Resveratrol Inhibits LPS-induced Apoptosis By Up-regulating SIRT3 In Bovine Mammary Epithelial Cells

Introduction: Mastitis is one of the most common and harmful diseases in modern dairy industry.The occurrence and development of dairy mastitis is accompanied by the apoptosis of mammary epithelial cells.Inhibiting the apoptosis of mammary epithelial cells is of great significance for restoring mammary function and improving milk yield.Escherichia coli is one of the most common pathogens infecting mammary gland of dairy cows.Its pathogenicity stems from the virulence factor Lipopolysaccharide(LPS).LPS can induce apoptosis of mammary epithelial cells.Resveratrol(Res)is a natural polyphenol compound that can inhibit LPS-induced tissue damage in mice.Res is also an agonist of silent Information Regulator 3(SIRT3),a key deacetylase that regulates cell energy metabolism and oxidation-antioxidation balance.We hypothesized that Res could inhibit LPS-induced apoptosis of bovine mammary epithelial cells by up-regulating SIRT3 expression.In this study,bovine mammary alveolar cell line(MAC-T)was used as experimental material.Cell apoptosis was induced by LPS,then Res was added,and SIRT3 gene was silenced to explore the role and mechanism of Res in inhibiting LPS-induced apoptosis.The present study will provide an in vitro experimental basis for developing drugs or additives to alleviate mastitis.Methods:(1)MAC-T was treated with 0,100,250 and 400 μg/m L LPS for 12 h,and cell viability was detected by CCK-8 kit.The expression levels of Bax,Bcl-2 and Caspase-3 proteins were detected by Western-blot.TUNEL kit was used to detect the degree of apoptosis.(2)MAC-T with different concentrations of Res was pretreated for 12 h and then treated with 250 μg/m L LPS for 12 h.Cells were divided into 5 groups: 0 μM Res + 0 μg/m L LPS group,0 μM Res + 250 μg/m L LPS group,10 μM Res + 250 μg/m L LPS group,25 μM Res + 250 μg/m L LPS group,and 50 μM Res + 250 μg /m L LPS group.Cell viability was detected.The expression levels of Bax,Bcl-2 and Caspase-3 proteins were detected by Western-blot.Apoptosis was detected by TUNEL assay.(3)Using 50 μM Res pretreatment for 12 h,then Si-RNA/Si-SIRT3 treatment for 12 h,and finally 250 μg/m L LPS treatment for 12 h,cells were divided into 5 groups: 0 μM Res +0 μg/m L LPS group,0 μM Res + 250 μg/m L LPS group,50 μM Res + 250 μg/m L LPS group,Si-SIRT3 + 50 μM Res + 250 μg/m L LPS group and Si-RNA + 50 μM Res + 250 μg/m L LPS group.The SIRT3 m RNA expression level was detected by q PCR.Cell viability was also detected.The protein expression levels of SIRT3,Bax,Bcl-2 and Caspase-3 were detected by Western-blot.Apoptosis was detected by TUNEL assay.Results:(1)The MAC-T activity decreased linearly with the increase of LPS concentration(Linear P < 0.01).The level of pro-apoptotic protein Bax increased linearly and quadratically with the increase of LPS concentration(P < 0.01);levels of anti-apoptotic protein Bcl-2decreased linearly with the increase of LPS concentration(Linear P < 0.01);Bax/Bcl-2increased linearly and quadratically with the increase of LPS concentration(P < 0.01);levels of Caspase-3 increased linearly with LPS concentration(Linear P < 0.01).TUNEL assay showed that the fluorescence intensity increased linearly and quadratically with the increase of LPS concentration(P < 0.01).(2)The cell viability of MAC-T increased linearly with the increase of Res concentration(Linear P < 0.01).Compared with 250 μg/m L LPS group,Bax level,Bax/Bcl-2 and Caspase-3 levels in 50 μM Res + 250 μg/m L LPS group decreased(P < 0.01),while the level of Bcl-2 increased(P < 0.01).TUNEL assay showed that the fluorescence intensity decreased linearly and quadratically with the increase of Res concentration(P <0.01),and the fluorescence intensity of 250 μg/m L LPS group was significantly higher than that of 50 μM Res + 250 μg/m L LPS group(P < 0.01).(3)Compared with Res + Si-RNA + LPS group,levels of SIRT3,cell viability,and Bcl-2 in Res + Si-SIRT3 + LPS group decreased(P < 0.05),while levels of Bax,Caspase-3and Bax/ Bcl-2 increased(P < 0.01).TUNEL assay showed that the fluorescence intensity of Res + Si-SIRT3 + LPS group increased compared with Res + Si-RNA + LPS group(P <0.01).Conclusion:(1)Res inhibits LPS-induced MAC-T apoptosis.(2)Res alleviates LPS-induced MAC-T apoptosis by upregulating SIRT3.