The Mechanism Of Nongsuo Danggui Wan Combined With Human Umbilical Cord Mesenchymal Stem Cells To Improve Premature Ovarian Insufficiency Through Nrf2 Antioxidant Pathway

Objective This study used 4-vinylcyclohexane dioxide（VCD）induced ovarian dysfunction（POI）model rats as experimental subjects,Study the improvement of ovarian function by combining Nongsuo Danggui Wan with h UCMSCs;Exploring the mechanism of improving ovarian function in VCD induced POI model rats through the Nrf2 pathway,providing new reference and basis for clinical treatment of POI,and providing innovative guidance for female reproductive health care and ovarian biological function research.Methods For the normal（n=9）and model（n=33）groups were randomly divided from 42SPF female SD rats.The establishment of premature ovarian failure model in the rats of modeling group was achieved by intraperitoneal injection of VCD at 80mg/kg for 14consecutive days.After that,model identification was performed by randomly selecting 3 rats from each of the two groups.There were 5 groups separated from the remaining rats:model group,Femoston group,Nongsuo Danggui Wangroup,and human umbilical cord mesenchymal stem cells group,Nongsuo Danggui Wan combined with h UCMSCs group,with 6 rats in each group.The next 28 days after the completion of modeling,continuous treatment was given.During the modeling period and the last two weeks of treatment,smear of vaginal exfoliated cells was taken continuously and weighed weekly.After treatment,samples were taken uniformly,and serum sex hormone and oxidative factor levels were determined by ELISA method and thiobarbital method;HE staining can enable the ovarian tissue structure to be observed;In ovarian tissue,the manifestation of SOD1,SOD2,NQO1,HO-1,Nrf2 and Keap1 protein was detected by immunohistochemistry.The expression of SOD1,SOD2,NQO1,HO-1,Cyto Nrf2,Nu Nrf2,Nrf2 and Keap1 protein in ovarian tissue was detected by Western blot.The data were statistically analyzed with SPSS 22.0 and Graph Pad Prism 9.0 software.Results 1.Evaluation of model constructionThe estrous cycle of rats in the all POI modeling group appeared different degrees of disturbance contrast with that of the normal group.The HE results showed that In the normal group,follicles at all levels were present in the ovarian tissue of rats,and the granular layer was dense.And the ovaries of the POI modeling preparation group showed a certain degree of atrophy,the granulosa cell layer descend,the atresia follicles appeared,and the number of follicles at all levels descend.2.Effect of Nongsuo Danggui Wan combined with h UCMSCs on ovarian function of POI model rats（1）General situation and estrous cycle condition:The rats in model group contrast with the normal group elevated slowly in weight and has disordered estrous cycle.They were depressed,and had little movement,and their hair fell off in disorder.After treatment,contrast with the model group,the rats in the Nongsuo Danggui Wan combined with h UCMSCs group elevated prominently in weight,and their hair color,smoothness and activity were prominently improved.Besides,5 of them resumed their normal estrous cycle.（2）Ovarian wet weight,ovarian index and ovarian volume:The ovarian volume of rats in the model group contrast with the normal group were descend,and the ovarian wet weight and ovarian index of the model group contrast with the normal group were prominently descend（P<0.01）.Compared with those in other group,the ovarian volume,ovarian wet weight and ovarian index of rats in the Nongsuo Danggui Wan combined with h UCMSCs group was much higher（P<0.01）.（3）Serum sex hormones:The levels of serum E₂and AMH in the model group contrast with the normal group were prominently descend（P<0.01）,and their serum LH and FSH were elevated（P<0.01）;The levels of serum E₂and AMH in rats of each treatment group contrast with those of the model group were elevated,and their serum LH and FSH were serum LH and FSH were elevated（P<0.05）.Among them,the serum hormone level of Nongsuo Danggui Wan combined with h UCMSCs group obviously ameliorated（P<0.01）.（4）Ovarian tissue structure:Contrast with the normal group,the number of all kinds of follicles at all levels in the ovarian cortex descend prominently,the number of atretic follicles elevated,and the granular layer arranged loosely Contrast with the model group,the ovarian volume of rats in the Nongsuo Danggui Wan combined with h UCMSCs group elevated,and the number of follicles at all levels in the ovary elevated,and the granular layer was dense.3.Serum oxidation factor level and Nrf2 pathway related protein expression of Nongsuo Danggui Wan combined with h UCMSCs（1）Serum oxidative factors:Serum SOD and GSH concentrates in the model group descend prominently contrast with the normal group,the content of MDA increased significantly（P<0.01）.The serum GSH and SOD concentrates elevated,and the serum MDA concentrate descend in each treatment group relative to the model group（P<0.05）.Among them,the Nongsuo Danggui Wan combined with h UCMSCs group prominently improved the level of serum oxidation factor（P<0.01）。（2）Expression of SOD1,SOD2,NQO1,HO-1,Nrf2 and Keap1 proteins in ovarian tissue:The manifestation of Keap1 protein in ovarian tissue contrast with the normal group,the positive manifestation of SOD1,SOD2,NQO1,HO-1,Nrf2 protein in ovarian tissue of model group rats descend prominently,and the positive manifestation of Keap1 protein elevated prominently（P<0.01）.Contrast with all of other treatment groups except normal group,the positive manifestation of Keap1 in the ovarian tissue of rats in the Nongsuo Danggui Wan combined with h UCMSCs group was prominently descend（P<0.01）,and the positive manifestation of SOD1,SOD2,NQO1,HO-1,Nrf2 protein was prominently elevated（P<0.01）;（3）Western blot detection of the manifestation of SOD1,SOD2,NQO1,HO-1,Nrf2,Nu Nrf2,Cyto Nrf2,Keap1 protein:The manifestation of SOD1,SOD2,NQO1,HO-1,Nrf2,Nu Nrf2 protein in the ovarian tissue of the model group,which contrast with the normal group,is descend.And the manifestation of Keap1,Cyto Nrf2 protein elevated（P<0.01）;the manifestation levels of SOD1,SOD2,NQO1,HO-1 and Nrf2 in each treatment group,which contrast with the model group,is elevated to varying degrees,and the manifestation levels of Keap1 and Cyto Nrf2 are descend（P<0.05）.The Nongsuo Danggui Wan combined with human umbilical cord mesenchymal stem cells group was the most obvious（P<0.05）.Conclusion（1）Nongsuo Danggui Wan combined with h UMSCs can improve the hormone level and function of rats with premature ovarian insufficiency.（2）The effect of Nongsuo Danggui Wan combined with h UMSCs is better than using Nongsuo Danggui Wan or h UCMSCs alone for treatment.（3）Nongsuo Danggui Wan with h UMSCs can down-regulate the expression of Keap1factor in ovarian tissue,up-regulate the expression of Nrf2 factor as a whole and in the nucleus,and up-regulate the expression of HO-1,NQO1,SOD2,SOD1 proteins,improve the ability of ovarian tissue to resist oxidative stress damage in rats,thus improving POI.Its mechanism may be related to the activation of Nrf2 antioxidant signal pathway.