| Chemerin gene is also known as tazarotene-induced gene 2(TIG2)or retinoic acid receptor responder 2(RARRES2),and it is a newly discovered adipokine with chemotaxis in 2007.Chemerin and its receptor chemokine receptor 1(CMKLR1)are widely distributed in a variety of tissues,including adipose tissue(High expression),liver,placenta and ovary.Recent studies have shown that Chemerin and its receptors are not only related to a variety of diseases such as obesity and metabolic syndrome,but also play an important role in regulating the reproductive function of animals.Apoptosis of ovarian granulosa cells directly affects oocyte development and ovulation.Oxidative stress is a common factor causing apoptosis of ovarian granulosa cells.As a classic signal pathway of apoptosis,ERK1/2 also plays an important role in the proliferation and apoptosis of ovarian granulosa cells.However,the specific role and mechanism of Chemerin and its receptor CMKLR-1 in the apoptosis of ovarian granulosa cells induced by oxidative stress are not clear.Therefore,in this study,bovine granulosa cells were used as experimental materials,the effect of Chemerin and its receptor on the apoptosis of bovine ovarian granulosa cells induced by oxidative stress was investigated.The specific contents and results were as follows:1.Isolation and culture of bovine ovarian granulosa cellsOvarian granulosa cells were isolated from ovarian follicle fluid(The diameter of the follicle was 2-6 mm),bovine ovarian granulosa cells were identified by specific receptor-follicle-stimulating hormone receptor(FSHR)in vitro.Results:bovine ovarian granulosa cells grew well,and FSHR identified positive granulosa cells to reach more than 95%.2.Expression of Chemerin and its receptors CMKLR1 in ovarian granulosa cells under oxidative stress.In this experiment,the bovine ovarian granulosa cells were treated with 600 ?mol/mL H2O2 for 2 h.Under oxidative stress(normal culture group as control),we determined the gene and protein expression levels of Chemerin and CMKLR-1 by the RT-qPCR and the Western blot methods.The results showed that Chemerin and its receptor CMKLR-1 were expressed in bovine ovarian granulosa cells.The expression of Chemerin protein was significantly up-regulated in the cells by oxidation treatment(P<0.01),and the expression of Chemerin and its receptor CMKLR-1 mRNA was also significantly increased(P<0.01).It is suggested that Chemerin axis is involved in the oxidative stress process of bovine ovarian granulosa cells.3.Effects of Chemerin on bovine ovarian granulosa cells induced by oxidative stress.Under oxidative stress,the expression of Chemerin in bovine ovarian granulosa cells increased with Chemerin recombinant protein.The results showed that the content of reactive oxygen species(ROS)and malondialdehyde(MDA)increased significantly in ovarian granulosa cells(P<0.05),and the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-px)decreased significantly(P<0.05).In addition,compared with the control group,the high expression of Chemerin significantly increased the rate of apoptosis induced by oxidative stress(P<0.01),and the protein level of Bax and Cleaved-caspase-3 increased significantly,while the protein level of Bcl-2 decreased.Meanwhile,the Caspase-3 mRNA and ratios of Bax/Bcl-2 increased significantly(P<0.05).It is suggested that Chemerin may initiate apoptosis pathway by regulating the expression of Bax,Bcl-2 and Caspase-3,and affect the apoptosis of bovine ovarian granulosa cells.Moreover Chemerin gene was silenced by transfection of siRNA-Chemerin,the results showed that the content of ROS and malondialdehyde(MDA)decreased,and superoxide dismutase(SOD)and glutathione peroxidase(GSH-px)increased significantly(P<0.01),In addition,the protein level of Bax and Cleaved-caspase-3 decreased significantly,while the expression of Bcl-2 protein increased,and the Caspase-3 mRNA and radios of Bax/Bcl-2 mRNA decreased.It indicates that inhibiting the expression of Chemerin can rescue the apoptosis of ovarian granulosa cells induced by oxidative stress.It is further confirmed that Chemerin promotes the apoptosis of bovine ovarian granulosa cells induced by oxidative stress.4.Chemerin regulates oxidative stress induced apoptosis of bovine ovarian granulosa cells via ERK1/2 signaling pathwayChemerin recombinant protein was used to treat oxidative stress ovarian granulosa cells.At the same time,the content of ROS and the expression of apoptosis key genes and ERK1/2 phosphorylation were down-regulation by adding ERK1/2 pathway inhibitor U0126,and we analyzed that the effect of Chemerin on the oxidative stress induced apoptosis of bovine ovarian granulosa cells after the inhibition of ERK1/2 signaling pathway.The results showed that high expression of Chemerin increased the level of ERK1/2 of phosphorylation in cells under oxidative stress,and the expression of silent Chemerin could significantly reduce the level of ERK1/2 phosphorylation in cells(P<0.05).It indicates that high expression of Chemerin can activate ERK1/2 signaling pathway.After the inhibitor U0126 inhibited the ERK1/2 pathway,the content of ROS decreased significantly after addition Chemerin protein or the silent Chemerin gene in cells;the pro-apoptosis related proteins of Bax and Cleaved-caspase-3 significantly down-regulated,and the expression of Bcl-2 protein in the anti-apoptosis key genes significantly up-regulated(P<0.05),and the ratio of Caspase-3 mRNA and Bax/Bcl-2 mRNA was decreased.In addition,after inhibition of ERK1/2 phosphorylation level,the expression of Chemerin and its receptor CMKLR-1 in bovine ovarian granulosa cells was significantly lower than that in the oxidative stress group(P<0.05).It suggests that silencing Chemerin gene and inhibiting ERK1/2 pathway can save Chemerin and oxidative stress induced apoptosis of bovine ovarian granulosa cells.The results showed that Chemerin at least partially regulates the apoptosis of bovine ovary granulosa cells under oxidative stress through ERK1/2 signaling pathway. |
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