Clinical Efficacy And Mechanism Study Of Qianyang Fengsui Pills In Treating Diminished Ovarian Reserve(Deficiency Syndrome Of Both Kidney Yin And Yang)

Objective:1 Systematically to evaluate the clinical efficacy and safety of Qianyang Fengsui Pills in treating diminished ovarian reserve（DOR）（kidney yin and yang deficiency syndrome）.2 To explore the improvement effect of Qianyang Fengsui Pills on ovarian reserve function in DOR rats at;Starting from granulosa cells（GCs）,screen for the material basis of Qianyang Fengsui Pills to improve ovarian reserve function,and explore the mechanism of action of Qianyang Fengsui Pills in treating DOR.Methods:1 Clinical researchA prospective collection of 72 patients diagnosed with DOR（deficiency syndrome of both kidney yin and yang）was conducted.The observation group was treated with Qianyang Fengsui Pills,while the control group was treated with Fenmotong.Both groups were treated for 3 menstrual cycles.The clinical efficacy rate,the modified Kupperman index（KI）score,traditional Chinese medicine syndrome score（total score and single symptom score）,M3 antral follicle count（AFC）,mid menstrual（maturation date of follicles）endometrial thickness,menstrual volume changes,and changes in basal follicle-stimulating hormone（FSH）,serum luteinizing hormone（LH）,and anti mullerian hormone（AMH） before and after treatment were compared,evaluating the therapeutic effect of Qianyang Fengsui Pills on DOR.2 Experimental research（1）Using Ultra performance Liquid Chromatography（UPLC）to analyze the fingerprint of Qianyang Fengsui Pills and evaluate its stability.Selecting 8-week-old female SD rats as experimental subjects,a DOR rat Model was established using cyclophosphamide.By observing the changes in fur,behavior,mental state,and estrus cycle of each group of rats,the establishment of the DOR Model was understood,and the improvement of fur,behavior,and estrus cycle by Qianyang Fengsui Pills was observed;Observe cellular morphological changes,including the numbers of growing follicles at all levels atresia follicles,through HE staining to evaluate their impact on follicular development;Detect serum levels of AMH,FSH and LH using enzyme linked immunosorbent assay（ELISA）to investigate the effect of Qianyang Fengsui Pills on hormone expression in DOR rats;By using immunohistochemistry,the positive rate of follicle-stimulating hormone receptor（FSHR）was observed under an optical microscope,and combined with HE staining,the morphology and thickness of the granulosa cells layer in secondary and antral follicles were observed to evaluate their impact on the number and morphology of GCs;Detection of iron deposition in ovarian tissue using prussian blue staining,combined with immunofluorescence detection of Nuclear receptor coactivator 4（NCOA4）and ferritin heavy chain 1（FTH1）expression,to evaluate their effect on improving autophagy dependent iron death.The above results comprehensively elucidate the effect of Qianyang Fengsui Pills on improving ovarian function,and it is related to autophagy dependent ferroptosis.By gavage to rats,prepare the medicated serum of each group,as well as the medicated serum of single herbs such as phellodendri Chinensis Cortex,Babury Wolfberry Fruit,Common Macrocarpium Fruit,Carapax Etplastrum Testudinis,Aconiti Lateralis Radix praeparata,Anemarrhenae Rhizoma,Schisandrae Chinensis Fructus,Cassia Twig.（2）Human ovarian granulosa cell（KGN）were selected as experimental subjects.A KGN injury Model was established using 4-Hydroperoxycyclophosphamide（4-HC）.Using CCK8 and ELISA to detect the effects of Qianyang Fengsui Pills compound and various single herbs on the proliferation and secretion of GCs,and screen for effective single herbs and main active ingredients;Finally,by using liquid chromatography-mass spectrometry technology,the absolute content of the active ingredients in the Qianyang Fengsui Pills compound was determined,verifying that it is one of the substances that exert therapeutic effects.（3）A Model of KGN injury was established using 4-HC.Intervention with a single active ingredient of traditional Chinese medicine（berberine）was administered,and Fe²⁺,lipid peroxide levels,mitochondrial membrane potential,mitochondrial ultrastructure,as well as protein expression of FTH1,glutathione peroxidase 4（GPX4）,NCOA4,autophagy related 5（ATG5）,autophagy related 7（ATG7）,the autophagy-related protein microtubule-associated protein 1 light chain 3（LC3B）,p62 were detected through flow cytometry,confocal microscopy,transmission electron microscopy,WB,etc.to evaluate berberine’s ability to alleviate autophagy dependent ferroptosis in GCs;Further utilizing gene editing technology,NCOA4 overexpression plasmids were transfected and combined with WB method to detect protein expression of FTH1,NCOA4,GPX4,etc.,to evaluate the effect of Qianyang Fengsui Pills targeting NCOA4 in alleviating autophagy dependent ferroptosis.Results:1 Clinical research results（1）Menstrual changes:the menstrual cycle,menstrual period,menstrual volume,and color quality score of the observation group decreased compared to before treatment（there was no significant difference in improving menstrual color）,the international menstrual bleeding chart score increased,and the thickness of the endometrium increased.The therapeutic effect was comparable to that of the control group.The Qianyang Fengsui Pills can significantly improve menstrual disorders;（2）TCM syndrome:compared with the control group,the observation showed a slight advantage in improving the symptoms of cold limbs,low back pain,and abdominal cold yin depression,the therapeutic effect in improving symptoms of vexing heat in the five hearts,sweating,and insomnia was comparable to that of the control group,indicating that Qianyang Fengsui Pills can effectively alleviate the symptoms of kidney yin and yang deficiency;（3）Ovarian function:FSH and LH were decreased in both groups compared to before treatment,while AMH showed an increasing trend.However,the control group had a greater advantage in reducing FSH.The improvement level of LH and AMH in the two groups is similar.After treatment,both groups showed an increase in AFC,indicating that Qianyang Fengsui Pills can effectively improve serum hormone levels and promote follicular development;（4）KI index:the total score of the improved kupperman Index（KI）decreased in both groups of patients.Among them,the observation group was significantly better than the control group in alleviating symptoms such as sensory abnormalities and temperament（p<0.05）.The therapeutic effect in relieving hot flashes,sweating,insomnia,sexual activity,and urinary system symptoms is comparable to that of the observation group,indicating that Qianyang Fengsui Pills has a certain improvement effect on physical,physiological,and psychological symptoms.（5）Efficacy rate:the clinical efficacy rate of the observation group was 79.41%,while it was 72.73%in the control group;The efficacy rate of traditional Chinese medicine syndrome was 85.29%,while it was 78.79%in the control group.（6）Safety:there were no serious adverse reactions and it had high safety in this study.2 Experimental research results（1）Qianyang Fengsui Pills can restore estrus cycle disorder,delay ovarian atrophy,promote follicular development,maintain hormone balance,and improve DOR in rats.（2）Qianyang Fengsui Pills has a promoting effect on proliferation and secretion of GCs.Through the screening of individual drugs in the formula,it was found that phellodendri Chinensis Cortex has a repairing effect on GCs damage.And the active ingredients berberine（BBR）and phellodendrine were screened,the results showed that BBR has a positive effect on cell proliferation and hormone secretion.At the same time,the liquid chromatography-mass spectrometry technology was used to determine the composition of the compound and analyze the components entering the bloodstream,confirming that BBR can serve as the one of the effective components of Qianyang Fengsui Pills.（3）BBR can inhibit Fe²⁺overload in damaged GCs,reduce lipid peroxide content,increase mitochondrial membrane potential,restore mitochondrial ultrastructure,alter autophagy dependent ferroptosis protein expression,and elucidate that BBR improves DOR by regulating autophagy dependent ferroptosis.After transfection with NCOA4overexpression plasmid,it was found that the expression of FTH1 and GPX4 proteins was significantly reduced.After intervention with BBR,the expression of NCOA4 protein was reduced,while the expression of FTH1 and GPX4 protein was significantly increased.This indicates that BBR targets NCOA4 to alleviate autophagy dependent ferroptosis and improve DOR.Conclusion:1 Clinical studies have found that Qianyang Fengsui Pills can significantly alleviate symptoms of kidney yin and yang deficiency,effectively regulate menstruation,increase the number of antral follicles,and maintain hormone balance.2 In terms of animal phenotype,this formula can significantly restore the estrus cycle,promote follicular development,improve hormone secretion,and alleviate DOR,of which is associated with autophagy dependent ferroptosis.3 Using the KGN cell injury Model,the efficacy of single drugs and active ingredients in the formula of Qianyang Fengsui Pills was screened.Combined with liquid chromatography-mass spectrometry（LC-MS）technology for content determination,it is speculated that BBR may be one of the representative substances exerting therapeutic effects.4 BBR can improve DOR by targeting NCOA4 to inhibit iron overload,reduce lipid peroxidation,and enhance mitochondrial function,thereby alleviating autophagy dependent ferroptosis.