Study On The Treatment Of Oligoasthenozoospermia And The Regulation Of The Proliferation And Apoptosis Of Spermatogenic Cells By The Method Of Invigorating The Kidney And Producing Sperm

OBJECTIVEHuman infertility has become a medical and sociological issue that affects global development,and male infertility(MI)accounts for 40%-50%of it.Among all the causes of MI,oligoasthenozoospermia is one of the most common causes.Western medicine trealment is not very effective,especially for idiopathic semen abnormalities,while traditional Chinese medicine(TCM)has unique advantages and good clinical efficacy.TugouZhuyu Decoction is created from Wuziyanzong pills.It has been used for treating oligoasthenozoospermia for more than 20 years,however,there is no related research about it.Therefore,this study contains a clinical trial and an animal experiment,the former one is to observe and evaluate the clinical efficacy of TugouZhuyu Decoction in patients with oligoasthenozoospermia,and the latter one is to evaluate the therapeutic effect of semen cuscutae and fructus lycii(SC-FL,the core herbal pair of TugouZhuyu Decoction)in glucosides of Tripterygium wilfordii Hook.f(GTW)-induced dyszoospermia rats and elucidate the underlying molecular mechanism.METHODSClinical trial:76 cases of oligoasthenozoospermia were collected,and no case-control method was used to observe the clinical efficacy of TugouZhuyu Decoction on oligoasthenozoospermia patients.Observation indicators include semen quality(semen liquefaction status,semen volume,sperm motility,sperm concentration and sperm count)and TCM syndrome scores.The patients were given daily treatment of TugouZhuyu Decoction in granules for a total of 90 days.Clinic visits were conducted on day0,day30,day60,and day90,respectively,and the TCM syndromes were recorded in the meantime.Semen examination of each patient was carried out before and after treatment and case report form(CRF)of each patient was fill out.Safety observation were performed in the whole process,and any kind of adverse events were recorded.Evaluation of shedding and rejection cases were strictly performed at each visit,and finally all data were applied for statistical treatment.Animal experiment:1.Seventy-eight Sprague-Dauley(SD)rats were randomly divided into five groups:normal control group(NC group),GTW group(treated with saline),GTW+LEV group(treated with levocarnitine),GTW+SCFL group(treated with SC-FL),and LY+SCFL group(treated with SC-FL and LY294002,the PI3K inhibitor).2.Rats of GTW group,GTW+LEV group,GTW+SCFL group,and LY+SCFL group were intragastrically administered GTW(40 mg/kg/d)for 4 weeks to establish dyszoospermia model.3.From the fifth week,rats in the GTW+LEV group were daily administered levocarnitine,and rats of GTW+SCFL and LY+SCFL groups were daily administered SC-FL.Besides,from the start of the study,LY294002 was additionally injected into the tail vein of rats of the LY+SCFL group once a week.Rats of NC and GTW groups were administered with deionized water everyday.4.After 8 weeks,semen quality and organ coefficient were determined and sex hormone,inhibin B,and epididymal carnitine levels were measured.Testicular tissue and spermatogenic cells were observed using optical microscope and electron microscope.Immunohistochemistry,western blotting,and quantitative reverse transcription polymerase chain reaction(qRT-PCR)were used to determine the protein and mRNA expression of SCF,c-kit,PI3K,p-Akt,Bad,Bcl-2,and Bax in rat testis.RESULTSClinical trial:After 90 days' treatment of TugouZhuyu Decoction,the sperm liquefaction status,PR sperm percentage,PR+NP sperm percentage,sperm concentration,and sperm count were significantly improved in oligoasthenozoospermia patients(P<0.05 or P<0.01);The score of TCM syndromes decreased significantly along with the progress of treatment(P<0.05 or P<0.01),There were 4 cases shed during the period,no cases were excluded,and no adverse reactions were reported.Animal experiment:Compared with the GTW group,semen quality,the epididymal organ coefficient,follicle stimulating hormone(FSH),luteinizing hormone(LH),prolactin(PRL),and epididymal carnitine levels were significantly improved in the GTW+SCFL group(P<0.05 or P<0.01).Histomorphology and testicular ultrastructural evaluation showed that in the GTW+SCFL group,the structure and arrangement of seminiferous tubules were better,the amount of spermatogenic cells increased significantly,the morphology of spermatogenic cells improved,and the mitochondria increased,compared to those in the GTW group.Immunohistochemistry,western blotting,and qRT-PCR results showed that compared with the GTW group,the expression of SCF,c-kit,PI3K,p-Akt,and Bcl-2 in the GTW+SCFL group was increased,while that of Bax and Bad was decreased.The expression of p-Akt and Bcl-2 decreased,while that of Bad and Bax increased in the LY+SCFL group compared with the GTW+SCFL group.CONCLUSION1.TugouZhuyu Decoction has significant effects in improving semen parameters and kidney deficiency syndrome of TCM scores in oligoasthenozoospermia patients,thus it can improve the spermatogenic function of patients.2.The core herbal pair of TugouZhuyu decoction(SC-FL)can improve the general state,epididymal coefficients,sperm quality,FSH,LH,PRL and epididymal carnitine of dyszoospermia rats.SCFL has a good repairing effect on the structure and function damage of testicular tissue and spermatogenic cells caused by GTW.3.SC-FL can effectively regulate the protein and mRNA expression of SCF,c-kit,PI3K,p-Akt,Bad,Bcl-2 and Bax in dyszoospermia rats,and can promote the proliferation of spermatogenic cells and inhibit apoptosis of spermatogenic cells through the SCF/c-kit--PI3K--Bcl-2 pathway.Above all,TugouZhuyu Decoction can effectively improve the spermatogenic function of MI patients with oligoasthenospermia,and its clinical effect is significant.Its mechanism may be promoting spermatogenic cell proliferation and inhibiting their apoptosis through the SCF/c-kit--PI3K--Bcl-2 pathway.