| Diosgenin mainly comes from monocotyledonous herbaceous twining vine Dioscorea,such as the rhizome of Chinese yam, yellow ginger, airpotato yam and Rhizoma Dioscoreae Nipponicae,which is an important raw material for the production of steroid hormone drugs.In addition, Diosgenin can synthesize hydrocortisone, prednisone,norethindrone, fluocinolone acetonide, dexamethasone and other steroids drugs. Researchs show that diosgenin has the effect of antioxidant, anti vascular calcification, anti-tumor,anti-inflammatory, anti-allergic, anti-viral and anti-shock. Even though that, researchs about Diosgenin on the reproduction are quite little.Preliminary studies have demonstrated that Diosgenin can improve mild male reproductive disorders of aging, improve sperm activity; but study about the role of Diosgenin on Sertoli cells(SCs) has not been reported. Therefore, further study the impact of Diosgenin on male reproduction may provide new ideas on prevention and treatment of male infertility.Mature Sertoli cells(SCs) function as ‘‘nurse cells,’’ which play crucial roles in supporting spermatogenesis through establishing a unique and essential environment inthe male reproductive tract. Sertoli cells have a variety of functions,not only can they provide structure and nutrition for spermatogenic cells,but also have the functions of immunity, phagocytosis and secretion.Furthermore, Estrogen plays an irreplaceable role in the maintenance of the male reproductive.SCs is the main source of immature animal estrogen.Estrogen can directly promote SCs proliferation, differentiation and maturation.It can also regulates SCs mitotic activity by monitor FSH indirectly. At the same time,because estrogen function its role mainly through Estrogen receptor(ESR) signaling pathway to complete the regulation of male spermatogenesis.Therefore, oue study on the effect of Diosgenin on SCs and its role in the regulation of ESR signal transduction pathway, not only to explore the role and mechanism of Diosgenin on male infertility treatment, but also provide the experimental basis for further drug development.Part one :The influence of Diosgenin on SCs proliferationObjective:To explore the effect of diosgenin on the proliferation of SCs through the observation of Diosgenin on mouse testis derived SCs(TM4 cells) proliferation, cycle distribution and apoptosis.Methods:1) Using MTT detection method and the Brdu method to detect the influence of Diosgenin on the proliferation of TM4 cells.2) Flow cytometry analysis of diosgenin intervention TM4 cells cycle distribution;Western Blot method for detection the effects of TM4 cells in G0/G1 phase of CDK4/CDK6、cyclin D1、CDK2 and cyclin E,the expression of G2/M phase marker protein cdc2 and cyclin B.3) TUNEL method to detect on apoptosis of mouse TM4 cells after Diosgenin intervention; Western Blot method for detection of diosgenin on TM4 cells Bax,AIF, Caspase 3, Caspase 9 and Bcl-2 protein expression.Results:1) Different Diosgenin intervention concentration could stimulate proliferation of TM4cells(P<0.05), of which, 2.5μM most significant; 2.5μM Diosgenin intervention on TM4 cells 6-48 h can make the cell viability significantly increased, which reached the maximum value at 24h(P<0.05).2) 2.5μM Diosgenin intervention on TM4 cells for 24 h, the number of cells in G0/G1 phase decreased significantly, while S phase cell mass increased significantly(P<0.05); no significant change in G2/M cells mass(P<0.05); G0/G1 phase marker protein CDK4/CDK6, D1, Cyclin CDK2 and Cyclin E expression was significantly increased, and no obvious changes on the expression of G2/M phase marker proteins cdc2 and Cyclin B(P > 0.05). Expression of the CDKs inhibitor P27 decreased slightly(P > 0.05).3) After 2.5μM Diosgenin intervention on TM4 cells for 24 h, cell apoptosis were significantly inhibited; the expression of apoptosis related factors Bax, AIF, Cleaved Caspase3 and Cleaved Caspase9 protein were inhibited obviously, the expression of Bcl-2 protein was increased(P<0.05).Conclusion:Diosgenin can promote the proliferation of SCs in mice,inhibit the apoptosis of SCs,and there was a dose-time effect relationship as well.Part two:The mechanism of Diosgenin regulate ESR and ERK/Akt signaling pathway to promote the proliferation of TM4 cells.Objective:To explore the mechanism of Diosgenin promote SCs proliferation through observing the influence of Diosgenin on estrogen receptor and ERK/Akt pathway.Methods:1) Western Blot to detect the expression of nucleus and plasma membrane ESR1, ESR2 protein after 2.5μM Diosgenin intervention on TM4 cells; and the nuclear andplasma membrane ESR1, ESR2 protein expression were measured after adding 1nM the anti-estrogen ICI and 5n M tyrosine kinase inhibitor PP2 using the same method.Based on intervention above, MTT and Brdu were used to detect TM4 cells proliferation; flow cytometry analysis was used to detect cell cycle;TUNEL was used to measured the apoptosis of TM4 cells.2) Western Blot was used to assay the phosphorylation level of ERK and Akt at different time points after Diosgenin intervent on TM4 cells.In addition, the same target parameters were detected after adding related inhibitors of ICI, PP2, U0126 and LY294002; MTT and Brdu labeling method were used to detect TM4 cells proliferation.3) After Diosgenin intervention,Western Blot was used to detect the expression of ESR in the nucleus of TM4 cells.Luciferase reporter gene to assay ESR transcription activity of TM4 cells.4) CHIP was used to detect the effect of ESR late activity of TM4 cells after diosgenin intervention.Results:1) After 2.5 μM diosgenin intervention TM4 cell plasma membrane 0-30 min, ESR1 and ESR2 protein expression increased significantly, which reached the peak at the15min(P<0.05). While nuclear ESR1 and ESR2 protein expression was significantly decreased, which reached the lowest at 15min(P<0.05).The expression level of ESR1, ESR2 cell membrane was inhibited(P<0.05) after joining ICI and PP2;meanwhile, the expression level of nuclear ESR1 and ESR2 were increased(P<0.05).The effects of Diosgenin promotes the proliferation of TM4 cells was significantly inhibited after adding ICI and PP2(P<0.05);At the same time, G0/G1 phase cell populations are on the rise while the number of S phase cells decreased(P<0.05);and cell apoptosis index was significantly up-regulated(P<0.05).2) After 2.5μM Diosgenin intervention TM4 cells 0-30 min, the phosphorylation level of ERK and Akt were increased at 15 min, which reached the highest(P<0.05); thephosphorylation level of ERK and Akt were inhibited after joining inhibitor PP2,U0126, ICI, and LY294002, ERK, Akt phosphorylation was significantly inhibited(P<0.05). Among them,the proliferation of TM4 cells decreased after adding U0126 ICI, and LY294002(P<0.05).3) After Diosgenin intervent on TM4 cells 0-48 h, nuclear expression of ESR1 and ESR2 of TM4 cells were significantly increased,of which the 24 h treatment is of most significant(P<0.05).When PP2 is added, the nucleus expression level of ESR1 and ESR2 were improved(P<0.05). Under the same conditions, the transcriptional activity of TM4 cells ESR increased significantly, of which the 24 h treatment is of most significant(P<0.05); ESR transcription activity of TM4 cells was significantly inhibited after adding the inhibitor ICI, PP2, U0126 and LY294002(P<0.05).4) The level of ESR1, ESR2 protein binding with Cyclin D1 and Bcl-2 gene were increased significantly after 2.5 μM Diosgenin add to TM4 cells(P<0.05).Conclusion:The possible mechanism of Diosgenin promote SCs cell proliferation may be this:Diosgenin induced instant and transient SRC-dependent plasma membrane translocation of ESR and subsequent activation of ERK/Akt signaling, followed by late activation of nuclear ESR transcriptional activity and direct transcriptional regulation of cell cycle and apoptosis regulators, such as cyclin D and Bcl-2, resulting in promotion of the G1/S phase transition and inhibition of apoptosis, finally leading to SC proliferation. |
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