The Mechanism Of Scutellarin Against ZEA-induced Mouse Ovarian Granulosa Cells Apoptosis

Reproductive disorders of female animals cause great economic losses to livestock and poultry production.Ovarian granulosa cells are one of the important somatic cells in the ovarian follicles which plays a key role in the follicle’s formation and development.The ovarian granulosa cells are often the target of various external stimuli,which could induce reproductive disorders.Zearalenone（ZEA）is a mycotoxin widely found in natural grains which could cause granulosa cells damage.At present,there is still a lack of effective drugs on the treatment of reproductive diseases caused by granulosa cells injury.Therefore,in this study,a model of mouse ovarian granulosa cell injury induced by ZEA was established,and the monomer compounds of traditional Chinese medicine with anti-ZEA activity were screened.The selected candidate compound（Scutellarin,Scu）was used to explore the mechanism of apoptosis in mouse ovarian granulosa cell model and mouse model,respectively.i TARQ quantitative proteomics technique was used to further screen the key proteins of mouse ovarian granulosa cells treated with scutellarin,and to identify the anti-injury target of scutellarin.Results:1.Mouse ovarian granulosa cells were isolated by mechanical method,and the isolated cells were identified through the observation of cell morphology,HE staining and indirect immunofluorescence analysis.The appearance of the isolated cells was spindle or polyangular,and indirect immunofluorescence analysis confirmed that these cells were follicle-stimulating hormone receptor（FSHR）positive.These results indicated that the mouse ovarian granulosa cells were successfully isolated in vitro.The effect of ZEA on granulosa cell viability was detected by MTT,and the results showed that ZEA inhibited cells viability in a dose-and time-dependent manner,60 μM ZEA inhibited about 50% of granulosa cells viability（p<0.05）,and this concentration was selected to establish mouse model of granulosa cell injury in vitro.2.The effects of 21 traditional Chinese medicine monomer compounds such as icariin,glycyrrhizic acid,chlorogenic acid,and scutellarin on the viability of granulosa cells,and the effective monomer compounds that could rescue the injury of granulosa cells induced by ZEA were determined by MTT assay.Among the 21 compounds,mangiferin（1000 μg/mL）,chlorogenic acid（250,500 and 1000 μg/mL）and Scu（500,1000 and 2000 μg/mL）were successfully identified which can demolish the ZEA-induced mouse ovarian granulosa cell injury（p<0.05）.Among these three,Scu showed the best effects to against ZEA-induce granulosa cells injury,and the cell increment rate was more than 100%.The flow cytometric analysis was used to detected the effects of scutellarin on cell cycle distribution of granulosa cells,and the results showed that Scu promoted the cells transformation from S phase to G2/M phase（p<0.05）.Flow cytometric analysis also showed that ZEA significantly arrested granulosa cells in S phase（p<0.05）,while Scu treatment restored ZEA-induced S-phase arrest（p<0.05）.These results suggested that Scu may alleviate ovarian granulosa cell injury by affecting cell cycle distribution.3.The mouse ovarian injury model was established by intragastric administration of 40mg/kg ZEA.ZEA was administered intra-gastrically for 2 h,followed by 100 mg/kg Scu for 3d.The serum of mouse was collected to determine the contents of E₂ and P,and the mouse ovarian tissue was fixed for TUNEL staining,and the localization and expression of apoptosis-related proteins were detected by immunohistochemistry and western blot analysis.The results showed that the contents of serum E₂ and P in ZEA group were lower than those in the control group（p>0.05）,but there was no significant effect on the expression of E₂ and P in scutellarin group（p>0.05）.Ovarian apoptosis was detected by TUNEL staining,and the results showed that compared with the control group,a large number of positive stained cells appeared in ZEA group,mainly in follicle granulosa layers,while the number of apoptotic cells were decreased in scutellarin group.The location of cleaved-caspase-3 was detected by immunohistochemistry analysis,the results showed that compared with control group,the positive signal of cleaved-caspase-3 in ZEA group was significant in follicle granulosa layers,theca layer and ovary stroma,and among these three positions,the granulosa layer possessed the strongest positive signal.While the protein expression levels of cleaved-caspase-3,especially in follicle granulosa layers,were decreased in Scu treated mouse.The expression of apoptosis-related proteins in ovary was detected by western blot analysis,and the results showed that compared with control group,the protein expression levels of cleaved-caspase-3,cleaved-PARP and the Bax/Bcl-2 ratio were significantly increased in ZEA group（p<0.001）.While Scu could significantly inhibit cleaved-caspase-3 and cleaved-PARP expression and decrease the ratio of Bax/Bcl-2（p<0.05）.It is concluded that Scu inhibited mouse ovarian granulosa cell apoptosis through mitochondrial apoptotic pathway in vivo,thereby exerting reproductive protection.4.TUNEL and western blot assays were performed to detect cell apoptosis and expression of apoptosis-related proteins in mouse ovarian granulosa cell injury model.The results showed that ZEA exposure significantly increased the apoptosis rate of granulosa cells,while was decreased by Scu treatment（p<0.001）.And ZEA exposure significantly increased the protein expression levels of cleaved-caspase-3,cleaved-PARP and the Bax/Bcl-2 ratio（p<0.001）,while the Scu has significantly inhibited the apoptosis induced by ZEA via inhibiting the up-regulation protein expression levels of cleaved-caspase-3 and cleaved-PARP and reducing the ratio of Bax/Bcl-2（p<0.001）.5.Comparative proteomic analysis was carried out between cell group（control）,ZEA treatment group（model）,Scu and ZEA co-treatment group（Scu）by iTRAQ quantitative proteomics technology.Total of 415 differential proteins were identified in the cell group compared with the ZEA group（control vs model）,including 131 up-regulated proteins and284 down-regulated proteins.Total of 415 differential proteins were identified in the Scu group compared with the ZEA group（Scu vs model）,including 128 up-regulated proteins and287 down-regulated proteins.From these two compare groups（control vs model and Scu vs model）,141 differential proteins were identified,including HSPs,26 s proteasome and key proteins of MAPK pathway.GO enrichment analysis found that the differences between the two compare groups of proteins were mainly concentrated in biosynthesis,metabolism and translation.KEGG pathway enrichment is mainly concentrated in drug metabolism,chemical carcinogenesis,pentose phosphate pathway,gap junction and other signal transduction pathways.The RIPK1 has the largest fold difference in Scu vs model.ZEA increased the expression of cleaved-caspase-3 in RIPK interference cells compared with si RNA-NC group,but there was no significant change in Scu group（p<0.05）,indicating that RIPK1 may be involved in inhibiting ZEA-induced granulosa cells apoptosis,but does not play a role in the anti-apoptosis of Scu.Conclusion:1.The mouse ovarian granulosa cells were treated with 60 μ M ZEA for 24 h to establish the cells injury model.2.Among the 21 monomer compounds screened,we have successfully identified 3compounds that can rescue the injury caused by ZEA with the Scu as the most effective compound as it can significantly rescue the ZEA caused damage on mouse ovarian granulosa cells,and alleviate the S-phase arrest caused by ZEA.3.Scu can inhibit ZEA-induced mouse ovarian granulosa cell apoptosis through the mitochondrial apoptotic pathway in vivo and in vitro.4.The proteomic assay suggested that Scu exerted protective action of reproductive toxicity via modulating multiple proteins,targets and pathways.RIPK1 did not play a role in the anti-apoptosis of Scu,but was involved in the inhibition of ZEA-induced mouse ovarian granulosa cells apoptosis.