Protective Effects Of Propolis Against Bacterial Lipopolysaccharide-induced Mastitis

Propolis is an adhensive substance formed by worker bees(Apis mellifera L.)collecting one or more plant resins and mixing the secretions of their maxillary and wax glands.The ingredients of propolis are complex and varied.At present,there are more than 20 types over 600 chemical components have been identified from propolis.Research reportes that propolis is widely used for its varous biological activities,such as antiinflammatory,antibacterial,antioxidant,enhanceing immune and protecting liver.Mastitis is an important disease caused by a variety of factors,seriously affecting the development of the dairy industry.So far,there is no complete solution.LPS(Lipopolysaccharide)is the main component of the cell wall of Escherichia coli.Once in the body,it can cause the cascade reaction of immune stimulation and the body's toxic pathophysiological activities,activate NF-?B and MAPK signaling pathways as well as release inflammatory cytokines that cause inflammation.Studies imply that propolis can effectively kill mastitis pathogens and can be used as an adjuvant to enhance the immunity of vaccines,and some studies have used propolis to make breast wipes and nipple protection films to against mastitis.In this paper,the purpose is to investigate the effect of propolis on LPS-induced mastitis in vivo and in vitro.The main contents and results are as follows:(1)EECP(Ethanol extract of Chinese propolis)component analysis: Folinol method and aluminum nitrate method were used to determine the content of total phenols and total flavonoids in EECP.The results demonstrated that the total phenolic acid was 106.35 mg GAE(gallic acid equivalent)/g and the total flavonoids was 320.85 mg RE(rutin equivalent)/g.The accurate content of 17 major phenolic flavonoids in EECP were detected by UHPLC-QQQ-LC/MC.The highest contents were galangin(12.88±0.57 ?g/mg),3-O-Acetylpinobanksin(12.93±0.59 ?g/mg),pinocembrin(8.56±0.27 ?g/mg)and pinobanksin(8.52±0.25 ?g/mg).(2)Screening of safe concentration of EECP on MAC-T cells and its effect on cell viability under LPS stimulation: 0 ?g / m L,2.5 ?g / m L,5 ?g / m L,10 ?g / m L,15 ?g / m L and 20 ?g / m L of EECP were selected and treated for 24 hours,and the cell survival rates is measured by CCK-8.Results indicated the safe concentration range of EECP was 0-15 ?g /m L.After the addition of 0-15 ?g / m L EECP pretreated cells for 2 h,measure the viability of MAC-T cells were stimulated by 1 ?g / m L LPS for 24 h.The viability of MAC-T cells in the LPS model group was significantly lower than that in the control group(P<0.05),while EECP effectively increased the viability of cells.The results revealed that EECP had a protective effect on MAC-T cells damage induced by LPS.(3)EECP analysis of transcriptome data of LPS-induced MAC-T cells inflammation model: RNA-seq method is used to compare the LPS group and EECP + LPS group cell transcriptome analysis,evaluation of EECP in MAC-T cells inflammation induced by LPS.Gene expression profile demonstrated that compared with the LPS group,2490 genes were significantly differentially expressed in the EECP+LPS group(P<0.05),which 1130 were up-regulated and 1360 were down-regulated.There are 79 genes related to inflammation and immunity were detected in DEGs(differentially expressed genes),31 genes are up-regulated and 48 genes are down-regulated.In addition,DEGs enriched the most genes in GO term such as cell process,biological regulation,cell components and binding activity.KEGG pathway analysis illustrated that DEGs were mainly enriched in MAPK(mitogen-activated protein kinase)signaling pathway,interleukin-17 signaling pathway,the cell growth-related Fox O signaling pathway and apoptosis-related p53 signaling pathway.(4)Anti-inflammatory effects of EECP on LPS-induced MAC-T cells inflammation model: RT-q PCR technology was used to detect inflammation-related factors and tight junction genes.It was found that EECP inhibited the m RNA transcription levels of IL-6,IL-8,IL-1?,TNF-a and improved the tight junction protein genes(occludin,ZO-1)m RNA transcription levels.The results of cellular immunofluorescence and western blotting showed that EECP could enhance the distribution and expression of occludin and ZO-1.In addition,western blotting results suggest that EECP reduced the expression levels of p-P65,p-Ikb and p-JNK,and inhibited the activation of NF-?B and MAPK signaling pathways.(5)Effect of EECP on LPS-induced mammary gland inflammatory injury in mouse: The anti-mastitis activity of EECP in vivo was evaluated by LPS-induced mastitis model in mouse with nipple duct perfusion.The tests were divided into blank control group,model group(LPS,1 mg / kg),EECP group(100 mg / kg),and positive control group(DEX,5 mg / kg).After delivery,EECP or positive control was administered to the female rats for 7 days,and then the rats were injected with 1 mg / kg LPS via the fourth and fifth pairs of nipple tubes and sacrificed 24 hours later to induce a model of acute mastitis in mouse.The samples were stained with HE,ELISA was used to detect inflammatory factors,RT-q PCR was used to detect the m RNA transcription level of tight junction protein genes,and immunohistochemical analysis of breast tissue.The results indicated that EECP can reduce the infiltration of inflammatory cells and relieve the damage to the acinar structure of the breast under LPS stimulation.Inhibits the overexpression of cytokins such as IL-1?,IL-6 and IL-10 in mouse breast tissue.Increase the m RNA transcription level of occludin,cluadin-1 and ZO-1 as well as enhances the expressions of occludin and ZO-1 on the cell membrane of breast tissues.In conclusion,EECP has a good protective effect on bacterial lipopolysaccharide-induced mastitis,which provides a test basis for the clinical application of Chinese propolis to prevent mastitis disease.