Anti-oxidative And Anti-inflammatory Effects Of Puerarin On Bovine Mammary Epithelial Cells

During the perinatal period,the mammary epithelial cells of dairy cows have vigorous metabolism and generate a large amount of reactive oxygen species（ROS）.Disruption of redox balance leads to oxidative stress,one of the main causes of mastitis.Milk production and milk quality are adversely affected by mastitis in dairy cows.Antibiotics are the mainstay of clinical treatment for mastitis.The application of a large number of antibiotics will lead to the emergence of drug-resistant bacteria,and the problem of bacterial resistance in high-yielding dairy cows is particularly serious.In addition,antibiotic residues are present in milk from cows treated with antibiotics.Puerarin（PUE）,a natural flavonoid in Pueraria,has attracted widespread attention as a potential antioxidant.However,it is unknown whether puerarin can act as anti-inflammatory and anti-oxidative stress in bovine mammary epithelial cells.Therefore,the purpose of this study was to investigate whether puerarin had anti-inflammatory and anti-oxidative stress effects on cow mammary epithelial cells.In this study,the relieving effect of Pueraria lobata on bovine mastitis was first investigated through in vivo experiments.Pick 20 cows.There are 10 normal cows and 10 cows with mastitis.Divided into normal cows group,normal cows fed puerariae powder group,mastitis cows group,mastitis cows fed puerariae powder group.5 cows per group.The feeding experiment was carried out for a total of 7days.The contents of TNF-α,IL-6 and IL-1βin milk and serum were detected before and after feeding the experimental cows with Pueraria lobata（P<0.01）.In vivo experiments confirmed that feeding puerariae powder can reduce the expression of inflammatory factors in milk and serum of mastitis cows.Next,we tested at the cellular level whether puerarin could alleviate inflammation in cow mammary epithelial cells.First,a model of oxidative stress and inflammation in cow mammary epithelial cells was constructed by H₂O₂treatment.The effect of different concentrations of H₂O₂（0μM,200μM,400μM,600μM,800μM and 1000μM）on cell viability for 24 h was detected by CCK8 assay.The results showed that the cell viability of bovine mammary epithelial cells decreased at the concentrations of 200μM and 400μM H₂O₂（P<0.05）;600μM and higher concentrations of H₂O₂significantly decreased the viability of bovine mammary epithelial cells（P<0.01）.600μM H₂O₂was subsequently chosen to establish inflammation and model of oxidative stress in cow mammary epithelial cells.Reactive oxygen species（P<0.01）and MDA（malondialdehyde）levels were increased（P<0.01）after H₂O₂treatment of cow mammary epithelial cells.T-AOC（total antioxidant capacity）（P<0.01）,SOD（superoxide dismutase）（P<0.01）,CAT（catalase）（P<0.05）,GSH（reduced glutathione）)（P<0.01）and other indicators decreased.The results of real-time quantitative PCR showed that the m RNA expression of inflammatory factors（CCL5,IL-8,and IL-6）in cow mammary epithelial cells was significantly increased under H₂O₂treatment（P<0.01）.The m RNA expression levels of blood-milk barrier-related genes Occludin,Symplekin,Claudin-4 and ZO-1 were significantly decreased under H2O2 treatment（P<0.01）.This study further explored whether puerarin could alleviate H₂O₂-induced oxidative damage and inflammation in bovine mammary epithelial cells,and elucidate its molecular mechanism.The bovine mammary epithelial cells were divided into 4 treatment groups:control group,stimulation group（H₂O₂）,rescue group（puerarin+H₂O₂）and puerarin group.The growth of bovine mammary epithelial cells in each group was observed,and oxidative stress-related indicators were detected.Fluorescence quantitative PCR（QRT-PCR）was used to detect the expression of tight junction genes,antioxidant genes and inflammatory factors,and Western blot was used to detect the expression of NF-κB protein.The results showed that puerarin treatment increased the activities of glutathione（GSH）（P<0.01）,catalase（CAT）（P<0.05）and superoxide dismutase（SOD）in bovine mammary epithelial cells（P<0.05）.<0.01),alleviated H₂O₂-induced oxidative stress.Reactive oxygen species（P<0.01）and malondialdehyde（MDA）（P<0.01）levels were decreased.Total antioxidant capacity（T-AOC）increased（P<0.01）.Puerarin also inhibited H₂O₂-induced expression of NF-κB protein and related inflammatory factors（IL-6 and IL-8）and chemokine CCL5 in bovine mammary epithelial cells（P<0.01）.Puerarin treatment of bovine mammary epithelial cells abolished the inhibition of H₂O₂expression of antioxidant genes and tight junction genes,and increased the enrichment of NRF-2,HO-1,x CT,Claudin4,Occludin,ZO-1 and Symplekin（P<0.01）.In conclusion,feeding pueraria root powder to dairy cows with mastitis can reduce the contents of TNF-α,IL-6 and IL-1βin serum and whey.Puerarin can strengthen tight junctions between cells,reduce oxidative stress in bovine mammary epithelial cells,and exert anti-inflammatory effects.This study provides a theoretical basis for puerarin to alleviate mastitis and oxidative stress.Consider using pueraria powder as a feed additive in future dairy farming.