Effects Of Catechins On LTA-induced Inflammation And Oxidative Stress Response In Bovine Mastitis

Dairy mastitis is one of the most common infectious diseases of dairy cows,which will have a serious impact on the performance of dairy cows and lead to a large number of production losses in the dairy industry.Staphylococcus aureus is one of the main pathogenic microorganisms causing mastitis in dairy cows.Lipoteichoic acid(LTA),the main component of its cell wall,can cause inflammation and oxidative stress.Antibiotics are generally preferred in the treatment of cow mastitis in many countries around the world.However,the abuse of antibiotics will cause bacterial resistance and antibiotic residues in milk,which seriously harm the health of consumers.The active ingredient of traditional Chinese medicine has low toxicity,no residue and is not easy to develop drug resistance,which has been widely concerned in the prevention and treatment of cow mastitis.Catechins,as the tea polyphenol component with the highest content and the most widely studied in tea,have the effect of controlling inflammation and reducing oxidative stress.However,the effect of catechins on LTA induced mastitis and its regulatory mechanism remain to be clarified.This study aims to explore whether catechin can improve mammary inflammation and oxidative stress induced by LTA and analyze its possible molecular mechanism through the combination of in vivo and in vitro experiments,so as to confirm the therapeutic effect of catechin on LTA-induced mastitis of dairy cows and provide reference and basis for clinical medication of mastitis.(1)Establishment of MAC-T cell inflammation and oxidative stress model in vitroTo ensure the success of subsequent tests the optimal inflammatory concentration of LTA should be determined first.CCK-8 was used to detect cell activity,and MAC-T cells were significantly affected when the concentration of LTA was greater than 20 μg/mL.The expression of pro-inflammatory cells(IL-1β,IL-6,TNF-α)was detected by RT-qPCR,and the results showed that the expression levels of pro-inflammatory factors were increased 1.3,1.3,and 7.2 times,respectively.Reactive oxygen species(ROS)activity detection kit,malondialdehyde detection kit and antioxidant enzyme detection kit were used to further detect the expression levels of oxidative stress indexes in LTA-induced MAC-T cells.The results showed that ROS and MDA were produced in large quantities,ROS expression was increased by 9.5 times,MDA content was increased from 1.19 nmol/mg to 4.62nmol/mg,the expression level was increased by 3.9 times,and SOD and CAT antioxidant enzymes were inhibited.According to the test results,20 μg/mL LTA concentration was selected in this study to construct the in vitro model.(2)Study on the mechanism of catechin alleviating LTA induced inflammation and oxidative stress in MAC-T cellsIn order to explore whether catechin has a alleviating effect on LTA induced MAC-T cells,the safe concentration of MAC-T cells treated with catechin was measured by CCK-8method.The results showed that 80 μg/mL catechin significantly affected cell viability(P<0.05).Therefore,in this study,blank control group(CG group),inflammatory model group(LTA group),D1 group(LTA+20 μg/mL catechin),and D2 group(LTA+40 μg/mL catechin)were set for follow-up tests.RT-qPCR was used to detect the inhibitory effect of catechin on inflammatory mediating factors IL-1β,IL-6,and TNF-α.The results showed that catechin reduced the level of inflammatory factors in LTA-induced MAC-T cells,and40 μg/mL dose group had the best inhibitory effect.Western blot assay was used to detect the effects of catechin on the expression and phosphorylation of NF-κB,MAPK pathway,Nrf2 pathway and pro-inflammatory cytokine protein in MAC-T cells induced by LTA.The results showed that compared with the LTA group,catechin could inhibit the phosphorylation levels of p38,ERK and JNK in MAPK signaling pathway.Inhibit the phosphorylation of p65 and IκBA in NF-κB signaling pathway,activate the Nrf2 pathway and the expressions of HO-1,NQO1 signal and SOD,CAT and other antioxidant enzymes,and reduce the accumulation of ROS and MDA.The results of this study showed that catechin could relieve LTA induced inflammation and oxidative stress in MAC-T cells.(3)Mouse model to verify the regulation effect of catechin on mammary gland inflammation and oxidative stressAs a kind of large economic animal,the economic cost of dairy cow is too high,and it is inconvenient in practical application and operation,so it is not suitable for scientific research directly as the research object.In this experiment,mice are used as the test animal to carry out the experiment.Blank control group,inflammation model group(LTA group),low(LTA+10 mg/kg catechin),medium(LTA+20 mg/kg catechin),high(LTA+30 mg/kg catechin)dose group and dexamethasone(LTA+5 mg/kg)dose group were set.The average body weight of mice induced inflammation by LTA decreased by about 6.5 g,and that of the LTA+20 mg/kg catechin treatment group decreased by about 3.6 g.Obvious breast nodules and symptoms of vascular tension and swelling were observed in the LTA biopsy group,and pathological reactions were alleviated after treatment with different concentrations of catechin.H&E staining showed that acinus hyperplasia,cell exhalation and necrosis,and neutrophil infiltration were observed after LTA inflammation,and the situation was improved after different concentrations of catechin treatment compared with LTA inflammation group.TLR2 receptor can be recognized in mouse mammary tissue induced by LTA,and MAPK,NF-κB signaling pathway can be activated to increase the release of TNF-α,IL-1β and IL-6.Different concentrations of catechins promoted the expression of Nrf2 protein,increased downstream antioxidant genes,inhibited the activation of MAPK and NF-κB signaling pathways,and reduced the release of pro-inflammatory factors.The results showed that catechin could relieve inflammation and oxidative stress of mastitis in mice.