| Herba Epimedium(EP)is a traditional Chinese medicine which has been used for more than 2000 years.It is one of the most widely used and long-standing traditional Chinese medicines(TCM).As a kind of tonic,EP has many effects,such as enhancing kidney function,strengthening muscles and bones,and dispelling wind dampness.In addition,it also has effects on protein synthesis promotion,anti-aging,improving immune function,and inhibiting tumor.Many pharmacological effects of EP have been reported by scholars at home and abroad.At present,more than 270 compounds have been extracted from EP.It has been found that icariin(ICA)and Epimedium Polysaccharide(EPS)are also effective active components of EP.The puberty of male mice lasts for about 20 days.Therefore,in this study,5-week-old male Kunming mice were treated with EP,EPS,and ICA via oral gavage for 20 days.After the experiment,the mice were sacrificed and their testis,epididymis,and blood samples were collected.Haematoxylin and eosin(HE)staining was used to observe morphological changes of testis.Sperm density of epididymis,serum testosterone(T),and follicle stimulating hormone(FSH)levels,as well as expression levels of testosterone synthesis regulatory genes 3β-HSD,17β-HSD,and StAR were also detected.The objective of this study also included an evaluation of the effects of EP,EPS,and ICA on antioxidant capacity of testis and expression of apoptosis related genes Bax and Bcl-2.These will help to better understand the effects and mechanism of EP,EPS,and ICA on reproductive development of puberty male mice.Male Kunming mice reached sexual maturity at about 8 weeks old,and spermatogenesis cycle of mice lasts for about 35 days.Therefore,9-weeks-old male mice were treated with ICA and nicotine for 35 consecutive days.According to our results,the optimal drug(ICA)dose for improving sperm density and antioxidant capacity was 3 mg(75 mg/kg BW).Furthermore,the effects of ICA on spermatogenesis,serum testosterone level,and antioxidant capacity of testis were studied.Meanwhile,the protective effect of ICA on nicotine-induced reproductive toxicity was also evaluated while sperm density of epididymis and serum testosterone level was also investigated.The relative expression of testosterone synthesis regulatory genes and antioxidant capacity of testis were further examined.To facilitate understanding of the protective effect and mechanism of ICA on nicotine induced reproductive toxicity,and provide basic information on ICA regarding its’ potential to reduce or improve nicotine-induced reproductive function decline,this study includes four parts as follows.Experiment 1.Effects of Epimedium and its main active ingredients on reproductive organ development,spermatogenesis,and testosterone secretion in puberty male miceThe aim of this study was to investigate the effects of EP,EPS,and ICA on reproductive organ development,epididymal sperm density,and testosterone secretion in puberty male Kunming mice.Ninety(90)4-week-old healthy male Kunming mice were randomly divided into EP,EPS,and ICA groups after one week adaptive feeding.The control group was given 0.1 ml normal physiological saline.Mice in each experimental group were given different doses of EP,EPS,and ICA each day for 20 consecutive days.When continuous drug administration ended,the mice were sacrificed by cervical dislocation.Testis,epididymis,and blood samples were collected.The whole blood sample was placed in room temperature for 2 h and later centrifuged to obtain blood serum.Subsequently,serum testosterone and FSH levels were analyzed by radioimmunoassay(RIA).Testicular tissue 3β-HSD,17β-HSD,and StAR relative mRNA expression levels were detected by real-time PCR SYBRGREEN method.Compared with the control group,10 mg EP,6 mg EPS,3 mg,and 5 mg ICA increased testicular and epididymal indices of mice;except 14 mg EP group.Sperm density of epididymis in other treatment groups was significantly higher than that of the control group.There was no significant difference in serum testosterone concentration between 14 mg group and the control group,and it was lower than that of 2 mg,6 mg,and 10 mg groups,while serum FSH concentration was significantly lower than that of the control group;serum testosterone and FSH concentration of 6 mg EPS group were significantly higher than those of the control group;3 mg EPS group was significantly higher than that of the control group.The serum testosterone concentration of ICA group was also significantly higher than that of the control group,and that of other ICA groups.Serum FSH concentration of ICA groups was significantly higher than that of the control group.The results showed that EP,EPS,and ICA could promote development of puberty male mice,increase reproductive organ index,and sperm density of epididymis.Meanwhile,EP,EPS,and ICA could also promote secretion of testosterone and FSH,and increase the expression of 3β-HSD,17β-SD,and StAR mRNA.Experiment 2.Effects of Epimedium and its main active ingredients on antioxidant and apoptosis related gene expression in testis of adolescent male miceThe aim of this study was to investigate the effects of EP,EPS,and ICA on antioxidant enzyme activity and expression of apoptosis related genes Bax and Bcl-2 in the testis of puberty male Kunming mice,as well as to explore the mechanism of how EP,EPS,and ICA affect antioxidants of testicular tissue and apoptosis of testicular cells.5-week-old male Kunming mice were given different doses of EP,EPS,and ICA by oral gavage,and the control group was given normal physiological saline.After 20 days,the mice were sacrificed by cervical dislocation.Testis,epididymis,and blood samples were collected,and the testes were cryopreserved at-80℃.The activity of SOD,GSH-Px,and content of MDA in the testicular tissue were analyzed by SOD,GSH-Px,and MDA kits,while relative expression of SOD1,SOD2,SOD3,and Gpx1 mRNA in mouse testicular tissue was detected by real-time PCR SYBRGREEN method.Real time PCR SYBRGREEN method was also used to detect relative expression of 3β-HSD,17β-HSD,StAR,Bax,and Bcl-2 mRNAs in the mice testes.Western blot was used to detect the expression of apoptosis related proteins(Bax and Bcl-2).The results showed that activities of SOD and GSH-Px in EP 6 mg,10 mg,EPS 6 mg,8 mg,and ICA 3 mg groups were higher than that of the control group.RT-PCR results showed that EP,EPS,and ICA increased the relative expression of SOD1,SOD2,SOD3,and Gpx1 mRNA in testis;meanwhile,EP,EPS,and ICA could increase the relative expression of Bcl-2 mRNA and reduce Bax mRNA expression.The expression of Bcl-2 and Bax proteins increased with increased Bcl-2/Bax ratio.Experiment 3.Icariin alleviates nicotine-induced reproductive toxicity on spermatogenesis and reduced testosterone secretionThe aim of this study was to investigate the effect of ICA on decreased sperm number and testosterone secretion induced by nicotine treatment in male Kunming mice,and to explore the mechanism of how ICA alleviates nicotine-induced spermatogenesis and testosterone secretion reduction.Forty healthy mice were randomly divided into control group,nicotine group,ICA group and nicotine+ICA group.After 35 days of continuous administration,the mice were sacrificed by cervical dislocation.Testis,epididymis,and blood samples were collected and frozen at-80℃.The expression of 3β-HSD mRNA was detected by real-time PCR.The results showed that sperm density and serum testosterone content in the ICA group were significantly higher than those in the control group.Sperm density and serum testosterone content in the nicotine group were significantly lower than those in the control group,sperm density and serum testosterone content in nicotine+ICA group were significantly higher than those in nicotine group.The expression of 3β-HSD,17β-HSD and StAR mRNA in the ICA group was significantly higher than that in the control group.The expression of 3β-HSD and StAR mRNA in nicotine group was significantly lower than that in the control group,while a decreased expression trend of 3β-HSD and StAR mRNA was reversed in the nicotine+ICA group.Experiment 4.Icariin alleviates nicotine-induced oxidative stress in testisThe aim of this study was to investigate the effect of ICA on nicotine induced oxidative stress in testis of male Kunming mice and explore the mechanism of how ICA alleviates nicotine-induced oxidative stress in testicular tissues of mice.The mice were randomly divided into nicotine+ICA group and control group.After 35 days of continuous administration,the mice were sacrificed by cervical dislocation.Testis,epididymis,and blood samples were collected and frozen at-80℃.The activity of SOD and GSH-Px as well as content of MDA in testicular tissue was analyzed by SOD,GSH-Px and MDA kits.Relative expression of SOD1,SOD2,SOD3,and Gpx1 mRNA in mouse testicular tissue was detected by real-time PCR SYBRGREEN method.The results showed that activities of SOD and GSH-Px in the ICA group were significantly higher than in the control group,and the content of MDA was significantly lower than in the control group;the activities of SOD and GSH-Px in the nicotine group were significantly lower than those in the control group,and the MDA content was significantly higher than that in the control group;the SOD activity of the testicular tissue in the nicotine+ICA treatment group was significantly higher than that in the control group,and there was no significant difference in GSH-Px activity and MDA content between the two groups.The results of RT-PCR showed that relative expression of SOD1,SOD3,and Gpx1 mRNA in testis of ICA group was significantly higher than that in the control group.Also,relative expression of SOD1 in testis of nicotine group was significantly lower than that in the control group,while its expression in the testis of nicotine+ICA group was significantly higher than in the control group.These results showed that ICA could alleviate decrease antioxidant enzyme activity and increase MDA production in nicotine-induced testicular tissues of mice.This will improve the antioxidant capacity of testicular tissues and alleviate oxidative stress caused by nicotine treatment. |
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