Study On The Therapeutic Effect Of Caffeic Acid On LPS-induced Mastitis In Mice

Caffeic acid(CA)is a natural phenolic compound,which is rich in medicinal plants and has anti-inflammatory and antioxidant effects.In this study,the mouse mastitis model induced by lipopolysaccharides(LPS)was used to investigate the therapeutic effect of CA on this model,and the inhibitory effect of CA on lps-induced mouse macrophages(RAW264.7)on inflammation and immune chemoattractant and its molecular mechanism,so as to provide certain theoretical basis for the treatment of escherichia coli dairy cow mastitis with CAMethods:(1)48 female ICR mice on day 7-11 of lactation were randomly divided into Control,LPS,LPS+CA5,LPS+CA10,LPS+CA15 and CA15 groups,8 in each group.Except the Control group and the CA group,mice in the other groups were injected with 50 mL LPS(200 mL g/mL)diluent in the milk region(the fourth pair),and 50 mL CA(5,10,15 mg/kg)diluent in the CA treatment group after 1 hour.The Control group was injected with 50 mL normal saline,and the CA group was injected with 50 mL CA(15 mg/kg)diluent.After 24 hours,the mice in each group were sacrificed by cervical dissection and breast tissue was collected.(2)RAW264.7 was divided into 6 groups:Control,LPS,LPS+CA10,LPS+CA25,LPS+CA50 and CA50.Except the Control group and the CA group,the other groups were given LPS(1 mL)stimulation,and the CA treatment group was given CA(10?25?50 ?g/mL)treatment for 11h after 1 h.The Control group was cultured normally,and the CA group was treated with CA(50 ?g/mL)for 12 h.After the end of the experiment,the cells were fixed and total protein and RNA samples were extracted.The research adopted the HE stain and immunofluorescence to observe the tissue structure damage or locate the protein and detected the intracellular ROS content?mitochondrial membrane potential and cell apoptosis by flow cytometry.Also RT-PCR and ELISA were used to test the change of the inflammatory factor?chemotactic factor?REDOX index which were included in the tissue and cell.By using western blot,the changes of the protein expression of the tissue and intracellular apoptosis-related proteins?the key protein of the NF-?B signal path expression changes were monitoredResults:(1)LPS group mice breast hyperemia,swelling,acinar structure damage,a large number of inflammatory cell infiltration,tight junction protein(Claudin-1)significantly reduced.The expression levels of iL-1 ?,iL-6,TNF-?,MIF,CXCL-2 were significantly increased.The content of MPO and MDA increased significantly,and the activity of SOD and GSH-Px decreased significantly.The expression levels of Bax/Bcl-2,p-p65 and p-I?B? were significantly increased.The degree of breast tissue injury was significantly reduced in the CA treatment group.The expression levels of inflammation and chemokines were significantly decreased.The contents of MPO and MDA were significantly decreased,and the activities of SOD and gsh-px were significantly increased.Bax/Bcl-2 expression was significantly decreased,and Bax/Bcl-2,p-p65,p-I?B? expression levels were significantly decreased,among which the high-dose group had the best effect.(2)the combination of Park7 and P47phox in LPS group increased significantly;The intracellular ROS content significantly increased,the mitochondrial membrane potential significantly decreased,and the apoptosis rate significantly increased.The expression levels of iL-1?,iL-6,TNF-?,MIF,CXCL-2 were significantly increased.The content of MPO and MDA increased significantly,and the activity of SOD and gsh-px decreased significantly.The expression of Bax/Bcl-2,p-p65,p-I?B? was significantly increased.The combination of Park7 and P47phox was significantly reduced in the CA treatment group.The intracellular ROS content significantly decreased,the mitochondrial membrane potential significantly increased,and the apoptosis rate significantly decreased.The expressions of inflammation and chemokines were significantly decreased.The contents of MPO and MDA were significantly decreased,and the activities of SOD and GSH-Px were significantly increased.The expression of Bax/Bcl-2,p-p65,p-I?B? was significantly decreased.Conclusion:In vivo and in vitro studies have shown that CA inhibits Park7 binding to P47phox and regulates NF-?B signaling pathway,thereby inhibiting inflammation and oxidative stress between mouse mammary tissue and RAW264.7 and reducing LPS-induced mammary tissue damage in mice.