To Investigate The Effect Of Kidney-tonifying Herbs On The Outcome Of Infertility In Elderly Patients With Kidney-qi Deficiency Based On Drp1/Mitophagy Pathway

Objective:To observe the changes of DRP1,FUNDC1 and LC3 B in ovarian granulosa cells of elderly patients with kidney qi deficiency type undergoing in vitro fertilization-embryo transfer(IVF-ET),and to explore the possible regulatory mechanism of Erzhi Tiangui Granule,a traditional Chinese medicine for tonifying kidney,on mitochondrial dynamics(mitophagy)and mitophagy angle in elderly patients with kidney qi deficiency type undergoing IVF-ET and its effect on clinical outcomes.Methods:A total of 66 elderly patients with kidney qi deficiency who underwent assisted reproductive technology in the Reproductive and Genetic Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine were collected and divided into the traditional Chinese medicine treatment group(Erzhi Tiangui Granule +IVF/ICSI)and the placebo group(placebo Granule + IVF/ICSI)according to the random number table,with 33 cases in each group.Another normal control group(placebo Granule + IVF/ICSI)was selected for 33 women with male factors who underwent assisted reproductive technology and the women were younger than 35 years.Antagonist protocol(Gn RH-ant protocol)was used in all three groups.The changes of syndrome score of kidney qi deficiency syndrome in the two groups of elderly patients were observed,and the number of oocytes retrieved,number of 2PN fertilization,2PN fertilization rate,number of available embryos,high-quality embryo rate,and clinical pregnancy rate per oocyte retrieval cycle among the three groups were compared.On the day of oocyte retrieval,follicular fluid was collected from the dominant follicles of patients in each group and granulosa cells were extracted to detect the relative expression of DRP1,FUNDC1,and LC3 B m RNA contents in granulosa cells by real-time quantitative PCR;the relative expression of DRP1,FUNDC1,and LC3 B protein contents in granulosa cells was determined by Western blot.Results:1.The symptoms of kidney qi deficiency were significantly improved before and after treatment in the TCM treatment group,and the difference was significant(P<0.01);compared with the placebo group,the symptoms of kidney qi deficiency were significantly improved,and the difference of kidney qi deficiency syndrome scores before and after treatment was significant(P<0.01).2.The number of oocytes retrieved in the normal control group was significantly higher than that in the elderly group,and the difference was significant(P<0.01);there was no significant difference between the two groups(P>0.05),but the number of oocytes retrieved showed an increasing trend.3.The 2PN fertilization rate was higher in the TCM treatment group than in the placebo group,and the difference was statistically significant(P<0.05);the normal control group was significantly higher than the placebo group,and the difference was significant(P<0.01).4.The excellent embryo rate in the normal control group was higher than that in the placebo group,and the difference was statistically significant(P<0.05);the difference in the excellent embryo rate in the placebo group was not statistically significant(P>0.05),but the excellent embryo rate increased.5.The clinical pregnancy rate per oocyte retrieval cycle in the normal control group was higher than that in the placebo group,and the difference was statistically significant(P<0.05);the difference in the placebo group was not statistically significant(P>0.05),but the clinical pregnancy rate showed an increasing trend.6.RT-PCR results showed that the relative expression of DRP1,FUNDC1 and LC3B m RNA in ovarian granulosa cells showed an increasing trend in the elderly group compared with the normal control group;the relative expression of DRP1,FUNDC1 and LC3 B m RNA in the elderly group was lower than that in the placebo group after treatment,and the difference had statistical significance(P<0.05).In the relative expression of DRP1 m RNA,the placebo group was higher than the normal control group,and the difference was statistically significant(P<0.05);in the relative expression of FUNDC1 m RNA,the TCM treatment group was higher than the normal control group and the difference was statistically significant(P<0.05),and the placebo group was significantly higher than the normal control group and the difference was significant(P<0.01);in the relative expression of LC3 B m RNA,the placebo group was significantly higher than the normal control group,and the difference was significant(P<0.01).7.Western-blot results showed that the relative expression of DRP1,FUNDC1,and LC3 B proteins in ovarian granulosa cells showed an increasing trend in the elderly two groups compared with the normal control group;the relative expression of FUNDC1 and LC3 B after TCM treatment was lower in the elderly two groups than in the placebo group,and the difference was statistically significant(P<0.05).In the relative expression of DRP1 protein,the elderly group was higher than the normal control group,the differences were statistically significant(P<0.05),the traditional Chinese medicine treatment group was not significantly different from the normal control group,but showed a decreasing trend;in the relative expression of FUNDC1 protein,the placebo group was significantly higher than the normal control group,the differences were significant(P<0.01);in the relative expression of LC3 B protein,the traditional Chinese medicine treatment group was higher than the normal control group,the differences were statistically significant(P<0.05),the placebo group was significantly higher than the normal control group,the differences were significant(P<0.01).Conclusion:1.In the IVF-ET treatment cycle of elderly infertile women with kidney qi deficiency,the application of Erzhi Tiangui Granule,a traditional Chinese medicine for tonifying kidney,can significantly improve the symptoms of kidney qi deficiency,increase the fertilization rate of 2PN,and improve the high-quality embryo rate and clinical pregnancy rate.2.The levels of mitochondrial fission and autophagy in ovarian granulosa cells of elderly women were increased,and the levels of the two showed a certain degree of downward trend after treatment with traditional Chinese medicine,even close to the level of normal controls.Erzhi Tiangui Granule may improve the state of excessive mitochondrial division and excessive autophagy in ovarian granulosa cells of elderly women by regulating the expression of Drp1,FUNDC1,and LC3 B proteins,which are key factors in the mitochondrial division and autophagy pathway in ovarian granulosa cells,maintain the stability of mitochondrial morphology and function,promote the normal development of granulosa cells and oocytes,improve the normal fertilization rate and embryo quality,and ultimately improve the conception rate.3.According to the relatively consistent expression changes of the three expression factors in the same group and the relationship between mitochondrial division and autophagy,we speculated that DRP1-mediated mitochondrial fission caused subsequent changes in autophagy,and similarly,the regulation of mitochondria by traditional Chinese medicine may be achieved through DRP1-mediated mitodivision/FUNDC1-mediated mitophagy,a complete sequential path.