Anti-inflammatory Effect And Its Mechanism Of Glycyrrhizic Acid On LPS-induced Mastitis

Object: To establish LPS-induced mouse mastitis animal model and cell model and mastitis origin animal cell model.The anti-mastitis mechanism of glycyrrhizic acid was further analyzed.Methods:(1)Bacteriostasis of glycyrrhizic acid in vitro by MIC testThe bacteriostasis and bactericidal effects of glycyrrhizin on Escherichia coli,Streptococcus agalactiae and Staphylococcus aureus,the main pathogens of dairy mastitis,were tested.(2)Establishment of LPS-induced mammary inflammatory injury model in mice(1)The pathological changes of mammary gland tissues of female mice stimulated by LPS were observed.(2)Histopathological section and immunohistochemical section of myeloperoxidase(MPO)were used to observe the histopathological changes and the distribution and activity of MPO in the mammary tissues of female mice.(3)The expression level of TNF-α in mammary gland tissue was detected by enzyme linked immunosorbent assay(ELISA).(3)The protective effect of glycyrrhizin on LPS-induced mastitis injury in mice(1)The pathological changes of breast tissue and the distribution and activity of MPO were observed.(2)The expression levels of TNF-α,IL-1β and IL-6 in mammary gland tissues were detected by ELISA,and the mRNA expression levels of each cytokine were detected by fluorescence quantitative PCR.(3)Western blot and fluorescence quantitative PCR were used to detect the effects of glycyrrhizin on the protein expression levels and gene expression levels of IκBα and P65 in TLR4/NF-κB pathway and P38,JNK and ERK in MAPK pathway.(4)The protective effect of glycyrrhizin on LPS-stimulated mammary epithelial cells of mouse(1)The toxicity of glycyrrhizin on mouse mammary epithelial cells was detected by MTT assay.(2)The expression levels of TNF-α,IL-1β,IL-6 and other inflammatory factors in mouse mammary epithelial cells were detected by GLYcyrrhizin BY ELISA,and TNF-α was analyzed by fluorescence quantitative PCR.(3)The effects of glycyrrhizin on the expression levels of IκBα,P65 and P38,JNK and ERK proteins in NF-κB pathway and the expression levels of mRNA in MAPK pathway were detected by Western blot and fluorescence quantitative PCR.(5)The protective effect of glycyrrhizin on LPS-stimulated bovine mammary epithelial cells(1)The toxicity of glycyrrhizin on bovine mammary epithelial cells was detected by MTT assay.(2)The expression levels of TNF-α,IL-1β and IL-6 in mammary epithelial cells of dairy cows were detected by glycyrrhizin ELISA method,and TNF-αwas analyzed by fluorescence quantitative PCR.(3)The effects of glycyrrhizin on the expression levels of IκBα,P65 and P38,JNK and ERK in NF-κB pathway and mRNA expression levels in MAPK pathway were detected by Western blot and fluorescence quantitative PCR.Results: Glycyrrhizin can inhibit E.coli,S.aureus and S.agalactiae in vitro.In LPS-induced mouse mastitis model,histopathology,immunohistochemistry,ELISA,fluorescence quantitative PCR and Western blot experiments showed that:(1)different concentrations of glycyrrhizin can inhibit LPS-induced mouse mastitis;(2)the secretion and expression of TNF-α,IL-1β and IL-6 in LPS-stimulated cells(including mouse mammary epithelial cells and cow mammary epithelial cells)were decreased by different concentrations of glycyrrhizin;and(3)glycyrrhizin could inhibit the key molecules of LPS-induced NF-κB pathway(IκBα,P65)and MAPK pathway(P38,JNK and ERK)by the analysis of protein expression and mRNA transcription.Conclusions: Glycyrrhizin can inhibit l PS-induced inflammatory response in the level of experimental animals,cells and molecules,respectively.The mechanism plays an important role in the prevention and treatment of dairy cow mastitis.