| Objective:1.This study was conducted to investigate the effectiveness and safety of Yuzi Formula for the treatment of weak spermatozoa through clinical observation to confirm the clinical efficacy of Yuzi Formula for the treatment of weak spermatozoa;2.To observe the effect of Yuko Formula on the mitochondrial autophagy signaling pathway by observing the quantitative relationship between mitochondria,autophagic vesicles and autophagic lysosomes through transmission electron microscopy;3.To conduct a preliminary study on the apoptotic mechanism of Yuko Formula in improving sperm viability of patients by observing the expression of relevant regulatory proteins that can promote mitochondrial autophagy and thus inhibit spermatogenic cell apoptosis,using mitochondrial autophagy as an entry point.Methods:1.A total of 65 patients were selected according to the inclusion,exclusion,exclusion,and shedding criteria consistent with weak spermatozoa infertility of the kidney deficiency and blood stasis type.Dosage:The fertility formula was decocted uniformly by the decoction machine of the decoction room of Yunnan Provincial Hospital of Traditional Chinese Medicine,and 150 ml/time was taken orally,divided into three doses in the morning,midday and evening,and the course of treatment was three months.To compare the clinical treatment effect of Chinese medicine,semen quality analysis was performed by CASA system,and the semen quality judgment criteria in the fifth edition of the WHO Laboratory Manual for Examination and Processing of Human Semen were used as reference,and the changes of sperm motility before,one month,two months and three months after treatment were strictly collected from patients(abstinence for 3-7 days before each examination).A computer-assisted sperm analysis system was applied to do a self-control study on the sperm quality of the included patients before and after treatment.2.5 patients were randomly selected among 65 cases included in the outpatient clinic that met the inclusion criteria,and the number of mitochondria,the distribution of autophagic vesicles and the number of autophagic lysosomal vacuoles in each group were observed by transmission electron microscopy.3.After purification of sperm from 5 randomly selected patients,the following indexes were detected:(1)apoptosis rate of sperm cells in patients before and after treatment with the fertility formula;(2)changes in m RNA expression of LC3 and LAMP2,mitochondrial autophagy-related genes in spermatozoa of patients before and after treatment with the fertility formula,during the improvement of weak spermatozoa.(3)Western blotting detected the changes of mitochondrial autophagy lysosomal proteins and mitochondrial autophagy signature proteins LAMP2 and LC3 levels in sperm cells of patients before and after the treatment of fertility formula.Results:1.Effect of Yuzi formula on sperm motility in patients with weak spermatozoa with kidney deficiency and blood stasisThe statistical data of the percentage of forward motility sperm before and after treatment in 65 cases were in accordance with normal distribution.The paired t test showed that the percentage of forward motility sperm after 1 month(23.74±4.75%),2months(27.50±5.85%)and 3 months(30.31±6.88%)was significantly different from that before treatment(19.08±4.51%)(P<0.05).The statistical data of forward motility sperm and non-forward motility sperm before and after treatment in 65 cases were in accordance with the normal distribution.The paired t test showed that the percentage of forward motility sperm and non-forward motility sperm after 1 month(34.35±6.47%),2months(38.94±7.91%)and 3 months(42.56±9.31%)were significantly different from those before treatment(29.45±6.19%)(P<0.05).The statistical data of sperm concentration before and after treatment in 65 cases were in accordance with the normal distribution.The paired t test showed that the sperm concentration of 1 month(48.67±9.47*10~6/m L),2months(49.05±9.97*10~6/m L)and 3 months(49.24±9.49*10~6/m L)After 3 months of treatment,6 cases were cured(9.22%),16 cases were markedly effective(24.62%),34cases were effective(52.31%),9 cases were ineffective(13.85%),and the total effective rate was 86.15%.2.Changes in mitochondrial number,autophagic vesicle distribution and autophagic lysosomal vacuole-like counts of spermatozoa affected by kidney deficiency and blood stasis type of weak spermatozoa in Yukozi formulaA small amount of autophagic vesicles could be detected in 5 included cases,mitochondria were relatively more with autophagic lysosomes,mitochondria were unevenly divided,and most autophagic lysosomal vacuoles were seen around mitochondria,and the number of mitochondria and lysosomes tended to increase after treatment,and the study indicated the increase of mitochondrial autophagy in male spermatozoa during the treatment of Yukio formula.3.Effect of Yuko formula on the apoptosis rate of sperm cells in patients with weak spermatozoa with kidney deficiency and blood stasis,and on the expression of autophagy-related indexes LC3、LAMP2 protein and m RNAThe statistical data of sperm cell apoptosis rate before and after treatment in 5included cases conformed to normal distribution,and paired t-test was performed,and the results showed that there was a significant difference(P<0.05)in sperm cell apoptosis rate expression in patients before treatment(34.44±4.42%)and after treatment(25.96±4.79%);5 included cases The statistics of LC3 and LAMP2 expression in spermatozoa detected by fluorescence fixed PCR before and after treatment conformed to normal distribution,and paired t-test showed that there was a significant difference(P<0.05)in LC3 expression in patients before treatment(27.51±0.43)and after treatment(29.09±0.55)compared with that before treatment.There was a significant difference(P<0.05)in the expression of LAMP2 in patients before treatment(0.97±0.34)and after treatment(1.26±0.28)compared with that before treatment.5 included cases had LC3 and LAMP2 expression in spermatozoa detected by WB before and after treatment statistically conformed to normal distribution,and paired t-test showed that:LC3 before treatment(2.62±0.23),after treatment(3.09±0.23)patients LC3 expression was significantly different than before treatment(P<0.05);LAMP2(0.73±0.29)before treatment,after treatment(1.17±0.37),patients LAMP2 expression was significantly different than before treatment(P<0.05).Conclusions:1.Yuzi formula soup can effectively improve sperm vitality of patients with weak spermatozoa after 3 months of treatment.2.The level of mitochondrial autophagy in spermatozoa of patients with kidney deficiency and blood stasis increased during the treatment of patients with weak spermatozoa by Yuzi Formula.3.Yuzi Formula improves sperm motility by decreasing the rate of sperm apoptosis,upregulates the expression of autophagy-related indexes LC3,LAMP2 protein and m RNA,and promotes mitochondrial autophagy,which play a positive role in the regulation of male sperm motility. |
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