Mechanism Of Forsythiaside A Inhibiting LPS Induced Inflammatory Response In MAC-T Cells

Cow mastitis is an increasingly severe health problem for dairy cows,which affects the welfare and milk production of dairy cows.Dairy cow mammary gland bacterial infection can lead to a series of clinical results,from acute and life-threatening to chronic and subclinical,which will affect the integrity of mammary gland tissue and reduce the production performance of dairy cows.Escherichia coli is one of the main mastitis pathogens causing clinical mastitis in dairy cows.Lipopolysaccharide(LPS)is a significant component of the outer membrane of E.coli,which is a potent initiator of inflammation and endotoxic shock.Therefore,how to detect and remove bacteria early and control inflammation is very important.At present,antibiotics are still an effective method for the treatment of cow mastitis,but due to the increasingly serious problem of drug resistance in recent years and people’s concerns about food safety,the uses of antibiotics has been limited.It is necessary to conduct new research on pharmacological methods for the treatment of this pathology.Since natural molecules and their derivatives are safer for animals,biological molecules are preferred.At present,a variety of biological molecules or products,results have been tested for the treatment of cow mastitis.Forsythia suspense is a traditional Chinese medicine,which has been used to treat mastitis for a long time.Forsythiaside A(FTA),as its main pharmacodynamic component,has many biological activities such as anti-inflammatory and anti-oxidation.In this study,we used cow mammary epithelial cells(MAC-T)as the experimental object,established MAC-T cell inflammation model by LPS induction.We detected cell viability by CCK-8 method,qRT-PCR,ELISA and Western blot.To evaluate the anti-inflammatory effect of FTA on MAC-T cells induced by LPS and elucidate its potential anti-inflammatory mechanism,the mRNA and protein expression levels of related inflammatory factors and NF-B and MAPK pathway were detected by blot,which provided theoretical basis for the effective screening of drugs for the treatment of mastitis.The experiments are divided into four parts:Part 1:The effect of cell culture and FTA on the activity of MAC-T cellsTo study the effect of FTA on the viability of MAC-T cells,the viability of MAC-T cells was detected after treated with FTA at different concentrations(2.5,5,10,20,40,80,160 μg/mL)for 12h.CCK-8 assay showed that FTA at the concentration of 0-40 μg/mL had no significant effect on the activity of MAC-T cells,while FTA at the concentration of 80 and 160 μg/mL significantly decreased the activity of MAC-T cells(P<0.01).According to the experimental results,FTA concentrations of 7.5,15 and 30 μg/mL were selected to study the protective effect of FTA on LPS induced inflammatory injury of MAC-T cells.Part 2:Establishment of MAC-T cell inflammation modelTo determine the effect of LPS on MAC-T cells’ inflammatory injury,different concentrations of LPS(0,50,100,150,200,250 μg/mL)were used to treat MAC-T cells for different time(12,24,48 h).The results of CCK-8 assay showed that LPS could reduce the cell viability in a time and dose-dependent manner.qRT-PCR showed that the expression levels of IL-6,IL-8 and TNF-α were the highest at 100 μg/mL after LPS treatment at different concentrations(0,50,100,150,200 and 250 μg/mL)for 12 h.According to the experimental results,100μg/mL LPS was selected as the injury model condition for 12 h.Part 3:Sequencing analysis of the transcriptome of MAC-T cells induced by LPSA reference genome transcriptome sequencing analysis showed a total of 139 differential genes in the model group and the control group,including 121 up-regulated genes and 18 down-regulated genes.GO analysis showed that differential genes were mainly concentrated in binding,extracellular region,cytokine-mediated signaling pathways,etc.,which had a more significant impact on physiological functions.KEGG enrichment analysis found that multiple differential genes are mainly enriched in the IL-17 signaling pathway,etc.Therefore,it is preliminarily clarified that the activated signaling pathway induces the release of many of inflammatory factors,resulting in inflammatory damage.Part 4:Molecular mechanism of FTA on LPS induced MAC-T cell inflammationFTA at different concentrations inhibited the activity of NF-κB and the transduction of MAPK signal pathway by reducing the degradation of IkBa and the phosphorylation levels of p65,p38 and ERK1/2,thus reducing the expression levels of IL-6,IL-8 and TNF-αinduced by LPS,and t hen weakening the LPS induced inflammatory injury.The results s how that FTA can protect MAC-T cells from inflammatory injury by interfering with the activation of NF-κB and MAPK signaling pathways and reducing the expression of IL-6,IL-8 and TNF-a.