| BackgroundDiminished ovarian reserve(DOR)is a key disease in the field of gynecological reproductive endocrinology.It can be divided into physiological DOR related to age and pathological DOR caused by environment,iatrogenic and social psychology.The main clinical manifestations are decreased fertility(including difficulty in conception,repeated abortion,poor ovarian response,repeated transplantation failure).Clinical use of drugs to promote ovulation,self-gametes or in vitro fertilization-embryo transfer,mesenchymal stem cells and platelet-rich plasma and other treatments.The resulting adverse reactions,ethics,laws and offspring health problems have caused many problems to patients and families.Patients urgently need safer and more effective treatments to improve ovarian reserve function and fertility.Traditional Chinese medicine treatment can improve ovarian function,improve the number and quality of oocytes obtained by DOR patients,increase embryo implantation rate and clinical pregnancy rate.It is recommended as grade ⅡB evidence and has unique advantages in improving the fertility potential of patients.At the same time,it has a long history and rich experience.The clinical efficacy is definite,safe and reliable.Bushen Tianjing Chinese Medicine in this study is the clinical prescription of Professor Cai Lianxiang,a famous doctor of traditional Chinese medicine in the capital.Professor Cai believes that the basic pathogenesis of DOR is deficiency of kidney essence,and the treatment is tonifying kidney and filling essence,nourishing blood and nourishing yin.Previous randomized controlled studies have found that Bushen Tianjing Chinese Medicine can improve AMH level,reduce FSH level in basal follicular phase and improve ovarian reserve function in DOR patients.Retrospective clinical studies have found that Bushen Tianjing Chinese Medicine can promote the conception of DOR patients with fertility requirements.Network pharmacology found that Nrf2 signaling pathway is an important target for the treatment of DOR with Bushen Tianjing Chinese Medicine.Knowledge map analysis suggested that pyroptosis is closely related to oxidative stress.Keap1-Nrf2 signaling pathway is the most important pathway in anti-oxidative stress.Based on the previous basis,clinical studies have clarified the clinical efficacy and safety of Bushen Tianjing Chinese Medicine in improving the fertility of DOR patients.The experimental study takes oxidative stress and pyroptosis as the starting point to explore the mechanism of Bushen Tianjing Chinese Medicine in protecting ovarian reserve function and improving fertility.Objective1.A double-blind,single-simulation randomized controlled clinical study was conducted to clarify the clinical efficacy of Bushen Tianjing Chinese Medicine in the treatment of DOR-related infertility,and to confirm its effectiveness and safety in improving ovarian reserve function and fertility.2.Through animal experiments,it is clarified that Bushen Tianjing Chinese Medicine can improve ovarian oxidative damage,inhibit ovarian granulosa cell scorch,and reduce abnormal follicular atresia by regulating the Keapl-Nrf2 signal pathway in the body,thereby improving ovarian reserve function and improving fertility.Methods1.Clinical researchIn this study,a double-blind,single-simulated randomized controlled study was conducted.A total of 68 patients with DOR-related infertility of liver-kidney yin deficiency type were included.All of them were admitted to Xiyuan Hospital of China Academy of Chinese Medical Sciences from December 2020 to December 2022,which met the inclusion and exclusion criteria of DOR-related infertility of liver-kidney yin deficiency type and passed the ethical review.Random numbers were generated by SAS and randomly divided into two groups according to 1:1.All patients took clomiphene citrate tablets 50mg/time,once a day,for 5 days from the 5th day of menstruation,and estradiol valerate tablets 1 mg/time,once a day,for 10 days on the 9th day of menstruation.On the 10 th day of the menstrual cycle,the follicles were monitored,and the diameter of the follicles was 1.8cm-2.0cm by ultrasound.The ovulation was induced by intramuscular injection of 10000U of chorionic gonadotropin.On this basis,34 cases in the experimental group were given Bushen Tianjing Chinese medicine granules(drug composition:Zhiguiban,Shudi,Tusizi,Ligustrum lucidum,etc.),and the control group was given Bushen Tianjing Chinese medicine granules placebo,1 bag/time,2 times/day,for 21 days.If the patient is pregnant,the treatment is ended,and the non-pregnant woman continues the above treatment after menstruation,for 3 consecutive menstrual cycles,and is followed up for 3 months after the treatment.The main efficacy index was clinical pregnancy rate;the secondary efficacy indicators were comprehensive efficacy,basic follicular phase serum hormones(AMH,FSH,LH,E2),basic follicular phase ultrasound indicators(antral follicle count AFC,ovarian stromal blood flow resistance index RI),ovulation period B-ultrasound(ovulation score,endometrial thickness,morphology,blood flow score),fertility score and TCM syndrome score and safety indicators(blood routine,urine routine,liver and kidney function,electrocardiogram).2.Animal experiments72 ICR mice were randomly divided into blank group,model group,western medicine group,Chinese medicine group,ML385(Nrf2 inhibitor)+Chinese medicine group,MCC950(NLRP3 inhibitor)+Chinese medicine group,12 in each group.In addition to the blank group,the other 5 groups were prepared DOR mouse model.After successful modeling,the blank group and the model group were given normal saline by gavage,and the other groups were given corresponding drugs by gavage,once a day.ML385 and MCC950 were given by intraperitoneal injection once a day.After 14 days of administration,the rats were euthanized and detected.The general state of mice was observed,and the body weight was recorded.The estrous cycle was observed by vaginal smear.The number of uterus,ovarian tissue and follicles at all levels was observed by HE staining.The levels of sex hormones AMH,INHB,FSH,LH and E2 were detected by ELISA.The levels of serum ROS,MDA and SOD were detected by biochemistry.The expression of IL-1β and IL-18 in ovarian tissue was detected by immunohistochemistry.The protein expression of pyroptosis-related proteins NLRP3,Caspase-1,GSDMD and key factors Keapl and Nrf2 were detected by Western Blot.The expression levels of NLRP3,Caspase-1,GSDMD mRNA,Keap1 and Nrf2 mRNA were detected by RT-PCT,and the pyroptosis morphology of ovarian granulosa cells was observed by transmission electron microscopy.Results1.Clinical research(1)A total of 68 patients with DOR type of liver and kidney yin deficiency were enrolled in this study,and 3 patients were lost.There was no significant difference in baseline data such as age,course of disease,menstrual history,marriage and childbearing history,sex hormone level,ultrasound index,fertility related score and TCM syndrome score between the two groups(P>0.05).(2)Hormone level:Compared before and after treatment in the experimental group,the level of AMH increased,and the difference was statistically significant(P<0.05).The increase of AMH in the experimental group was better than that in the control group,and the difference was statistically significant(P<0.05).There was no significant difference in FSH,LH and E2 between the two groups before and after treatment(P>0.05).(3)Ultrasound indicators:Compared with before and after treatment in the experimental group,the level of AFC increased,and the difference was statistically significant(P<0.05).The increase of AFC in the experimental group was better than that in the control group,and the difference was statistically significant(P<0.05).There was no significant difference in ovarian stromal blood flow resistance index(RI)between the two groups before and after treatment(P>0.05).(4)Fertility related score:There was no significant change in ovulation score,endometrial morphology score,endometrial thickness score and endometrial blood flow score before and after treatment in the two groups(P>0.05).The total fertility score of the experimental group was increased before and after treatment,and the difference was statistically significant(P<0.05).(5)TCM syndrome score:Compared with the two groups before and after treatment,the TCM syndrome score of the two groups decreased,and the difference was statistically significant(P<0.05).The TCM syndrome score of the experimental group was lower than that of the control group,and the difference was statistically significant(P<0.05).(6)Clinical pregnancy rate:After treatment,the experimental group and the control group were 35.29%(12/34)and 12.90%(4/31),respectively.The experimental group was superior to the control group,and the difference between the two groups was statistically significant(P<0.05).(7)Comprehensive efficacy:The total effective rates of the experimental group and the control group after treatment were 70.59%and 45.16%,respectively.The experimental group was better than the control group,and the difference was statistically significant(P<0.05).(8)Safety indicators:There were no abnormalities in the experimental group and the control group before and after treatment(blood routine,urine routine,liver and kidney function,electrocardiogram).A total of 3 mild adverse events were observed during the study(1 patient in the control group had diarrhea,and 2 patients in the experimental group had nausea).The patients were improved after adjusting the medication method,and the remaining patients had no adverse reactions.2.Animal experiments(1)General condition of mice:Compared with the blank group,the mice in the model group had dull hair,reduced activity and weight loss after cisplatin modeling.The weight of D21 and D28 in each group decreased compared with the blank group(P<0.05).After intervention with traditional Chinese medicine,western medicine,ML385+traditional Chinese medicine and MCC950+traditional Chinese medicine,the weight of mice increased.(2)Mouse estrous cycle:Compared with the blank group,the mice in each group showed estrous cycle disorder after cisplatin modeling(P<0.05).After drug intervention in each group,compared with the blank group,the estrous cycle of the mice in the model group was disordered(P<0.05),and the mice in the Chinese medicine group,the western medicine group and the MCC950+Chinese medicine group recovered the regular estrous cycle(P<0.05).Compared with the Chinese medicine group,the estrous cycle of the western medicine group was stagnant(P<0.05).(3)Hormone levels in mice:Compared with the blank group,the levels of FSH and LH in the model group increased(P<0.05),and the levels of E2,AMH and INHB decreased(P<0.05).Compared with the model group,the levels of FSH and LH in the western medicine group,the Chinese medicine group and the MCC950+Chinese medicine group decreased(P<0.05),and the levels of E2,AMH and INHB increased(P<0.05).Compared with the Chinese medicine group,the level of INHB in the ML385+Chinese medicine group was lower(P<0.05).(4)Gonadal index of mice:Compared with the blank group,the uterus and ovary index of the model group decreased(P<0.05).Compared with the model group,the uterus and ovary index of the Chinese medicine group,the western medicine group and the MCC950+Chinese medicine group increased(P<0.05).The uterus index of the ML385+Chinese medicine group showed an upward trend(P>0.05).Compared with the Chinese medicine group,the uterine and ovarian index trends of the western medicine group and the MCC950+Chinese medicine group were similar(P>0.05).(5)The morphology and follicle count of uterus and ovary in mice:the volume and morphology of uterus and ovary in the blank group were normal,the glands were abundant,and follicles at different stages could be seen.In the model group,the uterus and ovary were atrophied,the glands were thin,the primordial follicles and antral follicles were reduced,the preantral follicles were increased,and the atresia follicles were increased.The volume and morphology of uterus and ovary in the Chinese medicine group and MCC950+Chinese medicine group were similar to those in the blank group.The ovarian interstitial fibrosis in the western medicine group was severe,and the ML385+Chinese medicine group was similar to the model group.The gonadal index was consistent with the results of histomorphology.The uterus and ovary index of the model group decreased compared with the blank group(P<0.05).The uterus and ovary index of the mice increased after treatment with traditional Chinese medicine,MCC950+traditional Chinese medicine and western medicine(P<0.05).The ML385+traditional Chinese medicine group was similar to the model group(P>0.05).Compared with the blank group,the primordial follicles,antral follicles and corpus luteum in the model group decreased(P<0.05,P<0.01),and the atresia follicles increased(P<0.05).Compared with the model group,the primordial follicles,antral follicles and corpus luteum in the Chinese medicine group and the MCC950+Chinese medicine group increased(P<0.05,P<0.01),and the atresia follicles decreased(P<0.01).The primordial follicles in the western medicine group increased(P<0.05).The primordial follicles,antral follicles and corpus luteum in the ML385+Chinese medicine group were similar to those in the model group,and the number of atresia follicles showed a decreasing trend(P>0.05).Compared with the Chinese medicine group,the corpus luteum in the western medicine group decreased(P<0.05).(6)Oxidative stress molecules in mouse ovary:Compared with the blank group,the levels of ROS and MDA in the model group increased(P<0.01),and the level of SOD decreased(P<0.05).After treatment with Chinese medicine group and MCC950+Chinese medicine group,the levels of ROS and MDA decreased(P<0.05),and the level of SOD increased(P<0.05).The level of SOD in the western medicine group increased(P<0.05),and there was no significant difference between the ML385+Chinese medicine group and the model group(P>0.05).Anti-oxidative stress Keap1/Nrf2 signaling pathway in mice:Compared with the blank group,the relative expression of Keapl and Nrf2 protein in the model group decreased(P<0.05);compared with the model group,the values of Keap1 and Nrf2 in the Chinese medicine group and the MCC950+Chinese medicine group increased after intervention(P<0.05),and the relative expression of Keapl in the western medicine group decreased(P<0.05).There was no significant difference between the ML385+Chinese medicine group and the model group(P>0.05).The mRNA expression levels of Keap1 and Nrf2 were consistent with the results of protein expression levels.(7)Mouse ovarian inflammatory factors:IL-18 and IL-1 β immunohistoche mistry showed that compared with the blank group,the expression of IL-18 an d IL-1 β molecules in the ovarian tissue of the model group was higher;afte r the intervention of Chinese medicine and MCC950+Chinese medicine group,t he expression of IL-18 and IL-1 β was lower than that of the model group,an d there was no significant improvement in the western medicine group and M L385+Chinese medicine group..(8)Expression of ovarian pyroptosis in mice:Compared with the blank group,the values of NLRP3,Caspase-1 and Gasdermin D in the model group increased(P<0.05);compared with the model group,the values of NLRP3,Caspase-1 and Gasdermin D in the Chinese medicine group,MCC950+Chinese medicine group and western medicine group decreased after intervention(P<0.01),and the values of NLRP3,Caspase-1 and Gasdermin D in the ML385+Chinese medicine group decreased after intervention(P>0.05).The mRNA expression levels of NLRP3,Caspase-1 and Gasdermin D were consistent with the results of protein expression levels.(9)Transmission electron microscopy of mouse ovary:Compared with the blank group,the cell membrane of some granulosa cells in the model group was ruptured,the organelles were deformed,and the cell contents were overflowed.After the intervention of traditional Chinese medicine and MCC950+traditional Chinese medicine,the number of normal granulosa cells increased,and the number of ruptured and deformed cells decreased.There was no significant difference between the ML385+Chinese medicine group and the model group after intervention.Conclusion1.Clinical studies have shown that Chinese medicine for tonifying kidney and replenishing essence can improve the clinical pregnancy rate of DOR-related infertility patients with liver and kidney yin deficiency,regulate hormone levels,increase AMH and AFC,improve ovarian reserve function,and relieve TCM symptoms.2.Animal experiments confirmed that kidney-tonifying and essence-filling C hinese medicine reduced ovarian oxidative damage,inhibited granulosa cell pyr optosis,and reduced follicular atresia by activating the Keap1/Nrf2 signaling pa thway,thereby improving ovarian reserve function and fertility. |
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