| Spermatogenesis is a process which start with spermatagonial stem cells(SSCs)and become mature sperm through mitosis and meiosis in the seminiferous tubule.The process is regulated by variety of cell types,hormones,paracrine factors,genes,and epigenetic regulation factors.Spermatogenesis directly affects sperm count and quality.Eucommiae Folium is the dry leaves of Eucommia ulmoides Oliv.It is pungent and warm in flavor,tonifying the liver and kidney and strengthening bones and muscles,attending the deficiency of liver and kidney.It,which mainly contained chlorogenic acid,genipin,quercetin etc.,has founction of antibacterial,antioxidant and anti-inflammatory.Studies have shown that Eucommiae Folium extract could improve male mice reproductive hormone secretion and promote the spermatogenesis in mice.However,there are few studies on active compounds and their mechanism.The purposes of this study to detect the spermatogenic activity and mechanism of Eucommiae Folium ethanol extracts in mice,the main active compounds and their mechanism through network pharmacology active compounds analysis,screening of active compounds to enhance SSCs proliferation and promote testosterone secretion of leydig cells in vitro,detection of active compounds in spermatogenic activity,and inhibition of active compounds in cyclophosphamide reproductive toxicity.The following results have been achieved so far:(1)Preparation of Eucommiae Folium ethanol extract and detection of the extract in spermatogenesis promotion.The extract of Eucommiae Folium was prepared by ethanol extraction method,and the quality control component chlorogenic acid was detected by HPLC.The spermatogenic activity of the extract was detected by spermatogenesis activity test in mice.The results showed that the yield of ethanol extract was 41.25%,and the content of chlorogenic acid was 26.67 mg/g.Ethanol extracts(0.8 and1.2 g/kg)could effectively promote the secretion of testosterone and FSH(P<0.05),and increase sperm count in testis and epididymis(P<0.05).The results indicated that Eucommiae Folium ethanol extract could promote spermatogenesis in mice.(2)Screening of active components from Eucommiae Folium ethanol extract regulating SSCs proliferation and self-renewal,and its mechanism.The compounds related to spermatogenesis in Eucommiae Folium were screened by network pharmacology.The compounds were screened by the promotion of C18-4 SSCs proliferation.The effect of compounds on key genes in SSCs proliferation and self-renewal were analyzed by network pharmacology and q RT-PCR,and the interference of sh RNA with key gene expression was verified by detection of cell cycle,and western blot,immunofluorescence and q RT-PCR analysis of SSCs self-renewal related gene expression.The results showed that quercetin,rutin,kaempferol and chlorogenic acid in Eucommiae Folium were correlated with spermatogenesis,and the chlorogenic acid(50 μmol/L)had better proliferation effect on C18-4 SSCs than the others(P<0.05).Inhibition of Shp2 gene expression reduced the proliferation of C18-4(P<0.05),and chlorogenic acid had no significant promoting effect on it.Further studies showed that chlorogenic acid(50 μmol/L)promoted the expression of Bcl6 b,Etv5,and Plzf and phosphorylation of ERK1/2 by up-regulating Shp2 expression(P<0.05),and inhibited the expression of c-Kit(P<0.05).These results indicated that chlorogenic acid promoted the proliferation and self-renewal of spermatogonial stem cells by activating MAPK signaling pathway and up-regulating Plzf,Bcl6 b and Etv5 gene expression mediated by Shp2 gene.(3)Screening of active components from Eucommiae Folium ethanol extract regulating Leydig cells testosterone synthesis,and its mechanism.Four compounds were screened by the promotion of TM3 Leydig cells testosterone synthesis.The effects of compounds on key genes in testosterone synthesis were analyzed by network pharmacology and q RT-PCR,and the interference of sh RNA with key gene expression was verified by detection of cell cycle and Elisa,western blot analysis and q RT-PCR analysis of testosterone synthesis related gene expression.The results showed that the chlorogenic acid(1 μmol/L)had better testosterone synthesis effect on Leydig cells than the others(P<0.05).Inhibition of Shp2 gene expression reduced the testosterone synthesis of TM3 cells(P<0.05),and chlorogenic acid had no significant promoting effect on it.Further studies showed that chlorogenic acid(1 μmol/L)promoted the expression of Star and phosphorylation of ERK1/2 by up-regulating Shp2 expression(P<0.05).The results indicated that chlorogenic acid promoted the testosterone synthesis and proliferation of Leydig cells by activating MAPK signaling pathway and up-regulating Star gene expression mediated by Shp2 gene.(4)Effects of chlorogenic acid on spermatogenesis in mice.The results of testicular coefficient,sperm count,testosterone synthesis,and q RT-PCR showed that the chlorogenic acid(20 and 40 mg/kg)significantly enhanced the testicular coefficient,promoted testosterone secretion(P<0.05)and increased sperm count(P<0.05)in mice.Furthermore,chlorogenic acid(20 and 40 mg/kg)could promote the expression of SSCs proliferation and self-renewal related genes Plzf and Bcl6 b,and testosterone synthesis gene Star(P<0.05).The results indicated that chlorogenic acid could promote spermatogenesis in mice,but its effect was weaker than Eucommiae Folium ethanol extract.The results,which were from detection of cytotoxicity and apoptosis in Leydig cells,and apoptosis related genes Bax,Caspase3 and Bcl2 genes expression,showed that chlorogenic acid(100 and150 mg/kg)can effectively improve the inhibitory effect of cyclophosphamide on testosterone and FSH secretion in mice(P<0.05),and increased the number of sperm in testis and epididymis(P<0.05).Chlorogenic acid(10 μmol/L)could alleviate the cytotoxicity of acrolein to Leydig cells(P<0.05),apoptosis assay showed that chlorogenic acid(10 and 20 μmol/L)could inhibit the expression of apoptosis genes Caspase3 and Bax(P<0.05),and promote the expression of anti-apoptosis gene Bcl2(P<0.05),thus inhibiting acrolein induced testicular interstitial cell apoptosis.These results suggested that chlorogenic acid could alleviate the reproductive toxicity of cyclophosphamide by inhibiting Leydig cell apoptosis.In conclusion,this study showed that Eucommiae Folium ethanol extract had the effect of promoting spermatogenesis.Four compounds were predicted by network pharmacology and screened by activity of SSCs proliferation and Leydig cell testosterone secretion,among which chlorogenic acid was more effective.Further studies showed that chlorogenic acid regulates the SSCs self-renewal and proliferation and Leydig cells testosterone secretion through promoting the expression of Shp2 gene.Furthermore,chlorogenic acid mitigated the reproductive toxicity of cyclophosphamide by inhibiting the Leydig cell apoptosis.The results of this study provide theoretical basis for Eucommiae Foliums and their components to improve the efficiency of livestock reproduction and the treatment of related diseases,and provide ideas for the development and application of tonifying kidney in Traditional Chinese Medicine. |
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