05).There was no significant difference in AFC between Chinese medicine group and western medicine group after treatment(P>0.05).There was no statistical difference between the two groups before and after AFC treatment(P>0.05).6.Comparison of pregnancy rates between the two groups:there were 12 cases of infertility in the TCM group and 13 cases of infertility in the western medicine group.After treatment,there were 5 cases of pregnancy in the TCM group and 3 cases in the western medicine group.7.Comparison of the effects of two groups:The total effective rate index of two groups had no significant difference(P>0.05),the difference between the efficacy index distribution was not statistically significant(P>0.05).Conclusion1.Jiajianyijing Decoction and estrogen progesterone therapy can both treat DOR patients,and the comprehensive efficacy of the two groups is similar;It can improve the clinical symptoms of DOR patients,decrease bFSH and bLH,increase bE2 and AMH,increase AFC,increase Treg,decrease Th17,increase IL-10,decrease IL-17A,TNF-? and IFN-?.The effect of Th17,IL-17A,TNF-? and IFN-? in Chinese medicine group was more obvious than that in western medicine group.2.Both Jiajianyijing Decoction and estrogen progesterone therapy can reduce the scores of various TCM symptoms,but the TCM group has advantages in increasing menstrual volume and improving menstrual blood quality,lumbosacral pain,chest and hypochal pain,and breast pain.The total scores of TCM symptoms in the TCM group after treatment are significantly lower than those in the western medicine group.3.Jiajie Yijing Decoction may improve ovarian reserve function by regulating Th17/Treg immune balance;To a certain extent,the fertility and pregnancy rate of patients can be improved.part ? Experimental researchObjective(1)To investigate the mechanism of Th17/Treg,SDF-1/CXCR4 gene and protein levels in ctX-induced DOR rats;(2)By comparing western medicine Bujialle and AMD3100,the improvement of ovarian function of DOR rats induced by CTX was detected and observed,as well as the effects of Jiajie Yijing Decoction on Th17/Treg,SDF-1/CXCR4 gene and protein levels of DOR rats.Further explore the efficacy and possible mechanism of Jiajianyijing Decoction in the treatment of DOR,so as to provide theoretical basis for the clinical treatment of DOR by Jiajianyijing Decoction.MethodsSixty female rats with estrous cycles of 4 to 5 days(2 consecutive estrous cycles)were selected to carry out the experiment.The animals in the experimental group were randomly divided into ?blank control group,?model group,?Bujiele group,?Plus or minus Yijing Decoction group and ?AMD3100 group according to body weight(random number table method),with 12 animals in each group.The blank control group was intraperitoneally injected with 0.9%normal saline 1ml a day for 15 consecutive days.The other groups were given a single intraperitoneal injection of CTX 50mg/kg on the first day,and then 8mg/kg CTX intraperitoneal injection every day for 14 consecutive days,a total of 15 days.On the second day of modeling(i.e.the first day of intraperitoneal injection of CTX 8mg/kg),the other groups were given intraperitoneal injection,once a day,for consecutive 28 days,except group ?.In addition,11.79g crude drug·kg-1 and 0.9%normal saline were mixed into 10ml·kg-1 by gavage.Bujiale group was given 0.09mg/kg intragastric administration;AMD3100 group was intraperitoneally injected 10 mg/kg CXCR4 antagonist(10 mg/kg dissolved in 200?L phosphate buffered saline)three times a week.Blank control group and model group were given distilled water by gavage of l0mL/kg.On the 15th day of the experiment,6 rats were sacrificed in each group.On the 29th day of the experiment(and the day when all drugs were used),the remaining 6 rats in each group were sacrificed,and blood was collected from abdominal aorta and related tissues were collected.The general condition and estrous cycle of rats were observed.The changes of body weight and ovarian index and uterus index were observed.Serum hormone detection:The levels of E2,LH,FSH,AMH and IL-10 and IL-17 in serum of rats before and after administration were determined by enzyme-linked immunosorbent assay(ELISA).The mRNA expression of CXCR-4 and SDF-1 in ovarian tissue was detected by PCR.Western blot was used to detect cxCR-4 and SDF-1 protein expression in ovary of rats.The ratio of Th17,Treg and Th17/Treg cells were detected by flow cytometry.Results1.General observation results of ratsAll the rats in each group survived during the experiment,and some of the rats in each model group showed unmoist hair color,slightly worse cleanliness and smoothness,and were easy to be irritated.In the fourth week of treatment,there was no significant difference between the overall state of rats in each administration group and that in the blank control group.2.Effect on estrous cycle in ratsAfter 2 weeks of administration,the percentage of normal cycles in model group decreased significantly compared with blank control group,and the difference was statistically significant(P<0.05).Compared with the model group,the percentage of normal cycle in the yijingtang plus or minus group and bujiele group was significantly increased,with statistical significance(P<0.05),while the percentage of normal cycle in the AMD3100 group was increased,but there was no statistical significance(P>0.05).Compared with the AMD3100 group,the percentage of normal cycles in the plus or minus Yijing Decoction group and the bujiale group was significantly higher,with statistical significance(P<0.05),while there was no statistical significance between the plus or minus Yijing Decoction group and the bujiale group(P>0.05).Compared with the blank group,the percentage of normal cycle in yijing Tang group,Bujiele group and AMD3100 group was significantly decreased,with statistical significance(P<0.05).After 4 weeks of administration,the percentage of normal cycles in the model group increased compared with the blank control group,but the difference was still statistically significant(P<0.05),indicating that the rats in the model group had self-recovery ability,but had not returned to normal.Compared with the model group,the percentage of normal cycle in yijing Tang group,Bujiele group and AMD3100 group was significantly increased,with statistical significance(P<0.05).Compared with AMD3100 group,there was no statistical significance in adding or subtracting Yijingtang group and Bujiale group(P>0.05).Compared with blank group,there was no statistical significance in the percentage of normal cycle in yijing Tang group,Bujiele group and AMD3100 group(P>0.05).3.Comparison of ovarian index and uterine index in ratsAfter 2 weeks of administration,ovarian index and uterine index in model group were decreased compared with blank control group,with statistical significance(P<0.05).Compared with model group,the ovarian index and uterus index of bujiele group,Plus or minus Yijing Decoction group and AMD3100 group were increased,with statistical significance(P<0.05);Compared with AMD3100 group,there was no statistical difference between the plus or minus Yijing Decoction group and the bujiale group(P>0.05).Compared with the blank group,the ovarian index and uterus index of bujiele group,Plus or minus Yijing Decoction group and AMD3100 group were decreased,with statistical significance(P<0.05).After 4 weeks of administration,ovarian index and uterine index in model group were decreased compared with blank control group,with statistical significance(P<0.05).Compared with model group,the ovarian index and uterus index of bujiele group,Plus or minus Yijing Decoction group and AMD3100 group were increased,with statistical significance(P<0.05);Compared with AMD3100 group,there was no statistical difference between yijingtang group and Bujiale group(P>0.05);There was no statistical difference between supplementing yijing Decoction group and bujiale group(P>0.05).Compared with blank group,there was no statistical significance in ovarian index and uterine index in bujiale group and Plus or Minus Yijing Decoction group(P>0.05);The ovarian index and uterine index of AMD3100 group were still decreased,and the differences were statistically significant(P<0.05).4.Comparison of SERUM IL-10 and IL-17Two weeks after administration,compared with blank control group,the expression level of IL-10 in model group was decreased and the expression level of IL-17 was increased,with statistical significance(P<0.05).Compared with model group,the expression level of IL-10 in Bujiele group,Plus or minus Yijing Decoction group and AMD3100 group was increased and the expression level of IL-17 was decreased,with statistical significance(P<0.05).Compared with bujiale group,there was no statistical difference between yijingtang group and AMD3100 group(P>0.05);There was no statistical significance between AMD3100 group and Yijing Decoction group(P>0.05).Compared with the blank group,the expression level of IL-10 was decreased and the expression level of IL-17 was increased in bujiele group,Plus or minus Yijing Decoction group and AMD3100 group,with statistical significance(P<0.05).After 4 weeks of administration,compared with blank control group,the expression level of IL-10 in model group was decreased and the expression level of IL17 was increased,with statistical significance(P<0.05).Compared with model group,the expression level of IL-10 in Bujiele group,Plus or minus Yijing Decoction group and AMD3100 group was increased and the expression level of IL-17 was decreased,with statistical significance(P<0.05).Compared with bujiele group,the expression level of IL17 in yijingtang group and AMD3100 group was decreased,and the difference was statistically significant(P<0.05).Compared with bujiele group,the expression level of IL10 in yijing Tang group and AMD3100 group had no statistical significance(P>0.05);After treatment,there was no statistical significance in the expression level of IL-10 and IL-17 in yijing Decoction group compared with AMD3100 group(P>0.05).Compared with blank group,the expression level of IL-10 and IL-17 in bujiele group,Plus or minus Yijing Decoction group and AMD3100 group had no statistical difference(P>0.05).5.Comparison of expressions of FSH,LH,E2 and AMH in serumAfter 2 weeks of administration,compared with blank control group,FSH and LH levels in model group were significantly increased,while E2 and AMH levels were significantly decreased,with statistical significance(P<0.05).Compared with model group,the levels of FSH and LH in bujiele group,Plus or minus Yijing Decoction group and AMD3100 group were significantly decreased,while the levels of E2 and AMH were significantly increased,with statistical significance(P<0.05).Compared with AMD3100 group,the levels of FSH and LH in yijingtang group and Bujiele group were significantly decreased,while the levels of E2 and AMH were increased,with statistical significance(P<0.05).Compared with the bujiale group,the level of E2 in the plus or Minus Yijingtang group was lower,and the difference was statistically significant(P<0.05).Compared with the blank group,the levels of FSH,LH and AMH in the plus or minus Yijing Decoction group,the plus or minus Yijing Decoction group and the AMD3100 group were increased,while the levels of E2 and AMH decreased,and the differences were statistically significant(P<0.05).After 4 weeks of administration,compared with blank control group,FSH and LH levels in model group were significantly increased,while E2 and AMH levels were significantly decreased,with statistical significance(P<0.05).Compared with model group,the levels of FSH and LH in bujiele group,Plus or minus Yijing Decoction group and AMD3100 group were significantly decreased,while the levels of E2 and AMH were significantly increased,with statistical significance(P<0.05).Compared with AMD3100 group,the levels of FSH and LH in yijingtang group and Bujiele group were significantly decreased,while the levels of E2 and AMH were increased,with statistical significance(P<0.05).Compared with the bujiale group,the level of E2 in the plus or Minus Yijingtang group was lower,and the difference was statistically significant(P<0.05).There were no significant differences in FSH,LH and AMH between the plus or minus Yijing Decoction group and the Bujiale group(P>0.05).Compared with the blank group,there were no statistically significant differences in FSH,LH,E2 and AMH in the bujiele group and the Plus or minus Yijing Decoction group(P>0.05),while the LEVELS of FSH and LH were increased and the levels of E2 and AMH were decreased in the AMD3100 group,with statistically significant differences(P<0.05).6.Comparison of CXCR-4 and SDF-1 mRNA expression in rat ovariesAfter 2 weeks of administration,the mRNA levels of CXCR-4 and SDF-1 in model group were increased compared with blank control group,and the difference was statistically significant(P<0.05).Compared with model group,the mRNA levels of CXCR-4 and SDF1 in Bujiale group,Plus or minus Yijing Decoction group and AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with the bujiale group,the mRNAlevels of CXCR-4 and SDF-1 in the plus or minus Yijingtang group and the AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with yijing Decoction group,cxCR-4 and SDF-1 mRNA levels in AMD3100 group were significantly decreased,with statistical significance(P<0.05).Compared with blank group,CXCR-4 and SDF-1 mRNA in bujiale group,Plus or minus Yijing Decoction group and AMD3100 group were increased,with statistical significance(P<0.05).After 4 weeks of administration,the mRNA levels of CXCR-4 and SDF-1 in model group increased compared with blank control group,and the difference was statistically significant(P<0.05).Compared with model group,the mRNA levels of CXCR-4 and SDF1 in Bujiale group,Plus or minus Yijing Decoction group and AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with the bujiale group,the mRNAlevels of CXCR-4 and SDF-1 in the plus or minus Yijingtang group and the AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with yijing Decoction group,cxCR-4 and SDF-1 mRNA levels in AMD3100 group were decreased,but the difference was not statistically significant(P>0.05).Compared with blank group,CXCR-4 and SDF-1 mRNA were increased in bujiale group,and the difference was statistically significant(P<0.05).Compared with blank group,there was no significant difference in CXCR-4 and SDF-1 mRNA between yijingtang group and AMD3100 group(P>0.05).7.Comparison of CXCR-4 and SDF-1 protein expression in rat ovariesTwo weeks after administration,cxCR-4 and SDF-1 protein levels in model group were increased compared with blank control group,and the difference was statistically significant(P<0.05).Compared with model group,the protein levels of CXCR-4 and SDF-1 in Bujiale group,Plus or minus Yijing Decoction group and AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with the bujiale group,the protein levels of CXCR-4 and SDF-1 in the plus or minus Yijing Decoction group and the AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with yijing Decoction group,cxCR-4 and SDF-1 protein levels in AMD3100 group were significantly decreased,with statistical significance(P<0.05).Compared with blank group,cxCR-4 and SDF-1 proteins in bujiale group,Plus or minus Yijing Decoction group and AMD3100 group were increased,and the differences were statistically significant(P<0.05).After 4 weeks of administration,cxCR-4 and SDF-1 protein levels in model group increased compared with blank control group,and the difference was statistically significant(P<0.05).Compared with model group,the protein levels of CXCR-4 and SDF-1 in Bujiale group,Plus or minus Yijing Decoction group and AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with the bujiale group,the protein levels of CXCR-4 and SDF-1 in the plus or minus Yijing Decoction group and the AMD3100 group were decreased,and the differences were statistically significant(P<0.05).Compared with yijing Decoction group,cxCR-4 and SDF-1 protein levels in AMD3100 group were decreased,but the difference was not statistically significant(P>0.05).Compared with blank group,cxCR-4 and SDF-1 proteins in bujiale group were increased,and the difference was statistically significant(P<0.05).Compared with blank group,cxCR-4 and SDF-1 proteins in yijing Decoction group and AMD3100 group had no statistical significance(P>0.05).8.Comparison of Th17,Treg and Th17/Treg cells ratio in ratsTwo weeks after administration,compared with blank control group,Th17 level and Th17/Treg ratio in model group increased,while Treg level decreased,with statistical significance(P<0.05).Compared with the model group,the Th17 level and Th17/Treg ratio of bujiele group,Plus or minus Yijing Decoction group and AMD3100 group were decreased and Treg level was increased,with statistical significance(P<0.05).Compared with bujiele group,Th17 level and Th17/Treg ratio were decreased and Treg level was increased in the Plus or minus Yijing Decoction group and AMD3100 group,and the differences were statistically significant(P<0.05).Compared with AMD3100 group,there were no significant differences in Th17 level,Treg level and Th17/Treg ratio in yijing Decoction group(P>0.05).Compared with the blank group,the Th17 level and Th17/Treg ratio increased and Treg level decreased in bujiele group,Plus or minus Yijing Decoction group and AMD3100 group,with statistical significance(P<0.05).After 4 weeks of administration,compared with blank control group,Th17 level and Th17/Treg ratio in model group increased,while Treg level decreased,with statistical significance(P<0.05).Compared with the model group,the Th17 level and Th17/Treg ratio of bujiele group,Plus or minus Yijing Decoction group and AMD3100 group were decreased and Treg level was increased,with statistical significance(P<0.05).Compared with bujiele group,Th17 level and Th17/Treg ratio were decreased and Treg level was increased in the Plus or minus Yijing Decoction group and AMD3100 group,and the differences were statistically significant(P<0.05).Compared with AMD3100 group,Treg level in yijing Decoction group was significantly increased(P<0.05);Compared with the AMD3100 group,the Th17 level and Th17/Treg ratio in the plus or minus Yijing Decoction group were significantly decreased,with statistical significance(P<0.05).Compared with blank group,Th17 level and Th17/Treg ratio increased and Treg level decreased in Bujiale group,but the differences were not statistically significant(P>0.05).Compared with the blank group,there were no significant difference in Th17 level and Th17/Treg ratio between yijing Decoction group and AMD3100 group(P>0.05).Conclusion1.Cyclophosphamide(CTX)can be used to establish an animal model of reduced ovarian reserve function,which can lead to upregulation of SDF-1/CXCR4,thereby disrupting the immune balance of Th17/Treg.2.After treatment with Jiajie Yijing Decoction and Bujiole,the mrna and protein levels of CXCR4 and SDF-1 in DOR model rats were significantly reduced,Th17/Treg balance was regulated,serum FSH,LH and IL-17 levels were decreased,E2,AMH and IL-10 levels were increased,and ovarian index and uterine index were increased.The ovarian function of DOR rats was improved,but the levels of SDF-1/CXCR4 and Th17 in the plus or minus Yijing Decoction group were reduced,and Th17/Treg in the plus or minus Yijing Decoction group were significantly better than those in the Bujiele group.The results showed that both Jiajie Yijing Decoction and Bujiale decoction could adjust the immune imbalance,but the effect of Jiajie Yijing Decoction was more significant.3.After treatment,the mrna and protein levels of CXCR4 and SDF-1,Th17/Treg balance,AMH,FSH,LH and IL-17 levels in serum were decreased and E2 and IL-10 levels were increased in BOTH AMD3100 group and Jianjianyijing Decoction group.Furthermore,the ovarian function of DOR rats was improved,but the formula of adding or subtracting Yijing Decoction to reduce FSH and LH levels and increase AMH and E2 levels was significantly better than that of AMD3100 group.These results indicate that both Jiajie Yijing Decoction and AMD3100 can improve ovarian function,but the effect of Jiajie Yijing Decoction is more significant.4.The decoction and AMD3100 group may regulate Treg/Th17 immune balance and improve ovarian function by blocking sdF-1/CXCR4 expression. |
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