Study On The Proliferation Induced By Icariin In Rat Sertoli Cells With Molecular Mechanism Research

Sertoli cell and its structure were first described by Italian organizationexpert, Enrico Sertoli, in1865. The structural integrity of the SCs ensure normalspermatogenesis. In the seminiferous tubules, SCs provide supportment andnutrition for germ cells. At the same time, the mature SCs that secrete steroidhormones, regulate protein, growth factors, paracrine factors, transport proteinsand so on. All of these provide a stable environment and energy substances forspermatogonium differentiation. In recent years, studies have confirmed thatFasL secreted by SCs is an important foundation of its immune privilege.Clinically, the function of SCs immune privilege has been used to treat humanneurodegenerative diseases. In recent years, scholars have successfullyconfirmed the SCs as a feeder layer can significantly promote the proliferationof a variety of stem cells in vitro. So, SCs in cell culture technology, tissueengineering and organ transplants and other areas has become a hotspot and willhave a broad application prospects in the field of clinical transplantation. Icariin derived from natural plants, is the main active ingredient oftraditional Chinese medicine Epimedium (Epimedium brevicornum Maxim).Modern pharmacological studies have shown that the ICA can promote thedevelopment and maturation of male reproductive system, protect thecardiovascular system, promote the proliferation and differentiation ofosteoblasts, improve immune function and anti-cancer, however, part of itsmechanism of action is not clear. Therefore, this study was to observe the impactof the ICA on SCs proliferation and discuss the corresponding mechanism. So,to study the culture and its proliferation has important clinical significance.This experiment is divided into three parts as follow：Section1The isolation, purification and identification ofPrimary Sertoli cellsObjective:To isolate and culture high vitality and purity primary Sertoli cells.Methods：8-9days SD rats were selected and two-step enzymatic was used to isolateprimary Sertoli cells. After48h, spermatogenic cells were removed byTris-HCL, PH7.4. Trypan blue staining was used to detect cell viability.Immunofluorescence and crystal violet staining methods were used in ourexperiment for cell identification.Results:The results showed that cell viability reached more than95%by trypan blue staining and morphological observation. After48h, immunofluorescence andcrystal violet staining showed that Sertoli cells’ purity was more than95%.Conclusions:1、Two-step enzymatic digestion to isolate and culture of primary Sertoli cells ismore simple and less contaminated cells.2、High vitality and purity primary Sertoli cells can be obtained via two-stepenzymatic digestion.Section2Effect of Icariin on Sertoli cells proliferation in vitroObjective:Use MTT, FCM (flow cytometry) and direct cell counting methods to detectthe effect of ICA on Sertoli cells proliferation in vitro.Methods：MTT assay was used to confirm ICA can promote SCs proliferation and todetermine the effective dose and time period. Sertoli cells adhered to culturedishes were collected and placed at a density of2×104cells/200μl in each wellof96-well plates. After24h, the cells were treated with or without variousconcentrations of Icariin (0μM,5μM,10μM,15μM,20μM,25μM) for differentperiods (0h,6h,12h,24h,48h). According to the result of above,10μM ICAwas added in cells for different periods. In order to avoid the deficiencies of theMTT assay, we used the more accurate and complicated measure than MTT,direct cell counting method. Cells in48-well plates at a lower density than in96-well of4×104cells/500μl, namely8×104/ml, were treated with the sameconditions as MTT assay. At the same time, CFSE-marked cells treated as MTT assay fluorescence intensity were detected by FCM. CFSE integrated into cellsand evenly distributed into each generation cells, so the fluorescence intensitygradually decreased with cell proliferation.Results:MTT, direct cell counting and fluorescence intensity to detect the effect ofICA on SCs proliferation. Compared with the control, cell viability wassignificantly promoted after treatment with10μM,15μM,20μM, or25μMIcariin for24h (p <0.05). Treatment with10μM Icariin increased SCs viabilityin a time-dependent manner, with a significant difference after12h incubation(p﹥0.05). Flow cytometric analysis further confirmed that Icariin stimulatesproliferation in SCs, It was clearly shown, compared with the control, thefluorescence intensity of cell proliferation decreased significantly by thetreatment with Icariin except for5μM (p﹥0.05).Conclusion:ICA promoted SCs proliferation both in concentration-effect andtime-dependent manners in vitro.Section3The proliferative mechnism study of ICA onSertoli cells in vitroObjective:To detect the mechnism of ICA on SCs proliferation in vitro.Methods：MAPK, mitogen-activated protein kinases is one of intracellularserine/threonine protein kinases. The study has confirmed that MAPKs transfer signal from extracellular to intracellular and play a crucial role in cellproliferation, differentiation, transformation and apoptosis, etc. MAPKsinclude three parallel signal pathways, namely ERK, JNK and p38. The ERKsignal pathway is mainly involved in the regulation of cell proliferation anddifferentiation, in contrast to ERK, JNK and p38involved in oxidative stress,inflammation, and apoptosis, etc. In the experiment, we designed groups asfollowing, control group, ICA, UO126, ICA+UO126, SP600125, ICA+SP600125, SB203580, and ICA+SB203580. UO126, SP600125and SB203580are respectively blockers of ERK, JNK and p38. The cells were treated with orwithout ICA for24h after each blocker incubated for30min before MTT assay,direct cell counting and flow cytometry. To illustrate the accurate role ofphosphorylated ERK1/2in Icariin-induced cells proliferation, Sertoli cells weretreated with Icariin in the presence or absence of inhibitor UO126. Thephosphorylation of ERK1/2was detected by Western blot after proteinextraction.Results:Pre-treatment and subsequent co-treatment with the ERK inhibitor UO126groups caused a statistically significant change in Icariin-induced SCsproliferation (p<0.05); Compared with control, the cells proliferation in othergroups have no significant change (p﹥0.05). To determine the levels of totaland phosphorylated ERK1/2, SDS-PAGE/Western blot was used. Treatmentwith various concentrations described above of Icariin induced phosphorylationof ERK1/2, which was not seen in control and5μM cultures. As shown inresults, the level of phosphorylated ERK1/2in cells treated with Icariin wasincreased in both dose-and time-dependent manners, and UO126significantly inhibited phosphorylation of ERK1/2(p﹤0.05). It may illustrate the role ofphosphorylated ERK1/2in Icariin-induced proliferation.Conclusions:1The inhibiter UO126of ERK significantly inhibited the Icariin-induced SCsproliferation, but JNK and p38blockers have no effect on Icariin-induced SCsproliferation.2ICA can significantly increase the expression level of phosphorylated ERK1/2,which suggested that the ERK signal pathway was involved in proliferativeeffect of ICA on SCs.