| Objective:The incidence of male infertility has increased year by year,especially in oligoasthenozoospermia.The treatment of Western medicine has some limitations,the curative effect is not ideal.Traditional Chinese medicine(TCM)is special in theoretical system and diagnostic and therapeutic strategies,which is empirical in the prevention and treatment of male infertility.Combined with the previous study,it has been found that zuoggui pill can obviously inhibit the apoptosis of spermatogenic cells and promote the proliferation of spermatogenic cells.The aim of this study is to further evaluate the clinical efficacy of zuigu pill on males with infertility oligoasthenozoospermia due to deficiency of kidney yin and further explore its mechanism of anti apoptosis,proliferation and differentiation.Methods:Clinical study:No case control method was used in the study.Males with infertility oligoasthenozoospermia due to deficiency of kidney yin who met the inclusion criteria were enrolled,whose conditions like general data,symptoms,signs,treatment history and complications were recorded,and TCM syndromes were scored and recorded.All of 44 patients received Zuogui Pill fonnula granules,with therapeutic cycle of 90 days.The TCM syndrome scales of each patient and all adverse reactions occurred during medication were recorded in detail on post-treatment d30,60 and 90.The quality of sperms was detected before treatment and on post-treatment d90,for consecutive twice within one week,and the results with the maximal sperm count were recorded.The clinical efficacy of Zuogui Pill in the treatment of oligoasthenozoospermia due to deficiency of kidney yin,the effect of Zuogui Pill on patients' sperm volume,sperm concentration,total sperm count,percentage of spenns with forward movement,and total vitality of sperms,and the efficacy of Zuogui Pill in improving total TCM syndrome scales and syndrome scales related to deficiency of kidney yin were observed.Experiments:1.A total of 60 SD male rats were randomized to blank control group(n=20)and molding group(n=40).Blank control group received deionized water for 4 weeks while molding group received GTW 40mg/(Kg·d)for 4 weeks,then 4 rats were randomly selected from each group for model validation.After successful molding,12 rats were selected from blank control group as blank control,while 24 rats were selected from molding group and randomized to GTW model group and Zuogui group by random number table,with 12 in each group.Blank control group and GTW model group were treated with gastric gavage of deionized water while Zuogui group with gastric gavage of Zuogui Pill 6 g/(Kg·d)(6 times human clinical dose)for 4 weeks;2.To evaluate the effect of Zuogui Pill on the sperm quality,gonadal hormone levels,pathological morphology of testis,and ultrastructure of testicular tissues of rats models with dyszoospermia;3.The expression levels of c-kit and Oct4 proteins and mRNA in testicular tissues in the three groups were detected using Western blot and real-time polymerase chain reaction(PCR).Results:Clinical study:Compared with the baseline level,44 patients' sperm volume(P<0.05),and other indexes(P<0.01)including total vitality of sperms,percentage of sperms with forward movement,sperm concentration and total sperm count were increased significantly.The scales of TCM syndromes and syndromes related to deficiency of kidney yin showed a decreasing tendency,with significant difference compared with the baseline levels.The overall response rate was up to 93.18%.None suffered any adverse reactions during study.Experiments:1.Compared with GTW model group,general status was significantly improved in Zuogui group,and there was a significant difference in organ coefficients of testicular tissues(P<0.05)and organ coefficients of epididymis tissues(P<0.01)between these two groups.Sperm levels,sperm motility,and percentage of forward moving sperms were significantly higher in Zuogui group than those in GTW model group(P<0.01).Compared with GTW model group,Zuogui group showed improvement of pathological tissue injury,obvious enlargement of lumen and increase of sperms,with partial testicular follicles restoring to normal.The ultra-structure injury of seminiferous epithelium of testicular tissues was recovered significantly in Zuogui group,and the recovered ultra-structure was similar to that in blank control group.In Zuogui group,the spermatogenic cells were intact in morphology and structure,few denatured organelles could be found,the number of mitochondria increased,and sperms and Golgi apparatus could be observed.2.The levels of follicle-stimulating hormone(FSH),estrogen(E2)and luteinizing hormone(LH)were notably higher in Zuogui group than those in GTW model group(P<0.01),but were not significantly different from those in blank control group(P>0.05).The levels of prolaction(PRL)in Zuogui group was significantly higher than that in other two groups(P<0.01).The Testosterone(T)level in Zuogui group was significantly improved compared with that in GTW model group(P<0.05),but was not significantly different from that in blank control group(P>0.05).3.In western blot rest,the comparison of expression levels of c-kit and Oct4 proteins in testicular tissues showed no significant difference between groups(P>0.05),but suggested that the levels were the highest in Zuogui group and the lowest in GTW model group.In real-time PCR test,expression level of c-kit mRNA in Zuogui group and GTW model group was decreased compared with that in blank control group respectively(P<0.05),and the level of c-kit mRNA was significantly higher in Zuogui group than in GTW model group(P<0.01).The expression level of Oct4 mRNA was decreased notably in GTW model group compared with that in blank control group(P<0.01),and the expression level of Oct4 mRNA increased significantly in Zuogui group compared with that in GTW model group(P<0.01),but was similar to that in blank control group(P>0.05).Conclusion:1.This clinical study has proved that Zuogui Pill is safe and effective in promoting the quality of sperms,improve the TCM syndrome score of males with infertility oli-goasthenozoospermia due to deficiency of kidney yin.2.Zuogui Pill can significantly improve the general status,notably promote the organ coefficients of testis and epididymis as well as quality of sperms,improve gonadal hormone levels and prominently improve the structure and ultrastructure of testicular tissues of rat models.3.Zuoggui pill can inhibit the apoptosis of spermatogenic cells and promote the proliferation of spermatogenic cells in the rat model of spermatogenesis disorder.The specific mechanism may be related to the increase of Oct4 expression by regulating the content of stem cell factor receptor c-kit on spermatogenic cells.To sum up,Zuogui pill is effective in the treatment of male infertility with deficiency of kidney yin,and its specific mechanism may be by regulating the expression of c-kit,Oct4 protein and mRNA,thus inhibiting apoptosis and promoting proliferation. |
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