| Objective: The purpose of this study was to screen Chinese medicine monomer compounds that can promote the proliferation of small yellow follicle granulosa cells in laying hens,then explored the effects of traditional Chinese medicine monomer compounds on the proliferation of granulosa cells and their mechanisms.Methods: The small yellow follicle granulosa cells were separated and isolated from the 200-day-old Hailan brown laying hens,the indirect immunofluorescence method was used to identify the granulosa cells.The MTT method was used to detect the safe concentration of each traditional Chinese medicine monomer compound on granulosa cells.In order to screen Chinese medicine monomer compounds that can promote the proliferation of small yellow follicle granulosa cells in laying hens,q PCR was used to detect the effect of each traditional Chinese medicine monomer compound on PCNA m RNA of granulosa cells,flow cytometry was used to detect the percentage of proliferating cells in granulosa cells.Then q PCR and Western Blot were used to detect the expression of PCNA to verify again.Flow cytometry was used to detect the effect of traditional Chinese medicine monomer compounds on the cell cycle of granulosa cells.The effects of Chinese medicine monomer compounds on granulosa cells cyclin E1,cyclin A2,cyclin-dependent kinase 2 was detected by q PCR and western blot.Results: 1.After the granulosa cells were cultured for 24 h,the cells are completely attached,and they aggregate growth in a good state.During 24 to 48 hours,the cells stretched and grew rapidly.After the granulosa cells were cultured for 48 h,the cells covered the bottom of the plate.FSHR was positively expressed in cells identified by indirect immunofluorescence.2.The safe concentration of angelicin on granulosa cells is 80 μg/m L,scutellarin is 125 μg/m L,chlorogenic acid is 125 μg/m L,astragaloside IV is 240 μg/m L,syringin is 2.4 mg/m L and icariin is 80 μg/m L.3.Compared with the blank control group,the angelicin of 1.25 μg/m L(p<0.001),the scutellarin of 1.953 μg/m L(p<0.001),the chlorogenic acid of 7.813 μg/m L(p<0.001),the astragaloside IV of 3.75 μg/m L(p<0.05),the syringin of 37.5 μg/m L(p<0.001),the icariin of 20 μg/m L and 5 μg/m L(p<0.001)all can increase the relative expression of PCNA m RNA in granulosa cells.Flow cytometry results showed that compared with the blank control group,only the icariin of 20 μg/m L treated granulosa cells for 24 h,the percentage of proliferating cells increased significantly by 10.24%(p<0.05).Based on the results of q PCR and flow cytometry,icariin can promote the proliferation of granulosa cells.4.Compared with the blank group,the relative expression of PCNA m RNA in the treatment group of 20 μg/m L was significantly increased(p<0.001)after 24 hours of icariin treatment,the protein relative expression of PCNA in the treatment groups of 40 μg/m L,20 μg/m L and 10 μg/m L was significantly increased(p<0.001).The treatment groups of 40 μg/m L and 10 μg/m L had no significant effect on the proportion of S-phase cells(p>0.05);the treatment group of 20 μg/m L significantly increased the proportion of S-phase cells(p<0.01).The results showed that icariin promotes the proliferation of granulosa cells by affecting the cell cycle distribution.5.The results of q PCR showed that: After 24 hours of icariin treatment,compared with the blank group,the cyclin E1 m RNA relative expression in the 20 μg/m L treatment group was significantly increased(p<0.001),but there was no significant change in the treatment group with 40 μg/m L and 10 μg/m L;icariin significantly increased the expression of cyclin A2 m RNA in 20 μg/m L(p<0.001)and 10 μg/m L(p<0.05),there was no significant change in the 40 μg/m L treatment group;the relative expression of CDK2 m RNA was significantly rised in 40 μg/m L(p<0.01),20 μg/m L(p<0.001)and 10 μg/m L(p<0.001),respectively.The results of western blot showed that: After 24 hours of icariin treatment,compared with the blank group,the protein expression of cyclin A2 was significantly increased in the 40 μg/m L and 20 μg/m L treatment groups(p<0.001),but was not significantly changed in the 10 μg/m L treatment group(p>0.05);the protein expression of cyclin E1 was significantly increased in the 40 μg/m L(p<0.05),20 μg/m L(p<0.001)and 10 μg/m L(p<0.001)treatment group;the protein expression of CDK2 was significantly increased in the 40 μg/m L and 20 μg/m L treatment groups(p<0.001);There was no significant change in the 10 μg/m L treatment group(p>0.05).Based on the results of q PCR and western blot,icariin promotes the progress of the S phase by up-regulating the relative expression of cyclin E1,cyclin A2,and CDK2 genes and proteins in laying hen small yellow follicle granulosa cells.Conclusion: 1.The maximum safe concentration of icariin,angelicin,scutellarin,chlorogenic acid,astragaloside IV and syringin were determined on the small yellow follicle granulosa cells of laying hens.Based on the results of q PCR and flow cytometry,it has been confirmed that only Icariin could promote the proliferation of small yellow follicle granulosa cells in laying hens.2.The icariin of 20 μg/m L promotes the S-phase progression of the cell cycle by promoting cyclin A2,cyclin E1 and CDK2 proteins expression to induce granulosa cells proliferation. |
PDF Full Text Request