Study On The Mechanism Of Treating Primary Ovarian Insufficiency With Huyang Yangkun Formulae Based On Transcriptomics

Primary Ovarian Insufficiency(POI)is a clinical syndrome caused by ovarian dysfunction in women younger than 40 y/o,characterized by menstrual disorders due to high levels of gonadotropin and low levels of estrogen.Most of the current alternative and complementary treatments for POI is inefficacy and lack of safety data.Therefore,it is important to explore a safe and effective alternative treatment for the disease.In recent years,there is certain progression in both clinical and experimental research of POI,due to the vigorous development of Chinese Traditional Medicine and the deeper understanding of the disease.The Huyang Yangkun Formulae(HYF)using in this study was originated by Yang Hongyan my PHD supervisor,who combined her own clinical experiences with the theories of the declined reproductive cycle of female,Sanyin Sanyang Closing and Pivoting Mechanisms and "Taiyin will be the main meridian post menopause" by Liu Wansu.It has been confirmed in clinical research that,this HYF can help with POI patients by alleviating the clinical symptoms,regulating hormone levels,resuscitating residual follicle and restoring menstrual cycle.However,the mechanism of using of the formula HYF to regulate genes as a treatment for POI is unclear at present.Based on the significant practical efficacy and previous clinical research of HYF,this study will furtherly analyze the potential mechanism of HYF for treating POI using Transcriptomics techniques in order to provide the experimental supporting and consummate the mechanism.Objective1.To preliminarily evaluate the safety of HYF prescription by observing the effects of HYF on ovarian function of the rats with VCD-induced POI.2.To screen the differentially expressed genes and signaling pathways using transcriptome sequencing technologies.3.To explore the possible mechanism of HYF on patient with POI by verifying the screened the Key genes on the signaling pathway using Real Time PCR technologiesMethods1.The study of Pharmacodynamic effect of HYF on treatment of POI ratsForty-four healthy female Sprague-Dawley(SD)rats were randomly divided into 2 groups with the ratio of 3:1.There are 33 rats in the VCD modeling group and 11 in the blank control group.The VCD model group was intraperitoneally injected with VCD solution 160 mg/kg daily for 15 consecutive days to establish a POI rat model.The blank control group was intraperitoneally injected with the same dose of sesame oil once a day for 15 consecutive days.On the 10th day after the end of VCD modeling,27 SD rats were randomly divided into 3 groups,with 9 in each group:VCD model group,HYF low dosage group,and HYF high dosage group.At the same time,9 SD rats with intraperitoneal injection of sesame oil were randomly selected as blank control group.The low-dose group was given 0.135g/kg of HYF with intragastric administration,bid for 25 days;the high-dose group was given 0.235g/kg of HYF same way with low-dose group;the VCD model group and the blank control group were given the same volume of normal saline twice a day for 25 days.Observe the general condition of the rats,including food and drink intake,body weight,bladder and bowel function,coordination,survival,mobility and hair condition,etc.Meanwhile,vaginal exfoliated cell smear was taken to observe the estrous cycle changes of rats in each group.Samples were taken with anesthesia,and E2,FSH,AMH,ALT,AST,BUN and Cr were detected by enzyme-linked immunosorbent assay.Morphological changes of ovarian tissue were observed,and follicles were counted.2.The study of HYF on treatment of POI rats based on the TranscriptomicsThree ovarian samples were randomly selected from each group(Blank control group,VCD model group,HYF low-dose group,HYF high-dose group)from the sate one experiment.RNA was extracted and quality detection was preformed from the samples.The sequencing library was constructed,the transcripts were assembled and combined,in order to screen the differentially expressed genes.To screen the Significantly enriched differentially expressed genes and signaling pathways analysing the GO function and KEGG function of the differentially expressed genes.3.The study of the effect on MAPK and NF-kB Signaling Pathway by HYFFilter the key genes in the signaling pathway of HYF treatment and verified the key genes by Real Time PCR.Analyse the mechanism of the HYF treatment for POI combining the result with the previous experiments and literature data.Results1.The study of Pharmacodynamic effect of HYF on treatment of POI rats(1)The General condition of rats Compared with the blank control group,the rats in VCD model group was stiff and sparse,dull,unresponsive,lying,chasing each other,biting or wilting,weakened behavioral activities,reduced feeding water,and hard stools;Compared with the model group,the the rats in HYF low-dose group and HYF high-dose was gradually reduced,the reaction was gradually sensitive,the behavioral activities returned to normal,and the food intake gradually increased.(2)Ovarian morphology Compared with the blank control group,the ovarian volume of the VCD model group was atrophied,the structure of follicles in all levels was incomplete or disappeared,the number of antral follicles and mature follicles was significantly reduced,the diameter of follicles was small and the number was small,and the number of granule cells was disordered and the number of layers was small.Decreased,nucleus pyknosis,increased follicles,rare corpus luteum,less interstitial blood vessels,fibrotic hyperplasia and inflammatory cell infiltration,dense medulla.Compared with VCD model group,follicular development level of rats in HYF low-dose group and HYF high-dose group Preferably,the number of follicles increased,the number of antral follicles and mature follicles did not decrease significantly,the granule cells were arranged neatly and the number of layers increased,and the number of atresia follicles decreased slightly.(3)Follicular count Compared with the blank control group,the percentage of follicles in the VCD model group decreased,and the percentage of the number of follicles in the locus increased.Compared with the VCD model group,the percentage of all stages follicles in the HYF low-dose group and HYF high-dose group increase,the percentage of atresia follicles decreases.(4)Rat hormone levels Compared with the blank control group,the levels of E2 and AMH in the VCD model group decreased,and the level of FSH increased.Compared with the VCD model group,the E2 and AMH levels in the HYF low-dose group and HYF high-dose group increased,and the FSH level increased.It is on a downward trend.(5)Safety index evaluation Research result from comparison of the blank control group,VCD model group,HYF low-dose group,HYF high-dose group shows that there were no significant differences in the levels of ALT,AST,BUN and Cr among those groups.2.The study of HYF on treatment of POI rats based on the Transcriptomics(1)Transcriptome sequencing quality The high-quality reads of the original reads of the four groups of ovary sequencing libraries were over 98%,and the base ratio of Q20 was about 95%.The base composition before and after filtration was good,and the sequencing amount reached the saturation standard.The degree of randomness of sample mRNA interruption was poor,and the Pearson correlation coefficient of the intra-group repetitive test was close to 1.(2)Compare the reference genome The reads of the reference genome of the single alignment in the four groups of samples accounted for about 80%of the total reads,and the proportion of the uncompared reads was less.(3)Gene statistics The percentages of known genes detected by blank control group,VCD model group,HYF low-dose group,HYF high-dose group were 76.09%,76.19%,76.25%,and 75.30%.The gene coverage of the four groups of rat samples was good,and the gene expression abundance was different.(4)Pairwise differential gene statistics The variations in gene expression were analyzed by comparing the blank control group,VCD model group,HYF low-dose group,HYF high-dose group.A total of 17 genes,including 5 up-regulated genes and 12 down-regulated,were identified in the VCD model group compared to blank control group.A total of 52 genes,including 35 up-regulated genes and 17 down-regulated,were identified in the HYF low-dose group compared to VCD model group.A total of 72 genes,including 19 up-regulated genes and 53 down-regulated,were identified in the HYF high-dose group compared to VCD model group.(5)The KEGG Pathway enrichment analysis Significantly expressed genes mainly involved 26 signaling pathways,including MAPK signaling pathway,PI3K-Akt signaling pathway,JAK signaling pathway,Toll-like receptor signaling pathway,NF-kB signaling pathway,AMPK signaling pathway,The RIG-I-like receptor signaling pathway was significantly enriched.3.The study of the effect on MAPK and NF-kB Signaling Pathway by HYF(1)There were 5 differentially expressed genes enrichment in MAPK signaling pathway.There were 3 differentially expressed genes enrichment in NF-kB signaling pathway.(2)The results of RT-PCR By regulating the expression of CIS,SOCS,TAK1,JAK,MAP2K4,MYD88,TLR5 and MAPKK3 genes,HYF affects the activity of MAPK signaling pathway and NF-kB signaling pathway.Conclusion1.VCD can successfully construct a POI animal model to destroy the Primordial follicle and Preantral follicles selectively.The mechanism of premature ovarian failure may be through the apoptotic pathway.2.HYF can improve the POI rat’s the general condition,the ovarian tissue structure,increase the proportion of follicles at all stages and reduce the proportion of atresia follicles at the same time,with high safety.3.The signaling pathways associated with the treatment of POI rats with HYF are mainly included MAPK signaling pathway,PI3K-Akt signaling pathway,Jak-STAT signaling pathway,Toll-like receptor signaling pathway,NF-kappa B signaling pathway,AMPK signaling pathway,RIG-I-like receptor signaling pathway.4.HYF can improve the ovarian function of the POI rats,the working Mechanism which may be effective via the expression of CIS、SOCS、TAK1、JAK、MAP2K4、MYD88、TLR5、MAPKK3 genes in NF-kB signaling pathway,MAPK signaling pathway.