The Effects And Mechanism Of Resveratrol In Relieving Oxidative Damage Of Mammary Gland Induced By Streptococcus Uberis Infection

In this study,resveratrol,Streptococcus uberis 0140J(S.uberis 0140J)were used as the research objects.By constructing mouse mammary epithelial cells(EpH4-Ev)and C57BL/6 mouse infection models,we explored the effects and mechanism of resveratrol on oxidative stress caused by S.uberis infection.This study elucidated how resveratrol alleviates the oxidative damage induced by S.uberis infection in vivo and in vitro by observing the changes in oxidative injury related indicators and related proteins as well as enzymes in the signaling pathways.The purpose is to provide new threads and methods for the prevention and control of S.uberis and other similar pathogens.The specific research content and results are as follows:1.The effects of resveratrol on oxidative stress of mammary epithelial cells Induced by Streptococcus uberis infectionIn order to investigate the effects of resveratrol on oxidative stress caused by S.uberis challenge,mouse mammary epithelial cells(EpH4-Ev)were used as the research object;then the relevant indicators were observed or detected by microscopes,related kits(colorimetry,micromethod,visible spectrophotometry,etc),flow cytometry,et al instruments/methods.The results showed that the safe concentration range of resveratrol for treating EpH4-Ev cells was 0-20 μmol·L-1,and the optimal concentration and time to regulate S.uberis infection of EpH4-Ev cells was 15 μmol·L-1 and 4 h.Compared with the control,S.uberis infection can significantly elevate the level of reactive oxygen species(ROS),the contents of nitric oxide(NO),glutathione(GSH)and malondialdehyde(MDA),the activities of inducible nitric oxide synthase(iNOS),N-acetyl-β-D-glucosaminidase(NAGase),lactate dehydrogenase(LDH)and superoxide dismutase(SOD)(P<0.05),and significantly reduce the level of total antioxidant capacity(T-AOC)(P<0.05)in EpH4-Ev cells.It also caused the shrinkage of EpH4-Ev cells.Resveratrol pretreatment markedly declined the level of ROS,the contents of NO and MDA,the activities of iNOS,NAGase and LDH(P<0.05),and significantly increased the level of T-AOC(P<0.05),the activity of SOD and the content of GSH(P<0.05)in EpH4-Ev cells after S.infection.In addition,the shrinkage morphology of cells was effectively relieved.The results demonstrated that 15 βmol·L-1 of resveratrol pretreatment for 4 h can significantly alleviate the oxidative damage of mammary epithelial cells caused by S.uberis challenge.2.Resveratrol activates Nrf2 signaling pathway to relieve oxidative stress caused by Streptococcus uberis infection in mammary epithelial cellsIn order to explore the mechanism of resveratrol alleviating the oxidative stress induced by S.uberis infection,cells were treated with 15 βmol·L-1 resveratrol for 4 h,then S.uberis was added and co-incubated for 3 h to collect samples(inhibiting Nrf2 expression with inhibitor ML385 and interfering p62 gene expression with siRNA at the according time).Western blot,RT-PCR,related kits and other methods were used to measure relevant indicators.The results showed that S.uberis infection significantly increased the protein expression levels of nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),NAD(P)H:quinone oxidoreductase 1(NQO1),SOD1 and p62(P<0.05);the mRNA expression leves of Nrf2,HO-1,NQO1,glutathione S-transferase(GST),catalase(CAT)and p62 were also significantly increased(P<0.05),while Kelch-like ECH-associated protein 1(Keapl)protein expression level was markedly reduced(P<0.05)in EpH4-Ev cells.Resveratrol pretreatment further increased the protein expression levels of Nrf2,HO-1,SOD1 and p62(P<0.05);NQO1 protein expression level showed an upward trend.At the same time,the mRNA expression levels of Nrf2,HO-1,NQO1,GST,CAT and p62 were significantly increased(P<0.05),but Keapl protein expression level was significantly decreased(P<0.05).Upon treatment with Nrf2 inhibitor(ML385),S.uberis infection and resveratrol pretreatment has a declined effect on the protein expression levels of HO-1,NQO1,SOD1;GSH content and SOD activity also effectively decreased(P<0.05);meanwhile,it was found that the above treatment significantly decreased the mRNA expression levels of CAT,HO-1,NQOl and GST(P<0.05),but have an upregulated effect on the protein expression levels of Keapl and p62(P<0.05);LDH activity and MDA content were also significantly increased(P<0.05)in EpH4-Ev cells.Compared with the control group,sip62 treatment significantly reduced the protein expression levels of Nrf2 and NQO1(P<0.05).while significantly increased the protein expression level of Keap1(P<0.05)after S.uberis infection and resveratrol pretreatment in EpH4-Ev cells.The findings suggested that resveratrol can mediate p62 to regulate the Keap1/Nrf2 signaling pathway,activate the expression of downstream antioxidant-related proteins,and alleviate the oxidative damage caused by S.uberis in mouse mammary epithelial cells.3.The effect and mechanism of resveratrol on mammary glands inflammation induced by Streptococcus uberis in miceIn order to investigate the effect and mechanism of resveratrol on mammary glands inflammation induced by S.uberis in mice,this study used C57BL/6 mice as the research object.Mice were administered by intragastric gavage daily 50 mg·kg-1 resveratrol or an equal volume of physiological saline from gestation day 14 until parturition.After delivery 48 h,the female mice were perfused with S.uberis(S.uberis,Resveratrol+S.uberis group)or a corresponding volume of sterile saline(Control,Resveratrol group)with a breast infusion needle.After 24 h,mammary glands tissue and plasma samples were collected for related indicators determination.The results indicated that S.uberis perfusion can induce severe inflammation response in mammary tissue;although there is no significant difference in the bacterial load,the NAGase activity and MDA content significantly increased(P<0.05);protein expression levels of p62 and HO-1 were also considerably up-regulated in breast tissue(P<0.05).In addition,the content of MDA increased while the level of T-AOC decreased significantly in plasma samples(P<0.05).Preconditioning with resveratrol can ameliorate the inflammatory response and significantly reduce the increase of NAGase activity and MDA content caused by S.uberis infection in mammary glands tissue(P<0.05).At the genetic level,the expression levels ofp62,Nrf2,HO-1 and GST enhanced significantly(P<0.05),NQO1 and CAT showed an increasing trend in mammary glands tissue;at the protein level,the above treatments had an increased effect on the expression levels of Nrf2,NQO1 and HO-1 in mammary tissue.In addition,it was found that the T-AOC level was significantly increased while the MDA content was significantly decreased in plasma(P<0.05).The above results confirmed that resveratrol preadministration to C57BL/6 mice can alleviate the oxidative damage caused by S.uberis in mammary glands tissue via activating p62,Nrf2 and its downstream signaling pathways.In summary,resveratrol mediates Keapl degradation by activating p62,induces the expression of Nrf2 and its downstream antioxidant signaling pathways,and alleviates oxidative damage caused by S.uberis.The research provides a theoretical basis for the application of resveratrol,a polyphenolic compound,in the prevention and control of dairy cow mastisis.