Mechanism Of Alginic Acid On Steroid Synthesis In Porcine Ovarian Granulosa Cells Through Oxidative Stress Pathway

Improving the reproductive performance of sows is very important for the development of animal husbandry in China.The follicle is the basic functional unit of the female reproductive system.Granulosa cells(GCs)is the largest cell population in the follicle,and they play a crucial part in the process of follicle development,maturation,apoptosis and atresia.Alginic acid(AA)is a kind of natural polysaccharide extracted from brown algae,which has been widely used in chemical industry and food industry.It has been proved that alginic acid has certain anti-inflammatory,anti-allergic and immunomodulatory biological activities,but the effects of alginic acid on female reproduction have rarely been reported.As alginic acid is more and more widespread in the environment,its effect on the reproductive development of domestic animals is worth studying.The purpose of this study was to investigate the effects of alginic acid on cell viability and steroid secretion of porcine ovarian granulosa cells,and to further explore the influencing mechanisms in terms of oxidative stress,apoptosis and autophagy.In this study,porcine ovarian granulosa cells were cultured with DMEM/F12 medium containing alginic acid,and the effects of alginic acid on cell viability and steroid secretion were detected by CCK8 assay,ROS fluorescent staining,ELISA,RT-q PCR and western blot.Furthermore,GSH fluorescent staining,GSH assay,TAOC assay,MDA assay,Annexin V/PI apoptosis detection,JC-1 staining and autophagic vesicle staining were used to investigate the effects of alginic acid on oxidative stress,apoptosis and autophagy.The results of this study are as follows:1.Alginic acid inhibited the activity of porcine ovarian granulosa cells.The results of CCK8 assay,ROS fluorescence staining and western blot analysis of caspase-3 and P62 proteins showed that the activity of porcine ovarian granulosa cells decreased with the increase of alginic acid concentration when the concentration was greater than or equal to20 μM.2.20 μM alginic acid disturbed the synthesis of steroid hormone in porcine ovarian granulosa cells.The results showed that after alginic acid treatment,the synthesis of estradiol(P<0.01)and progesterone(P<0.001)in the experimental group were significantly decreased.The result of western blot showed that the expression of CYP19A1(P<0.05),FSHr(P<0.05)and St AR(P<0.05),key proteins for steroid hormone synthesis,were obviously decreased,while the expression of CYP17A1 was notably increased(P<0.05).3.20 μM alginic acid increased the oxidative stress level of porcine ovarian granulosa cells.After alginic acid treatment,the levels of GSH(P<0.01)and T-AOC(P<0.05)were markedly decreased,and the levels of ROS(P<0.05)and MDA(P<0.05)were significantly increased in the experimental group.The result of western blot showed that the expression levels of SOD1(P<0.05),SOD2(P<0.01)and GPX5(P<0.05)were significantly decreased in the alginic acid treated group.4.20 μM alginic acid promoted mitochondrial-mediated apoptosis and autophagy in porcine ovarian granulosa cells.The result of Annexin V/PI apoptosis detection showed that the proportion of apoptotic cells in the experimental group was signally increased(P<0.05).The result of JC-1 staining showed that the mitochondrial membrane potential of cells was obviously decreased(P<0.001)in the experimental group.The result of western blot showed that the expression level of pro-apoptotic protein cleaved caspase-3 was obviously increased(P<0.05),and the expression level of pro-autophagy protein Beclin1 was significantly increased(P<0.05)in alginic acid treated group.5.Intraperitoneal injection of alginic acid solution(10 mg/Kg)for consecutive 35 days inhibited the estrus and the synthesis of steroid hormones in mice,increased the level of oxidative stress and induced apoptosis in mice ovaries.In conclusion,20 μM alginic acid treatment for 24 h significantly inhibited the cell viability of porcine ovarian granulosa cells,induced oxidative stress,induced mitochondria mediated apoptosis and autophagy,and ultimately inhibited the secretion of steroid hormones.