Impact And Mechanism Of Epimedium And Its Ingredients On Murine Female Reproductive Development

The traditional Chinese medicine Epimedium has been used for centuries to promote reproductive health.The Epimedium(EP)herb has been used to treat endocrine disorders,also,the herb is used to treat coldness,infertility,senile functional diseases,gonadal dysfunction,and male impotence and to improve female health.It has also been used as an aphrodisiac and has been reported to improve erectile function in aged male rats.It has also been used on both human beings and animals.In animals,it has been thought to promote reproductive function and has thus been given the name "horny goat weed".Epimedium polysaccharides(EPS)are a key ingredient in Epimedium,and Icariin(ICA)is a flavonoid present in Epimedium species,where it is used for herbal quality control.In the present study,we mainly investigated the effects and mechanisms of action of Epimedium extract,EPS and ICA on the female reproductive development in Kunming mice.We firstly used the reproductive index to assess the effects of Epimedium,EPS and ICA on the development of ovary and uterus.The in vitro maturation was adopted to assess the rate of first polar body(pbI)extrusion.We also used the radioimmunoassay method for analysis of serum,and analyzed the ovaries using real-time quantitative PCR(qRT-PCR),after which we analyzed the ovaries using immunohistochemistry(IHC)and western blot to evaluate for expression of proteins.We had 2 series of experiments,in the first series,which is discussed in Chapter 3 and 4;the experiment comprised 360 mice divided equally into 30 groups.Each group comprised 12 mice.Eight of these groups were orally administered with EP at 2,6,10,or 14 mg/0.1 mL daily for 7 or 14 d.An additional 8 groups were treated with EPS at dosages of 2,4,6,and 8 mg/0.1 mL daily for 7 or 14 d;another 8 groups were administered with ICA at 0.1,0.3,0.5,and 0.7 mg/0.1 ml daily for 7 or 14 d.The second series experiment comprised 480 mice divided equally into 12 groups.The first 2 groups were orally administered with 10 mg/0.1 mL of EP,the next 2 groups were orally administered with 6 mg/0.1 mL of EPS and the last 2 groups were orally administered 0.5 mg/0.1 ml of ICA for either 7 or 14 d while each group had a control which was orally administered with 0.9%physiological saline(sodium chloride)solution for 7 or 14 d.Experiment 1.Effects of Epimedium and its active ingredients on mice ovary developmentGenerally,the results of reproductive index from Epimedium(EP),EPS and ICA groups showed increase compared to the control.After EP treatment in mice the highest reproductive index was 10 mg in the 7-d group and 2mg in the 14-d group.This may be explained by the fact that the 14-d group of mice had by this time more dosage to have gained more weight.The 10 and 14 mg groups showed higher changes in the 7 d while the 6,10 and 14 mg showed increased reproductive index compared to control.In 14-d group the 10 mg was the second highest.The reproductive index for EPS showed that the 4 mg,7 d group had the highest reproductive index,followed by the 4 mg 14 d group.The reproductive index after ICA treatment was highest at 0.5 mg followed by 0.7 mg dosage for 7 d.Under in vitro maturation of oocytes,following the Epimedium treatment,7-d group the highest rate was noted in the 10 mg group at 75.4%(p<0.05),while the maturation rate from 14 d in the 6 mg group was 70.7%(p<0.05).After EPS treatment,the 7-d group showed the 8 mg being 80%(p<0.05),while 14-d group was the 6 mg with 60%.Furthermore after ICA treatment,the 7-d group highest was the 0.5 mg with 75%and the 14-d group it was the 0.3 mg with 68.1%(p<0.05).Experiment 2.Steroid hormonal alterations after Epimedium and its ingredients treatment in female miceThe alterations that Epimedium and its ingredients had on hormone serum levels were evaluated.After EP treatment,we detected no significant difference(p>0.05)in the 17?-E2 or P4 serum levels when compared to control.The EP results showed no significant differences(p>0.05)for either 17?-E2 or P4 between the 7-d and 14-d samples.When mice were treated with EPS,we detected no significant difference(p>0.05)in the 17?-E2 or P4 serum levels when compared to control.However,there were significant differences after EPS treatment when the 7-d and 14-d groups were compared.Administration of EPS at 2 mg resulted in a significant decrease in both 17?-E2 and P4(p<0.05)between the 7-d and 14-d groups.The ICA treatment eventually led to no significant difference(p>0.05)in the 17?-E2 or P4 blood serum levels when fed to mice orally.Comparison of the 7-d and 14-d treatment groups,however,revealed some differences.After treatment with ICA,there was a statistically significant difference(p<0.05)with 17?-E2 in the 0.7 mg group increasing after 14-d of the experiment.There was as well a statistically significant difference(p<0.05)for P4 levels in the 0.1 mg group,which was not the case in the 14-d group.These findings resulted in the choosing of the dosage level to be used.These were 10 mg EP,6 mg EPS and 0.5 mg ICA.Experiment 3.Effect of Epimedium on gene and protein expression in mice ovaryTo evaluate the mechanisms that effect changes of EP on the ovary,the real-time RT-qPCR was used to assess the changes in gene expression of steroidogenic genes(Cyp19a1,Hsdl7bl or Star),growth factor genes(Egf,Igf-2,Ptgs-2,Vegfa,and Tyk-2),and immunohistochemistry and western blot for receptors(ER?,ER? and PRA).The results from the 7-d group showed the significant changes were Hsdl7bl(2.5-fold),Igf-2(2.4-fold),and Ptgs-2(3.4-fold),while for the 14-d group these were Cypl9al(8-fold),Egf(10-fold),and Igf-2(69-fold).The general range was 0.5-to 3.4-fold change in gene expression for the 7-d treatment group and 0.6-to 69.4-fold for the 14-d treatment group.After immunohistochemistry,we found that ERa was more highly expressed in the 7-d EP treatment group compared to control group;ER?,on the other hand,was expressed at a higher intensity in the control compared to the 7-d treatment,as was PRA.In the 14-d treatment group,ER? was expressed at higher intensities compared to ERa.In western blot we analyzed the expression pattern of ERa,ERP and PRA.After EP treatment,there was no significant difference(p>0.05)in ERa expression between the 7-d and 14-d control and treatment groups.However,ER? and PRA showed significant decreases(p<0.05)from control in both the 7-d and 14-d treatment groups.Epimedium treatment resulted in the increase of Igf-2 and Egf.Considering that there exists crosstalk between EGF and ERa,EGF could result to changes in the ovary via binding and activating ERa.Experiment 4.Impact of Epimedium polysaccharide on gene and protein expression in mice ovaryTo evaluate the effect of EPS on the ovary,the genes up-regulated more than twofold were considered significant and they are:Cypl9a1(2.7-fold),Hsd17b1(2.2-fold),Igf-2(2.8-fold),and Tyk-2(2.1-fold)in the 7-d treatment group and Cypl9al(4.5-fold),Hsdl7bl(3.2-fold),Igf-2(15-fold),and Ptgs-2(85-fold)in the 14-d treatment group.The changes in gene expression ranged from 0.8-to 2.8-fold in the 7-d treatment group and from 0.5-to 85-fold in the 14-d treatment group.When the treatment groups were compared with the control,the 7-d treatment stained much darker for ERa than the control.In the 14-d treatment group,the granulosa cells of the tertical follicles stained much darker for ER?compared to ERa.The ERa in the 14-d treatment group consisted of a few scattered granulosa cells showing some expression.Both ER expressions were stronger in the 7-d treatment group compared to the 14-d treatment group.PRA was weakly expressed in the cytoplasm of thecal cells.After EPS treatment,ERa expression in both the 7-d and 14-d treatment groups was significantly increased(p<0.05)compared with control.On the other hand,ER(3 was increased significantly(p<0.05)in the 14-d treatment group when compared with the 14-d control group.There was,however,no significant difference(p>0.05)between 7-d control group and the 7-d treatment group.PRA expression was significantly decreased(p<0.05)in both the 7-d and 14-d treatment groups.EPS was found to utilize a mode of action slightly different from its parent plant extract Epimedium.In this study,we demonstrated that EPS acted on steroidogenesis and follicular development genes by altering mRNA levels and protein expression of estrogen and progesterone receptors.EPS increasingly enhanced mRNA expression of Ptgs-2 and Igf-2.Experiment 5.Icariin affects gene and protein expression in mice ovaryHere evaluation of the action of ICA on the ovary was mainly focused on the gene and protein expression.Using the method discussed above,we found that ICA treatment led to the up-regulation of various genes;those over twofold magnitude after the 7-d treatment were as follows:Cyp19a1(34-fold),Hsd17b1(3.2-fold),Igf-2(33-fold),and Ptgs-2,19-fold.On the other hand,14-d treatment resulted in the up-regulation of Egf(4-fold),Ptgs-2(3.2-fold),and Tyk-2(10-fold).No major differences were observed for Star(7 d,1.3-fold;14 d,1.4-fold),Hsd17b1(14 d,0.9-fold),and Tyk-2(7 d,0.99-fold).Vegfa(7 d,0.89-fold;14 d,0.49-fold)and Egf(7 d,0.7-fold)were down-regulated.The expression pattern of ERa in the 7-d treatment in tertical follicles was such that the cells surrounding the oocyte showed the most expression,and it decreased toward the basement membrane and thecal cells.ERa was expressed in the oocyte,granulosa cells of the primary,secondary,and tertical follicles,and the corpus luteum(CL)of the 7-d and 14-d control and 7-d and 14-d treatment groups.However,the thecal cells did not exhibit any expression of ER?,ER?,and PRA.PRA was mildly expressed in the cytoplasm of some CL cells in the treatment group.ERa expression was higher in the treatment groups compared with control.However,PRA was expressed more in the control than in the treatment groups.We found that after ICA treatment,using western blot that ERa expression was significantly increased(p<0.05)after 7-d treatment,but decreased significantly(p<0.05)after 14-d treatment.ER?expression was significantly decreased(p<0.05)in both the 7-d and 14-d treatment groups.Similarly,PRA was significantly decreased(p<0.05)in both treatment groups.Our experiment confirmed that Ptgs-2 expression was increased and Igf-2,Egf and Tyk-2 expressions varied depending on duration of treatment.Icariin could possibly regulate ovarian follicular development through the estrogen receptor pathway by increasing mRNA expression of some genes associated with steroidogenesis(e.g.Hsd17b1 and Cyp19a1)or others.ERa expression was evidently increased for those groups treated with ICA for 7 d.Longer treatment with ICA may have negative effects as shown by the decreased receptor expression.In summary,we found that Epimedium and its main ingredients EPS and ICA did impact the reproductive development in mice.They could positively improve the reproductive index and in vitro maturation rates and act on the development of ovarian follicles.The action pathway of Epimedium and its ingredients EPS and ICA may have some differences,but they acted through changing the expressions of associated genes and proteins,such as Ptgs-2,Igf-2,Egf and ERa.