| Objective:To observe the regulatory effects of Zishen Quyujiedu Decoction on Wnt5a,SIAH2,β-catenin and caspase-3 in ovarian granulosa cells(OGCs)of patients with kidney-deficiency and blood-stasis endometriosis(EMS),and to explore the efficacy of Chinese medicine in treating EMs with kidney deficiency and blood stasis type and the molecular mechanism of their effects from the perspective of apoptosis.Methods:A total of 72 patients between 21-35 years of age who met the criteria for EMs infertility and were to be assisted with in vitro fertilization and embryo transfer(IVF-ET)were collected and randomly divided into the treatment group(TG)(Zishen Quyujiedu Granule)(n=36),the placebo group(PG)(placebo)(n=36),and another 36 patients who were assisted with IVF-ET due to tubal factors alone in the same period were selected as the control group(CG)(placebo).All three groups took each group orally for three months prior to egg retrieval,and all were subjected to controlled ovarian hyperstimulation using a long luteal phase regimen(GnRH-a regimen).The changes of serum carbohydrate antigen 125(CA125)levels and the kidney deficiency and blood stasis syndrome score were compared between the TG and PG before and after treatment.The dosage of days of gonadotropin(Gn)use and Gn dosage in the three groups were counted;the serum estradiol(E2),luteinizing hormone(LH)and progesterone(P)levels on the day of injection of chorionic gonadotropin(HCG)were measured;the number of eggs retrieved,the fertilization rate of double protoplast(2PN),the number of available embryos and the highquality embryo rate were counted and compared among the three groups.The levels of Wnt5a,SIAH2,β-catenin,caspase-3 mRNA and protein in OGCs of three groups of patients were determined by quantitative real-time PCR(qPCR)and Western blot;TUNEL staining was used to detect the apoptosis of OGCs and the apoptosis rate was calculated.Results:1.The symptoms of kidney deficiency and blood stasis improved significantly in the TG,and the difference of the kidney deficiency and blood stasis syndrome scores before and after treatment was greater than that in the PG(P<O.01).2.The serum CA125 levels decreased in both the TG and PG after treatment(P<0.01),and the decrease was greater in the treatment group(P<0.01).3.There was no significant difference in Gn treatment days and dosage between the three groups(P>0.05).4.The levels of E2,LH and P on HCG day were basically the same in the three groups(P>0.05).5.The number of eggs obtained and high quality embryo rate were higher in the TG and the CG than in the PG(P<0.05);the number of eggs obtained and high quality embryo rate were not significantly different in the TG compared with the CG(P>0.05);the fertilization rate of 2PN and the number of available embryos were higher in the CG than in the PG(P<0.05);the fertilization rate of 2PN and the number of available embryos were not significantly different in the TG compared with the PG(P>0.05).6.The expression of Wnt5a,SIAH2 and caspase-3 mRNA in OGCs of the three groups of patients was lower in the TG and the CG than in the PG,and the expression of β-catenin mRNA was higher in the CG(P<0.01);the expression of Wnt5a and caspase-3 mRNA was lower in the CG than in the TG(P<0.05).The expression of β-catenin mRNA in the CG was higher than that in the TG(P<0.01);there was no significant difference in the expression of SIAH2 mRNA between the TG and the CG(P>0.05).7.In the three groups of patients with OGCs Wnt5a,SIAH2,and caspase-3 protein expressions were lower in the TG and CG than in the PG,and β-catenin protein expressions were higher than in the PG(P<0.01);SIAH2 and caspase-3 protein expressions were lower in the CG than in the TG(P<0.05);β-catenin protein expressions were higher in the CG(P<0.01);there was no significant difference in Wnt5a protein expression between the TG and CG(P>0.05).8.The apoptosis rates were(30.67±9.33)%,(45.56±12.06)%and(25.87±12.63)%in the treatment group,placebo group and blank control group,respectively.The apoptosis rate in the CG was lower than that in the PG(P<0.01);the apoptosis rate in the TG was lower than that in the PG(P<0.05);there was no significant difference in the apoptosis rate between the TG and CG(P>0.05).Conclusions:1.Zishen Quyujiedu Decoction can significantly improve the symptoms of kidney deficiency and blood stasis in EMs patients with kidney deficiency and blood stasis type in IVF cycles,improve the quality of oocytes,and thus increase the fertilization rate and embryonic development potential.2.Higher apoptosis in OGCs of patients with EMs compared to normal subjects.High levels of Wnt5a may antagonize the typical Wnt pathway by promoting β-catenin degradation,inhibiting the gonadotropin signaling pathway,causing upregulation of caspase-3 protein expression and increased apoptosis in OGCs compared with normal subjects,resulting in decreased oocyte quality in EMs patients.3.Zishen Quyujiedu Decoction may increase the expression of the key effector molecule βcatenin gene and related proteins by decreasing the expression of Wnt5a and SIAH2 in OGCs of patients with EMs,and lowered the level of caspase3,thus inhibiting oocyte apoptosis,4.Bushen sputum detoxification method may play a role in inhibiting granulosa cell apoptosis through the Wnt pathway,and "nourishing the kidneys to remove blood stasis and detoxify the body,and promoting the circulation and relieving pain" to improve egg and embryo quality,further enriching the study of " ovarian retention and drainage " and " Blood stasis and toxin accumulation " theory. |
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