| Background:In the world,about 72.40 million people are vexed at fertility problem,24%～30%of which is idiopathic asthenospermia.At present,modern medicine has a certain limitation in treatment of idiopathic asthenospermia;and there is no etiological treatment.Chinese medicine possesses certain superiority in treatment of idiopathic asthenospermia.Jingui Yulin Formula is an empirical Formula of Clinic of Andrology in Xiyuan Hospital of China Academy of Chinese Medical Sciences.This Formula possesses the following functions:kidney invigorating and essence replenishing;dispersion of liver stagnation and regulation of qi flow.It has a better efficacy for idiopathic asthenospermia(syndrome of kidney deficiency and liver stagnation).However,its safety and effectiveness are not evaluated systematically;its action mechanism is yet unclear.Therefore,this study was designed to verify its safety,effectiveness and possible action mechanism.This study was divided into three sub-studies:Part I Clinical trial on effectiveness and safety of Jingui Yulin Formula for treatment of idiopathic asthenospermia(syndrome of kidney deficiency and liver stagnation)Objective:To evaluate the effectiveness and safety of this Formula for treatment of idiopathic asthenospermia(syndrome of kidney deficiency and liver stagnation).Methods:This sub-study was a randomized controlled trial.A total of 124 patients were enrolled.From December 2019 to December 2020,they visited the Clinic of Andrology in Xiyuan Hospital of China Academy of Chinese Medical Sciences.They conformed to inclusion/exclusion criteria for idiopathic asthenospermia(syndrome of kidney deficiency and liver stagnation).Through SAS software,random code was generated.According to the proportion of 1:1,these patients were randomly allocated into study group(62 cases)and control group(62 cases).In study group,this Formula was given,with the following Chinese medicines:prepared tortoise shell 15 g,turmeric root tuber 12 g,Chinese thorowax root 12 g,prepared rehmannia root 15 g,antler glue 6 g,dodder seed 10 g,raspberry seed 10 g,fresh malt 12 g,suberect spatholobus stem 10 g,and fresh liquorice 6 g.This Formula was given twice a day,1 bag each time.In control group,Levocarnitine Oral Liquid was given:GuoYaoZhunZi:H19990372;10 mL each time,3 times a day.In both groups,the drug was given for 12 weeks;and the patients were followed up for 4 weeks.Primary efficacy indices included:forward motility of sperm(PR);and overall motility of sperm(PR+NP).Secondary efficacy indices included:score of syndrome of Chinese medicine;fertilization rate;the concentration of sperm and semen volume.Before and after the treatment,the following examinations of safety indices were performed once respectively:blood routine examination,urine routine examination,liver functional examination(ALT and AST),kidney functional examination(BUN and CREA)and ECG examination.Results:A total of 124 patients were enrolled.There were 11 cases of dropout,including:4 cases in study group and 7 cases in control group;overall dropout rate was 8.87%.The effectiveness was analyzed significantly for the data of per-protocol set(PPS).1.Primary efficacy indices:(1)The forward motility of sperm:① Intra-group comparison:As compared with that before the treatment in control group,the forward motility of sperm changed not significantly after treatment for 4 weeks(P>0.05),but increased after treatment for 8 and 12 weeks(P<0.05);as compared with that before treatment in study group,the forward motility of sperm increased significantly after treatment for 4,8 and 12 weeks(P<0.05).②Inter-group comparison in the same period:after treatment for 4 weeks,the increase in forward motility of sperm was not significantly different between two groups(P>0.05);after treatment for 8 and 12 weeks,the increase in forward motility of sperm in study group was superior to that in control group(P<0.05).(2)Overall motility of sperm:①Intra-group comparison:As compared with that before the treatment in control group,the overall motility of sperm increased not significantly after treatment for 4 weeks(P>0.05),but increased after treatment for 8 and 12 weeks(P<0.05);as compared with that before the treatment in study group,the overall motility of sperm increased after treatment for 4,8 and 12 weeks(P<0.05).②Inter-group comparison in the same period:After treatment for 4 weeks,the increase in overall motility of sperm was not significantly different between two groups(P>0.05);after treatment for 8 and 12 weeks,the increase in overall motility of sperm in study group was superior to that in control group(P<0,05),2.Secondary efficacy indices:(1)Score of syndrome of Chinese medicine:①Intra-group comparison:As compared with that before the treatment,the score of syndrome of Chinese medicine decreased not significantly after treatment for 4,8 and 12 weeks in control group(P>0.05);the score of syndrome of Chinese medicine decreased significantly after treatment for 4,8 and 12 weeks in study group(P<0.05).②Inter-group comparison in the same period:After treatment for 4,8 and 12 weeks,the decrease in score of syndrome of Chinese medicine in study group was superior to that in control group(P<0.05).(2)Semen volume:As compared with that before the treatment,the change in semen volume was not significantly different after treatment for 4,8 and 12 weeks in both groups(P>0.05).(3)Concentration of sperm:As compared with that before the treatment,the change in the concentration of sperm was not significantly different after treatment for 4,8 and 12 weeks in both groups(P>0.05).(4)Fertilization rate:As compared with that before the treatment,the change in fertilization rate was not significantly different after treatment for 4,8 and 12 weeks in both groups(P>0.05).3.Evaluation of safety:There were 7 cases of mild adverse events,including:4 cases in study group and 3 cases in control group;after the guidance on administration method,these discomfort symptoms were relieved spontaneously.No adverse reactions were noted.In both groups,obvious abnormality was not shown at liver functional examination(ALT and AST)and kidney functional examination(BUN and Cr).Conclusion:For increasing the forward motility and overall motility of sperm and for reducing the score of syndrome of Chinese medicine in the patients with idiopathic asthenospermia,Jingui Yulin Formula is superior to Levocarnitine Oral Liquid,and does not cause obvious toxic effect.Therefore,the effectiveness and safety of this Formula is verified for treatment of idiopathic asthenospermia(syndrome of kidney deficiency and liver stagnation).Part Ⅱ Pharmacodynamic test on Jingui Yulin Formula in rat model of Ornidazole-induced asthenospermiaObjective:To observe the influence of this Formula on the quality of semen,the pathomorphology of testis/epididymis,sex hormone and liver/kidney function in rat model with Ornidazole-induced asthenospermia.To verify the effectiveness and safety of this Formula for improving asthenospermia in rats.Methods:60 male SD rats of SPF grade were first numbered.According to the random number table,they were randomly allocated into six groups:control group,model group,study groups(including:low-dose group,middle-dose group and high-dose group)and Levocarnitine group.In each group,there were 10 rats.In control group,0.5%CMC-Na Solution-was given at 1 mL/100 g;in model group,Ornidazole was given at 400 mg/(kg·d);in low-dose group,the rats were treated through Jingui Yulin Formula 4.9 g/(kg·d)(crude drug)+Ornidazole 400 mg/(kg.d);in middle-dose group,the rats were treated through Jingui Yulin Formula 9.7 g/(kg·d)(crude drug)+Ornidazole 400 mg/(kg.d);in high-dose group,the rats were treated through Jingui Yulin Formula 19.4 g/(kg·d)(crude drug)+Ornidazole 400 mg/(kg.d);in Levocarnitine group,the rats were treated through Levocarnitine 100 mg/(kg.d)+Ornidazole 400 mg/(kg.d).As for the time of intragastric lavage in study groups,Ornidazole 400 mg/(kg.d)was given in the morning;this Formula was given in the afternoon;both drugs were given for 28 days.After the fasting of food for 24 hours at the last administration,intraperitoneal anesthesia was performed;body weight of rats and weight of testis and epididymis were measured;visceral coefficient of testis and epididymis was calculated.The cauda of left epididymis was homogenated;after warm bath,the motility of sperm was assessed.A fixed staining was made on right epididymis and testis of rats.Blood was collected from abdominal aorta;the following indices were detected through ELISA method and biochemical analyzer:sex hormone(FSH,LH and T),liver function(ALT and AST)and renal function(UREA,BUN and CREA).Results:1.General condition of rats:In control group,food intake changed not obviously;hair was dense and lustrous;activity degree was good;mental state was moderate;urine and stool were not obviously abnormal.In model group,food intake decreased;hair was loose,and luster of hair decreased;less and slow activity occurred in some rats;the rats were tired,listless or irritable;there was a loose semiliquid stool and yellowish urine.In Levocarnitine group,food intake increased;mental state was good;activity was flexible;urine was of normal color;stool was occasionally too loose.Study groups were compared as follows with model group:food intake increased;hair was loose but lustrous;activity degree increased;mental state was flexible;some rats were irritable;the texture of stool was improved;the color of urine was restored to normal state.In middle-dose group and high-dose group,mental state and food intake were obviously improved than that in low-dose group.2.Influence of this Formula on the quality of sperm:(1)Motility of sperm:As compared with that in model group,the increase in forward motility and motility of sperm was significantly different in middle-dose group,high-dose group and Levocarnitine group(P<0.05);as compared with that in Levocarnitine group,the increase in overall motility of sperm was superior in high-dose group(P<0.05).(2)Density of sperm:As compared of model group with control group and other groups,the concentration of sperm was not significantly different(P>0.05).3.Change in weight and visceral coefficient of testis and epididymis:(1)Change condition of testis:As compared of model group with control group and other groups,the weight and visceral coefficient of testis was not significantly different(P>0.05).(2)Change condition of epididymis:As compared with that in control group,the weight and visceral coefficient of epididymis decreased in model group(P<0.05);as compared with that in model group,the weight and visceral coefficient of epididymis increased in high-dose group(P<0.05).4.Influence of this Formula on liver/kidney function:As compared of model group with control group and other groups,liver function(ALT and AST)and renal function(UREA,BUN and CREA)were not significantly different(P>0.05).Conclusion:1.After the intragastric lavage of Ornidazole 400 mg/(kg.d)for 28 days,body weight of rats,weight and visceral coefficient of testis and concentration of sperm are not obviously influenced;epididymis is injured to cause a decrease in motility of sperm.2.In rat model of Ornidazole-induced asthenospermia,this Formula improves the injury of epididymis,and increases the motility of sperm.In high-dose group,the increase in motility of sperm is superior to that in Levocarnitine group.After the administration of this Formula,liver/kidney function(ALT,AST,UREA,BUN and CREA)and sex hormone(FSH,LH and T)were not obviously influenced.This Formula is safe and effective.PartⅢ Exploring action mechanism of Jingui Yulin Formula based on Pink1/Parkin signal pathway for regulating mitophagy to improve the rats in rat model of Ornidazole-induced asthenospermiaObjective:To explore the possible mechanism of this Formula for asthenospermia treatment from the angle of mitophagy mediated by Pinkl/Parkin signal pathway.To further reveal the possible mechanism of Ornidazole for asthenospermia induction in rats and the protective effect of this Formula for rats.Methods:40 rats were randomly allocated into four groups:control group,model group and study groups(including:low-dose group and high-dose group).After the intragastric lavage,the samples for test were the same as those in sub-study 2.The oxidative stress of sperm was determined through activity test kit of SOD,MDA and GSH-Px;MMP for mitochondrial function of sperm was examined through JC-1 mitochondrial membrane potential test kit and flow cytometer;ATP content in mitochondrion of sperm was detected through test kit.Expression level of mRNA and protein related to mitophagy of sperm was determined through Real-time PCR and Western Blot:Pink1,Parkin,p62,LC3Ⅱ/Ⅰ,TOM20 and Beclin1.Results:1.Change in indices for oxidative stress of sperm:As compared with that in control group,MDA content in sperm of epididymis in model group rose(P<0.01)but the content of SOD and GSH-Px decreased in model group(P<0.01).As compared with that in model group,MDA content decreased(P<0.05)but the content of SOD and GSH-Px rose in each study group(P<0.01,P<0.05).2.Change in indices for mitochondrial function of sperm:(1)Mitochondrial membrane potential(MMP):As compared with that in control group,MMP in mitochondrion of sperm decreased obviously and early apoptosis rate of sperm increased in model group,whose difference was statistically significant(P<0.01,P<0.001);as compared with that in model group,MMP rose and early apoptosis rate of sperm decreased in each study group(P<0.01,P<0.05).(2)ATP content in mitochondrion:As compared with that in control group,ATP content in mitochondrion of sperm decreased significantly in model group,which was statistically different(P<0.001);as compared with that in model group,ATP content in mitochondrion of sperm increased not significantly in low-dose group(P>0.05),but increased significantly in high-dose group increase(P<0.01).3.Change in mRNA and protein related to mitophagy:(1)As compared with that in control group,relative expression level of mRNA and protein of Pink1 increased in model group(P<0.01);as compared with that in model group,relative expression level of mRNA and protein of Pink1 decreased in each study group(P<0.01,P<0.05).(2)As compared with that in control group,relative expression level of mRNA and protein of Parkin increased in model group(P<0.01);as compared with that in model group,relative expression level of mRNA and protein of Parkin decreased in each study group(P<0.01,P<0.05).(3)As compared with that in control group,relative expression level of mRNA and protein of p62 decreased in model group(P<0.01);as compared with that in model group,relative expression level of mRNA and protein of p62 increased in each study group(P<0.01,P<0.05).(4)As compared with that in control group,relative expression level of mRNA and protein of LC3Ⅱ/Ⅰ increased in model group(P<0.01);as compared with that in model group,relative expression level of mRNA and protein of LC3Ⅱ/Ⅰ decreased in each study group(P<0.01,P<0.05).(5)As compared with that in control group,relative expression level of mRNA and protein of TOM20 decreased in model group(P<0.01,P<0.05);as compared with that in model group,relative expression level of mRNA of TOM20 increased in each study group(P<0.01,P<0.05),and relative expression level of protein of TOM20 increased in low-dose group(P<0.05).(6)As compared with that in control group,relative expression level of mRNA and protein of Beclinl increased in model group(P<0.01,P<0.05);as compared with that in model group,relative expression level of mRNA and protein of Beclinl decreased in each study group(P<0.01,P<0.05).Conclusion:1.Possibly through the injury of oxidative stress in rat model of Ornidazole-induced asthenospermia,expression level of mRNA and protein of Pink1,Parkin,LC3-Ⅱ/Ⅰ and Beclinl is up-regulated;but expression level of mRNA and protein of p62 and TOM20 is down-regulated.Therefore,the mitophagy of sperm is over-activated by Ornidazole possibly through the injury of oxidative stress.2.After the administration of this Formula in rat model of Ornidazole-induced asthenospermia,expression level of mRNA and protein of Pink1,Parkin,LC3-Ⅱ/Ⅰ and Beclinl is down-regulated;but expression level of mRNA and protein of p62 and TOM20 is up-regulated.Therefore,asthenospermia is improved by this Formula possibly by inhibiting the mitophagy pathway of Pink1/Parkin. |
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